Leslie I Gold, Ph.D.

Calreticulin: non-endoplasmic reticulum functions in physiology and disease
Gold, Leslie I; Eggleton, Paul; Sweetwyne, Mariya T; Van Duyn, Lauren B; Greives, Matthew R; Naylor, Sara-Megumi; Michalak, Marek; Murphy-Ullrich, Joanne E
2010 Mar;24(3):665-683, FASEB journal
Calreticulin (CRT), when localized to the endoplasmic reticulum (ER), has important functions in directing proper conformation of proteins and glycoproteins, as well as in homeostatic control of cytosolic and ER calcium levels. There is also steadily accumulating evidence for diverse roles for CRT localized outside the ER, including data suggesting important roles for CRT localized to the outer cell surface of a variety of cell types, in the cytosol, and in the extracellular matrix (ECM). Furthermore, the addition of exogenous CRT rescues numerous CRT-driven functions, such as adhesion, migration, phagocytosis, and immunoregulatory functions of CRT-null cells. Recent studies show that topically applied CRT has diverse and profound biological effects that enhance cutaneous wound healing in animal models. This evidence for extracellular bioactivities of CRT has provided new insights into this classically ER-resident protein, despite a lack of knowledge of how CRT exits from the ER to the cell surface or how it is released into the extracellular milieu. Nonetheless, it has become clear that CRT is a multicompartmental protein that regulates a wide array of cellular responses important in physiological and pathological processes, such as wound healing, the immune response, fibrosis, and cancer.-Gold, L. I., Eggleton, P., Sweetwyne, M. T., Van Duyn, L. B., Greives, M. R., Naylor, S.-M., Michalak, M., Murphy-Ullrich, J. E. Calreticulin: non-endoplamic reticulum functions in physiology and disease
— id: 107766, year: 2010, vol: 24, page: 665, stat: Journal Article,

UNLOCKING BIOMARKER DISCOVERY FOR EARLY DETECTION OF LUNG CANCER
Ostroff, R.; Bigbee, W.; Franklin, W.; Gold, L.; Mehan, M.; Miller, Y.; Pass, H.; Rom, W.; Siegfried, J.; Stewart, A.; Walker, J.; Weissfeld, J.; Williams, S.; Zichi, D.; Brody, E.
2010 AUG ;31:S14-S14, Tumour biology
— id: 132750, year: 2010, vol: 31, page: S14, stat: Journal Article,

Non-ER outside-in functions of the ER chaperone calreticulin in diabetic wound repair
Samra F.; Naylor S.-M.; Gorovets D.; Pavlides S.; Murphy-Ullrich J.E.; Levine J.P.; Warren S.M.; Gold L.I.
2010 ;18(2):A28-A28, Wound repair & regeneration
We previously reported that topically applied calreticulin (CRT), a calcium-binding ER chaperone protein comprising N, P, and C domains, markedly enhances diabetic murine (db/db) and porcine cutaneous wound healing. Consistent with the potent wound healing effects, we further showed, in vitro, that exogenous CRT stimulated proliferation of keratinocytes and fibroblasts, induced concentration-dependent migration of these cells and monocytes and macrophages, and upregulated protein expression of collagen, fibronectin, and TGF-beta-3 in fibroblasts. Notably, all these broad-ranging effects purport novel non-ER functions for CRT that act from outside the cell inward. The current studies address: 1) whether the ER chaperone function of CRT is required for its extracellular functions, 2) the molecular structure(s) of CRT that function in its biological activities and 3) the in vitro effects of CRT on diabetic compared to normal mouse and human fibroblasts. Using CRT null mouse embryo fibroblasts (K42) compared to wild type (K41) in proliferation and migration assays (scratch plate and chamber), we show that exogenous CRT stimulates proliferation of null K42 cells to a similar extent as K41 cells (2-fold at 10 pg/ml). However, K42 cells require 100 times more CRT for a peak migratory response (1 vs 100 ng/ml), with a 20% decreased response. We also show that the C domain stimulates fibroblast proliferation to the same extent and peak response as the entire molecule. Finally, we show that fibroblasts isolated from db/db mouse skin and human fibroblasts cultured in high glucose, to simulate type II diabetes, respond to CRT by migration and proliferation albeit with 1/3 less robust response requiring 10-fold more CRT for peak responses compared to controls. The breath of novel non-ER functions of CRT, structure-function relationships, and effects on diabetic cells in vitro underscore this molecule as a potential potent agent for the topical treatment for healing diabetic wounds
— id: 135597, year: 2010, vol: 18, page: A28, stat: Journal Article,

TGFbeta prevents proteasomal degradation of the cyclin-dependent kinase inhibitor p27kip1 for cell cycle arrest
Lecanda, Jon; Ganapathy, Vidya; D'Aquino-Ardalan, Christine; Evans, Brad; Cadacio, Caprice; Ayala, Aidee; Gold, Leslie I
2009 Mar 1;8(5):742-756, Cell cycle
TGFbeta mediates cell cycle arrest in late G(1) phase of the cell cycle with a simultaneous peak in the levels of the cyclin-dependent kinase inhibitor, p27(kip1) (p27). In this report, we show that whereas p27 resides in the cytoplasm in the endometrial carcinoma (ECA) cell line HEC-1A, TGFbeta increases the total levels and translocation of p27 into the nucleus. Concomitantly, TGFbeta activates the transcription factors Smad2 and Smad3, inhibits proliferation, and blocks Cdk2 activity; all these events are blocked by an inhibitor of TbetaRI serine kinase activity (SD208). In addition, we show that inhibiting p27 transcription with a specific siRNA completely blocks TGFbeta-mediated growth inhibition in these cells. These data suggest that TGFbeta inhibits cellular proliferation by increasing p27 levels through Smad2/3 signaling in HEC-1A cells. We further show that TGFbeta decreases the levels of components of the SCF(Skp2) targeting complex for ubiquitin-mediated degradation of p27 in proteasomes, at the protein but not the mRNA level. Therefore, TGFbeta accumulates nuclear p27 by preventing its degradation to enable G(1) arrest in HEC-1A cells. Importantly, these data suggest a novel mechanism for TGFbeta/Smad mediated growth inhibition that might be inoperable in the numerous human cancers demonstrating early dysregulated TGFbeta signaling and loss of growth inhibition. The TGFbeta/p27 axis might provide novel therapeutic targets for cancer
— id: 93786, year: 2009, vol: 8, page: 742, stat: Journal Article,

Calreticulin, a multi-process calcium-buffering chaperone of the endoplasmic reticulum
Michalak, Marek; Groenendyk, Jody; Szabo, Eva; Gold, Leslie I; Opas, Michal
2009 Feb 1;417(3):651-666, Biochemical journal
Calreticulin is an ER (endoplasmic reticulum) luminal Ca2+-buffering chaperone. The protein is involved in regulation of intracellular Ca2+ homoeostasis and ER Ca2+ capacity. The protein impacts on store-operated Ca2+ influx and influences Ca2+-dependent transcriptional pathways during embryonic development. Calreticulin is also involved in the folding of newly synthesized proteins and glycoproteins and, together with calnexin (an integral ER membrane chaperone similar to calreticulin) and ERp57 [ER protein of 57 kDa; a PDI (protein disulfide-isomerase)-like ER-resident protein], constitutes the 'calreticulin/calnexin cycle' that is responsible for folding and quality control of newly synthesized glycoproteins. In recent years, calreticulin has been implicated to play a role in many biological systems, including functions inside and outside the ER, indicating that the protein is a multi-process molecule. Regulation of Ca2+ homoeostasis and ER Ca2+ buffering by calreticulin might be the key to explain its multi-process property
— id: 93787, year: 2009, vol: 417, page: 651, stat: Journal Article,

Calreticulin induces matrix proteins and integrins in keratinocytes and fibroblasts for enhanced wound healing
Gold, LI; Greives, MR; Woodrell, C; Chesser, AS; Bancroft, TA; Murphy-Ullrich, JE
2008 MAR-APR ;16(2):A15-A15, Wound repair & regeneration
— id: 76408, year: 2008, vol: 16, page: A15, stat: Journal Article,

Calreticulin enhances porcine wound repair by diverse biological effects
Nanney, Lillian B; Woodrell, Christopher D; Greives, Mathew R; Cardwell, Nancy L; Pollins, Alonda C; Bancroft, Tara A; Chesser, Adrianne; Michalak, Marek; Rahman, Mohammad; Siebert, John W; Gold, Leslie I
2008 Sep;173(3):610-630, American journal of pathology
Extracellular functions of the endoplasmic reticulum chaperone protein calreticulin (CRT) are emerging. Here we show novel roles for exogenous CRT in both cutaneous wound healing and diverse processes associated with repair. Compared with platelet-derived growth factor-BB-treated controls, topical application of CRT to porcine excisional wounds enhanced the rate of wound re-epithelialization. In both normal and steroid-impaired pigs, CRT increased granulation tissue formation. Immunohistochemical analyses of the wounds 5 and 10 days after injury revealed marked up-regulation of transforming growth factor-beta3 (a key regulator of wound healing), a threefold increase in macrophage influx, and an increase in the cellular proliferation of basal keratinocytes of the new epidermis and of cells of the neodermis. In vitro studies confirmed that CRT induced a greater than twofold increase in the cellular proliferation of primary human keratinocytes, fibroblasts, and microvascular endothelial cells (with 100 pg/ml, 100 ng/ml, and 1.0 pg/ml, respectively). Moreover, using a scratch plate assay, CRT maximally induced the cellular migration of keratinocytes and fibroblasts (with 10 pg/ml and 1 ng/ml, respectively). In addition, CRT induced concentration-dependent migration of keratinocytes, fibroblasts macrophages, and monocytes in chamber assays. These in vitro bioactivities provide mechanistic support for the positive biological effects of CRT observed on both the epidermis and dermis of wounds in vivo, underscoring a significant role for CRT in the repair of cutaneous wounds
— id: 93788, year: 2008, vol: 173, page: 610, stat: Journal Article,

Tumor-specific efficacy of transforming growth factor-beta RI inhibition in Eker rats
Laping, Nicholas J; Everitt, Jeffrey I; Frazier, Kendall S; Burgert, Mark; Portis, Melisa J; Cadacio, Caprice; Gold, Leslie I; Walker, Cheryl L
2007 May 15;13(10):3087-3099, Clinical cancer research
PURPOSE: Transforming growth factor beta (TGF-beta), which generally stimulates the growth of mesenchymally derived cells but inhibits the growth of epithelial cells, has been proposed as a possible target for cancer therapy. However, concerns have been raised that whereas inhibition of TGF-beta signaling could be efficacious for lesions in which TGF-beta promotes tumor development and/or progression, systemic pharmacologic blockade of this signaling pathway could also promote the growth of epithelial lesions. EXPERIMENTAL DESIGN: We examined the effect of a TGF-beta inhibitor on mesenchymal (leiomyoma) and epithelial (renal cell carcinoma) tumors in Eker rats, which are genetically predisposed to develop these tumors with a high frequency. RESULTS: Blockade of TGF-beta signaling with the ALK5/type I TGF-beta R kinase inhibitor, SB-525334, was efficacious for uterine leiomyoma; significantly decreasing tumor incidence and multiplicity, and reducing the size of these mesenchymal tumors. However, SB-525334 was also mitogenic and antiapoptotic for epithelial cells in the kidney and exacerbated the growth of epithelial lesions present in the kidneys of these animals. CONCLUSION: Although pharmacologic inhibition of TGF-beta signaling with SB-525334 may be efficacious for mesenchymal tumors, inhibition of this signaling pathway seems to promote the development of epithelial tumors
— id: 76284, year: 2007, vol: 13, page: 3087, stat: Journal Article,

Transforming growth factor-beta, estrogen, and progesterone converge on the regulation of p27Kip1 in the normal and malignant endometrium
Lecanda, Jon; Parekh, Trilok V; Gama, Patricia; Lin, Ke; Liarski, Vladimir; Uretsky, Seth; Mittal, Khush; Gold, Leslie I
2007 Feb 1;67(3):1007-1018, Cancer research
Hormones and growth factors regulate endometrial cell growth. Disrupted transforming growth factor-beta (TGF-beta) signaling in primary endometrial carcinoma (ECA) cells leads to loss of TGF-beta-mediated growth inhibition, which we show herein results in lack of up-regulation of the cyclin-dependent kinase inhibitor p27(Kip1) (p27) to arrest cells in G(1) phase of the cell cycle. Conversely, in normal primary endometrial epithelial cells (EECs), TGF-beta induces a dose-dependent increase in p27 protein, with a total 3.6-fold maximal increase at 100 pmol/L TGF-beta, which was 2-fold higher in the nuclear fraction; mRNA levels were unaffected. In addition, ECA tissue lysates show a high rate of ubiquitin-mediated degradation of p27 compared with normal secretory-phase endometrial tissue (SE) such that 4% and 89% of recombinant p27 added to the lysates remains after 3 and 20 h, respectively. These results are reflected in vivo as ECA tissue lacks p27 compared with high expression of p27 in SE (P < or = 0.001). Furthermore, we show that estrogen treatment of EECs causes mitogen-activated protein kinase-driven proteasomal degradation of p27 whereas progesterone induces a marked increase in p27 in both normal EECs and ECA cells. Therefore, these data suggest that TGF-beta induces accumulation of p27 for normal growth regulation of EECs. However, in ECA, in addition to enhanced proteasomal degradation of p27, TGF-beta cannot induce p27 levels due to dysregulated TGF-beta signaling, thereby causing 17beta-estradiol-driven p27 degradation to proceed unchecked for cell cycle progression. Thus, p27 may be a central target for growth regulation of normal endometrium and in the pathogenesis of ECA
— id: 70879, year: 2007, vol: 67, page: 1007, stat: Journal Article,

Overview of the role for calreticulin in the enhancement of wound healing through multiple biological effects
Gold, Leslie I; Rahman, Mohammad; Blechman, Keith M; Greives, Matthew R; Churgin, Samara; Michaels, Joseph; Callaghan, Matthew J; Cardwell, Nancy L; Pollins, Alonda C; Michalak, Marek; Siebert, John W; Levine, Jamie P; Gurtner, Geoffrey C; Nanney, Lillian B; Galiano, Robert D; Cadacio, Caprice L
2006 Sep;11(1):57-65, Journal of investigative dermatology symposium proceedings
Calreticulin (CRT), an intracellular chaperone protein crucial for the proper folding and transport of proteins through the endoplasmic reticulum, has more recent acclaim as a critical regulator of extracellular functions, particularly in mediating cellular migration and as a requirement for phagocytosis of apoptotic cells. Consistent with these functions, we show that the topical application of CRT has profound effects on the process of wound healing by causing a dose-dependent increase in epithelial migration and granulation tissue formation in both murine and porcine normal and impaired animal models of skin injury. These effects of CRTare substantiated, in vitro, as we show that CRT strongly induces cell migration/wound closure of human keratinocytes and fibroblasts, using a wound/scratch plate assay, and stimulates cellular proliferation of human keratinocytes, fibroblasts, and vascular endothelial cells, providing mechanistic insight into how CRT functions in repair. Similarly, in both animal models, the histology of the wounds show marked proliferation of basal keratinocytes and dermal fibroblasts, dense cellularity of the dermis with notably increased numbers of macrophages and well-organized collagen fibril deposition. Thus, CRT profoundly affects the wound healing process by recruiting cells essential for repair into the wound, stimulating cell growth, and increasing extracellular matrix production
— id: 69252, year: 2006, vol: 11, page: 57, stat: Journal Article,

Overview of the Role for Calreticulin in the Enhancement of Wound Healing through Multiple Biological Effects
Gold, Leslie I; Rahman, Mohammad; Blechman, Keith M; Greives, Matthew R; Churgin, Samara; Michaels, Joseph; Callaghan, Matthew J; Cardwell, Nancy L; Pollins, Alonda C; Michalak, Marek; Siebert, John W; Levine, Jamie P; Gurtner, Geoffrey C; Nanney, Lillian B; Galiano, Robert D; Cadacio, Caprice L
2006 Sep;126 Suppl:57-65, Journal of investigative dermatology
Calreticulin (CRT), an intracellular chaperone protein crucial for the proper folding and transport of proteins through the endoplasmic reticulum, has more recent acclaim as a critical regulator of extracellular functions, particularly in mediating cellular migration and as a requirement for phagocytosis of apoptotic cells. Consistent with these functions, we show that the topical application of CRT has profound effects on the process of wound healing by causing a dose-dependent increase in epithelial migration and granulation tissue formation in both murine and porcine normal and impaired animal models of skin injury. These effects of CRT are substantiated, in vitro, as we show that CRT strongly induces cell migration/wound closure of human keratinocytes and fibroblasts, using a wound/scratch plate assay, and stimulates cellular proliferation of human keratinocytes, fibroblasts, and vascular endothelial cells, providing mechanistic insight into how CRT functions in repair. Similarly, in both animal models, the histology of the wounds show marked proliferation of basal keratinocytes and dermal fibroblasts, dense cellularity of the dermis with notably increased numbers of macrophages and well-organized collagen fibril deposition. Thus, CRT profoundly affects the wound healing process by recruiting cells essential for repair into the wound, stimulating cell growth, and increasing extracellular matrix production.Journal of Investigative Dermatology Symposium Proceedings (2006) 11, 57-65. doi:10.1038/sj.jidsymp.5650011
— id: 69462, year: 2006, vol: 126 Suppl, page: 57, stat: Journal Article,

The activation function-1 domain of estrogen receptor alpha in uterine stromal cells is required for mouse but not human uterine epithelial response to estrogen
Kurita, Takeshi; Medina, Roanna; Schabel, Alex B; Young, Peter; Gama, Patricia; Parekh, Trilok V; Brody, Joel; Cunha, Gerald R; Osteen, Kevin G; Bruner-Tran, Kaylon L; Gold, Leslie I
2005 Jul;73(6):313-322, Differentiation
The activation function-1 (AF-1) domain of the estrogen receptor alpha (ERalpha) in stromal cells has been shown to be required for epithelial responses to estrogen in the mouse uterus. To investigate the role of the stroma in estrogenic responses of human uterine epithelium (hUtE), human/mouse chimeric uteri composed of human epithelium and mouse stroma were prepared as tissue recombinants (TR) that were grown in vivo under the renal capsule of female nude mouse hosts. In association with mouse uterine stroma (mUtS), hUtE formed normal glands surrounded by mouse endometrial stroma and the human epithelium influenced the differentiation of stroma into myometrium, such that a histologically normal appearing uterine tissue was formed. The hUtE showed a similar proliferative response and increase in progesterone receptors (PR) in response to 17beta-estradiol (E2) in association with either human or mUtS, as TRs. However, under identical endocrine and micro-environmental conditions, hUtE required 5-7 days exposure to E2 rather than 1 day, as shown for mouse uterine epithelium, to obtain a maximal proliferative response. Moreover, this extended length of E2 exposure inhibited mouse epithelial proliferation in the presence of mouse stroma. In addition, unlike the mouse epithelium, which does not proliferate or show regulation of PR expression in response to E2 in association with uterine stroma derived from mice that are null for the AF-1 domain of ERalpha, hUtE proliferates and PR are up-regulated in response to E2 in association genetically identical ERalpha knock-out mouse stromal cells. These results clearly demonstrate fundamental differences between mouse and human uterine epithelia with respect to the mechanisms that regulate estrogen-induced proliferation and expression of PR. Moreover, we show that genetically engineered mouse models could potentially aid in dissecting molecular pathways of stromal epithelial interactions in the human uterus
— id: 62762, year: 2005, vol: 73, page: 313, stat: Journal Article,

Gene expression changes induced by estrogen and selective estrogen receptor modulators in primary-cultured human endometrial cells: signals that distinguish the human carcinogen tamoxifen
Pole, Jessica C M; Gold, Leslie I; Orton, Terry; Huby, Russell; Carmichael, Paul L
2005 Jan 5;206(1):91-109, Toxicology
Tamoxifen has long been the endocrine treatment of choice for women with breast cancer and is now employed for prophylactic use in women at high risk from breast cancer. Other selective estrogen receptor modulators (SERMs), such as raloxifene, mimic some of tamoxifen's beneficial effects and, like tamoxifen, exhibit a complex mixture of organ-specific estrogen agonist and antagonistic properties. However, accompanying the positive effects of tamoxifen has been the emergence of evidence for an increased risk of endometrial cancer associated with its use. A more complete understanding of the mechanism(s) of SERM carcinogenicity and endometrial effects is therefore required. We have sought to compare and characterise the transcript profile of tamoxifen, raloxifene and the agonist estradiol in human endometrial cells. Using primary cultures of human endometria, to best emulate the in vivo responses in a manageable in vitro system, we have shown 230 significant changes in gene expression for epithelial cultures and 83 in stromal cultures, either specific to 17beta-estradiol, tamoxifen or raloxifene, or changed across more than one of the treatments. Considering the transcriptome as a whole, the endometrial responses to raloxifene or tamoxifen were more similar than either drug was to 17beta-estradiol. Treatment of endometrial cultures with tamoxifen resulted in the largest number of gene changes relative to control cultures and a high proportion of genes associated with regulation of gene transcription, cell-cycle control and signal transduction. Tamoxifen-specific changes that might point towards mechanisms for its proliferative response in the endometrium included changes in retinoblastoma and c-myc binding proteins, the APCL, dihydrofolate reductase (DHFR) and E2F1 genes and other transcription factors. Tamoxifen was also found to give rise to the highest number of gene expression changes common to those that characterise malignant endometria. It is anticipated that this study will provide leads for further and more focused investigation into SERM carcinogenicity
— id: 62763, year: 2005, vol: 206, page: 91, stat: Journal Article,

Differential expression of transforming growth factor-beta type I and II receptors by pulmonary cells in bleomycin-induced lung injury: correlation with repair and fibrosis
Khalil, Nasreen; Parekh, Trilok V; O'Connor, Robert N; Gold, Leslie I
2002 Apr-May;28(3):233-250, Experimental lung research
In a rat model of lung injury induced by the antineoplastic antibiotic, bleomycin, there is loss of type I alveolar epithelial cells (AECs) followed by infiltration of activated inflammatory cells, type II AEC proliferation, and fibrosis. At 4 and 7 days after bleomycin administration alveolar macrophages have increased production and release of active transforming growth factor (TGF)-beta1, an inhibitor of epithelial cell proliferation. Paradoxically at these same time intervals there is a concomitant induction of type II AEC proliferation. For TGF-beta-mediated signal transduction to occur, the expression of both TCF-beta receptor types I (TbetaR-I) and II (TbetaR-II) must be present. Using immunohistochemistry and in situ hybridization, 4 and 7 days after bleomycin administration the expression of TbetaR-I on AECs was reduced whereas that of TbetaR-II was unaltered. However, 14 and 28 days after bleomycin injury, when there is decreased proliferation and induction of differentiation of type II AECs, there was a return of TbetaR-I expression on AECs. In contrast, TbetaR-I and TbetaR-II were observed on interstitial fibroblasts at all time intervals after bleomycin administration. Because both TbetaR-I and TbetaR-II are required for signal transduction, the reduction of TbetaR-I levels on the alveolar epithelium may alter the sensitivity of AECs to the antiproliferative effects of TGF-beta1 present in increased quantities following bleomycin injury. The loss of an antiproliferative response to TGF-beta1 may be important for the regeneration of the alveolar epithelium by proliferation while the expression of both receptors onfibroblasts would result in TGF-1 signaling for the synthesis of connective tissue proteins. Ourfindings suggest that during bleomycin-induced pulmonary fibrosis, the effects of TGF-beta1 on cells may be regulated by the expression of TbetaRs
— id: 62764, year: 2002, vol: 28, page: 233, stat: Journal Article,

Transforming growth factor beta signaling is disabled early in human endometrial carcinogenesis concomitant with loss of growth inhibition
Parekh, Trilok V; Gama, Patricia; Wen, Xie; Demopoulos, Rita; Munger, John S; Carcangiu, Maria-Luisa; Reiss, Michael; Gold, Leslie I
2002 May 15;62(10):2778-90, Cancer research
Transforming growth factor beta (TGF-beta), a potent ubiquitous endogenous inhibitor of epithelial cell growth, is secreted as a latent molecule (LTGF-beta)requiring activation for function. TGF-beta signals through the type I(TbetaRI) and type II (TbetaRII) receptors, which cooperate to phosphorylate/activate Smad2/3, the transcriptional regulators of genes that induce cell cycle arrest. That carcinomas grow in vivo suggests that they are refractory to TGF-beta. However, this has been difficult to prove because of an inability to analyze the functional status of TGF-beta in vivo as well as lack of close physiological paradigms for carcinoma cells in vitro. The current studies demonstrate that whereas primary cultures of endometrial epithelial cells derived from normal proliferative endometrium (PE; n = 10) were dose-dependently and maximally growth inhibited by 55% +/- 5.3% with 10 pM TGF-beta1, endometrial epithelial cells derived from endometrial carcinomas (ECAs; n = 10) were unresponsive (P < or = 0.0066). To determine the mechanism of TGF-beta resistance in ECAs, we analyzed the TGF-beta signaling pathway in vivo by immunohistochemistry using specific antibodies to TbetaRI and TbetaRII, Smads, and to the phosphorylated form of Smad2 (Smad2P), an indicator of cells responding to bioactive TGF-beta. Smad2P was expressed in all of the normal endometria (n = 25), and was localized to the cytoplasm and nucleus in PE, and only nuclear in the secretory endometrium. In marked contrast, Smad2P immunostaining was weak or undetectable in ECA (n = 22; P < or = 0.001) and reduced in glandular hyperplasia (n = 25) compared with normal endometrium. However, total Smad2 and Smad7 (which inhibits Smad2 activation) levels were identical in ECA and normal tissue. Consistent with loss of downstream signaling, both TbetaRI (n = 19) and TbetaRII (n = 22) protein expression were significantly reduced in ECA compared with PE (n = 11; P < or = 0.05). By in situ hybridization, the mRNA levels of TbetaRI and TbetaRII were decreased in the carcinoma cells compared with normal PE glands, suggesting that receptor down-regulation occurs at the transcriptional level. Furthermore, a somatic frameshift mutation in the polyadenine tract at the 5' end of the TbetaR-II gene was detected in two of six cases examined. Finally, the ability of explants of ECA to activate endogenous LTGF-beta was deficient compared with normal tissue (23.5% versus 7.4%). Therefore, our results suggest that loss of Smad2 signaling in ECA may be because of down-regulation of TbetaRI and TbetaRII, and/or decreased activation of LTGF-beta. Because disruption of TGF-beta signaling occurred independent of grade or degree of invasion and was evident in premalignant hyperplasia, we conclude that inactivation of TGF-beta signaling leading to escape from normal growth control occurs at an early stage in endometrial carcinogenesis, thereby defining novel molecular targets for cancer prevention
— id: 32907, year: 2002, vol: 62, page: 2778, stat: Journal Article,

Quantitative assessment of cranial defect healing and correlation with the expression of TGF-beta
Gosain, A K; Song, L; Yu, P; Mehrara, B J; Maeda, C Y; Gold, L I; Longaker, M T
2001 Jul;12(4):401-404, Journal of craniofacial surgery
Circular parietal defects from 3 to 12 mm in diameter were made in 45 6-month old skeletally mature guinea pigs, and animals were sacrificed after survival periods of 3 days to 12 weeks. The original defect was harvested in continuity with a rim of surrounding bone and the adjacent dura and pericranium. After 12 weeks, all 3 and 5 mm defects were completely covered by a bridge of bone, while residual defects were noted within the 8 and 12 mm wounds. Percentage of new bone formation was significantly higher within 3 mm defects, than in all larger defects at each time interval from 1 week on (P < .05), reaching a mean of 93% in 3 mm defects and remaining below a mean of 31% in the remaining defect sizes. Immunolocalization demonstrated an osteogenic front in which the osteoblasts stained strongly for all isoforms of TGF-beta, with the intensity decreasing after the majority of the defects had reossified; this front was located at the advancing bone edge of the defect as well as the endocranial side adjacent to the dura. In conclusion, isoforms of TGF-beta are upregulated during a limited 'window' of time corresponding to the period of calvarial reossification, and are localized to osteoblasts within an osteogenic front at the periphery and dural surfaces of the defects
— id: 76286, year: 2001, vol: 12, page: 401, stat: Journal Article,

Biological effects of transforming growth factor-beta(1) in idiopathic pulmonary fibrosis may be regulated by the activation of latent transforming growth factor-beta(1) and the differential expression of transforming growth factor-beta receptors
Khalil, N; O'Connor, R; Gold, L I; Parekh, T; Raghu, G
2001 Jul;120(1 Suppl):48S-48S, Chest
— id: 76287, year: 2001, vol: 120, page: 48S, stat: Journal Article,

Regulation of the effects of TGF-beta 1 by activation of latent TGF-beta 1 and differential expression of TGF-beta receptors (T beta R-I and T beta R-II) in idiopathic pulmonary fibrosis
Khalil, N; Parekh, T V; O'Connor, R; Antman, N; Kepron, W; Yehaulaeshet, T; Xu, Y D; Gold, L I
2001 Dec;56(12):907-915, Thorax
BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is characterised by subpleural fibrosis that progresses to involve all areas of the lung. The expression of transforming growth factor-beta1 (TGF-beta 1), a potent regulator of connective tissue synthesis, is increased in lung sections of patients with IPF. TGF-beta 1 is generally released in a biologically latent form (L-TGF-beta 1). Before being biologically active, TGF-beta must be converted to its active form and interact with both TGF-beta receptors type I and II (T beta R-I and T beta R-II). TGF-beta latency binding protein 1 (LTBP-1), which facilitates the release and activation of L-TGF-beta 1, is also important in the biology of TGF-beta 1. METHODS: Open lung biopsy samples from patients with IPF and normal controls were examined to localise T beta R-I, T beta R-II, and LTBP-1. Alveolar macrophages (AM) and bronchoalveolar lavage (BAL) fluid were examined using the CCL-64 bioassay to determine if TGF-beta is present in its active form in the lungs of patients with IPF. RESULTS: Immunoreactive L-TGF-beta 1 was present in all lung cells of patients with IPF except for fibroblasts in the subepithelial regions of honeycomb cysts. LTBP-1 was detected primarily in AM and epithelial cells lining honeycomb cysts in areas of advanced IPF. In normal lungs LTBP-1 immunoreactivity was observed in a few AM. AM from the upper and lower lobes of patients with IPF secreted 1.6 (0.6) fmol and 4.1 (1.9) fmol active TGF-beta, respectively, while AM from the lower lobes of control patients secreted no active TGF-beta (p< or =0.01 for TGF-beta in the conditioned media from AM obtained from the lower lobes of IPF patients v normal controls). The difference in percentage active TGF-beta secreted by AM from the lower lobes of patients with IPF and the lower lobes of control patients was significant (p< or =0.01), but the difference between the total TGF-beta secreted from these lobes was not significant. The difference in active TGF-beta in conditioned media of AM from the upper and lower lobes of patients with IPF was also not statistically significant. BAL fluid from the upper and lower lobes of patients with IPF contained 0.7 (0.2) fmol and 2.9 (1.2) fmol active TGF-beta, respectively (p< or =0.03). The percentage of active TGF-beta in the upper and lower lobes was 17.6 (1.0)% and 78.4 (1.6)%, respectively (p< or =0.03). In contrast, BAL fluid from control patients contained small amounts of L-TGF-beta. Using immunostaining, both T beta R-I and T beta R-II were present on all cells of normal lungs but T beta R-I was markedly reduced in most cells in areas of honeycomb cysts except for interstitial myofibroblasts in lungs of patients with IPF. TGF-beta 1 inhibits epithelial cell proliferation and a lack of T beta R-I expression by epithelial cells lining honeycomb cysts would facilitate repair of the alveoli by epithelial cell proliferation. However, the presence of both T beta Rs on fibroblasts is likely to result in a response to TGF-beta 1 for synthesis of connective tissue proteins. Our findings show that biologically active TGF-beta 1 is only present in the lungs of patients with IPF. In addition, the effects of TGF-beta 1 on cells may be further regulated by the expression of T beta Rs. CONCLUSION: Activation of L-TGF-beta 1 and the differential expression of T beta Rs may be important in the pathogenesis of remodelling and fibrosis in IPF
— id: 76285, year: 2001, vol: 56, page: 907, stat: Journal Article,

TGF-beta isoforms are differentially expressed in increasing malignant grades of HaCaT keratinocytes, suggesting separate roles in skin carcinogenesis
Gold LI; Jussila T; Fusenig NE; Stenback F
2000 Apr;190(5):579-588, Journal of pathology
The three mammalian isoforms of transforming growth factor-beta (TGF-beta1, -beta2, and -beta3) are potent regulators of cell growth, differentiation, and extracellular matrix deposition. To study their role in skin carcinogenesis, normal human keratinocytes, early (31) and late (310) passage immortalized keratinocytes (HaCaT cells), and five HaCaT-ras clones exhibiting benign (A-5, I-7), malignant (II-4, A-5 RT1), and highly aggressive (A-5 RT3) tumourigenic phenotypes were examined for the expression of TGF-beta isoforms, by immunohistochemistry. This was performed under in vivo conditions, in surface transplants and subcutaneously growing tumours in nude mice. Generally, all tissues that formed keratinized epithelia demonstrated an immunostaining pattern similar to normal human skin. TGF-beta1 was localized to the upper differentiated layers, the stratum granulosum and corneum, in a perimembranous pattern, whereas TGF-beta2 and, weaker, TGF-beta3 immunostaining was present in all suprabasal layers of normal keratinizing epithelia. In contrast, non-keratinizing transplants of non-tumourigenic or highly aggressive cells showed little to no immunoreactivity for TGF-beta1. Whereas TGF-beta2 expression was moderate in the upper layers of non-tumourigenic epithelia, large tumour cells of the malignant HaCaT-ras clones, particularly at the invasion front, were strongly positive for TGF-beta2. TGF-beta3 immunostaining was most pronounced in the stroma of malignant tumours, implying its paracrine induction by the malignant tumour transplants. These results suggest differential functions for each TGF-beta isoform in epidermal carcinogenesis, such that TGF-beta1 is associated with the more differentiated state, TGF-beta2 with highly malignant and invading cells, and TGF-beta3 with tumour stroma formation and angiogenesis. Furthermore, the expression of TGF-betas by both early- and late-stage tumours implies that the isoforms may have distinct functions at different stages of malignancy, supporting their dual role in skin carcinogenesis.
— id: 11793, year: 2000, vol: 190, page: 579, stat: Journal Article,

Osteogenesis in cranial defects: reassessment of the concept of critical size and the expression of TGF-beta isoforms
Gosain, A K; Song, L; Yu, P; Mehrara, B J; Maeda, C Y; Gold, L I; Longaker, M T
2000 Aug;106(2):360-371, Plastic & reconstructive surgery
Transforming growth factor-betas (TGF-beta) have been demontstrated to be upregulated during osteoblast function in vitro and during cranial suture fusion in vivo. The authors hypothesized that spontaneous reossification of calvarial defects was also associated with upregulation of TGF-beta. The present study was designed to (1) evaluate the concept of a critical-size defect within the calvaria in an adult guinea pig model and (2) investigate the association between the ossification of calvarial defects and TGF-beta upregulation. Paired circular parietal defects with diameters of 3 and 5 mm and single parietal defects with diameters of 8 or 12 mm were made in 45 six-month-old skeletally mature guinea pigs. Three animals per defect size were killed after survival periods of 3 days, 1 week, 4 weeks, 8 weeks, or 12 weeks. New bone ingrowth was evaluated by assessing for linear closure by a traditional linear method and by a modified cross-sectional area method using an image analysis system in which the thickness of new bone was taken into account. Immunohistochemistry was performed using rabbit polyclonal antibodies to localize TGF-beta1, -beta2, and -beta3. All specimens were photographed, and the intensity of immunostaining was graded based on subjective photographic assessment by three independent reviewers. No defect demonstrated any measurable bone replacement after a survival period of 3 days. All 3- and 5-mm defects were completely reossified after 12 weeks based on the linear analysis of new bone, indicating these defects to be less than critical size. However, new bone formation in the 5-mm defects never exceeded a mean of 40 percent by cross-sectional area of new bone. Percent of new bone formation by cross-sectional area was significantly higher within 3-mm defects than in all larger defects 4 weeks after the craniotomy, reaching a mean of 89 percent new bone by 12 weeks. Persistent gaps were noted on linear analysis of the 8- and 12-mm wounds by 12 weeks, and mean percent new bone by cross-sectional area remained below 30 percent. Immunolocalization demonstrated osteogenic fronts at the advancing bone edge and the endocranial side, in which the osteoblasts stained strongly for all isoforms of TGF-beta. The intensity of osteoblast expression waned considerably after the majority of the defect had reossified. These data indicate that histometric analysis based on cross-sectional area more accurately reflects the osteogenic potential of a cranial defect than does linear inspection of defect closure. Although the interpretation of immunolocalization studies is highly subjective, independent assessment by three reviewers indicates that isoforms of TGF-beta were upregulated during a limited 'window' of time corresponding to the period of active calvarial reossification, and expression of TGF-beta corresponded to osteoblast activity within osteogenic fronts
— id: 76288, year: 2000, vol: 106, page: 360, stat: Journal Article,

Transforming growth factor betas and their signaling receptors in human hepatocellular carcinoma
Abou-Shady, M; Baer, H U; Friess, H; Berberat, P; Zimmermann, A; Graber, H; Gold, L I; Korc, M; Buchler, M W
1999 Mar;177(3):209-215, American journal of surgery
BACKGROUND: Transforming growth factor betas (TGF-betas) are multifunctional polypeptides that have been suggested to influence tumor growth. They mediate their functions via specific cell surface receptors (type I ALK5 and type II TGF-beta receptors). The aim of this study was to analyze the roles of the three TGF-betas and their signaling receptors in human hepatocellular carcinoma (HCC). METHODS: HCC tissue samples were obtained from 18 patients undergoing partial liver resection. Normal liver tissues from 7 females and 3 males served as controls. The tissues for histological analysis were fixed in Bouin's solution and paraffin embedded. For RNA analysis, freshly obtained tissue samples were snap frozen in liquid nitrogen and stored at -80 degrees C until used. Northern blot analysis was used in normal liver and HCC to examine the expression of TGF-beta1, -beta2, -beta3 and their receptors: type I ALK5 (TbetaR-I ALK5), type II (TbetaR-II), and type III (TbetaR-III). Immunohistochemistry was performed to localize the corresponding proteins. RESULTS: All three TGF-betas demonstrated a marked mRNA overexpression in HCC in comparison with normal controls, whereas the levels of all three TGF-beta receptors showed no significant changes. Intense TGF-beta1, TGF-beta2, and TGF-beta3 immunostaining was found in hepatocellular carcinoma cells and in the perineoplastic stroma with immunohistochemistry, whereas no or mild immunostaining was present in the normal liver. For TbetaR-I ALK5 and TbetaR-II, the immunostaining in both HCC and normal liver was mild to moderate, with a slightly higher intensity in the normal tissues. CONCLUSION: The upregulation of TGF-betas in HCC suggests an important role for these isoforms in hepatic carcinogenesis and tumor progression. Moreover, the localization of the immunoreactivity in both malignant hepatocytes and stromal cells suggests that TGF-betas act via autocrine and paracrine pathways in this neoplasm
— id: 76291, year: 1999, vol: 177, page: 209, stat: Journal Article,

Neonatal estrogen exposure alters the transforming growth factor-beta signaling system in the developing rat prostate and blocks the transient p21(cip1/waf1) expression associated with epithelial differentiation
Chang, W Y; Birch, L; Woodham, C; Gold, L I; Prins, G S
1999 Jun;140(6):2801-2813, Endocrinology
Exposure of male rats to estrogens during the neonatal period retards prostate branching morphogenesis, blocks epithelial differentiation, and predisposes the adult prostate to hyperplasia and dysplasia. The mechanism of neonatal estrogenization is not well understood. The present study evaluated transforming growth factor-beta (TGFbeta) in the neonatally estrogenized ventral prostate to determine whether this paracrine/autocrine factor may in part mediate the effects ofestrogen on the developing prostate gland. Immunocytochemistry using antibodies against active TGFbeta1 and its latency-associated peptide localized this molecule to the periductal smooth muscle cells in the developing prostate. Although neonatal estrogenization increased the accumulation of total and active TGFbeta1 in the smooth muscle layer as early as day 6 of life, it was physically separated from the epithelial ducts by a proliferating layer of fibroblasts surrounding the basement membrane. RT-PCR demonstrated that alterations in TGFbeta1 levels were not due to alterations in TGFbeta1 transcription. TGFbeta2 and TGFbeta3 were primarily immunolocalized to differentiating epithelial cells in developing prostates, and this was markedly dampened between days 10-30 after neonatal estrogen exposure. Immunocytochemistry for TGFbeta signaling components revealed that neonatal estrogenization transiently reduced TGFbeta type I receptor levels in the prostate epithelium, but not in stroma, between days 6-15, whereas there was no effect on TGFbeta type II receptor. Levels of the intracellular signal Smad2 (52 kDa) were detected in epithelial cells but were not altered after estrogenization. To analyze the functional status of the TGFbeta signaling pathway, immunocytochemistry was performed for p21(cip-1/waf-1), a cyclin-dependent kinase inhibitor that is inducible by TGFbeta1 in the prostate. Transient nuclear localization of p21(cip-1/waf-1) was normally observed in epithelial cells between days 6-15 and was associated with entry of cells into a terminal differentiation pathway. Neonatal estrogenization prevented this transient expression of p21(cip-1/waf-1). The present findings demonstrate that the TGFbeta signaling system is perturbed at several levels in the estrogenized prostate, which may in part account for the epithelial cell differentiation blockade as well as the proliferation of periductal fibroblasts in this model
— id: 76290, year: 1999, vol: 140, page: 2801, stat: Journal Article,

Connective tissue growth factor is a regulator for fibrosis in human chronic pancreatitis
di Mola, F F; Friess, H; Martignoni, M E; Di Sebastiano, P; Zimmermann, A; Innocenti, P; Graber, H; Gold, L I; Korc, M; Buchler, M W
1999 Jul;230(1):63-71, Annals of surgery
OBJECTIVE: To evaluate the parameters that mediate fibrogenesis in chronic pancreatitis (CP). BACKGROUND: Connective tissue growth factor (CTGF), which is regulated by transforming growth factor beta (TGF-beta), has recently been implicated in skin fibrosis and atherosclerosis. In the present study, the authors analyzed the concomitant presence of TGF-beta1 and its signaling receptors-TGF-beta receptor I, subtype ALK5 (TbetaR-I(ALK5)), and TGF-beta receptor II (TbetaR-II)-as well as CTGF and collagen type I in the pancreatic tissue of patients undergoing surgery for chronic pancreatitis. PATIENTS AND METHODS: CP tissue samples were obtained from 40 patients (8 women, 32 men) undergoing pancreatic resection. Tissue samples of 25 previously healthy organ donors (12 women, 13 men) served as controls. The expression of TGF-beta1, TbetaR-I(ALK5), TbetaR-II, CTGF, and collagen type I was studied by Northern blot analysis. By in situ hybridization and immunohistochemistry, the respective mRNA moieties and proteins were localized in the tissue samples. RESULTS: Northern blot analysis showed that CP tissue samples exhibited concomitant enhanced mRNA expression of TGF-beta1 (38-fold), TbetaR-II (5-fold), CTGF (25-fold), and collagen type I (24-fold) compared with normal controls. In addition, TbetaR-I(ALK5) mRNA was increased in 50% of CP tissue samples (1.8-fold). By in situ hybridization, TGF-beta1, TbetaR-I(ALK5), and TbetaR-II mRNA were often seen to be colocalized, especially in the ductal cells and in metaplastic areas where atrophic acinar cells appeared to dedifferentiate into ductal structures. In contrast, CTGF was located in degenerating acinar cells and principally in fibroblasts surrounding these areas. Moreover, CTGF mRNA expression levels correlated positively with the degree of fibrosis in CP tissues. CONCLUSION: The concomitant overexpression of CTGF, collagen type I, TGF-beta1, and its signaling receptors in CP suggests that these proteins contribute to enhanced extracellular matrix synthesis and accumulation, resulting finally in the fibrogenesis observed in CP
— id: 76289, year: 1999, vol: 230, page: 63, stat: Journal Article,

The role for transforming growth factor-beta (TGF-beta) in human cancer
Gold LI
1999 ;10(4):303-360, Critical reviews in oncogenesis
Uncontrolled cellular proliferation is a hallmark of cancer. Thus, a relevant and important question is how cancer cells have escaped from normal growth regulatory mechanisms to become malignant and, further, what events favor progression and metastasis. Growth regulatory proteins of the transforming growth factor-beta family (TGF-beta) are one of the few classes of endogenous inhibitors of cell growth. Contrary to the first notion that these proteins may be downregulated in cancer cells to promote their growth, generally it has been otherwise found that there is a marked increase in the expression of TGF-beta mRNA and protein in human cancers (in vivo), including those of the pancreas, colon, stomach, lung, endometrium, prostate, breast, brain, and bone. Furthermore, in many of these cancers high expression correlates with more advanced stages of malignancy and decreased survival. The increased expression of TGF-beta is usually accompanied by a loss in the growth inhibitory response to TGF-beta. For example, certain tumor cells in culture (i.e., colon carcinoma and glioblastoma multiforme) demonstrate a progressive loss of the growth inhibitory response to TGF-beta that varies directly with the malignant stage of the original tumor, and the most aggressive forms actually switch to being autocrine and/or paracrine growth stimulated by TGF-beta. The study of the molecular events associated with the escape of tumor cells from growth regulation by TGF-beta has provided insight into mechanisms underlying carcinogenesis. The mechanisms for upregulation of TGF-beta are unknown. However, once malignant cells lose their growth inhibitory response to TGF-beta and produce massive amounts of these proteins, the increased expression of TGF-beta provides a selective advantage for tumor cell survival as TGF-betas are also angiogenic and have potent immunosuppressive effects, including specifically inhibiting tumoricidal natural and lymphocyte-activated killer cells. In light of the significant role for TGF-betas in regulating cell growth, it is not surprising that in more recent years studies have shown that specific genetic alterations involved in the signaling pathway for TGF-beta-mediated growth inhibition have occurred in many human cancers. Specific defects in TGF-beta receptors, TGF-beta-related-signal transduction/gene activation, and TGF-beta-regulated cell cycle proteins, have all been implicated in the oncogenesis of many human cancers. In this context, components of the TGF-beta growth response pathway are considered to be tumor suppressor genes, as absence (or malfunction) of one or more receptors or signaling proteins would have the potential to cause loss of growth regulation. More recently, the posttranslational reduction of levels of the cyclin-dependent kinase inhibitor (CKI), p27kip1, which mediates TGF-beta growth inhibition, provides an additional means for cancer cells to escape negative growth regulation by TGF-beta. This review provides background information on TGF-beta and updates the status of our knowledge of the role for TGF-beta in specific human malignancies. Understanding the molecular events involved in TGF-beta function in normal cells and its lack of function in tumor cells should identify novel therapeutic targets in human cancers
— id: 11849, year: 1999, vol: 10, page: 303, stat: Journal Article,

Loss of growth regulation by transforming growth factor-beta (TGF-beta) in human cancers: studies on endometrial carcinoma
Gold LI; Parekh TV
1999 ;17(1):73-92, Seminars in reproductive endocrinology
Members of the Transforming Growth Factor-beta (TGF-beta) family are one of the few endogenous inhibitors of cell growth. As uncontrolled cellular proliferation is a hallmark of cancer, an important question to address is how cancer cells escape normal growth regulatory mechanisms to become malignant. In this context, components of the TGF-beta growth response pathway are considered to be tumor suppressor genes, as absence of one or more of TGF-beta receptor and signaling proteins cause loss of cell growth regulation through an inability to regulate proteins that directly block cells in G1 phase of the cell cycle. Endometrial carcinoma (ECA) provides an excellent paradigm to study the changes that accompany loss of TGF-beta-mediated growth, control as a function of neoplastic development, since it is generally preceded by complex hyperplasia. Type 1 ECA is characterized as an estrogen-induced cancer, which responds well to progestin therapy. Since it has become increasingly evident that steroids can regulate growth through growth factors, ECA is also an ideal model for investigating the role for gonadal steroids in the loss of TGF-beta growth regulation in the etiopathogenesis of ECA. Thus, hormonal carcinogenesis adds another level of complexity in studying loss of growth regulation in human cancers. The purpose of this review is to 1) provide the most current background information on how TGF-beta functions including its activation, receptors, signal transduction mechanisms, and control of the cell cycle. 2) present recent information that shows how malignant cells subvert the growth inhibitory effects of TGF-beta by incurring defects in every aspect of the pathway that mediates the TGF-beta growth inhibitory response, and 3) describe the putative role for TGF-beta in the oncogenesis of ECA, provided primarily by the results from our laboratory. Understanding the molecular events involved in TGF-beta function in normal cells and its lack of function in tumor cells should identify novel therapeutic targets in human cancers
— id: 11987, year: 1999, vol: 17, page: 73, stat: Journal Article,

Comparison of the fibrin-binding activities in the N- and C-termini of fibronectin
Rostagno AA; Schwarzbauer JE; Gold LI
1999 Mar 1;338 ( Pt 2):375-386, Biochemical journal
Fibronectin (Fn) binds to fibrin in clots by covalent and non-covalent interactions. The N- and C-termini of Fn each contain one non-covalent fibrin-binding site, which are composed of type 1 (F1) structural repeats. We have previously localized the N-terminal site to the fourth and fifth F1 repeats (4F1.5F1). In the current studies, using proteolytic and recombinant proteins representing both the N- and C-terminal fibrin-binding regions, we localized and characterized the C-terminal fibrin-binding site, compared the relative fibrin-binding activities of both sites and determined the contribution of each site to the fibrin-binding activity of intact Fn. By fibrin-affinity chromatography, a protein composed of the 10F1 repeat through to the C-terminus of Fn (10F1-COOH), expressed in COS-1 cells, and 10F1-12F1, produced in Saccharomyces cerevisiae, displayed fibrin-binding activity. However, since 10F1 and 10F1.11F1 were not active, the presence of 12F1 is required for fibrin binding. A proteolytic fragment of 14.4 kDa, beginning 14 residues N-terminal to 10F1, was isolated from the fibrin-affinity matrix. Radio-iodinated 14.4 kDa fibrin-binding peptide/protein (FBP) demonstrated a dose-dependent and saturable binding to fibrin-coated wells that was both competitively inhibited and reversed by unlabelled 14.4 kDa FBP. Comparison of the fibrin-binding affinities of proteolytic FBPs from the N-terminus (25.9 kDa FBP), the C-terminus (14.4 kDa) and intact Fn by ELISA yielded estimated Kd values of 216, 18 and 2.1 nM, respectively. The higher fibrin-binding affinity of the N-terminus was substantiated by the ability of both a recombinant 4F1.5F1 and a monoclonal antibody (mAb) to this site to maximally inhibit biotinylated Fn binding to fibrin by 80%, and by blocking the 90% inhibitory activity of a polyclonal anti-Fn, by absorption with the 25.9 kDa FBP. We propose that whereas the N-terminal site appears to contribute to most of the binding activity of native Fn to fibrin, the specific binding of the C-terminal site may strengthen this interaction
— id: 7438, year: 1999, vol: 338 ( Pt 2), page: 375, stat: Journal Article,

Increased levels of transforming growth factor-beta in HIV-associated nephropathy
Yamamoto T; Noble NA; Miller DE; Gold LI; Hishida A; Nagase M; Cohen AH; Border WA
1999 Feb;55(2):579-592, Kidney international
BACKGROUND: Human immunodeficiency virus-associated nephropathy (HIVAN) is a renal disease of unknown pathogenesis. Recent evidence suggests that the fibrogenic cytokine transforming growth factor-beta (TGF-beta) might be involved. We hypothesized that overproduction of TGF-beta in the kidney might be involved in the pathogenesis of HIVAN. METHODS: The mRNA and protein expression of TGF-beta isoforms, TGF-beta 1, TGF-beta 2, and TGF beta 3, deposition of matrix proteins induced by TGF-beta, and levels of HIV Tat protein were studied in HIVAN. Controls included normal and diseased kidneys from HIV-positive and -negative patients. The ability of Tat to induce production of TGF-beta and matrix proteins was also studied in human mesangial cells. RESULTS: Normal kidneys, thin basement membrane nephropathy, and minimal change disease were negative for the three TGF-beta isoforms and Tat. In HIVAN, levels of TGF-beta isoforms and Tat were significantly increased, along with the expression of TGF-beta mRNA and deposition of matrix proteins stimulated by TGF-beta. Increased levels of TGF-beta isoforms, but not Tat, were also found in other glomerular diseases characterized by matrix accumulation. HIV infection, in the absence of HIVAN, was not associated with an increase in TGF-beta or Tat expression. Tat stimulated the expression and production of TGF-beta 1 and matrix proteins by human mesangial cells. CONCLUSIONS: Our findings suggest that overproduction of TGF-beta is involved in the pathogenesis of HIVAN
— id: 7467, year: 1999, vol: 55, page: 579, stat: Journal Article,

Transforming growth factor betas and their receptors in human liver cirrhosis
Baer, H U; Friess, H; Abou-Shady, M; Berberat, P; Zimmermann, A; Gold, L I; Korc, M; Buchler, M W
1998 Dec;10(12):1031-1039, European journal of gastroenterology & hepatology
BACKGROUND: Transforming growth factor betas (TGF-betas) are a group of homologous polypeptides that exert pleiotropic effects on various cell types and stimulate the formation of extracellular matrix and fibrosis. To evaluate whether TGF-beta isoforms (TGF-beta1, TGF-beta2 and TGF-beta3) and their receptors (types I-III) are also of importance in the pathophysiology of liver cirrhosis, we analysed their concomitant expression and localization in human liver cirrhosis. PATIENTS: Cirrhotic liver tissue samples were obtained from 17 patients (four women, 13 men) with a median age of 41 years (range 22-67). Normal liver tissues from ten patients (seven women, three men) with a median age of 55 years (range 45-75) served as controls. METHODS: The tissues were fixed in Bouin's solution and paraffin-embedded for histological analysis. For RNA analysis, freshly obtained tissue samples were snap-frozen in liquid nitrogen and stored at -80 degrees C until analysed. Northern blot analysis was used to examine the expression of TGF-beta1, beta2 and beta3 and their receptors, type I (TbetaR-I), type II (TbetaR-II) and type III (TbetaR-III). Immunohistochemistry was performed to determine the localization of the corresponding proteins in the normal and the cirrhotic liver. RESULTS: Northern blot analysis revealed enhanced expression (P < 0.05) of TGF-beta1 (twofold increase), TGF-beta2 (threefold increase) and TGF-beta3 (8.5-fold increase) and of TbetaR-II (threefold increase) mRNA in liver cirrhosis in comparison with normal controls. In contrast, TbetaR-I (ALK-5) and TbetaR-III mRNA expression showed no significant changes. No TGF-beta isoform immunoreactivity was present in hepatocytes in either normal livers or in liver cirrhosis. However, in liver cirrhosis, intense TGF-beta1 immunoreactivity was present in bile duct and ductular epithelial cells (including ductular proliferations) and in inflammatory cells. In a few sinusoidal lining cells, faint TGF-beta1 and moderate TGF-beta2 immunoreactivity was present. TGF-beta3 immunostaining was present in bile duct and ductular epithelial cells, in inflammatory cells and in fibroblast-like spindle cells in liver cirrhosis. For TbetaR-I and TbetaR-II, the immunoreactivity was localized in hepatocytes and biliary cells in normal and cirrhotic liver tissues, with higher intensity for TbetaR-II in the cirrhotic liver. CONCLUSION: Enhanced expression of all three TGF-bea isoforms and of TbetaR-II in liver cirrhosis suggests their involvement in this fibrotic disorder. The higher immunoreactivity of the three TGF-beta isoforms in the bile duct epithelial cells in cirrhotic tissues suggests a possible role of these cells in the pathogenesis of liver cirrhosis
— id: 76292, year: 1998, vol: 10, page: 1031, stat: Journal Article,

Proteoglycan distribution in lesions of atherosclerosis depends on lesion severity, structural characteristics, and the proximity of platelet-derived growth factor and transforming growth factor-beta
Evanko SP; Raines EW; Ross R; Gold LI; Wight TN
1998 Feb;152(2):533-546, American journal of pathology
The accumulation of proteoglycans (PGs) in atherosclerosis contributes to disease progression and stenosis and may partly depend on local regulation by growth factors such as platelet-derived growth factor (PDGF) and transforming growth factor (TGF)-beta. In this study, the distribution of the major extracellular PGs is compared with that of PDGF and TGF-beta isoforms in developing lesions of atherosclerosis from hypercholesterolemic nonhuman primates. Strong immunostaining for decorin, biglycan, versican, and hyaluronan is observed in both intermediate and advanced lesions. Perlecan staining is weak in intermediate lesions but strong in advanced lesions in areas bordering the plaque core. Immunostaining for PDGF-B and TGF-beta1 is particularly prominent in macrophages in intermediate and advanced lesions. In contrast, TGF-beta2 and TGF-beta3 and PDGF-A are present in both macrophages and smooth muscle cells. Overall, PG deposits parallel areas of intense growth factor immunostaining, with trends in relative localization that suggest interrelationships among certain PGs and growth factors. Notably, decorin and TGF-beta1 are distributed similarly, predominantly in the macrophage-rich core, whereas biglycan is prominent in the smooth muscle cell matrix adjoining TGF-beta1-positive macrophages. Versican and hyaluronan are enriched in the extracellular matrix adjacent to both PDGF- and TGF-beta1-positive cells. These data demonstrate that PG accumulation varies with lesion severity, structural characteristics, and the proximity of PDGF and TGF-beta
— id: 7557, year: 1998, vol: 152, page: 533, stat: Journal Article,

Enhanced expression of TGF-betas and their receptors in human acute pancreatitis
Friess H; Lu Z; Riesle E; Uhl W; Brundler AM; Horvath L; Gold LI; Korc M; Buchler MW
1998 Jan;227(1):95-104, Annals of surgery
OBJECTIVES: To determine which mechanisms are involved in pancreatic remodeling, repair, and fibrosis after acute necrotizing pancreatitis (NP) in humans. SUMMARY BACKGROUND DATA: Transforming growth factor betas (TGF-betas) are multifunctional polypeptides that have been implicated in the regulation and formation of extracellular matrix and fibrosis. They exert their functions by binding to specific receptors. In this study, we analyze the expression of TGF-beta1, TGF-beta2, and TGF-beta3 and their receptors type I (Tbeta-RI [ALK5]), type II (Tbeta-RII), and type III (Tbeta-RIII) in NP. PATIENTS: Pancreatic tissue samples were obtained from 6 female and 8 male patients with a median age of 65 years (range, 37 to 77 years) undergoing surgery for NP. The median Ranson score of the patients was 6 (range, 2 to 9). The operation was performed a median 5.5 days (range, 4 to 17 days) after the onset of acute pancreatitis. Pancreatic tissue obtained from 12 previously healthy organ donors (6 male, 6 female; median age of 43 years) served as controls. METHODS: The expression of TGF-beta1, TGF-beta2, TGF-beta3, Tbeta-RI (ALK5), Tbeta-RII, Tbeta-RIII, and collagen type I mRNA was analyzed by Northern blot analysis. In addition, immunohistochemical analysis using polyclonal antibodies was performed to detect TGF-beta1, TGF-beta2, TGF-beta3, Tbeta-RI (ALK5), and Tbeta-RII. RESULTS: Northern blot analysis showed an increase in TGF-betas and their receptors in NP tissue samples compared with samples from normal controls. The increase was 3.5-fold for TGF-beta1 (p < 0.05), 2.7-fold for TGF-beta2 (p < 0.05), 3.5-fold for TGF-beta3 (p < 0.05), 10-fold for Tbeta-RI (ALK5) (p < 0.05), 5.7-fold for Tbeta-RII (p < 0.05), and 1.4-fold for Tbeta-RIII (not significant). Collagen type I mRNA was also markedly increased in NP samples and correlated with the level of TGF-betas. Immunohistochemical analysis demonstrated intense TGF-beta1, TGF-beta2, TGF-beta3, Tbeta-RI (ALK5), and Tbeta-RII immunoreactivity in the remaining acinar and ductal cells in most NP samples; in the normal control pancreas, there was weak to moderate immunoreactivity for these factors only in some acinar cells and a few ductal cells. CONCLUSION: The marked increase in expression of TGF-betas and their signaling receptors Tbeta-RI (ALK5) and Tbeta-RII suggests a role for TGF-betas in the repair process after the onset of NP in humans and raises the possibility that TGF-betas might be involved in tissue remodeling and the fibrotic reaction that occurs in the pancreas after necrosis
— id: 8028, year: 1998, vol: 227, page: 95, stat: Journal Article,

A subset of metastatic human colon cancers expresses elevated levels of transforming growth factor beta1
Picon A; Gold LI; Wang J; Cohen A; Friedman E
1998 Jun;7(6):497-504, Cancer epidemiology biomarkers & prevention
Although transforming growth factor (TGF)-beta1 is a potent growth inhibitor of normal epithelial cells including colonocytes, TGF-beta1 has also been implicated as an enhancer of colon cancer metastasis. Decreasing TGF-beta1 protein levels in the metastatic U9 colon cancer cell line by antisense methodology decreased both U9 cell metastasis to the liver and s.c. tumor formation in a nude mouse system, and the tumors that did arise had regained TGF-beta1 expression (F. Huang et al, Cell Growth Differ., 6: 1635-1642, 1995). In addition, in a clinical immunohistochemistry study, colon cancers with elevated TGF-beta1 protein levels were found to be 18 times more likely to recur as distant metastases than colon cancers expressing low TGF-beta1 levels, after resection of the primary tumor (E. Friedman et al, Cancer Epidemiol. Biomark. Prev., 4:549-554, 1995). Because both studies implicated TGF-beta1 in colon cancer metastasis, we wished to know whether a selection bias for TGF-beta1 was maintained in metastatic cells or was only a property of the primary site tumors that were likely to metastasize. TGF-beta1 levels were measured using two different antibodies in paired primary site cancers and their metastases by immunohistochemistry and, in selected cases, by Western blot analysis. In 16 of 21 cases (76%) with antibody G and 23 of 31 cases (74%) with antibody P, higher expression of TGF-beta1 was found in colon cancer cells invading local lymph nodes compared with primary site colon cancer cells, or (2 and 6 cases, respectively) high TGF-beta1 expression in the primary site cancer was maintained in invasive cells. Analysis by Western blotting using both antibodies also demonstrated that higher levels of TGF-beta1 protein were found in metastases compared with the primary site tumor or normal tissue. Additional cases of paired primary site colon cancer, local lymph node metastases, and cancer cells metastasizing to distant sites were examined. In six of eight such cases (75%), TGF-beta1 levels were increased in both invasive cell populations compared with the primary site cancer (five cases), or high levels in the primary site cancer were maintained in the metastatic cells (one case). These data suggest that TGF-beta1 plays a role in promoting colon cancer metastasis throughout the metastatic process in roughly 75% of cases. TGF-beta1 may increase metastasis by paracrine mechanisms, such as suppression of local immune response or increased angiogenesis, as was seen with the U9 cell line. In those cancers with nonmutated TGF-beta receptors and nonmutated smad proteins like U9 cells, TGF-beta1 could also act in an autocrine manner to increase invasion by increasing cell motility (Hsu et al., Cell Growth Differ., 5: 267-275, 1994)
— id: 8083, year: 1998, vol: 7, page: 497, stat: Journal Article,

Type I (RI) and type II (RII) receptors for transforming growth factor-beta isoforms are expressed subsequent to transforming growth factor-beta ligands during excisional wound repair
Gold LI; Sung JJ; Siebert JW; Longaker MT
1997 Jan;150(1):209-222, American journal of pathology
Transforming growth factor (TGF)-beta isoforms (TGF-beta 1, -beta 2, and -beta 3) regulate cell growth and differentiation and have critical regulatory roles in the process of tissue repair and remodeling. Signal transduction for TGF-beta function is transmitted by a heteromeric complex of receptors consisting of two serine/threonine kinase transmembrane proteins (RI and RII). We have previously shown that each TGF-beta isoform is widely expressed in a distinct spatial and temporal pattern throughout the processes of excisional and incisional wound repair. As the presence of TGF-beta receptors determines cellular responsiveness, we have currently examined, by immunohistochemistry, the localization of RI (ALK-1, ALK-5) and RII throughout repair of full-thickness excisional wounds up to 21 days after wounding. The expression of RI (ALK-5) and RII co-localized in both the unwounded and wounded skin and was present in the same cell types as TGF-beta ligands. However, immunoreactivity for TGF-beta receptors, throughout repair, occurred 1 to 5 days later than TGF-beta isoform immunostaining. This implies that the presence of TGF-beta ligands may up-regulate TGF-beta receptors for function and/or may reflect a lag due to local processing of latent TGF-beta. As observed for the immunohistochemical localization of TGF-beta isoforms in unwounded skin, RI and RII were expressed throughout the four layers of the epidermis, showing a wavy pattern of slight to moderate immunostaining, and hair follicles, sweat glands, and sebaceous glands were moderately immunoreactive. The extracellular matrix, fibroblasts, and blood vessels in the dermis were not immunoreactive. After injury, as observed for TGF-beta ligands, RI and RII expression was increased in the epidermis adjacent to the wound and the epithelium migrating over the wound was completely devoid of TGF-beta receptor immunoreactivity until re-epithelialization was completed by day 7 after wounding. The dermis was only slightly immunoreactive for RI and RII until day 5 when, immediately under the wound, immunostaining for fibroblasts, connective tissue cells, and newly forming vasculature began to increase and remained intense until day 14. Consistent with the role for TGF-beta in scarring, numerous fibroblasts, ostensibly active in the production of extracellular matrix components, continued to be slightly immunoreactive for RI and RII at 21 days. The ALK-1 (TSR-1) type I receptor, which binds both activin and TGF-beta, showed slight immunostaining early in repair (days 1 to 7) that progressively became more intense later in repair after day 10 and through day 21. This suggests that there may be a switch to a different type I receptor, implying different functions for the ALK-1 and ALK-5 receptors. The concomitant expression of TGF-beta isoforms and their signal-transducing receptors denote potential spatial and temporal activity of TGF-beta. Thus, although TGF-beta ligand is present, TGF-beta would not function in wound repair until a later time when RI and RII appear. This information should aid in the development of receptor antagonists as a therapeutic approach to scarring and fibrosis. In addition, these studies underscore the importance of defining the expression of proteins in vivo to establish a basis for the analysis of mechanisms in vitro
— id: 12427, year: 1997, vol: 150, page: 209, stat: Journal Article,

Immunohistochemical localization of transforming growth factor beta isoforms in asbestos-related diseases
Jagirdar J; Lee TC; Reibman J; Gold LI; Aston C; Begin R; Rom WN
1997 Sep;105 Suppl 5:1197-1203, Environmental health perspectives
Transforming growth factor beta (TGF-beta), a multifunctional cytokine and growth factor, plays a key role in scarring and fibrotic processes because of its ability to induce extracellular matrix proteins and modulate the growth and immune function of many cell types. These effects are important in inflammatory disorders with fibrosis and cancer. The asbestos-related diseases are characterized by fibrosis in the lower respiratory tract and pleura and increased occurrence of lung cancer and mesothelioma. We performed immunohistochemistry with isoform-specific antibodies to the three TGF-beta isoforms on 16 autopsy lungs from Quebec, Canada, asbestos miners and millers. There was increased immunolocalization of all three TGF-beta isoforms in the fibrotic lesions of asbestosis and pleural fibrosis. The hyperplastic type II pneumocytes contained all three isoforms. By contrast, there was differential spatial immunostaining for the TGF-beta isoforms in malignant mesothelioma, with TGF-beta 1 in the stroma but TGF-beta 2 in the tumor cells. These data are consistent with an important role for TGF-beta in accumulation of extracellular matrix and cell proliferation in asbestos-related diseases
— id: 12204, year: 1997, vol: 105 Suppl 5, page: 1197, stat: Journal Article,

Expression of transforming growth factor-beta (TGF-beta) isoforms in osteosarcomas: TGF-beta3 is related to disease progression
Kloen, P; Gebhardt, M C; Perez-Atayde, A; Rosenberg, A E; Springfield, D S; Gold, L I; Mankin, H J
1997 Dec 15;80(12):2230-2239, Cancer
BACKGROUND: Transforming growth factor-beta (TGF-beta) is a multipotent growth factor affecting development, homeostasis, and tissue repair. In addition, increased expression of TGF-beta has been reported in different malignancies, suggesting a role for this growth factor in tumorigenesis. METHODS: Using immunohistochemistry, the expression, prevalence, and distribution of TGF-beta isoforms were evaluated in 25 high grade human osteosarcomas. The Cox proportional hazards models and Kaplan-Meier curves were calculated correlating disease free survival with TGF-beta expression. RESULTS: Expression of one or more TGF-beta isoforms was found in all the osteosarcomas. Immunoreactivity for TGF-beta1 and TGF-beta3 generally was stronger than for TGF-beta2. The cytoplasm of the tumor cells showed stronger staining than their surrounding extracellular stroma. Most notably, osteoclasts showed strong to intense staining for all three isoforms. In 11 of 25 specimens angiogenic activity was noted with staining of multiple small vessels in the tumor stroma. Expression of TGF-beta3, but not of TGF-beta2 or TGF-beta1, related to disease progression, such that there was a statistically significant decrease in the disease free interval as the immunoreactivity for TGF-beta3 increased. CONCLUSIONS: All osteosarcomas expressed TGF-beta in the cytoplasm of the tumor cells as well as in their extracellular stroma. The presence of TGF-beta in the endothelial and perivascular layers of small vessels in the tumor stroma suggests angiogenic activity of this growth factor. The expression of TGF-beta3 was correlated strongly with disease progression (P = 0.027). These data suggest that increased expression of TGF-beta isoforms, especially TGF-beta3, may play a role in osteosarcoma progression
— id: 76293, year: 1997, vol: 80, page: 2230, stat: Journal Article,

Immunohistochemical localization of transforming growth factor-beta and insulin-like growth factor-I in asbestosis in the sheep model
Lee TC; Gold LI; Reibman J; Aston C; Begin R; Rom WN; Jagirdar J
1997 ;69(3):157-164, International archives of occupational & environmental health
Asbestosis is characterized by increased collagen deposition along the walls of terminal respiratory bronchioles that extends into the alveolar ducts and septae. Alveolar macrophages are activated and release growth factors that stimulate mesenchymal cell proliferation and enhanced formation of extracellular matrix. Both insulin-like growth factor-I (IGF-I), and transforming growth factor beta (TGF-beta) regulate cellular growth and promote matrix accumulation and are hypothesized to play important roles in asbestosis. We performed immunohistochemistry using polyclonal antibodies to specific synthetic peptides of the three mammalian isoforms of TGF-beta (TGF-beta 1, -beta 2, -beta 3) and to IGF-I on lungs of sheep treated intratracheally with chrysotile asbestos. All three TGF-beta isoforms were found in bronchial and bronchiolar epithelium, macrophages, and bronchial and vascular smooth muscle in control lungs. The distribution of TGF-beta was increased in these lung constituents as fibrotic lesions developed. Fibrotic lesions additionally demonstrated intense immunostaining of all three TGF-beta isoforms that localized to the extracellular matrix zones with little staining of interstitial cells. In the control sheep lungs, IGF-I staining was detected in bronchial and bronchiolar epithelium, bronchial glands, bronchial and vascular smooth muscle, endothelium, and macrophages. IGF-I immunostaining was detected in macrophages in peribronchial fibrosis and in fibroblasts along the periphery of and within lesions, but not in the extracellular matrix. Metaplastic proliferating epithelium and macrophages were strongly immunoreactive for IGF-I in advanced lesions. Our data demonstrate different immunostaining patterns for IGF-I and TGF-beta in asbestosis, with IGF-I in the cellular periphery and TGF-beta in the extracellular matrix consistent with a complementary role in stimulating interstitial fibroblast proliferation and new collagen deposition in areas of active fibrosis
— id: 12416, year: 1997, vol: 69, page: 157, stat: Journal Article,

Exogenous and endogenous transforming growth factors-beta influence elastin gene expression in cultured lung fibroblasts
McGowan, S E; Jackson, S K; Olson, P J; Parekh, T; Gold, L I
1997 Jul;17(1):25-35, American journal of respiratory cell & molecular biology
Elastin, an important structural protein of the extracellular matrix, confers elastic properties on the pulmonary alveolar interstitium. In the alveolar wall, elastin is primarily produced postnatally by fibroblasts. The mechanisms that regulate lung fibroblast (LF) elastin gene expression have not been completely defined, although both transcriptional and posttranscriptional mechanisms appear to be involved. Transforming growth factors-beta (TGF-beta s) have been shown to increase elastin production by cultured neonatal rat LF. Analyses of elastin gene transcription and mRNA stability indicate that exogenous TGF-beta 1 increases the half-life of tropoelastin mRNA by 1.5-fold and does not alter elastin gene transcription. Interference with the functions of endogenous TGF-beta 1 in cultured LF, through the addition of neutralizing antibodies or antisense oligodeoxynucleotides, decreases tropoelastin and tropoelastin mRNA production by these cells. The content of total (latent plus active) TGF-beta s was approximately 4.5-fold greater in lungs obtained from rats on postnatal day 8 than in lungs obtained from adults. These findings indicate that endogenous TGF-beta s, in cultured LF, regulate elastin gene expression, most likely by a posttranscriptional mechanism. Since others have shown that elastin mRNA appears to have a longer half-life in neonatal than in adult rat lungs, we hypothesize that the higher content of TGF-beta s could contribute to the greater elastin mRNA stability in neonatal lungs
— id: 76294, year: 1997, vol: 17, page: 25, stat: Journal Article,

Differential localization of transforming growth factor-beta isoforms in human gastric mucosa and overexpression in gastric carcinoma
Naef, M; Ishiwata, T; Friess, H; Buchler, M W; Gold, L I; Korc, M
1997 Apr 10;71(2):131-137, International journal of cancer
Transforming growth factor beta (TGF-beta) isoforms comprise a family of multifunctional polypeptide growth factors that either inhibit or stimulate cell proliferation. We examined TGF-beta expression in normal human gastric mucosa and carcinoma. The distribution and expression of TGF-beta isoforms in 4 normal mucosa samples from organ donors, in 12 normal mucosa samples adjacent to gastric cancer and in 12 gastric carcinomas were examined using immunohistochemistry and Northern blot analysis. Because TGF-beta s regulate collagen expression, collagen type I alpha1 mRNA amounts were also examined. Immunohistochemical analysis of normal human gastric tissue samples indicated that TGF-beta1 localized principally in parietal cells but also in some surface mucus cells, TGF-beta2 was present exclusively in chief cells and TGF-beta3 was present in parietal, chief and mucus cells. In the gastric cancers, strong colocalization of TGF-beta1, -beta2 and -beta3 was evident in the cancer cells. Northern blot analysis indicated that, compared to normal gastric tissue, gastric cancers showed a 4.8- and 6-fold increase in mRNA amounts encoding TGF-beta1 and TGF-beta3, respectively. In contrast, TGF-beta2 mRNA amounts were comparable in both groups. Northern blot analysis showed a 10-fold increase in human collagen type I alpha1 mRNA amounts compared to normal gastric tissue. These findings imply a role forTGF-beta s in normal human gastric mucosa function, and raise the possibility that the aberrant colocalization and overexpression of all 3 TGF-beta isoforms in human gastric cancer cells in vivo may contribute to the pathobiology of gastric carcinoma
— id: 76295, year: 1997, vol: 71, page: 131, stat: Journal Article,

Increased expression of transforming growth factor beta s after acute oedematous pancreatitis in rats suggests a role in pancreatic repair
Riesle, E; Friess, H; Zhao, L; Wagner, M; Uhl, W; Baczako, K; Gold, L I; Korc, M; Buchler, M W
1997 Jan;40(1):73-79, Gut: journal of the British Society of Gastroenterology
BACKGROUND: Transforming growth factor beta isoforms (TGF beta s) belong to a family of multifunctional regulators of cellular growth and differentiation. They are mitogenic and chemotactic for fibroblasts and are potent stimulators of extracellular matrix production (collagen) and deposition. Upregulation of TGF beta transcription has been reported for several in vivo systems during repair after injury. AIMS: To study the expression of the three mammalian isoforms of TGF beta (TGF beta 1-3) and their relation to collagen expression as a marker for fibroblast response in acute oedematous pancreatitis in rats. METHODS: Using northern blot analysis and immunohistochemistry, the expression and localisation of TGF beta isoforms, collagen, and amylase were analysed during the course of acute oedematous pancreatitis in rats, experimentally induced by intravenous caerulein infusion. RESULTS: Induction of acute pancreatitis resulted in a biphasic peak pattern of expression of TGF beta 1, beta 2, and beta 3 mRNA, with a pronounced increase from day 1 to day 3 (sixfold, 2.5-fold, fivefold, respectively) and again from day 5 to day 7 (three-fold, 2.3-fold, 3.5-fold, respectively). The temporal changes in TGF beta mRNA identically paralleled the expression in collagen mRNA. In contrast, amylase mRNA expression, used as a general indicator of acinar cell integrity, was slightly decreased after induction of acute pancreatitis. Immunohistochemical analysis of pancreatitis tissue showed that increased expression of TGF beta s was mainly present in the pancreatic acinar and ductal cells; this was evident within one day after pancreatitis induction. CONCLUSION: Overexpression of TGF beta s after induction of acute pancreatitis suggests a role for these proteins in pancreatic repair and remodelling. The increased levels of TGF beta s may help suppress immune activation, and may contribute to the increase in the extracellular matrix including collagen and to the repair of the pancreatic parenchyma
— id: 76296, year: 1997, vol: 40, page: 73, stat: Journal Article,

Studies in cranial suture biology: Part I. Increased immunoreactivity for TGF-beta isoforms (beta 1, beta 2, and beta 3) during rat cranial suture fusion [see comments]
Roth DA; Longaker MT; McCarthy JG; Rosen DM; McMullen HF; Levine JP; Sung J; Gold LI
1997 Mar;12(3):311-321, Journal of bone & mineral research
The mechanisms involved in normal cranial suture development and fusion as well as the pathophysiology of craniosynostosis, a premature fusion of the cranial sutures, are not well understood. Transforming growth factor-beta isoforms (TGF-beta 1, beta 2, and beta 3) are abundant in bone and stimulate calvarial bone formation when injected locally in vivo. To gain insight into the role of these factors in normal growth and development of cranial sutures and the possible etiology of premature cranial suture fusion, we examined the temporal and spatial expression of TGF-beta isoforms during normal cranial suture development in the rat. In the Sprague-Dawley rat, only the posterior frontal cranial suture undergoes fusion between 12 and 22 days of age, while all other cranial sutures remain patent. Therefore, immunohistochemical analysis of the fusing posterior frontal suture was compared with the patent sagittal suture at multiple time points from the fetus through adult. Whereas the intensity of immunostaining was the same in the posterior frontal and sagittal sutures in the fetal rat, there was increased immunoreactivity for TGF-beta isoforms in the actively fusing posterior frontal suture compared with the patent sagittal suture starting 2 days after birth and continuing until approximately 20 days. There were intensely immunoreactive osteoblasts present during fusion of the posterior frontal suture. In contrast, the patent sagittal suture was only slightly immunoreactive. A differential immunostaining pattern was observed among the TGF-beta isoforms; TGF-beta 2 was the most immunoreactive isoform and was also most strongly associated with osteoblasts adjacent to the dura and the margin of the fusing suture. Since the increased expression of TGF-beta 2 during suture fusion suggested a possible regulatory role, recombinant TGF-beta 2 was added directly to the posterior frontal and sagittal sutures in vivo to determine if suture fusion could be initiated. Exogenously added TGF-beta 2 stimulated fusion of the ectocranial surface of the posterior frontal suture. These data provide evidence for a regulatory role for these growth factors in cranial suture development and fusion. Additionally, the intense immunostaining for TGF-beta 2 in the dura mater underlying the fusing suture supports a role for the dura mater in suture fusion. It is possible that premature or excessive expression of these factors may be involved in the etiopathogenesis of craniosynostosis and that modulation of the growth factor profile at the suture site may have potential therapeutic value
— id: 12370, year: 1997, vol: 12, page: 311, stat: Journal Article,

Intracellular demonstration of active TGFbeta1 in B cells and plasma cells of autoimmune mice. IgG-bound TGFbeta1 suppresses neutrophil function and host defense against Staphylococcus aureus infection
Caver, T E; O'Sullivan, F X; Gold, L I; Gresham, H D
1996 Dec 1;98(11):2496-2506, Journal of clinical investigation
Infection remains a leading cause of morbidity and mortality in patients with SLE. To investigate this, previously we assessed the host defense status of autoimmune MRL/lpr mice and found that elaboration of active TGFbeta suppressed neutrophil function and decreased survival in response to Staphylococcus aureus infection. The purpose of the present work was to elucidate the molecular form and the cellular source of the active TGFbeta involved. Here, we report for the first time that TGFbeta1 is found in the active form inside B cells and plasma cells and that it circulates in the plasma complexed with IgG in two murine models of systemic autoimmunity and in some patients with SLE. IgG-bound active TGFbeta1 is many times more potent than uncomplexed active TGFbeta1 for suppression of neutrophil function in vitro and host defense against S. aureus infection in vivo. These data indicate that TGFbeta1 is in the active form inside B cells and plasma cells, that the formation of a complex of IgG and active TGFbeta1 is greatly accelerated in autoimmunity, and that this complex is extremely potent for suppression of PMN function and host defense against bacterial infection
— id: 76297, year: 1996, vol: 98, page: 2496, stat: Journal Article,

Upregulation of transforming growth factors betas and collagen suggest a role in pancreatic healing following edematous pancreatitis in rats
Friess, H; Riesle, E; Zhao, L; Baczako, K; Gold, LI; Korc, M; Buchier, MW
1996 APR ;110(4):A389-A389, Gastroenterology
— id: 98382, year: 1996, vol: 110, page: A389, stat: Journal Article,

Altered expression of transforming growth factor-beta S in chronic renal rejection
Horvath, L Z; Friess, H; Schilling, M; Borisch, B; Deflorin, J; Gold, L I; Korc, M; Buchler, M W
1996 Aug;50(2):489-498, Kidney international
We examined the altered expression of transforming growth factor-beta s in chronic renal rejection in humans, including transforming growth factor beta-1 (TGF-beta 1), TGF-beta 2, TGF-beta 3 and their receptors, transforming growth factor beta receptor type I (T beta R-I) and T beta R-II. Using Northern blot analysis and immunohistochemistry, 10 specimens of chronically rejected and 8 normal kidney samples were analyzed. By Northern blot analysis the expression of mRNA encoding TGF-beta 1, TGF-beta 2, TGF-beta 3 (P < 0.02), T beta R-I and T beta R-II (P < 0.02) was decreased in chronically rejected renal cortex samples, compared to normal controls. Immunohistochemical analysis of the normal renal cortex showed strong immunostaining for TGF-beta 1 and TGF-beta 3, and mild immunostaining for TGF-beta 2 in the proximal and distal tubulointerstitium, but no signal for any of the TGF-beta isoforms in the glomeruli or in the cortical vessels. In sharp contrast, the glomeruli and the cortical vessels of the rejected kidney specimens exhibited strong immunostaining for TGF-beta 1 and TGF-beta 3, whereas the tubules revealed a decrease in immunoreactivity. T beta RI and T beta RII immunostaining showed similar changes as observed with TGF-beta 1 and TGF-beta 3 antibodies. There was a concomitant increase in B-cell accumulation in the glomeruli, while T-cells and macrophages were diffusely abundant in the rejected samples. Since TGF-beta S are potent inducers of extracellular matrix proteins and have been shown to be involved in fibrotic disease, the increase in TGF-beta 1 and TGF-beta 3 immunoreactivity in the glomeruli suggests that there is a redistribution in TGF-beta expression in chronic renal allograft rejection. Together with changes affected by B-cell mediated immunity, the above alterations might contribute to the histopathological changes that occur in this disorder, such as intimal fibrosis, arteriosclerosis and glomerular and tubular sclerosis
— id: 76299, year: 1996, vol: 50, page: 489, stat: Journal Article,

Distribution of transforming growth factor-beta isoforms in human immunodeficiency virus-1 encephalitis
Johnson, M D; Gold, L I
1996 Jul;27(7):643-649, Human pathology
The transforming growth factor-beta family of polypeptides includes three related isoforms with pervasive effects on immune system function. In this study, the authors evaluated human brains with human immunodeficiency virus (HIV)-1 encephalitis for transforming growth factor beta (TGFbeta)1, TGFbeta2, and TGFbeta3 immunoreactivity using isoform-specific polyclonal antibodies and avidin-biotin immunohistochemistry. Normal brains and those with progressive multifocal leukoencephalopathy, toxoplasma encephalitis, and cryptococcal meningitis were used as controls. In normal controls, TGFbeta1, TGFbeta2, and TGFbeta3 immunoreactivity were confined to arachnoid cells and blood vessels. In 9 of 10 cases of HIV-1 encephalitis, all three isoforms were also detected in arachnoid cells. In addition, variable, predominantly TGFbeta2 and TGFbeta3 immunoreactivity were also detected in reactive astrocytes and mononuclear cells of white matter lesions. Extensive TGFbeta3 immunoreactivity was also detected in multinucleated giant cells in one case. In a case of cryptococcal meningitis, all three isoforms were detected in arachnoid cells and macrophages. Lesions of progressive multifocal leukoencephalopathy and toxoplasma encephalitis also exhibited TGFbeta1, TGFbeta2, and TGFbeta3 immunostaining in reactive astrocytes. These findings suggest that TGFbeta isoforms are present in HIV-1 encephalitis and may participate in the pathogenesis of this and other inflammatory central nervous system (CNS) lesions associated with acquired immunodeficiency syndrome (AIDS)
— id: 76301, year: 1996, vol: 27, page: 643, stat: Journal Article,

Inward growth of colonic adenomatous polyps
Moss, S F; Liu, T C; Petrotos, A; Hsu, T M; Gold, L I; Holt, P R
1996 Dec;111(6):1425-1432, Gastroenterology
BACKGROUND & AIMS: Most colon cancers arise from polypoid adenomas, but how these benign lesions develop into malignant neoplasms is not understood. This study examined the migration of epithelial cells within human adenomatous polyps by determining the distribution of proliferating and apoptotic cells and immunoreactivity to transforming growth factor beta (TGF-beta). METHODS: Sections of surgically resected normal (n = 10) and adenomatous (n = 22) formalin-fixed tissue were examined for proliferating cells and TGF-beta isoenzymes 1-3 by immunohistochemistry and apoptotic cells by terminal deoxyuridine nick end-labeling. RESULTS: The distribution of proliferating, apoptotic, and TGF-beta immunoreactive cells was strikingly reversed in adenomatous polyps compared with normal mucosa. Proliferating cells were located in the base of normal colonic crypts and TGF-beta immunoreactive and apoptotic cells near or at the luminal surface, corresponding to the normal migration of colonocytes. In adenomas, increased numbers of proliferating cells were mainly located at the luminal surface and TGF-beta immunoreactive and apoptotic cells were located principally at the crypt base. CONCLUSIONS: This distribution suggests that cell migration in adenomas is not toward the lumen but instead inward toward the polyp base
— id: 76298, year: 1996, vol: 111, page: 1425, stat: Journal Article,

Fibrillary glomerulonephritis related to serum fibrillar immunoglobulin-fibronectin complexes
Rostagno A; Vidal R; Kumar A; Chuba J; Niederman G; Gold L; Frangione B; Ghiso J; Gallo G
1996 Nov;28(5):676-684, American journal of kidney diseases
Fibrillary glomerulonephritis is a disease of uncertain origin and pathogenesis characterized by nonamyloidotic fibrils in glomeruli. We report immunohistological, immunochemical, and biochemical studies of a serum fibrillar cryoprecipitate obtained from a patient with fibrillary glomerulonephritis, that formed on prolonged storage at 4 degrees C. By Western blot and amino acid sequence analysis, the cryoprecipitated fibril components consisted of immunoglobulins, heavy chains gamma and mu, light chains kappa and lambda, and fibronectin, similar to the proteins identified by immunofluorescence and immunoelectron microscopy in the glomerular fibrils. These findings support the hypothesis that serum precursors may be the source of the fibrillar deposits and suggest a role for immunoglobulin-fibronectin complexes in the pathogenesis of fibrillary glomerulonephritis
— id: 7254, year: 1996, vol: 28, page: 676, stat: Journal Article,

Biochemical analysis of the interaction of fibronectin with IgG and localization of the respective binding sites
Rostagno A; Williams M; Frangione B; Gold LI
1996 Apr;33(6):561-572, Molecular immunology
Fibronectin (Fn), a mosaic protein composed of multiple copies of three different module types (Fl, F2 and F3), has been found associated with circulating immune complexes (ICs) and immunoglobulin (Ig) aggregates in a variety of IC diseases and myeloproliferative disorders. We have previously shown that a proteolytic fragment of Mr = 25,900 Da, from the NH2-terminal domain of Fn, composed of five type 1 modules (1Fl -5Fl) binds to the major Ig classes under physiologic conditions, suggesting that the presence of Fn in ICs and cryoglobulins results from a physicochemical binding interaction between these two molecules. Using an ELISA, we now show that the interaction between Fn and IgG is: (1) not influenced by any other constituent of plasma; (2) unaffected by temperature; and (3) has an estimated Kd of 3.77 x 10(-9) M. In addition, we have further delineated the respective sites involved in the interaction between Fn and IgG. Recombinant type l module pairs (1Fl.2Fl and 4Fl.5Fl) from the NH2-terminus of Fn, expressed in yeast, were employed in an ELISA and affinity chromatography and compared with the 25.9 kDa (1Fl - 5Fl) fragment and intact Fn for binding to IgG. The 4Fl.5Fl and the 25.9 kDa fragment bound to immobilized IgG and inhibited Fn binding to IgG to nearly the same extent as the intact molecule (IC50: Fn = 6.77 x 1O(-9) M; 25.9 kDa fragment = 5 x 10(-9) M; 4Fl.5Fl = 7.6 x 10(-9) M). Thus, the binding site for IgG on the Fn molecule is localized to and completely conferred by the 4Fl.5Fl module pair (residues 151-244). Similar experiments using papain-generated Fab and Fc fragments of IgG localized the Fn binding site on IgG to the Fe region of the IgG molecule. Fn bound to the Fc fragment with a nearly identical Kd of 3.69 x 10(-9) M, as to intact IgG (3.77 x 10(-9) M). These studies support the hypothesis that the interaction between Fn and Ig may contribute to the pathophysiology of immune complex related disorders
— id: 7043, year: 1996, vol: 33, page: 561, stat: Journal Article,

Differential expression of transforming growth factor-beta isoforms and receptors in experimental membranous nephropathy
Shankland, S J; Pippin, J; Pichler, R H; Gordon, K L; Friedman, S; Gold, L I; Johnson, R J; Couser, W G
1996 Jul;50(1):116-124, Kidney international
In membranous nephropathy (MN) overproduction of matrix by glomerular epithelial cells (GEC) is believed to be responsible for glomerular basement membrane thickening and spikes. We studied experimental MN in rats (passive Heymann nephritis, PHN) at 5, 10 and 30 days. PHN rats exhibited a marked increase in GEC immunostaining for TGF-beta 2 at all time points. TGF-beta 3 staining was increased at day 10 only, and TGF-beta 1 was unchanged. Glomerular mRNA for TGF-beta 2 and -beta 3 was increased by day 5 when urine protein increased, whereas TGF-beta 1 was not. TGF-beta 2 bioactivity was increased at day 5. There was also a marked increase in GEC immunostaining for TGF-beta receptor type I (T beta IR) and TGF-beta receptor type II (T beta IIR) at all time points in PHN. mRNA levels for both receptors increased at day 5. Increases in protein expression and mRNA levels for the TGF-beta 2 and -beta 3 isoforms, and T beta IR and T beta RII were prevented by complement depletion. We conclude that complement-mediated injury to the GEC in vivo is associated with the up-regulation of TGF-beta 2 and -beta 3 isoforms, an increase in TGF-beta 2 bioactivity, and an increase in T beta RI and T beta RII expression. This contrasts with changes in TGF-beta 1 reported in mesangial disease, suggesting that TGF-beta 2 and -beta 3 may be important in diseases of the GEC. The differential expression of TGF-beta isoforms and receptors may be important determinants of the GEC response to injury
— id: 76300, year: 1996, vol: 50, page: 116, stat: Journal Article,

Transforming growth factor-beta protein and messenger RNA expression is increased in the closing ductus arteriosus
Tannenbaum, JE; Waleh, NS; Mauray, F; Gold, L; Perkett, EA; Clyman, RI
1996 MAR ;39(3):427-434, Pediatric research
In full-term newborns, permanent closure of the ductus arteriosus is associated with the formation of a neointima that is characterized by extracellular matrix deposition and smooth muscle cell migration. Transforming growth factor-beta (TGF-beta), a potent modulator of extracellular matrix deposition and smooth muscle cell migration, has been found to play a role in the remodeling associated with several forms of vascular disease. We examined the protein and mRNA expression of the three mammalian isoforms of TGF-beta (TGF-beta(1), TGF-beta(2) and TGF-beta(3)) during ductus arteriosus closure in full-term lambs. We found that the temporal changes and cellular localization of the proteins and mRNAs of all three TGF-beta isoforms were similar. TGF-beta proteins and mRNAs were present in very low levels in the late-gestation fetal ductus. Within 24 h of delivery, there was enhanced expression of TGF-beta in the newly forming neointima and outer muscle media; this continued to increase over the next 10 d. Increased expression of TGF-beta in the inner muscle media and adventitia lagged behind that of the neointima and outer muscle media. TGF-beta was not found in the luminal endothelial cells at any time. In contrast to the pattern described above, the appearance of TGF-beta protein differed from that of mRNA in the vasa vasorum of the ductus wall. After delivery, there was an increase in TGF-beta immunoreactivity in the smooth muscle cell layers of the vasa vasorum without any concurrent mRNA expression. The appearance of TGF-beta at the time of ductus closure suggests an important role for this growth factor in the reorganization of the ductus wall after birth
— id: 98398, year: 1996, vol: 39, page: 427, stat: Journal Article,

Expression of transforming growth factor-beta isoforms in human glomerular diseases
Yamamoto, T; Noble, N A; Cohen, A H; Nast, C C; Hishida, A; Gold, L I; Border, W A
1996 Feb;49(2):461-469, Kidney international
Protein and mRNA expression of TGF-beta isoforms, TGF-beta 1, -beta 2 and -beta 3, and deposition of fibronectin containing extra domain A (fibronectin EDA+) and plasminogen activator inhibitor-1 (PAI-1) were studied in human chronic glomerulonephritis and diabetic nephropathy. Normal kidneys showed similar, weak immunostaining for all three TGF-beta isoforms. TGF-beta mRNA expression was weak for all isoforms with TGF-beta 1 > TGF-beta 3 >> TGF-beta 2. In thin basement membrane disease and minimal change disease, disorders where extracellular matrix accumulation is not a feature, immunoreactivity and mRNA expression did not differ from normal. In contrast, diseases characterized by extracellular matrix accumulation (IgA nephropathy, focal and segmental glomerulosclerosis, crescentic glomerulonephritis, lupus nephritis and diabetic nephropathy) all showed significantly increased expression of the three TGF-beta isoforms in glomeruli and the tubulointerstitium. While glomerular and tubulointerstitial deposition of two matrix components induced by TGF-beta, fibronectin EDA+ and PAI-1, was significantly elevated in all diseases with matrix accumulation, correlation analysis revealed a close relationship primarily with TGF-beta 1. We conclude that, for a spectrum of human glomerular disorders, increased protein expression of all three TGF-beta isoforms and proteins induced by TGF-beta is associated with pathological accumulation of extracellular matrix
— id: 76302, year: 1996, vol: 49, page: 461, stat: Journal Article,

ACTIVE MACROPHAGE-ASSOCIATED TGF-BETA COLOCALIZES WITH TYPE-I PROCOLLAGEN GENE-EXPRESSION IN ATHEROSCLEROTIC HUMAN PULMONARY-ARTERIES
BAHADORI, L; MILDER, J; GOLD, L; BOTNEY, M
1995 MAY ;146(5):1140-1149, American journal of pathology
Vascular remodeling is adult atherosclerotic pulmonary arteries is characterized by discrete areas of neointimal smooth muscle cell extracellular matrix gene expression is close proximity to non-foamy macrophages, suggesting regulation by local macrophage-associated factors. the purpose of these studies was to begin addressing the role of putative macrophage-associated factors such as transforming growth factor-beta (TGF-beta), by determining the spatial relationship between TGF-beta and neointimal matrix gene expression in human atherosclerotic pulmonary arteries. For example, the participation of TGF-beta in vascular remodeling could be inferred by its colocalization with non-foamy macrophages in areas of active matrix synthesis. In situ hybridization and immunohistochemistry demonstrated focal neointimal procollagen gene expression in close association with non;foamy but not foamy macrophages. Immunohistochemistry with isoform-specific anti-TGF-beta antibodies demonstrated all three isoforms of TGF-beta associated with non-foamy macrophages, but foamy macrophages were not immunoreactive. Neointimal and medial smooth muscle cells stained lightly In contrast, intense TGF-beta immunoreactivity was also associated with medial smooth muscle cells in normal nonremodeling vessels. Immunohistochemistry with antibodies specific for latent TGF-beta was similar to immunohistochemistry for mature TGF-beta in both remodeling and nonremodeling vessels. Finally, using an antibody specific for active TGF-beta(1), immunoreactivity was only seen in non-foamy neointimal macrophages but not in foamy macrophages or medial smooth muscle cells from hypertensive or normal vessels. These observations suggest non-foamy macrophages may participate in modulating matrix gene expression ill atherosclerotic remodeling via a TGF-beta-dependent mechanism
— id: 87301, year: 1995, vol: 146, page: 1140, stat: Journal Article,

Transforming growth factor beta mediates the progesterone suppression of an epithelial metalloproteinase by adjacent stroma in the human endometrium
Bruner, K L; Rodgers, W H; Gold, L I; Korc, M; Hargrove, J T; Matrisian, L M; Osteen, K G
1995 Aug 1;92(16):7362-7366, Proceedings of the National Academy of Sciences of the United States of America
Unlike most normal adult tissues, cyclic growth and tissue remodeling occur within the uterine endometrium throughout the reproductive years. The matrix metalloproteinases (MMPs), a family of structurally related enzymes that degrade specific components of the extracellular matrix are thought to be the physiologically relevant mediators of extracellular matrix composition and turnover. Our laboratory has identified MMPs of the stromelysin family in the cycling human endometrium, implicating these enzymes in mediating the extensive remodeling that occurs in this tissue. While the stromelysins are expressed in vivo during proliferation-associated remodeling and menstruation-associated endometrial breakdown, none of the stromelysins are expressed during the progesterone-dominated secretory phase of the cycle. Our in vitro studies of isolated cell types have confirmed progesterone suppression of stromal MMPs, but a stromal-derived paracrine factor was found necessary for suppression of the epithelial-specific MMP matrilysin. In this report, we demonstrate that transforming growth factor beta (TGF-beta) is produced by endometrial stroma in response to progesterone and can suppress expression of epithelial matrilysin independent of progesterone. Additionally, we find that an antibody directed against the mammalian isoforms of TGF-beta abolishes progesterone suppression of matrilysin in stromal-epithelial cocultures, implicating TGF-beta as the principal mediator of matrilysin suppression in the human endometrium
— id: 76303, year: 1995, vol: 92, page: 7362, stat: Journal Article,

The in vivo effect of hyaluronan associated protein-collagen complex on wound repair
Cabrera RC; Siebert JW; Eidelman Y; Gold LI; Longaker MT; Garg HG
1995 Sep;37(1):151-158, Biochemistry & molecular biology international
Fetal skin wounds heal without scarring, however the underlying mechanisms remain unknown. Immunohistochemical staining and biochemical studies indicate the deposition of a collagen repair matrix that is highly organized. We have previously described a unique hyaluronan associated protein-collagen complex (HA-PC) profile present during the period of scarless healing in the sheep fetus. In this study, we examined the biologic activity of this HA-PC in an in vivo model of adult rat wound repair. A total of 84 incisional and 84 excisional wounds were examined by histology, TGF-beta immunocytochemistry and computer planimetry (excisional wounds only). Planimetry of the excisional wounds demonstrated the mean wound area remaining at day 1 was 88.7% for the control and 63.6% for the treated (p<0.01). At day 2, mean wound area was 81.5% for the control and 63.6% for the treated (p<0.01). At day 4, mean wound area was 56.6% for the control and 41.9% for the treated (p<0.01). At day 7, mean wound area was 26.9% for the control and 16.8% for the treated (p<0.01). At day 14, mean wound area was 7.9% for the control and 3.4% for the treated (p<0.05). Collagen organization was judged to be greater in the treated compared to control wounds, with a mean organization score of 2.3 vs. 1.9 (p=0.0596; Wilcoxon Signed Rank Sum Test). There were significantly more neutrophils at the wound margin of the treated compared to control wounds, 4.0 vs. 2.7 (p=0.038; Paired Two Tailed Student's t-Test). There was no difference in the number of microphages at the wound margin of the treated compared to control wounds, 6.15 vs. 6.0 (p>0.05). TGFbeta1 and beta2 staining was decreased whereas TGFbeta3 staining was increased in the HA-PC treated wounds. These results suggest that compared to control wounds HA-PC treated wounds heal more quickly, with more organized collagen, more neutrophils at the wound margin and increased TGFbeta3 expression. Furthermore, these data suggest that the manipulation of scarring in adult wounds is possible by the addition of proteins present in fetal skin
— id: 6986, year: 1995, vol: 37, page: 151, stat: Journal Article,

High levels of transforming growth factor beta 1 correlate with disease progression in human colon cancer
Friedman, E; Gold, L I; Klimstra, D; Zeng, Z S; Winawer, S; Cohen, A
1995 Jul-Aug;4(5):549-554, Cancer epidemiology biomarkers & prevention
Several genes have identified that play a role in colon cancer development. However, less is known about factors that increase the rate of progression of colon cancers to metastasis. One candidate is transforming growth factor beta 1 (TGF beta 1), which can enhance the aggressiveness of human colorectal cell lines in vitro and in vivo. The amount of TGF beta 1, TGF beta 2, and TGF beta 3 protein isoforms expressed in primary site colorectal cancers were measured to determine whether any correlation existed between protein levels and disease recurrence in a series of Memorial Sloan-Kettering Hospital patients who underwent potentially curative resections. Intense staining for TGF beta 1 correlated significantly (P < 0.0013; odds ratio, 18) with disease progression to metastasis and was independent of nodal status and the degree of differentiation of the primary tumor. Therefore, in this study, patients with high TGF beta 1 protein levels in their primary site colorectal cancer were 18 times more likely to experience recurrence of their disease than were patients whose tumors exhibited low levels of TGF beta 1. In this case-control study, patients whose cancer recurred and those remaining cancer free were age and sex matched. The disease recurred at a mean of 26.8 +/- 4.3 (SE) months, whereas the mean follow-up time in patients whose disease did not recur was over twice as long, 57.3 +/- 6.6 months. Ninety-four % of the patients in each group were node positive at the time of resection, with equal mean numbers of positive nodes per patient.(ABSTRACT TRUNCATED AT 250 WORDS)
— id: 76305, year: 1995, vol: 4, page: 549, stat: Journal Article,

OVEREXPRESSION OF TGF-BETA-1, TGF-BETA-3 AND TGF-BETA RECEPTOR TYPE-II BUT NOT TO TGF-BETA-2 AND TGF-BETA RECEPTOR TYPE-III IN HUMAN LIVER-CIRRHOSIS
FRIESS, H; BERBERAT, P; DEFLORIN, J; BAER, HU; GOLD, LI; KORC, M; BUCHLER, MW
1995 APR ;108(4):A1067-A1067, Gastroenterology
— id: 86751, year: 1995, vol: 108, page: A1067, stat: Journal Article,

OVEREXPRESSION OF TRANSFORMING GROWTH FACTOR-BETA-S AND THEIR RECEPTORS IN ACUTE-PANCREATITIS IN HUMANS
FRIESS, H; BUCHLER, MW; HOFBAUER, B; UHL, W; GOLD, LI; KORC, M
1995 APR ;108(4):A354-A354, Gastroenterology
— id: 86749, year: 1995, vol: 108, page: A354, stat: Journal Article,

TRANSFORMING GROWTH FACTOR-BETA-3 LOCALIZATION DURING REPAIR OF LUNG INJURY
HOFF, CR; GOLD, LI; DAVIDSON, JM
1995 APR ;37(4):A392-A392, Pediatric research
— id: 86762, year: 1995, vol: 37, page: A392, stat: Journal Article,

TRANSFORMING GROWTH-FACTOR-BETA (TGF-BETA) ISOFORM REACTIVITY IN NORMAL AND NEOPLASTIC BREAST-TISSUE
JHALA, N; BALSARA, G; ALBO, D; GRANICK, M; GOLD, L; ATKINSON, BF; SOLOMON, M
1995 JAN ;72(1):A19-A19, Laboratory investigation
— id: 98426, year: 1995, vol: 72, page: A19, stat: Journal Article,

Spatial and temporal expression of transforming growth factor-beta isoforms during ovine excisional and incisional wound repair
McMullen, H; Longaker, M T; Cabrera, R C; Sung, J; Canete, J; Siebert, J W; Lorenz, H P; Gold, L I
1995 Apr-Jun;3(2):141-156, Wound repair & regeneration
To elucidate the role for transforming growth factor-beta isoforms (beta(1), -beta(2), and -beta(3)) in wound repair, we used isoform-specific antibodies to detect the spatial and temporal expression of the latent and mature/active transforming growth factor-beta isoforms by immunohistochemical localization through 21 days after excisional and incisional wounding of ovine skin. Although incisional and excisional wounds showed similar patterns of transforming growth factor-beta immunoreactivity, we found a differential temporal and spatial expression of the latent and mature transforming growth factor-beta isoforms throughout wound repair. Specifically, 1 day after wounding, there was a marked increase in transforming growth factor-beta isoforms in the epithelium adjacent to the wound, epidermal appendages, and the cells and matrix of the granulation tissue. At this time, transforming growth factor-beta(3) isoform was the most abundant. Most notably, the epidermis adjacent to the wound was intensely immunoreactive for all transforming growth factor-beta isoforms 1 day after injury. However, the migrating epithelium, derived from both the hair follicles and the wound margins, was completely devoid of immunoreactive transforming growth factor-beta until reepithelialization was complete. Within the inflammatory exudate, there was a distinct band of leukocytes that was immunoreactive for transforming growth factor-beta(2) and -beta(3) 1 day after injury and 1 day later for transforming growth factor-beta(1). Although transforming growth factor-beta(1) and -beta(2), latent transforming growth factor-beta(2), transforming growth factor-beta(3), and latent transforming growth factor-beta(3) immunostaining was present in the numerous fibroblasts and other dermal cells, latent transforming growth factor-beta(1) was only associated with the extracellular matrix. In general, immunoreactivity remained high until day 7 after wounding and slowly subsided over time. However, by day 21, immunostaining had not returned to normal and the original wound was replete with immunoreactive fibroblasts and a dense, immunostained extracellular matrix. Thus, although the dynamic presence of transforming growth factor-beta isoforms exemplifies its positive role in the wound repair process, its persistence together with its known potent effects on matrix accumulation, supports its role in scar formation
— id: 76307, year: 1995, vol: 3, page: 141, stat: Journal Article,

COMPLEX REGULATION OF TRANSFORMING GROWTH-FACTOR-BETA ISOFORMS IN HYPEROXIC LUNG
PIEDBOEUF, B; GOLD, LI; SUNG, J; KAZZAZ, J; HOROWITZ, S
1995 APR ;37(4):A345-A345, Pediatric research
— id: 98411, year: 1995, vol: 37, page: A345, stat: Journal Article,

Fibronectin/fibrin interaction: Localization of the C-terminal fibrin-binding site
Pollakis, G.; Rostagno, A.; Gold, L. I.
1995 ;6(SUPPL.):383A-383A, Molecular biology of the cell
— id: 101622, year: 1995, vol: 6, page: 383A, stat: Journal Article,

FIBRONECTIN/FIBRIN INTERACTION - LOCALIZATION OF THE C-TERMINAL FIBRIN-BINDING SITE
POLLAKIS, G; ROSTAGNO, A; GOLD, LI
1995 NOV ;6(6738):2225-2225, Molecular biology of the cell
— id: 73962, year: 1995, vol: 6, page: 2225, stat: Journal Article,

FIBRONECTIN BINDS TO CRYOGLOBULINS FOLLOWING THEIR COLD PRECIPITATION
ROSTAGNO, A; GOLD, LI
1995 NOV ;6(6):2224-2224, Molecular biology of the cell
— id: 73963, year: 1995, vol: 6, page: 2224, stat: Journal Article,

Increased expression of transforming growth factor beta isoforms (beta 1, beta 2, beta 3) in bleomycin-induced pulmonary fibrosis
Santana, A; Saxena, B; Noble, N A; Gold, L I; Marshall, B C
1995 Jul;13(1):34-44, American journal of respiratory cell & molecular biology
Evidence suggests that transforming growth factor beta (TGF-beta) may play a central role in a variety of fibroproliferative disorders via the induction of extracellular matrix accumulation. The three mammalian TGF-beta isoforms are present in the normal lung, but very little is known about their expression during lung injury and repair. To more fully understand the role of TGF-beta in lung repair, we investigated the expression of the TGF-beta 1, TGF-beta 2, and TGF-beta 3 isoforms in a bleomycin-induced model of pulmonary fibrosis using immunohistochemical and in situ hybridization techniques. We found expression of the three TGF-beta isoforms, in an identical pattern, widely distributed throughout the normal rat lung: in airways, blood vessels, lung parenchyma, and alveolar macrophages. In general, the distribution of TGF-beta mRNA and protein coincided; however, bronchial epithelial cells were a notable exception, exhibiting immunoreactivity but no mRNA expression. During the 'inflammatory' phase (days 1 and 3) of bleomycin-induced injury there was an increase in the mRNA and protein expression of all three TGF-beta isoforms in the injured areas, most prominently in parenchymal cells and alveolar macrophages. There was a further increase in TGF-beta isoform expression in the areas of developing fibrosis during the later reparative phase (days 7 and 14), and the bronchial epithelium, previously not expressing TGF-beta mRNA, showed strong expression of mRNA for the three isoforms concomitant with increased immunoreactivity. These findings implicate the three mammalian TGF-beta isoforms in the dysregulated repair process that results in pulmonary fibrosis. Furthermore, the pattern of TGF-beta mRNA and protein expression by the bronchial epithelium suggests that a transition may occur at this site from a paracrine mode of action in the normal lung to an autocrine mode of action during the 'reparative' phase of fibrosis
— id: 76306, year: 1995, vol: 13, page: 34, stat: Journal Article,

Transforming growth factor-beta and matrix protein expression in acute and chronic rejection of human renal allografts
Shihab, F S; Yamamoto, T; Nast, C C; Cohen, A H; Noble, N A; Gold, L I; Border, W A
1995 Aug;6(2):286-294, Journal of the American Society of Nephrology
Because the increased tissue expression of TGF-beta underlies fibrosis in many diseases, it was hypothesized that sustained elevated transforming growth factor (TGF)-beta overexpression might be responsible for fibrosis in chronic rejection of the renal allograft. To test this hypothesis, biopsies were obtained from 5 patients with acute rejection, 5 patients with chronic rejection, 10 normal individuals, and 10 patients with kidney disease. The tissues were examined by immunofluorescence for the three TGF-beta isoforms (1, 2, and 3) and the two matrix proteins induced by TGF-beta that serve as markers of fibrosis: fibronectin extradomain A positive (EDA+) and plasminogen activator inhibitor-1 (PAI-1). The tubulointerstitium from all cases of acute rejection and chronic rejection showed highly significant increases in immunostaining for the three TGF-beta isoforms (P < 0.001), fibronectin EDA+ (P < 0.005), and PAI-1 (P < 0.001). In the glomeruli, only TGF-beta 1 expression achieved statistical significance (P < 0.005) in acute rejection, whereas in chronic rejection, all three TGF-beta isoforms (p < 0.001) in addition to fibronectin EDA+ (p < 0.001) and PAI-1 (p < 0.001) were elevated. There was both cellular and matrix staining of the TGF-beta isoforms. In striking contrast, control kidney tissues were negative or only weakly positive. Because TGF-beta was present both in acute and in chronic rejection but not in control tissues and because acute rejection episodes are a good predictor for chronic rejection, these results suggest that TGF-beta may play a role in the pathogenesis of fibrosis in chronic rejection
— id: 76304, year: 1995, vol: 6, page: 286, stat: Journal Article,

Vascular remodeling in primary pulmonary hypertension. Potential role for transforming growth factor-beta
Botney, M D; Bahadori, L; Gold, L I
1994 Feb;144(2):286-295, American journal of pathology
Active exogenous transforming growth factor-beta s (TGF-beta s) are potent modulators of extracellular matrix synthesis in cell culture and stimulate matrix synthesis in wounds and other remodeling tissues. The role of endogenous TGF-beta s in remodeling tissues is less well defined. Vascular remodeling in the pulmonary arteries of patients with primary pulmonary hypertension is characterized, in part, by abnormal deposition of immunohistochemically detectable procollagen, thereby identifying actively remodeling vessels. We used this marker of active matrix synthesis to begin defining the in vivo role of TGF-beta in the complex milieu of actively remodeling tissues. Immunohistochemistry using isoform-specific anti-TGF-beta antibodies was performed to determine whether TGF-beta was present in actively remodeling hypertensive pulmonary arteries 20 to 500 microns in diameter. Intense, cell-associated TGF-beta 3 immunoreactivity was observed in the media and neointima of these hypertensive muscular arteries. Immunostaining was present, but less intense, in normal arteries of comparable size. TGF-beta 2 immunoreactivity was observed in normal vessels and was increased slightly in hypertensive vessels, in a pattern resembling TGF-beta 3 immunoreactivity. No staining was associated with the adventitia. TGF-beta 1 immunostaining was either faint or absent in both normal and hypertensive vessels. Comparison of procollagen and TGF-beta localization demonstrated that TGF-beta 2 and TGF-beta 3 colocalized at all sites of procollagen synthesis. However, TGF-beta was observed in vessels, or vascular compartments, where there was no procollagen synthesis. Procollagen immunoreactivity was not present in normal vessels that showed immunoreactivity for TGF-beta 2 and TGF-beta 3. These observations suggest: a) the stimulation of procollagen synthesis by TGF-beta in vivo is more complex than suggested by in vitro studies and b) a potential role for TGF-beta 2 or TGF-beta 3, but not TGF-beta 1, in hypertensive pulmonary vascular remodeling
— id: 76312, year: 1994, vol: 144, page: 286, stat: Journal Article,

Localization of transforming growth factor beta isoforms TGF-beta 1, TGF-beta 2, and TGF-beta 3 in surgically induced endometriosis in the rat
Chegini, N; Gold, L I; Williams, R S
1994 Mar;83(3):455-461, Obstetrics & gynecology
OBJECTIVE: To elucidate the presence and cellular distribution of transforming growth factor beta (TGF-beta) in surgically induced endometriosis in the rat. METHODS: Endometriosis was induced by implanting pieces of uterine fragments in the mesenteric region adjacent to a blood vessel for a period of 4-6 weeks. The endometrial implants were removed and processed for immunohistochemical localization using isoform-specific polyclonal antibodies to TGF-beta 1, TGF-beta 2, and TGF-beta 3. RESULTS: All the cell types in the endometrial implants with the exception of stromal cells immunostained for TGF-beta 1 and TGF-beta 3, but not TGF-beta 2. The inflammatory cells that were infiltrated among endometriotic stromal cells and implant-associated cysts contained the highest immunostaining intensity for TGF-beta s 1-3, followed by luminal and glandular epithelial cells, fibroblasts of the fibrous adhesions, and endothelial and smooth-muscle cells of arterioles. The immunostaining intensity of TGF-beta 1 was substantially higher than that of TGF-beta 3 and TGF-beta 2, and intensity was similar to the endometrial tissue from cycling rats in diestrus II for TGF-beta 1 and estrus for TGF-beta 2 and TGF-beta 3. In the cycling rats, the order of immunostaining intensity in the endometrium was TGF-beta 1, TGF-beta 3, and TGF-beta 2, respectively, with a higher intensity in diestrus II and proestrus than in diestrus I and estrus. CONCLUSION: The results indicate that endometrial implants in surgically induced endometriosis contain immunoreactive TGF-beta s, which may imply a possible paracrine/autocrine role for the action of TGF-beta in the maintenance of viable endometriotic tissue and the development of fibrous adhesions associated with the implants
— id: 76310, year: 1994, vol: 83, page: 455, stat: Journal Article,

Localization of transforming growth factor beta isoforms TGF-beta 1, TGF-beta 2, and TGF-beta 3 in surgically induced pelvic adhesions in the rat
Chegini, N; Gold, L I; Williams, R S; Masterson, B J
1994 Mar;83(3):449-454, Obstetrics & gynecology
OBJECTIVE: To investigate the presence and cellular distribution of transforming growth factor (TGF)-beta s in surgically induced pelvic fibrous adhesions in rat uterine horns subjected to burn, crush, and debridement injury. METHODS: Thirty injured and 20 uninjured rats were treated postoperatively with intraperitoneal administration of either 2 micrograms/mL of recombinant human TGF-beta, 10 micrograms/mL TGF-beta neutralizing antibody, or phosphate-buffered saline + 500 micrograms rat serum albumin for 5 consecutive days. The intact (uninjured) and fibrous tissues were analyzed immunohistochemically for the presence of TGF-beta s using polyclonal antibodies to TGF-beta s 1-3. RESULTS: The intact peritoneum immunostained with a lower intensity than fibrous adhesive tissues for TGF-beta 1, TGF-beta 2, and TGF-beta 3. The immunoreactive TGF-beta s were present in fibroblasts, inflammatory cells infiltrated into the fibrous adhesion, and endothelial and smooth-muscle cells of the arterioles. In the uterine tissue at the site of injury, the following immunostained for TGF-beta s: uterine serosal tissue, myometrial smooth-muscle cells, endometrial luminal and glandular epithelial cells, and inflammatory cells. However, endometrial stromal cells did not immunostain for TGF-beta s. There were no substantial differences in immunostaining intensities of fibrous adhesive tissues in the TGF-beta group, neutralizing TGF-beta antibody group, and the controls. CONCLUSION: The data suggest that TGF-beta s may play a role in the formation and maintenance of fibrous adhesions following intraperitoneal injury
— id: 76311, year: 1994, vol: 83, page: 449, stat: Journal Article,

Increased expression of transforming growth factor beta isoforms and basic fibroblast growth factor in complex hyperplasia and adenocarcinoma of the endometrium: evidence for paracrine and autocrine action
Gold LI; Saxena B; Mittal KR; Marmor M; Goswami S; Nachtigall L; Korc M; Demopoulos RI
1994 May 1;54(9):2347-2358, Cancer research
Endometrial carcinoma is associated with antecedent simple and complex hyperplasia, and the endometrium is a target tissue for the action of cytokines and growth factors. Transforming growth factor (TGF)-beta s are potent cellular growth and differentiation regulatory factors. Therefore, we investigated the potential role for TGF-beta s in the normal proliferative endometrium and its possible involvement in the transition to complex hyperplasia and progression to endometrial carcinoma. The angiogenic and mitogenic growth factor, basic fibroblast growth factor, was used for comparison. Differential TGF-beta isoform-specific immunoreactivity was observed in the normal endometrium, which is composed of glandular and stromal cells. There was an increase in TGF-beta 3 but not TGF-beta 1 or TGF-beta 2 in the glandular epithelium from the proliferative to the secretory phase of the menstrual cycle. Immunostaining for TGF-beta 2 was more intense in the stroma than the glands. In contrast, TGF-beta 1 and TGF-beta 3 were near equal intensity in these two endometrial compartments, TGF-beta 3 being the most intense. The glandular epithelium demonstrated a statistically significant stepwise increase in the expression of all three TGF-beta s progressing from the normal proliferative endometrium to simple hyperplasia and on to complex hyperplasia. However, the stromal cells maintained approximately the same level of immunoreactivity for TGF-beta in all these samples. In comparing proliferative endometrium with complex hyperplasia, there was a 5.1-, 3.4-, and 2.6-fold increase in immunostaining in the glands for TGF-beta 1, TGF-beta 2, and TGF-beta 3, respectively (P < or = 0.001). There was no further increase in immunoreactivity with progression from preneoplastic complex hyperplasia to carcinoma. Immunoreactive basic fibroblast growth factor was slight in normal endometrium and simple hyperplasia. There was a 4.6- and 4.2-fold increase in immunostaining observed in complex hyperplasia compared with proliferative endometrium in the glandular (P < or = 0.0054) and stromal (P < or = 0.0053) cells, respectively, with no further increase in carcinoma. By in situ hybridization, an increase in mRNA for all TGF-beta isoforms paralleled TGF-beta immunoreactivity. However, in contrast to the increased immunostaining in the glands in complex hyperplasia, there was remarkably more mRNA in the stromal cell compartment. The discordant expression of mRNA and protein was only observed in the pathological endometrium since both were more highly expressed in the stromal cells in normal proliferative endometrium.(ABSTRACT TRUNCATED AT 400 WORDS)
— id: 6386, year: 1994, vol: 54, page: 2347, stat: Journal Article,

Expression of transforming growth factor-beta-1,2, and 3 mRNA and protein in human cancers
Gold, Leslie I.; Korc, Murray
1994 ;11(3-6):150-156, Digestive surgery
Historically, the role for transforming growth factor-Ps (TGF-beta-s) in malignancy has been difficult to prove. TGF-beta-s are potent autocrine and paracrine negative regulators of the growth of normal epithelial and neuroectodermal cells in vitro. Therefore we hypothesized that the aberrant growth of tumor cells and neoplastic development, in general, may be due to a loss of growth regulation by TGF-beta. By immunohistochemistry, using isoform specific antibodies raised to synthetic peptides of each TGF-beta isoform, and by in situ hybridization, using non-cross-hybridizing cRNA probes to each TGF-beta isoform, we examined the protein and mRNA expression of TGF-beta, respectively, in a variety of carcinomas and gliomas compared to normal tissue. The analysis of the cell types that synthesized the message for TGF-beta isoforms compared to those which synthesized the protein indicated whether TGF-beta functioned by an autocrine or paracrine mechanism of action in these tumors
— id: 98809, year: 1994, vol: 11, page: 150, stat: Journal Article,

TGF-beta-1, -beta-2, -beta-3, and IGF-1 localization in rat cranial suture development and fusion
Longaker, Michael T.; Roth, Douglas A.; McMullen, Heather F.; Breitbart, Arnold S.; Wisoff, Jeffrey H.; Han, Victor K.; Gold, Leslie I.; McCarthy, Joseph G.
1994 ;45(0):589-591, Surgical forum
— id: 98811, year: 1994, vol: 45, page: 589, stat: Journal Article,

Neutrophil chemotaxis in response to TGF-beta isoforms (TGF-beta 1, TGF-beta 2, TGF-beta 3) is mediated by fibronectin
Parekh T; Saxena B; Reibman J; Cronstein BN; Gold LI
1994 Mar 1;152(5):2456-2466, Journal of immunology
TGF-beta isoforms regulate numerous cellular functions including cell growth and differentiation, the cellular synthesis and secretion of extracellular matrix proteins, such as fibronectin (Fn), and the immune response. We have previously shown that TGF-beta 1 is the most potent chemoattractant described for human peripheral blood neutrophils (PMNs), suggesting that TGF-beta s may play a role in the recruitment of PMNs during the initial phase of the inflammatory response. In our current studies, we demonstrate that the maximal chemotactic response was attained near 40 fM for all mammalian TGF-beta isoforms. However, there was a statistically significant difference in migratory distance of the PMNs: TGF-beta 2 (556 microM) > TGF-beta 3 (463 microM) > TGF-beta 1 (380 microM) (beta 2: beta 3, p < or = 0.010; beta 3: beta 1, p < or = 0.04; beta 2: beta 1, p < or = 0.0012). A mAb to the cell binding domain (CBD) of Fn inhibited the chemotactic response to TGF-beta 1 and TGF-beta 3 by 63% and to TGF-beta 2 by 70%, whereas the response to FMLP, a classic chemoattractant, was only inhibited by 18%. In contrast, a mAb to a C-terminal epitope of Fn did not retard migration (< 1.5%). The Arg-gly-Asp-ser tetrapeptide inhibited chemotaxis by approximately the same extent as the anti-CBD (52 to 83%). Furthermore, a mAb against the VLA-5 integrin (VLA-5; Fn receptor) also inhibited TGF-beta-induced chemotaxis. These results indicate that chemotaxis of PMNs in response to TGF-beta isoforms is mediated by the interaction of the Arg-gly-Asp-ser sequence in the CBD of Fn with an integrin on the PMN cell surface, primarily the VLA-5 integrin. TGF-beta isoforms also elicited the release of cellular Fn from PMNs; we observed a 2.3-fold increase in Fn (389 to 401 ng/ml) in the supernatants of TGF-beta-stimulated PMNs compared with unstimulated cells (173.6 ng/ml). The concentration of TGF-beta required to cause maximal release of Fn from PMNs (4000 fM) is a concentration at which TGF-beta is no longer chemotactic, suggesting that PMNs only use Fn that is constitutively expressed for migration. At higher concentrations of TGF-beta, the Fn released may accumulate basal to the cell, ultimately retarding cellular migration and modulating the chemotactic response
— id: 6480, year: 1994, vol: 152, page: 2456, stat: Journal Article,

Expression of transforming growth factor-beta mRNAs and proteins in pulmonary vascular remodeling in the sheep air embolization model of pulmonary hypertension
Perkett, E A; Pelton, R W; Meyrick, B; Gold, L I; Miller, D A
1994 Jul;11(1):16-24, American journal of respiratory cell & molecular biology
Transforming growth factor-beta (TGF-beta) has been suggested as one of the mediators of vascular remodeling in chronic pulmonary hypertension. We have previously shown a transient early increase in TGF-beta levels in lung lymph during the development of sustained pulmonary hypertension in a sheep model (12 days of air embolization). The present study examines expression and cellular localization of mRNA and protein of the three mammalian isoforms of TGF-beta in lung biopsy tissue taken during the development of pulmonary hypertension (0, 1, 4, 8, and 12 days of embolization). In control tissue, immunohistochemical techniques localized each of the TGF-beta proteins in an identical pattern in large preacinar airways--bronchial epithelium and subepithelial cells--and in the medial wall of muscular vessels; no protein was detected in intraacinar regions. Following air embolization, immunoreactivity appeared in peripheral lung. At day 1, immunoreactivity for TGF-beta 1 and TGF-beta 3 proteins was seen in edema fluid, in perivascular cells associated with nonmuscular intraacinar arteries, and in alveolar walls; no increased immunoreactivity was detected for TGF-beta 2. After 4, 8, and 12 days of embolization, immunoreactivity for all three TGF-beta proteins was associated with newly muscularized intraacinar arteries. With in situ hybridization, the three TGF-beta mRNAs co-localized in lung tissue from both control and air-embolized animals. In control tissue, hybridization was seen around preacinar airways and muscular vessels; no hybridization seen in intraacinar regions of the lung. After 1 day of embolization, the pattern of hybridization was similar to controls.(ABSTRACT TRUNCATED AT 250 WORDS)
— id: 76309, year: 1994, vol: 11, page: 16, stat: Journal Article,

Further characterization of the NH2-terminal fibrin-binding site on fibronectin
Rostagno A; Williams MJ; Baron M; Campbell ID; Gold LI
1994 Dec 16;269(50):31938-31945, Journal of biological chemistry
The fibronectin (Fn) monomer contains two major sites of fibrin binding affinity present within the NH2-terminal and COOH-terminal domains; they consist of five (1F1-5F1) and three (10F1-12F1) consecutive type 1 modules, respectively. Recently, we have reported that the fourth and fifth type 1 module pair (4F1.5F1) of the NH2-terminal domain of fibronectin demonstrated fibrin binding ability (Williams, M. J., Phan, I., Harvery, T. S., Rostagno, A., Gold, L. I., and Campbell, I. D. (1994) J. Mol. Biol. 235, 1303-1311). In an attempt to further localize fibrin binding activity and to characterize the nature of the interaction between different type 1 modules of Fn and fibrin, we have tested a range of recombinant proteins and subtilisin generated proteolytic fragments of Fn in an enzyme-linked immunosorbent assay (ELISA) and by fibrin affinity chromatography. Of the recombinant proteins, we found that only the 4F1.5F1 exhibited significant fibrin binding activity, while 1F1, 1F1.2F1, 7F1, and 10F1 had little to no affinity for fibrin. On a molar basis, 4-5 times more 4F1.5F1 than a proteolytic fragment, corresponding to 1F1-5F1 (25.9 kDa) was required to cause 50% inhibition (IC50) of intact biotinylated Fn binding to fibrin in a competitive ELISA. This suggests that all five type 1 modules in tandem engender higher fibrin binding activity than the 4F1.5F1 alone. Furthermore, since fibrin binding activity of the intact Fn molecule was inhibited, by 70-80%, by the 4F1.5F1, the 25.9-kDa fragment, and a MoAb mapped to an epitope on the 4F1.5F1, the fibrin-binding site within the 4F1.5F1 contributes greatly to the non-covalent interaction of intact Fn with fibrin. These results provide significant insight into the Fn/fibrin interaction, a major component of the processes of wound repair and fibrin matrix assembly
— id: 6734, year: 1994, vol: 269, page: 31938, stat: Journal Article,

Expression of transforming growth factor-beta 1 and -beta 2 in human papillomavirus (HPV)-associated lesions of the uterine cervix
Tervahauta, A; Syrjanen, S; Yliskoski, M; Gold, L I; Syrjanen, K
1994 Sep;54(3):349-356, Gynecologic oncology
Human papillomaviruses (HPV) are implicated in the multistep process of cervical carcinogenesis. Transforming growth factor beta(TGF-beta) inhibits the proliferation of epithelial cells, and it has also been found to inhibit HPV gene expression in nontumorigenic epithelial cell lines. In the present study, we examined the expression of TGF-beta 1 and TGF-beta 2 protein immunohistochemically (IHC) in a series of 95 HPV-positive and HPV-negative lesions of the uterine cervix, with special emphasis on HPV type, grade of cervical intraepithelial neoplasia (CIN), and the clinical course of the disease. Expression of TGF-beta 1 was found in 56/95 (59%) and that of TGF-beta 2 in 87/95 (92%) of the specimens. Cytoplasmic TGF-beta 2 staining was localized in the epithelial layers higher than that of TGF-beta 1, which showed also some nuclear staining and was located in the basal cells of the epithelium as well. TGF-beta 1 was expressed in 36/68 (53%) of HPV-positive samples and in 16/21 (76%) of HPV-negative samples; TGF-beta 2 expression was detectable in 63/68 (93%) and 18/21 (86%), respectively. TGF-beta 1 was present slightly more frequently in HPV-CIN lesions (23/41, 56%) than in HPV-NCIN (HPV without CIN) specimens (13/27, 48%). TGF-beta 2 expression was detected in 39/41 (95%) of HPV-CIN and in 24/27 (89%) of HPV-NCIN specimens. TGF-beta 2 expression was not related to the clinical course of the disease. TGF-beta 1 expression was most frequent in regressed and persistent lesions (> 60%), compared to 45% in progressed and 33% in the recurred lesions. The results suggest that TGF-beta (especially TGF-beta 2) expression is common in CIN lesions, but the pattern and intensity of TGF-beta expression examined by IHC are not clearly related to the grade of the lesions or their clinical course. Assessment of the biological activity of TGF-beta s and their influence on HPV genes may shed more light on HPV-associated carcinogenesis
— id: 76308, year: 1994, vol: 54, page: 349, stat: Journal Article,

Localization of transforming growth factor beta isoforms in the normal murine small intestine and colon
Barnard, J A; Warwick, G J; Gold, L I
1993 Jul;105(1):67-73, Gastroenterology
BACKGROUND: The transforming growth factor beta (TGF-beta) proteins are key regulators of cellular growth and differentiation. Previous studies have shown that TGF-beta 1 is a potent growth inhibitor of cultured jejunal epithelial cells. The reported distribution of TGF-beta 1 messenger RNA (mRNA) expression along the intestinal villus has been controversial. The purpose of the current study is to determine the loci of TGF-beta protein expression in the normal small intestine and colon. METHODS: Intestinal localization of TGF-beta isoform mRNA and protein was examined by Northern blot analysis and immunohistochemistry using isoform specific reagents. RESULTS: TGF-beta 1, TGF-beta 2, and TGF-beta 3 mRNA were found in homogenates from the intact mouse jejunum and colon. The three isoforms colocalized in these tissues. Expression in the small intestinal epithelium was most prominent in cells located on the villus tip, and no staining was detected in the crypt. Occasional lymphocytes in the lamina propria were immunopositive, and all layers of the muscularis were moderately stained. This pattern was seen in all regions of the small intestine. The surface epithelium of the colon was intensely immunopositive, whereas cells in the glands were only weakly stained. CONCLUSIONS: TGF-beta molecules may serve overlapping functions in the intestinal tract, and expression in the epithelium may function to arrest growth of cells emerging from the crypt and induce or maintain the terminally differentiated state
— id: 76316, year: 1993, vol: 105, page: 67, stat: Journal Article,

Enhanced expression of transforming growth factor beta isoforms in pancreatic cancer correlates with decreased survival
Friess, H; Yamanaka, Y; Buchler, M; Ebert, M; Beger, H G; Gold, L I; Korc, M
1993 Dec;105(6):1846-1856, Gastroenterology
BACKGROUND: Transforming growth factor beta s (TGF-beta s) constitute a family of bifunctional polypeptide growth factors that either inhibit or stimulate cell proliferation. Perturbations in TGF-beta expression and function may lead to loss of negative constraints on cell growth. In this study, we examined TGF-beta expression in human pancreatic cancer. METHODS: The distribution of TGF-beta isoforms in 60 human pancreatic cancers was examined using immunohistochemical, Northern blot, and in situ hybridization techniques. RESULTS: Immunohistochemical analysis showed the presence of TGF-beta 1 (47% of tumors), TGF-beta 2 (42% of tumors), and TGF-beta 3 (40% of tumors) in the cancer cells. The presence of TGF-beta 2 was associated with advanced tumor stage (P < 0.05). Furthermore, there was a significant correlation between the absence of TGF-beta s in the tumors and longer postoperative survival. Northern blot analysis indicated that, by comparison with the normal pancreas, pancreatic adenocarcinomas showed 11- (P < 0.001), 7- (P < 0.05), and 9-fold (P < 0.001) increases in the messenger RNA (mRNA) levels encoding TGF-beta 1, TGF-beta 2, and TGF-beta 3, respectively. By in situ hybridization, these mRNA moieties colocalized with their respective proteins in the cancer cells. CONCLUSIONS: These findings show that human pancreatic cancers show increased levels of TGF-beta isoforms and enhanced TGF-beta mRNA expression and suggest that the presence of TGF-beta s in pancreatic cancer cells may contribute to disease progression
— id: 76314, year: 1993, vol: 105, page: 1846, stat: Journal Article,

Further characterization of the binding of fibronectin to gelatin reveals the presence of different binding interactions
Garcia-Pardo, A; Gold, L I
1993 Jul;304(1):181-188, Archives of biochemistry & biophysics. ABB
The specific interaction between fibronectin and collagen has permitted the isolation of fibronectin from plasma using gelatin-Sepharose affinity matrices. In this study we obtained evidence suggesting the presence of an additional high affinity binding interaction between fibronectin and collagen. The following information supports this idea: (i) Successive isolations of fibronectin using the same regenerated gelatin matrix resulted in progressively increased yields of fibronectin, thus indicating the progressive occupation of high affinity fibronectin-binding sites within collagen. (ii) This tightly bound fibronectin could not be eluted from gelatin-Sepharose matrices with strong denaturing agents, such as 8.0 M urea or 6.0 M guanidium chloride. (iii) The fibronectin-occupied matrices supported fibroblast adhesion and spreading, indicating that the amount of tightly bound fibronectin was quantitatively sufficient for this function, and that the fibronectin that bound with high affinity retained its biologically active conformation. (iv) Two fibronectin-derived fragments, CB52kDa and T55 kDa, selectively bound to fresh gelatin-Sepharose but not to gelatin-Sepharose previously employed to purify fibronectin, suggesting that these fragments recognize only the high affinity binding sites in gelatin. N-terminal amino acid sequence analysis of T55 kDa showed that it contained the previously described collagen-binding domain and included the repeat III-1 of fibronectin. These results support the existence of multiple fibronectin-binding sites in collagen and implicate a high affinity site(s) with a propensity for tenacious binding
— id: 76317, year: 1993, vol: 304, page: 181, stat: Journal Article,

Chemoattraction of neutrophils by substance P and transforming growth factor-beta 1 is inadequately explained by current models of lipid remodeling
Haines KA; Kolasinski SL; Cronstein BN; Reibman J; Gold LI; Weissmann G
1993 Aug 1;151(3):1491-1499, Journal of immunology
'Classical' chemoattractants, such as FMLP, C5a, or leukotriene B4, not only elicit directed motility but also activate neutrophils (degranulation, release of active oxygen species). Signal transduction after ligation of receptors for these classical chemoattractants is mediated by pertussis toxin (PT)-sensitive, heterotrimeric G proteins and the early production of lipid messengers via phospholipases. In contrast, we have previously shown that substance P (SP) and transforming growth factor-beta 1 (TGF-beta 1) are 'pure' chemoattractants in that they elicit chemotaxis without activating neutrophils. Paradoxically, pure chemoattractants also activate G proteins (plasmalemmal GTPase activity) without eliciting increments in cytosolic calcium ([Ca]i) and thus inositol trisphosphate. We therefore determined lipid remodeling and signal transduction in response to pure chemoattractants. Increments in plasmalemmal GTPase activated by SP (0.1 microM) and TGF-beta 1 (40 fM), like that after FMLP, were PT-sensitive (SP = 6.6 +/- 2 pm/mg/min vs SP + PT = 1.1 +/- 0.9 over basal activity; TGF-beta 1 = 4.3 +/- 1.6 vs TGF-beta 1 + PT = 2.3 +/- 0.9). In parallel, treatment of PMN with PT (1 microgram/ml, 30 min) inhibited chemotaxis (under agarose) after FMLP (2175 +/- 176 (SEM) microns vs 726 +/- 267) and SP (411 +/- 99 microns vs 103 +/- 62 microns) and TGF-beta 1 (40 fM, 375 +/- 53 microns vs 83 +/- 47). However, G proteins coupled to receptors for SP and TGF-beta 1, unlike FMLP, did not appear to be linked to phospholipases in that neither increments in diacylglycerol were detected after receptor ligation (FMLP = 152 +/- 22% resting levels; SP = 101 +/- 5%; TGF-beta 1 = 105 +/- 4%) nor was alkylacylglycerol increased by exposure to SP or TGF-beta 1 (SP = 92 +/- 4%; TGF-beta 1 = 101 +/- 8%; FMLP = 226 +/- 40%). Moreover, polymorphonuclear leukocytes failed to generate phosphatidates (PA) of either species after SP (DA-PA = 79 +/- 9% resting at 60 s; EA-PA = 103 +/- 4%) or TGF-beta 1 (DA-PA = 101 +/- 5%; EA-PA = 98 +/- 9%) in contrast to FMLP (DA-PA = 155 +/- 22%; EA-PA = 149 +/- 16%). The data clearly contravene the current dogma that all chemoattractants use inositol trisphosphate and diglycerides as intracellular signals and suggest the presence of a unique subset of PT-sensitive G proteins, not coupled to 'classical' phospholipases, transduce chemoattraction
— id: 9821, year: 1993, vol: 151, page: 1491, stat: Journal Article,

Transforming growth factor-beta in neural embryogenesis and neoplasia
Johnson, M D; Jennings, M T; Gold, L I; Moses, H L
1993 May;24(5):457-462, Human pathology
The transforming growth factor-beta (TGF-beta) family of polypeptides includes three structurally and functionally related mammalian isoforms that influence cell proliferation, differentiation, and extracellular matrix production. Recent identification of these isoforms in the embryonic murine central nervous system suggests that these factors may regulate proliferation and differentiation of meningeal and neuroepithelial cells during development. Predominant expression of TGF-beta 1 in the leptomeninges compared with the brain of the murine and human central nervous system implicates this isoform in regulation of that mesodermal tissue. Thus, defective TGF-beta regulation may contribute to neoplastic transformation. Failure to activate latent TGF-beta s may contribute to the loss of autocrine regulation seen in meningiomas. Expression of TGF-beta 2 and TGF-beta 3 primarily in embryonic murine radial glia and adult human astrocytes suggests other roles for these isoforms, including glioblast differentiation and guidance of neuroblast migration. Although inhibitory to 'normal' astrocyte proliferation, TGF-beta s demonstrate autocrine growth stimulation in vitro among hyperdiploid malignant gliomas, medulloblastomas, primitive neuroectodermal tumors, and anaplastic ependymomas. Hence, synthesis and release of active TGF-beta s by malignant brain tumors may create aberrant stimulatory autocrine loops. The mechanism of TGF-beta-induced growth stimulation is poorly understood. Future studies will likely clarify and identify additional roles for the TGF-beta isoforms in neuro-embryogenesis and neoplasia
— id: 76319, year: 1993, vol: 24, page: 457, stat: Journal Article,

Spatial and temporal patterns of immunoreactive transforming growth factor beta 1, beta 2, and beta 3 during excisional wound repair
Levine, J H; Moses, H L; Gold, L I; Nanney, L B
1993 Aug;143(2):368-380, American journal of pathology
Transforming growth factor beta (TGF-beta) regulates cellular growth and differentiation and stimulates the synthesis and secretion of protein constituents of the extracellular matrix. Three isoforms of TGF-beta have been found in mammals. Although the biological activities of TGF-beta 1, TGF-beta 2, and TGF-beta 3 are similar at the level of cell culture, distinct in vivo functions for these molecules are emerging. To gain insight into the role of each isoform in wound repair, antibodies specific for each isoform of TGF-beta were used to examine excisional wound repair. Marked differences in the temporal and spatial relationships for immunoreactive TGF-beta 1, -beta 2, and -beta 3 were noted throughout the repair process. TGF-beta 2 and TGF-beta 3 were prevalent by 24 hours after excisional wounding, and strong immunoreactivity was observed in the migrating epidermis. Subtle changes in immunoreactivity occurred for TGF-beta 2 and TGF-beta 3 in cells of the epidermal appendages, mesenchymal derivatives, granulation tissue, and the underlying dermis throughout wound repair. In contrast, TGF-beta 1 was not associated with any undifferentiated cells and was not present in the dermis and most dermal structures in both nonwounded skin or wounds until day 5 after wounding, when re-epithelialization was complete. Following re-epithelialization, TGF-beta 2 and TGF-beta 3 were present in all four layers of stratum corneum of the differentiating epidermis. All three TGF-beta isoforms were present in mesenchymal cells and basal lamina, suggesting their role in the modulation of dermal-epidermal interaction during wound repair. Our observations support individual in vivo function for TGF-beta isoforms in cutaneous wound repair
— id: 76315, year: 1993, vol: 143, page: 368, stat: Journal Article,

DIRECT TRANSFER OF TRANSFORMING GROWTH-FACTOR-BETA-1 GENE INTO ARTERIES STIMULATES FIBROCELLULAR HYPERPLASIA
NABEL, EG; SHUM, L; POMPILI, VJ; YANG, ZY; SAN, H; SHU, HB; LIPTAY, S; GOLD, L; GORDON, D; DERYNCK, R; NABEL, GJ
1993 NOV 15 ;90(22):10759-10763, Proceedings of the National Academy of Sciences of the United States of America
The arterial wall responds to thrombosis or mechanical injury through the induction of specific gene products that increase cellular proliferation and connective tissue formation. These changes result in intimal hyperplasia that is observed in restenosis and the early phases of atherosclerosis. Transforming growth factor beta1 (TGF-beta1) is a secreted multi-functional protein that plays an important role in embryonal development and in repair following tissue injury. However, the function of TGF-beta1 in vascular cell growth in vivo has not been defined. In this report, we have evaluated the role of TGF-beta1 in the pathophysiology of intimal and medial hyperplasia by gene transfer of an expression plasmid encoding active TGF-beta1 into porcine arteries. Expression of TGF-beta1 in normal arteries resulted in substantial extracellular matrix production accompanied by intimal and medial hyperplasia. Increased procollagen, collagen, and proteoglycan synthesis in the neointima was demonstrated by immunohistochemistry relative to control transfected arteries. Expression of TGF-beta1 induced a distinctly different program of gene expression and biologic response from the platelet-derived growth factor B (PDGF B) gene: procollagen synthesis induced by TGF-beta1 was greater, and cellular proliferation was less prominent. These findings show that TGF-beta1 differentially modulates extracellular matrix production and cellular proliferation in the arterial wall in vivo and could play a reparative role in the response to arterial injury
— id: 98458, year: 1993, vol: 90, page: 10759, stat: Journal Article,

Inhibition of mammary duct development but not alveolar outgrowth during pregnancy in transgenic mice expressing active TGF-beta 1
Pierce, D F Jr; Johnson, M D; Matsui, Y; Robinson, S D; Gold, L I; Purchio, A F; Daniel, C W; Hogan, B L; Moses, H L
1993 Dec;7(12A):2308-2317, Genes & development
The transforming growth factors beta (TGFs-beta) are potent inhibitors of cell proliferation and are usually secreted in a latent form. TGF-beta 1, TGF-beta 2, and TGF-beta 3 are expressed in distinct but overlapping patterns in the developing mouse mammary gland. To study the role of transforming growth factor-beta 1 (TGF-beta 1) in normal mammary development and in mammary neoplasia, we have constructed three transgenic mouse lines that express a simian TGF-beta 1 s223/225 mutated to produce a constitutively active product under the control of the MMTV enhancer/promoter. Expression of the transgene, as confirmed by in situ hybridization, immunohistochemistry, and Northern blot analysis, was associated with marked suppression of the normal pattern of mammary ductal tree development in female transgenics. Reduction in total ductal tree volume was observed at 7 weeks, soon after estrous begins, and was most apparent at 13 weeks, as ductal growth in the normal mammary gland declines. This effect was seen in all three lines. However, during pregnancy, alveolar outgrowths developed from the hypoplastic ductal tree, and lactation occurred, therefore, all transgenic females could feed full litters. Unlike many other transgenic mouse models in which expression of growth factors or oncogenes under control of the MMTV promoter leads to mammary epithelial hyperplasia and increased tumor formation, the MMTV-TGF-beta 1S223/225 transgene causes conditional hypoplasia of the mammary ductal tree and no spontaneous tumors have been detected in the MMTV-TGF-beta 1S223/225 transgenic animals
— id: 76313, year: 1993, vol: 7, page: 2308, stat: Journal Article,

Studies on the mechanisms by which transforming growth factor-beta (TGF-beta) protects against allergic encephalomyelitis. Antagonism between TGF-beta and tumor necrosis factor
Santambrogio L; Hochwald GM; Saxena B; Leu CH; Martz JE; Carlino JA; Ruddle NH; Palladino MA; Gold LI; Thorbecke GJ
1993 Jul 15;151(2):1116-1127, Journal of immunology
Experimental allergic encephalomyelitis (EAE) is an autoimmune disease in which peripheral lymphoid cells are activated by immunization with myelin proteins and become effector cells that traverse the central nervous system (CNS) capillaries and initiate inflammatory demyelinating lesions. The administration of transforming growth factor-beta (TGF-beta) has been shown previously to decrease the incidence and severity of EAE. In our studies we have determined: 1) the effects of TGF-beta injected at different intervals after the EAE-inducing immunization; 2) the effect of TGF-beta on the development of sensitized T cells, as assayed by the proliferative responses of T cells from lymph nodes and peripheral blood; 3) the extent of lymphoid cell infiltration in CNS of TGF-beta-treated and control mice; and 4) the role of endogenous TGF-beta and TNF in determining the severity of both acute and relapsing EAE. The onset of acute-EAE in SJL mice, induced by immunization with spinal cord homogenate in CFA and pertussigen, is on days 10 to 15. Although daily i.p. injections of 0.2 to 2 micrograms TGF-beta 1 or TGF-beta 2 on days 5 to 9 after immunization are highly protective, injections on days 1 to 5 or 9 to 13 are not. Moreover, anti-TGF-beta accelerates and aggrevates EAE when given on days 5 and 9, but not on day 12. Anti-TNF, injected on days 5 and 9, provides a comparable degree of protection as does TGF-beta. Similarly, in relapsing EAE, anti-TGF-beta increases, whereas anti-TNF decreases the incidence and severity of relapses. TGF-beta treatment on days 5 to 9 does not influence the appearance of sensitized cells in peripheral blood and lymph nodes, but does prevent the accumulation of T cells in brain and spinal cord, as assayed on days 15 to 20. It is concluded that the protective effect of TGF-beta is exerted at the level of the target organ, CNS and/or its vascular endothelium, rather than through a direct effect on lymphoid cells, and that there is a small window of 4 days in which TGF-beta exerts its protective effect
— id: 8728, year: 1993, vol: 151, page: 1116, stat: Journal Article,

Synthesis and expression of transforming growth factor beta-1, beta-2, and beta-3 in the endocrine and exocrine pancreas
Yamanaka, Y; Friess, H; Buchler, M; Beger, H G; Gold, L I; Korc, M
1993 May;42(5):746-756, Diabetes
The actions of transforming growth factor-beta isoforms as potent regulators of growth and differentiation have led to the examination of their presence in the human pancreas. The cellular localization of TGF-beta 1, TGF-beta 2, and TGF-beta 3 was assessed in the normal human pancreas by using immunohistochemical and in situ hybridization techniques. Although cytoplasmic immunoreactivity for TGF-beta 1, TGF-beta 2, and TGF-beta 3 was found in islet cells, acinar cells, and ductal cells, a differential immunostaining pattern for TGF-beta isoforms was observed. In the endocrine pancreas, the islet cells demonstrated diffuse cytoplasmic immunostaining for TGF-beta 1, TGF-beta 2, and TGF-beta 3. However, only TGF-beta 2 and TGF-beta 3 exhibited an intense pattern of immunostaining in a few endocrine cells. Most of the positive islet cells coexpressed insulin. In contrast, in the exocrine pancreas, a greater number of acinar cells showed immunoreactivity for TGF-beta 1 than for TGF-beta 2 and TGF-beta 3. In the ductal cells, all three TGF-beta isoforms showed a similar intensity and pattern of immunostaining and were observed more frequently in the smaller distal ductules than in the larger pancreatic ducts. TGF-beta 1 and TGF-beta 3, but not TGF-beta 2, immunostaining was detected strongly in the smooth muscle cells and weakly in the endothelial cells of the blood vessels, whereas the fibroblasts of the interstitium were completely negative. In situ hybridization revealed that mRNA encoding all three TGF-beta isoforms colocalized with their respective proteins in islets, acinar cells, and ductal cells. In contrast, mRNA expression was absent in the smooth muscle cells and endothelium of the vessels. These results suggest that TGF-beta isoforms may act by both autocrine and paracrine mechanisms in the pancreas. The differential pattern of expression observed for each TGF-beta isoform implies unique roles for these proteins in the regulation of the endocrine and exocrine pancreas
— id: 76318, year: 1993, vol: 42, page: 746, stat: Journal Article,

Localization of the cleavage sites on fibronectin following digestion by urokinase
Gold LI; Rostagno A; Frangione B; Passalaris T
1992 Dec;50(4):441-452, Journal of cellular biochemistry
Urokinase (u-PA) proteolytically cleaves both human plasma (pFn) and cellular (cFn) dimeric fibronectin (M(r) 440,000) into four major polypeptides of approximately M(r) 210,000, 200,000, 25,000, and 6,000. Amino acid sequence analysis of the polypeptide fragments indicated that the enzymatic cleavage of Fn occurs at two sites: 1) between an arginine/alanine peptide bond located C-terminal to residue 259; this cleavage liberates the N-terminal M(r) 25,000 fragment and the M(r) 210,000 and M(r) 200,000 polypeptides derived from the A and B chains of Fn, respectively; and 2) between an arginine/threonine peptide bond located C-terminal to residue 2,299, thereby yielding an M(r) 6,000 dimeric fragment containing the C-terminal interchain disulfide bonds. Predigestion of Fn with u-PA increased the molecule's vulnerability to further attack by the enzymes plasmin and cathepsin D. These data provide further biochemical evidence for the proteolytic cleavage of fibronectin by plasminogen activators and substantiate that u-PA digestion of Fn may be an initial event in the local degradation of the extracellular matrix by malignant cells, possessing elevated levels of these enzymes
— id: 9532, year: 1992, vol: 50, page: 441, stat: Journal Article,

Immunohistochemical staining for transforming growth factor beta 1 associates with disease progression in human breast cancer
Gorsch, S M; Memoli, V A; Stukel, T A; Gold, L I; Arrick, B A
1992 Dec 15;52(24):6949-6952, Cancer research
The transforming growth factor beta s (TGF-beta) comprise a family of M(r) 25,000 pluripotent growth factors which have been implicated in the development and progression of human breast cancer. Conflicting data suggest that TGF-beta has the potential to either inhibit or promote the progression of mammary neoplasia. We therefore examined a pathological library of malignant breast biopsy specimens to determine the prevalence and distribution of immunoreactivity with antibodies specific for the three mammalian isoforms of TGF-beta (beta 1, beta 2, and beta 3). We found that intense staining for TGF-beta 1 was positively associated with rate of disease progression, and that this was independent of age, stage, nodal status, or estrogen receptor status (P = 0.009)
— id: 76320, year: 1992, vol: 52, page: 6949, stat: Journal Article,

Transforming growth factor-beta and transforming growth factor beta-receptor expression in human meningioma cells
Johnson MD; Federspiel CF; Gold LI; Moses HL
1992 Sep;141(3):633-642, American journal of pathology
The transforming growth factor-beta (TGF beta) family in mammals includes three closely related peptides that influence proliferation and numerous physiologic processes in most mesenchymal cells. In this study, Northern blots, immunohistochemistry, TGF beta radioreceptor assays, TGF beta receptor affinity labeling and [3H] thymidine incorporation were used to evaluate whether primary cell cultures of human meningiomas synthesize the three TGF beta isoforms, bear TGF beta receptors, and respond to TGF beta. Transcripts for TGF beta 1 and 2 were detected in the three cases analyzed. Transforming growth factor-beta 1 immunoreactivity was detected in three of six cases, and TGF beta 2 and 3 immunoreactivity were detected in each case analyzed. Media conditioned by cells cultured from six meningiomas also contained latent TGF beta-like activity. Transforming growth factor-beta receptor cross-linking studies identified TGF beta binding sites corresponding to the type 1, type 2, and type 3 receptors on meningioma cells. Treatment with active TGF beta 1 produced a statistically significant reduction in [3H] thymidine incorporation after stimulation with 10% fetal calf serum and epidermal growth factor in all six cases studied
— id: 63198, year: 1992, vol: 141, page: 633, stat: Journal Article,

Evidence for transforming growth factor-beta expression in human leptomeningeal cells and transforming growth factor-beta-like activity in human cerebrospinal fluid
Johnson MD; Gold LI; Moses HL
1992 Sep;67(3):360-368, Laboratory investigation
BACKGROUND: Little is known about the factors regulating growth and maintenance of human leptomeningeal cells. The influence of cerebrospinal fluid on these functions is also unknown. Possible mediators include the transforming growth factor-beta (TGF beta) family, three closely related peptides that regulate proliferation and numerous other physiologic processes in most mesenchymal cells. EXPERIMENTAL DESIGN: Expression of both mRNA and protein for TGF beta isoforms TGF beta 1, TGF beta 2, and TGF beta 3 as well as TGF beta-competing activity were evaluated in primary human leptomeningeal cultures by Northern blot analysis, immunohistochemistry, and a radioreceptor assay, respectively. TGF beta 1, TGF beta 2, and TGF beta 3 immunoreactivity was also evaluated in brain sections containing leptomeninges from which these cell cultures were established. An additional study analyzed human cerebrospinal fluid for TGF beta-like activity. RESULTS: Transcripts for TGF beta 1, TGF beta 2 and TGF beta 3 were detected in RNA from each of the eight leptomeningeal cultures. Significant TGF beta 1 immunoreactivity was detected in leptomeningeal tissue from five of eight cases. TGF beta 2 and TGF beta 3 immunostaining was seen in eight and seven of the cases, respectively. Similarly, cells cultured from these meninges exhibited variable TGF beta 1 and extensive TGF beta 2 and TGF beta 3 immunoreactivity. Radioreceptor assays of conditioned media from four cultures demonstrated significant latent TGF beta-like activity. TGF beta radioreceptor competing activity was also detected by radioreceptor assay in normal blood-free cerebrospinal fluid from 32 patients without neurological disease. In addition, pooled cerebrospinal fluid (from six additional patients) exhibited dose dependent TGF beta-like activity in the radioreceptor assay, stimulation of AKR-2B cell growth in soft agar and inhibition of growth in CCL-64 cell assays suggesting that cerebrospinal fluid contains TG beta-like activity. CONCLUSIONS: These findings suggest that the human leptomeninges synthesize TGF beta 1, TGF beta 2 and TGF beta 3 and secrete latent TGF beta s at least in vitro. Human cerebrospinal fluid may also contain TGF beta isoforms. Collectively, these observations raise the possibility that members of the TGF beta family contribute to biologic processes of the leptomeninges
— id: 62336, year: 1992, vol: 67, page: 360, stat: Journal Article,

Effect of topical retinoic acids on the levels of collagen mRNA during the repair of UVB-induced dermal damage in the hairless mouse and the possible role of TGF-beta as a mediator
Kim, H J; Bogdan, N J; D'Agostaro, L J; Gold, L I; Bryce, G F
1992 Mar;98(3):359-363, Journal of investigative dermatology
Topically applied retinoic acids have been found to enhance the gene expression for collagen types I and III in the skin of UVB-irradiated hairless mice. Prior damage is required because the effect is not observed in the skin of age-matched, non-irradiated control animals. Immunochemical methods have shown an increase in TGF-beta 1 and, to a lesser extent, of TGF-beta 2 in the epidermis following retinoic acid treatment. There were no changes in mRNA levels for any of the isotypes of TGF-beta induced by retinoic acid treatment. This study suggests that TGF-beta may mediate the effect of retinoic acids on dermal repair through the stimulation of collagen gene expression
— id: 76321, year: 1992, vol: 98, page: 359, stat: Journal Article,

FURTHER LOCALIZATION OF THE TWO BINDING SITES FOR FIBRIN WITHIN FIBRONECTIN
GOLD L; ROSTAGNO A
1991 ;17(15 PART F):180-180, Journal of cellular biochemistry. Supplement
— id: 101624, year: 1991, vol: 17, page: 180, stat: Journal Article,

Expression of transforming growth factor-beta 1, -beta 2, and -beta 3 mRNA and protein in the murine lung
Pelton, R W; Johnson, M D; Perkett, E A; Gold, L I; Moses, H L
1991 Dec;5(6):522-530, American journal of respiratory cell & molecular biology
Evidence has accumulated suggesting that the various isoforms of beta-type transforming growth factors (TGF-beta s) regulate important functions in the lung; however, the cellular source of these proteins is not well defined. Northern blot analysis of murine lung tissue demonstrates that mRNA transcripts for all three TGF-beta isoforms are found from birth through adulthood. Although the level of expression for each TGF-beta is variable during the first 2 wk post partum, all three isoforms are equal in the adult lung. Using in situ hybridization and immunohistochemical analysis, we have localized both mRNA and protein expression for all three isoforms of TGF-beta in the adult murine lung. At low magnification, immunohistochemical localization of TGF-beta proteins appears coincident in their pattern of expression with TGF-beta mRNAs in the large proximal conducting airways of the lung. However, on closer analysis, protein expression of all three TGF-beta isoforms is confined to the bronchiolar epithelium, while TGF-beta mRNA transcripts for each of the TGF-beta genes are found in smooth muscle cells and connective tissue fibroblasts lying subjacent to the epithelium. Although the levels of both TGF-beta mRNA and protein expression are high in the proximal bronchiolar tree, their signal intensities completely disappear as the terminal bronchioles progress to respiratory bronchioles. Additionally, in the lung vasculature, there is very high expression of all three TGF-beta mRNA transcripts in the smooth muscle cells of the large vessels. TGF-beta2 and TGF-beta but not TGF-beta1 proteins are expressed in these same smooth muscle cells.(ABSTRACT TRUNCATED AT 250 WORDS)
— id: 76322, year: 1991, vol: 5, page: 522, stat: Journal Article,

Immunohistochemical localization of TGF beta 1, TGF beta 2, and TGF beta 3 in the mouse embryo: expression patterns suggest multiple roles during embryonic development
Pelton, R W; Saxena, B; Jones, M; Moses, H L; Gold, L I
1991 Nov;115(4):1091-1105, Journal of cell biology
Isoform-specific antibodies to TGF beta 1, TGF beta 2, and TGF beta 3 proteins were generated and have been used to examine the expression of these factors in the developing mouse embryo from 12.5-18.5 d post coitum (d.p.c.). These studies demonstrate the initial characterization of both TGF beta 2 and beta 3 in mammalian embryogenesis and are compared with TGF beta 1. Expression of one or all three TGF beta proteins was observed in many tissues, e.g., cartilage, bone, teeth, muscle, heart, blood vessels, lung, kidney, gut, liver, eye, ear, skin, and nervous tissue. Furthermore, all three TGF beta proteins demonstrated discrete cell-specific patterns of expression at various stages of development and the wide variety of tissues expressing TGF beta proteins represent all three primary embryonic germ layers. For example, specific localization of TGF beta 1 was observed in the lens fibers of the eye (ectoderm), TGF beta 2 in the cortex of the adrenal gland (mesoderm), and TGF beta 3 in the cochlear epithelium of the inner ear (endoderm). Compared to the expression of TGF beta mRNA transcripts in a given embryonic tissue, TGF beta proteins were frequently colocalized within the same cell type as the mRNA, but in some cases were observed to localize to different cells than the mRNA, thereby indicating that a complex pattern of transcription, translation, and secretion for TGF beta s 1-3 exists in the mouse embryo. This also indicates that TGF beta 1, beta 2, and beta 3 act through both paracrine and autocrine mechanisms during mammalian embryogenesis
— id: 76323, year: 1991, vol: 115, page: 1091, stat: Journal Article,

Transforming growth factor beta 1, a potent chemoattractant for human neutrophils, bypasses classic signal-transduction pathways
Reibman J; Meixler S; Lee TC; Gold LI; Cronstein BN; Haines KA; Kolasinski SL; Weissmann G
1991 Aug 1;88(15):6805-6809, Proceedings of the National Academy of Sciences of the United States of America
Transforming growth factor beta 1 (TGF-beta 1), a homodimeric polypeptide (Mr 25,000), derives from inflammatory cells and acts as a chemoattractant for monocytes and fibroblasts. We report here that TGF-beta 1 is also the most potent chemoattractant yet described for human peripheral blood neutrophils. Recombinant TGF-beta 1 elicited dose-dependent directed migration of neutrophils under agarose that was inhibited in the presence of a neutralizing antibody to TGF-beta 1. Maximal chemotaxis was evoked by TGF-beta 1 at femtomolar concentrations, whereas conventional chemoattractants act at nanomolar concentrations: on a molar basis, TGF-beta 1 was 150,000 times more potent than fMet-Leu-Phe. In contrast, TGF-beta 1 provoked neither exocytosis nor the production of superoxide by neutrophils. We further analyzed the mechanism by which TGF-beta 1 elicits chemotaxis (GTPase activity, [Ca2+], and actin polymerization). In contrast to the conventional chemoattractant fMet-Leu-Phe, TGF-beta neither activated classic heterotrimeric guanine nucleotide-binding proteins nor provoked global mobilization of intracellular Ca2+. Chemoattraction by both fMet-Leu-Phe and TGF-beta 1 was inhibited by cycloheximide and actinomycin D. Moreover, chemotaxis in response to TGF-beta 1 was associated with the polymerization of actin. The selectivity and potency of TGF-beta 1 as a chemoattractant suggest that it elicits directed cell migration by means of a pathway that depends not on classic intracellular signals but on protein synthesis
— id: 9831, year: 1991, vol: 88, page: 6805, stat: Journal Article,

Biochemical studies on the interaction of fibronectin with Ig
Rostagno AA; Frangione B; Gold L
1991 Apr 15;146(8):2687-2693, Journal of immunology
We have previously biochemically characterized three separate sites on the fibronectin (Fn) molecule that interact with IgG. These studies have been extended to examine the interaction of Fn with other classes and subclasses of Ig. By ELISA, a preferential quantitative binding order of Fn to the major Ig classes and subclasses was obtained as follows: IgG greater than IgM greater than IgA, IgG1 greater than IgG3 = IgG4 greater than IgG2, and IgA1 = IgA2. Using fragments of Fn obtained by subtilisin digestion followed by IgM and IgA affinity chromatography, immunoblot analysis using monospecific antisera to separate regions of the Fn molecule, and amino acid sequence analysis, these studies indicate that polyclonal IgA and IgM interact with Fn in the same three regions and under the same ionic conditions as previously described for IgG. Site 1 is a 22-kDa fragment that commences at residue 1 of the Fn molecule. Sites 2 (16 kDa) and 3 (26-29 kDa) begin at residues 588 and 1597, respectively. Under physiological conditions a monoclonal antibody that recognizes site 1 completely inhibited the interaction of intact Fn with IgG, IgM, and IgA. Therefore, this is the only physiologically active site in the intact molecule. Aggregated but not monomeric IgG competitively inhibited the binding of Fn to IgG-coated microtiter ELISA plates; thus, this interaction can take place in a fluid-phase system. These results indicate that Fn can potentially interact with immune complexes and aggregates of all Ig in the circulation and thus may play a significant role in both their clearance and deposition in Fn-containing tissues, such as occurs in immune-complex-related disorders
— id: 9547, year: 1991, vol: 146, page: 2687, stat: Journal Article,

TRANSFORMING GROWTH FACTOR-BETA-1 - THE MOST POTENT CHEMOATTRACTANT FOR HUMAN NEUTROPHILS (PMN)
Reibman, J; Meixler, SM; Lee, TC; Cronstein, BN; Gold, LI; Weissmann, G
1990 Apr;38(2):A406-A406, Clinical research
— id: 31965, year: 1990, vol: 38, page: A406, stat: Journal Article,

Human plasma fibronectin as a substrate for human urokinase
Gold LI; Schwimmer R; Quigley JP
1989 Sep 1;262(2):529-534, Biochemical journal
An early event in malignant transformation is the increased expression of proteases, such as plasminogen activator, which can degrade surrounding extracellular matrices, thereby conferring an advantage for tumour cell invasion and metastasis. The present studies provide evidence that plasma fibronectin (Fn), which is a component of the extracellular matrix, is a direct substrate for the plasminogen activator urokinase (UK). Human plasma Fn was incubated with human UK under plasminogen-free conditions. Fn cleavage was both time- and dose-dependent and was evident within 30 min. The proteolytic digestion was limited and complete within 12 h at an enzyme/substrate ratio of 1:20. Analysis of the final proteolytic digestion products demonstrated the disappearance of the native dimeric 440 kDa structure of Fn with the concomitant appearance of three proteolytic fragments of 210, 200 and 25 kDa. Since two large fragments of similar size to the 220 kDa monomeric chains of Fn were obtained following proteolysis, it is proposed that UK cleaves Fn at two sites, one towards the N-terminal and one close to the C-terminal, but N-terminal to its interchain disulphide bonds. These studies suggest that the local proteolytic digestion and release of Fn from the extracellular matrix by tumour cells possessing high levels of UK may involve the direct proteolytic breakdown of Fn by UK
— id: 10517, year: 1989, vol: 262, page: 529, stat: Journal Article,

Biochemical characterization of the fibronectin binding sites for IgG
Rostagno AA; Frangione B; Gold LI
1989 Nov 15;143(10):3277-3282, Journal of immunology
Plasma fibronectin (Fn) is a constituent of cryoglobulins and has been shown to interact with immune complexes. In a previous report we demonstrated that Fn specifically bound to IgG immobilized on a solid matrix. To localize and biochemically characterize the sites on the Fn molecule involved in this interaction, Fn was enzymatically cleaved with subtilisin and subjected to IgG affinity chromatography. Three major polypeptide fragments of 16 kDa, 22 kDa, and a triplet of 26- to 29-kDa bound IgG. They were localized to three separate regions of the molecule by Western blot analysis using antisera to specific regions of the Fn molecule, by amino acid sequencing, and by their previously described heparin binding affinities. The 22-kDa fragment interacted with IgG under physiologic conditions and it is localized at the N-terminal of the Fn molecule. The 16-kDa and 26- to 29-kDa fragments bound to IgG under conditions of lower ionic strength; the former commences at residue 588, carboxyl-terminal to the collagen binding region and the latter begins at residue 1597, carboxyl-terminal to the cell binding domain. The interaction of Fn with Ig has significant implications in host defense and also in immune complex disease where basement membrane Fn may sequester immune complexes from the circulation
— id: 9560, year: 1989, vol: 143, page: 3277, stat: Journal Article,

BIOCHEMICAL CHARACTERIZATION OF UROKINASE PROTEOLYTIC CLEAVAGE OF FIBRONECTIN
GOLD L I; ROSTAGNO A; PASSALARIS T
1988 ;107(6 PART 3):373A-373A, Journal of cell biology
— id: 101625, year: 1988, vol: 107, page: 373A, stat: Journal Article,

FIBRONECTIN IGG INTERACTION CHARACTERIZATION OF THE BINDING SITES IN FIBRONECTIN MOLECULE
ROSTAGNO A; FRANGIONE B; GOLD L I
1988 ;107(6 PART 3):599A-599A, Journal of cell biology
— id: 101626, year: 1988, vol: 107, page: 599A, stat: Journal Article,

Limited cleavage of cellular fibronectin by plasminogen activator purified from transformed cells
Quigley, J P; Gold, L I; Schwimmer, R; Sullivan, L M
1987 May;84(9):2776-2780, Proceedings of the National Academy of Sciences of the United States of America
The substrate specificity and direct catalytic activity of plasminogen activator (PA) was examined under conditions where its natural substrate, plasminogen, was missing or inhibited. PA, purified from cultures of transformed chicken fibroblasts, was incubated with purified preparations of potential substrates. The adhesive glycoprotein fibronectin, isolated from normal chicken fibroblast extracellular matrix, underwent limited but specific cleavage by PA in the absence of plasminogen. Analysis of the cleavage products by polyacrylamide gels under both reducing and nonreducing conditions indicated that PA-mediated cleavage occurred near the carboxyl terminus of fibronectin but on the amino-terminal side of the interchain disulfide bridge, thus disrupting the native dimeric fibronectin molecule. Under the identical conditions, chicken ovalbumin was not cleaved while the established substrate, chicken plasminogen, was extensively converted to plasmin. A monoclonal antibody, directed against avian PA and shown to inhibit plasminogen-free, cell-mediated matrix degradation, specifically inhibited the fibronectin cleavage. A human PA, urokinase, also cleaved fibronectin under plasminogen-free conditions yielding a limited number of high molecular weight cleavage products
— id: 76324, year: 1987, vol: 84, page: 2776, stat: Journal Article,

Biochemical and immunological characterization of three binding sites on human plasma fibronectin with different affinities for heparin
Gold LI; Frangione B; Pearlstein E
1983 Aug 16;22(17):4113-4119, Biochemistry
— id: 9620, year: 1983, vol: 22, page: 4113, stat: Journal Article,

HUMAN-PLASMA FIBRONECTIN (FN) BINDING DOMAINS FOR COLLAGEN, HEPARIN, AND FIBRIN LOCALIZED BY AMINO-ACID-SEQUENCE DETERMINATION
Gold, LI; Frangione, B; Pearlstein, E
1982 ;95(2):A127-A127, Journal of cell biology
— id: 30349, year: 1982, vol: 95, page: A127, stat: Journal Article,

LOCALIZATION AND BIOCHEMICAL-CHARACTERIZATION OF THE COLLAGEN, FIBRIN AND HEPARIN-BINDING DOMAINS OF FIBRONECTIN (FN)
Gold, LI; Pearlstein, E; Franklin, EC; Frangione, B
1981 ;91(2):A164-A164, Journal of cell biology
— id: 30404, year: 1981, vol: 91, page: A164, stat: Journal Article,

Fibronectin-collagen binding and requirement during cellular adhesion
Gold LI; Pearlstein E
1980 Feb 15;186(2):551-559, Biochemical journal
— id: 64086, year: 1980, vol: 186, page: 551, stat: Journal Article,

Fibronectin: a review of its structure and biological activity
Pearlstein E; Gold LI; Garcia-Pardo A
1980 Feb 8;29(2):103-128, Molecular & cellular biochemistry
— id: 64087, year: 1980, vol: 29, page: 103, stat: Journal Article,

Subtilisin and cyanogen bromide cleavage products of fibronectin that retain gelatin-binding activity
Gold LI; Garcia-Pardo A; Frangione B; Franklin EC; Pearlstein E
1979 Oct;76(10):4803-4807, Proceedings of the National Academy of Sciences of the United States of America
The gelatin-binding region of fibronectin has been obtained by subtilisin digestion and cyanogen bromide cleavage of the molecule. Enzymatic digestion yielded two fragments of molecular weights 50,000 (S50K) and 30,000 (S30K) which were isolated by elution from gelatin-Sepharose affinity columns. Because the S50K fragment also mediated the adhesion of fibroblasts to collagen, it contains both the collagen and cell binding sites on the fibronectin molecule. Both fragments had valine as the NH2-terminal residue, were enriched in half-cystine and methionine residues compared to the whole molecule, and were identical by immunodiffusion. The S50K fragment begins with the sequence Val-Tyr-Gln-Pro-Gln-Pro-His-Pro-Gln-Pro-(Pro)-(Gly)-Tyr-Gly-His-( )-Val, a region with an extended conformation which is susceptible to proteolysis and connects this domain to the remainder of the fibronectin molecule. The S50K fragment appears to be located in the COOH-terminal one-third of the fibronectin molecule but does not contain the interchain disulfide bridge(s); the S30K fragment is probably derived from the NH2-terminal region of S50K.
— id: 9650, year: 1979, vol: 76, page: 4803, stat: Journal Article,

Stability of fibronectin biological activity following chemical modification
Gold LI; Pearlstein E
1979 Dec 14;581(2):237-251, Biochimica & biophysica acta
Fibronectin isolated from human plasma functions in vitro as a mediator of adhesion and spreading of trypsinized fibroblasts on native or denatured collagen. As a means of elucidating structural characteristics which might contribute to fibronectin's biological activity, we have modified and digested the protein with several chemicals. Following various treatments, the protein was utilized to mediate cell adhesion and spreading on collagen to determine which alteration disrupted its activity. Fibronectin remained functionally intact after partial or complete reduction and alkylation, oxidation of 59% of the carbohydrates with sodium periodate, citraconylation, carbodiimide-catalyzed amide formation, and oxidation of 35.2 residues of tryptophan/molecule with N-bromosuccinimide. Dinitrofluorobenzene treatment, which phenylated ten residues/molecule of fibronectin, successfully inactivated fibronectin's in vitro biological function. Effective modification of the protein was determined by appropriate analytical procedures. Since fibronectin retained its biological function after several treatments that presumably affected its molecular conformation, we concluded that its secondary or tertiary structure appears not to be essential for its in vitro activity, or alternatively that the protein possesses a biologically active domain relatively resistant to chemical modification
— id: 64084, year: 1979, vol: 581, page: 237, stat: Journal Article,

Functional and structural studies in fibronectin
Gold, Leslie I
[S.l. : s.n.], 1979,
— id: 1045, year: 1979, vol: , page: , stat: ,

EXPRESSION OF MOUSE AND HUMAN FIBRONECTIN IN HYBRID-CELLS
Smith, M; Gold, LI; Pearlstein, E; Krinsky, A
1979 ;25(1-4):205-205, Cytogenetics & cell genetics
— id: 28003, year: 1979, vol: 25, page: 205, stat: Journal Article,

High-molecular-weight glycorprotein as a mediator of cellular adhesion
Pearlstein E; Gold LI
1978 Jun 20;312:278-292, Annals of the New York Academy of Sciences
— id: 64085, year: 1978, vol: 312, page: 278, stat: Journal Article,

CYANOGEN-BROMIDE FRAGMENT OF FIBRONECTIN WHICH RETAINS COLLAGEN BINDING-SITE
Pearlstein, E; Garciapardo, A; Gold, LI
1978 ;79(2):A228-A228, Journal of cell biology
— id: 29760, year: 1978, vol: 79, page: A228, stat: Journal Article,