John S. Munger, M.D.

Cross Talk among TGF-beta Signaling Pathways, Integrins, and the Extracellular Matrix
Munger, John S; Sheppard, Dean
2011 Nov;3(11):a005017 L-a005017 L, Cold Spring Harbor perspectives in biology
The growth factor TGF-beta is secreted in a latent complex consisting of three proteins: TGF-beta, an inhibitor (latency-associated protein, LAP, which is derived from the TGF-beta propeptide) and an ECM-binding protein (one of the latent TGF-beta binding proteins, or LTBPs). LTBPs interact with fibrillins and other ECM components and thus function to localize latent TGF-beta in the ECM. LAP contains an integrin-binding site (RGD), and several RGD-binding integrins are able to activate latent TGF-beta through binding this site. Mutant mice defective in integrin-mediated activators, and humans and mice with fibrillin gene mutations, show the critical role of ECM and integrins in regulating TGF-beta signaling
— id: 141722, year: 2011, vol: 3, page: a005017 L, stat: Journal Article,

Depletion of T Cells Potentiates Radiation-induced Acute Pneumonitis
Cheng, S. K.; Formenti, S.; Munger, J.
2010 OCT 13 ;78(3):S37-S38, International journal of radiation oncology biology physics
— id: 114014, year: 2010, vol: 78, page: S37, stat: Journal Article,

Mice that lack activity of alphavbeta6- and alphavbeta8-integrins reproduce the abnormalities of Tgfb1- and Tgfb3-null mice
Aluwihare, Poshala; Mu, Zhenyu; Zhao, Zhicheng; Yu, Dawen; Weinreb, Paul H; Horan, Gerald S; Violette, Shelia M; Munger, John S
2009 Jan 15;122(Pt 2):227-232, Journal of cell science
The arginine-glycine-aspartate (RGD)-binding integrins alphavbeta6 and alphavbeta8 activate latent TGFbeta1 and TGFbeta3 in vivo, but it is uncertain whether other RGD-binding integrins such as integrins alphavbeta5 and alphavbeta3 activate these TGFbeta isoforms. To define the combined role of alphavbeta6- and alphavbeta8-integrin in TGFbeta activation, we analyzed mice lacking function of both integrins by means of gene deletion and/or pharmacologic inhibition. Most Itgb6-/-;Itgb8-/- embryos die at mid-gestation; those that survive develop cleft palate-as observed in Tgfb3-/- mice. Itgb8-/- mice treated with an anti-alphavbeta6-integrin antibody develop severe autoimmunity and lack Langerhans cells-similar to Tgfb1-null mice. These results support a model in which TGFbeta3-mediated palate fusion and TGFbeta1-mediated suppression of autoimmunity and generation of Langerhans cells require integrins alphavbeta6 and alphavbeta8 but not other RGD-binding integrins as TGFbeta activators
— id: 95543, year: 2009, vol: 122, page: 227, stat: Journal Article,

Inhibition of Integrin beta 6 Prevents Radiation-induced Apoptosis of Intestinal Crypt Cells
Cheng, SK; Horan, GS; Weinreb, PH; Violette, SM; Formenti, SC; Munger, JS
2009 NOV ;75(3):S169-S170, International journal of radiation oncology biology physics
— id: 106170, year: 2009, vol: 75, page: S169, stat: Journal Article,

Anti-integrin beta 6 Therapy Reverses Radiation-induced Lung Fibrosis
Munger, J; Cheng, SK; Horan, GS; Weinreb, PH; Violette, SM; Formenti, SC
2009 NOV ;75(3):S173-S173, International journal of radiation oncology biology physics
— id: 106171, year: 2009, vol: 75, page: S173, stat: Journal Article,

What the lung has taught us about latent TGF-beta activation
Aluwihare, Poshala; Munger, John S
2008 Nov;39(5):499-502, American journal of respiratory cell & molecular biology
— id: 93380, year: 2008, vol: 39, page: 499, stat: Journal Article,

TGFbeta1 and TGFbeta3 are partially redundant effectors in brain vascular morphogenesis
Mu, Zhenyu; Yang, Zhiwei; Yu, Dawen; Zhao, Zhicheng; Munger, John S
2008 May-Jun;125(5-6):508-516, Mechanisms of development
Gene deletion experiments have shown that the three TGFbeta isoforms regulate distinct developmental processes. Recent work by our group and others showed that the integrins alphavbeta6 and alphavbeta8 activate latent forms of TGFbeta1 and TGFbeta3. This raises the possibility that TGFbeta1 and TGFbeta3 act redundantly in developmental processes where both isoforms are expressed and activation is by integrins. To investigate this issue, we generated mice with defective integrin-mediated TGFbeta1 activation (Tgfb1(RGE/RGE)) that were also homozygous for a null mutation in the TGFbeta3 gene. Tgfb1(RGE/RGE); Tgfb3(-/-) mice have severely perturbed development of the brain vasculature that is highly similar to that in mice lacking alphavbeta8. Some Tgfb1(RGE/RGE); Tgfb3(+/-) and Tgfb1(RGE/RGE); Tgfb3(+/+) mice have milder, background-dependent versions of the phenotype. In addition, we found that Tgfb3 gene status influences embryonic lethality due to TGFbeta1 deficiency after limited backcrossing to the BALB/c background. Conversely, Tgfb1 gene status modifies the extent of palate fusion in Tgfb3(-/-) mice after limited backcrossing to the ICR background. Our results are consistent with a functional connection between TGFbeta1 and TGFbeta3 during development based on a shared mechanism of activation
— id: 81571, year: 2008, vol: 125, page: 508, stat: Journal Article,

Inhibition of integrin alpha(v)beta6, an activator of latent transforming growth factor-beta, prevents radiation-induced lung fibrosis
Puthawala, Khalid; Hadjiangelis, Nicos; Jacoby, Steven C; Bayongan, Emmanuel; Zhao, Zhicheng; Yang, Zhiwei; Devitt, Mary Louise; Horan, Gerald S; Weinreb, Paul H; Lukashev, Matvey E; Violette, Shelia M; Grant, Kristen S; Colarossi, Cristina; Formenti, Silvia C; Munger, John S
2008 Jan 1;177(1):82-90, American journal of respiratory & critical care medicine
RATIONALE: In experimental models, lung fibrosis is dependent on transforming growth factor (TGF)-beta signaling. TGF-beta is secreted in a latent complex with its propeptide, and TGF-beta activators release TGF-beta from this complex. Because the integrin alpha(v)beta6 is a major TGF-beta activator in the lung, inhibition of alpha(v)beta6-mediated TGF-beta activation is a logical strategy to treat lung fibrosis. OBJECTIVES: To determine, by genetic and pharmacologic approaches, whether murine radiation-induced lung fibrosis is dependent on alpha(v)beta6. METHODS: Wild-type mice, alpha(v)beta6-deficient (Itgb6-/-) mice, and mice heterozygous for a Tgfb1 mutation that eliminates integrin-mediated activation (Tgfb1(+/RGE)) were exposed to 14 Gy thoracic radiation. Some mice were treated with an anti-alpha(v)beta6 monoclonal antibody or a soluble TGF-beta receptor fusion protein. Alpha(v)beta6 expression was determined by immunohistochemistry. Fibrosis, inflammation, and gene expression patterns were assessed 20-32 weeks postirradiation. MEASUREMENTS AND MAIN RESULTS: Beta6 integrin expression increased within the alveolar epithelium 18 weeks postirradiation, just before onset of fibrosis. Itgb6-/- mice were completely protected from fibrosis, but not from late radiation-induced mortality. Anti-alpha(v)beta6 therapy (1-10 mg/kg/wk) prevented fibrosis, but only higher doses (6-10 mg/kg/wk) caused lung inflammation similar to that in Itgb6-/- mice. Tgfb1-haploinsufficient mice were also protected from fibrosis. CONCLUSIONS: Alpha(v)beta6-mediated TGF-beta activation is required for radiation-induced lung fibrosis. Together with previous data, our results demonstrate a robust requirement for alpha(v)beta6 in distinct fibrosis models. Inhibition of alphavbeta6-mediated TGF-beta activation is a promising new approach for antifibrosis therapy
— id: 75454, year: 2008, vol: 177, page: 82, stat: Journal Article,

The integrin-TGF beta axis: Inhibition of integrin alpha v beta 6 prevents radiation-induced lung fibrosis
Munger, J; Cheng, SK; Puthawala, K; Hadjiangelis, N; Jacoby, S; Formenti, SC
2007 JAN ;69(3):S1-S1, International journal of radiation oncology biology physics
— id: 87190, year: 2007, vol: 69, page: S1, stat: Journal Article,

Absence of integrin-mediated TGF{beta}1 activation in vivo recapitulates the phenotype of TGF{beta}1-null mice
Yang, Zhiwei; Mu, Zhenyu; Dabovic, Branka; Jurukovski, Vladimir; Yu, Dawen; Sung, Joanne; Xiong, Xiaozhong; Munger, John S
2007 Mar 12;176(6):787-793, Journal of cell biology
The multifunctional cytokine transforming growth factor (TGF) beta1 is secreted in a latent complex with its processed propeptide (latency-associated peptide [LAP]). TGFbeta1 must be functionally released from this complex before it can engage TGFbeta receptors. One mechanism of latent TGFbeta1 activation involves interaction of the integrins alphavbeta6 and alphavbeta8 with an RGD sequence in LAP; other putative latent TGFbeta1 activators include thrombospondin-1, oxidants, and various proteases. To assess the contribution of RGD-binding integrins to TGFbeta1 activation in vivo, we created a mutation in Tgfb1 encoding a nonfunctional variant of the RGD sequence (RGE). Mice with this mutation (Tgfb1(RGE/RGE)) display the major features of Tgfb1(-/-) mice (vasculogenesis defects, multiorgan inflammation, and lack of Langerhans cells) despite production of normal levels of latent TGFbeta1. These findings indicate that RGD-binding integrins are requisite latent TGFbeta1 activators during development and in the immune system
— id: 71138, year: 2007, vol: 176, page: 787, stat: Journal Article,

Inhibition of TGF-beta activation by integrin-beta6 is protective against acute radiation skin injury
Cheng, S; Jacoby, S; Grant, K; Xavier, S; Munger, J; Formenti, S
2005 NOV 16 ;63(2):S461-S462, International journal of radiation oncology biology physics
— id: 58996, year: 2005, vol: 63, page: S461, stat: Journal Article,

Antibody to avb6 integrin prevents radiation-induced lung fibrosis
Formenti, SC; Jacoby, S; Grant, K; Horan, G; Weinreb; Devitt, M; Munger, J
2005 ;63(2):2408-2408, International journal of radiation oncology biology physics
— id: 109266, year: 2005, vol: 63, page: 2408, stat: Journal Article,

Integrin alphaVbeta6-mediated activation of latent TGF-beta requires the latent TGF-beta binding protein-1
Annes, Justin P; Chen, Yan; Munger, John S; Rifkin, Daniel B
2004 Jun 7;165(5):723-734, Journal of cell biology
Transforming growth factor-betas (TGF-beta) are secreted as inactive complexes containing the TGF-beta, the TGF-beta propeptide, also called the latency-associated protein (LAP), and the latent TGF-beta binding protein (LTBP). Extracellular activation of this complex is a critical but incompletely understood step in TGF-beta regulation. We have investigated the role of LTBP in modulating TGF-beta generation by the integrin alphaVbeta6. We show that even though alphavbeta6 recognizes an RGD on LAP, LTBP-1 is required for alphaVbeta6-mediated latent TGF-beta activation. The domains of LTBP-1 necessary for activation include the TGF-beta propeptide-binding domain and a basic amino acid sequence (hinge domain) with ECM targeting properties. Our results demonstrate an LTBP-1 isoform-specific function in alphaVbeta6-mediated latent TGF-beta activation; LTBP-3 is unable to substitute for LTBP-1 in this assay. The results reveal a functional role for LTBP-1 in latent TGF-beta activation and suggest that activation of specific latent complexes is regulated by distinct mechanisms that may be determined by the LTBP isoform and its potential interaction with the matrix
— id: 44942, year: 2004, vol: 165, page: 723, stat: Journal Article,

A genetic screen to identify latent transforming growth factor beta activators
Annes, Justin; Vassallo, Melinda; Munger, John S; Rifkin, Daniel B
2004 Apr 1;327(1):45-54, Analytical biochemistry
The mechanisms by which latent transforming growth factor beta (TGFbeta) is converted to the active cytokine are largely unknown. Here we present a genetic screen that combines retroviral mutagenesis and cDNA expression cloning to reveal proteins involved in the extracellular regulation of latent TGFbeta activation. The screen employs a cell line engineered to express green fluorescent protein (GFP) in response to TGFbeta. The cells produce their own latent TGFbeta. Therefore, after transduction with a retroviral cDNA library that contains an insert for an activator of latent TGFbeta, cells expressing the activator are GFP-bright. These cells are enriched by fluorescence-activated cell sorting and grown as individual clones. The isolated clones are cocultured with a second TGFbeta reporter cell line that produces luciferase in response to TGFbeta. Cells that have acquired the ability to activate latent TGFbeta induce luciferase expression in the absence but not in the presence of neutralizing antibodies to TGFbeta. The activator expressed by the positive clones can be identified by retrieval of the retrovirus cDNA insert
— id: 42347, year: 2004, vol: 327, page: 45, stat: Journal Article,

The integrin alpha v beta 6-knockout mouse is protected from radiation-induced lung fibrosis: Implications for the clinic
Munger, J; Hadjiangelis, N; Emmanuel, B; Devitt, M; Formenti, SC
2004 ;60(1):S327-S327 #1120, International journal of radiation oncology biology physics
— id: 109271, year: 2004, vol: 60, page: S327, stat: Journal Article,

Langerhans cell differentiation is dependent on integrin-mediated activation of TGF-beta 1
Sung, JJ; Habib, JZ; Munger, JS
2004 MAR ;122(3):A103-A103, Journal of investigative dermatology
— id: 46579, year: 2004, vol: 122, page: A103, stat: Journal Article,

Making sense of latent TGFbeta activation
Annes, Justin P; Munger, John S; Rifkin, Daniel B
2003 Jan 15;116(Pt 2):217-224, Journal of cell science
TGFbeta is secreted as part of a latent complex that is targeted to the extracellular matrix. A variety of molecules, 'TGFbeta activators,' release TGFbeta from its latent state. The unusual temporal discontinuity of TGFbeta synthesis and action and the panoply of TGFbeta effects contribute to the interest in TGF-beta. However, the logical connections between TGFbeta synthesis, storage and action are obscure. We consider the latent TGFbeta complex as an extracellular sensor in which the TGFbeta propeptide functions as the detector, latent-TGFbeta-binding protein (LTBP) functions as the localizer, and TGF-beta functions as the effector. Such a view provides a logical continuity for various aspects of TGFbeta biology and allows us to appreciate TGFbeta biology from a new perspective
— id: 35175, year: 2003, vol: 116, page: 217, stat: Journal Article,

The integrin alphaVbeta6 binds and activates latent TGFbeta3
Annes, Justin P; Rifkin, Daniel B; Munger, John S
2002 Jan 30;511(1-3):65-68, FEBS letters
Transforming growth factors-beta (TGFbeta1, 2 and 3) are secreted in a complex with their propeptides (latency-associated peptide 1 (LAP1), 2 and 3). TGFbeta signaling requires the dissociation of LAP and TGFbeta, a process termed latent TGFbeta activation. This process is a critical but incompletely understood step in the regulation of TGFbeta function. In particular, the extent to which activation mechanisms differ among the three TGFbeta isoforms is relatively unexplored. We show here that alphaVbeta6 binds and activates latent TGFbeta3
— id: 28180, year: 2002, vol: 511, page: 65, stat: Journal Article,

Integrin alpha8beta1 mediates adhesion to LAP-TGFbeta1
Lu, Min; Munger, John S; Steadele, Melissa; Busald, Christina; Tellier, Marinka; Schnapp, Lynn M
2002 Dec 1;115(Pt 23):4641-4648, Journal of cell science
The development of fibrosis is a common response to a variety of injuries and results in the net accumulation of matrix proteins and impairment of normal organ function. We previously reported that the integrin alpha8beta1 is expressed by alveolar interstitial cells in normal lung and is upregulated during the development of fibrosis. TGFbeta1 is an important mediator of the inflammatory response in pulmonary fibrosis. TGFbeta1 is secreted as a latent protein that is non-covalently associated with latency-associated peptide (LAP) and requires activation to exert its effects. LAP-TGFbeta1 and LAP-TGFbeta3 contain the tripeptide sequence, arginine-glycine-aspartic acid (RGD), a known integrin recognition motif. The integrin alpha8beta1 binds to several ligands such as fibronectin and vitronectin through the RGD sequence. Recent reports demonstrate that the integrins alphavbeta1, alphavbeta6 and alphavbeta8 adhere to LAP-TGFbeta1 through the RGD site. Therefore, we asked whether LAP-TGFbeta1 might be a ligand for alpha8beta1 and whether this may be important in the development of fibrosis. We found that cell lines transfected with alpha8 subunit were able to spread on and adhere to recombinant LAP-TGFbeta1 significantly better than mock transfected cell lines. alpha8-transfected cells were also able to adhere to LAP-TGFbeta3 significantly better than mock transfected cells. Adhesion to LAP-TGFbeta1 was enhanced by activation of alpha8beta1 by Mn(2+), or 8A2, an integrin beta1 activating antibody. Furthermore, cell adhesion was abolished when we used a recombinant LAP-TGFbeta1 protein in which the RGD site was mutated to RGE. alpha8beta1 binding to LAP-TGFbeta1 increased cell proliferation and phosphorylation of FAK and ERK, but did not activate of TGFbeta1. These data strongly suggest that LAP-TGFbeta1 is a ligand of alpha8beta1 and interaction of alpha8beta1 with LAP-TGFbeta1 may influence cell behavior
— id: 35176, year: 2002, vol: 115, page: 4641, stat: Journal Article,

The integrin alpha(v)beta8 mediates epithelial homeostasis through MT1-MMP-dependent activation of TGF-beta1
Mu, Dezhi; Cambier, Stephanie; Fjellbirkeland, Lars; Baron, Jody L; Munger, John S; Kawakatsu, Hisaaki; Sheppard, Dean; Broaddus, V Courtney; Nishimura, Stephen L
2002 Apr 29;157(3):493-507, Journal of cell biology
Integrins, matrix metalloproteases (MMPs), and the cytokine TGF-beta have each been implicated in homeostatic cell behaviors such as cell growth and matrix remodeling. TGF-beta exists mainly in a latent state, and a major point of homeostatic control is the activation of TGF-beta. Because the latent domain of TGF-beta1 possesses an integrin binding motif (RGD), integrins have the potential to sequester latent TGF-beta (SLC) to the cell surface where TGF-beta activation could be locally controlled. Here, we show that SLC binds to alpha(v)beta8, an integrin expressed by normal epithelial and neuronal cells in vivo. This binding results in the membrane type 1 (MT1)-MMP-dependent release of active TGF-beta, which leads to autocrine and paracrine effects on cell growth and matrix production. These data elucidate a novel mechanism of cellular homeostasis achieved through the coordination of the activities of members of three major gene families involved in cell-matrix interactions
— id: 28181, year: 2002, vol: 157, page: 493, 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,

Measurement of active TGF-beta generated by cultured cells
Mazzieri R; Munger JS; Rifkin DB
2000 ;142:13-27, Methods in molecular biology
— id: 11706, year: 2000, vol: 142, page: 13, stat: Journal Article,

The integrin alpha v beta 6 binds and activates latent TGF beta 1: a mechanism for regulating pulmonary inflammation and fibrosis
Munger JS; Huang X; Kawakatsu H; Griffiths MJ; Dalton SL; Wu J; Pittet JF; Kaminski N; Garat C; Matthay MA; Rifkin DB; Sheppard D
1999 Feb 5;96(3):319-328, Cell
Transforming growth factor beta (TGF beta) family members are secreted in inactive complexes with a latency-associated peptide (LAP), a protein derived from the N-terminal region of the TGF beta gene product. Extracellular activation of these complexes is a critical but incompletely understood step in regulation of TGF beta function in vivo. We show that TGF beta 1 LAP is a ligand for the integrin alpha v beta 6 and that alpha v beta 6-expressing cells induce spatially restricted activation of TGF beta 1. This finding explains why mice lacking this integrin develop exaggerated inflammation and, as we show, are protected from pulmonary fibrosis. These data identify a novel mechanism for locally regulating TGF beta 1 function in vivo by regulating expression of the alpha v beta 6 integrin
— id: 7411, year: 1999, vol: 96, page: 319, stat: Journal Article,

Proteolytic control of growth factor availability
Rifkin DB; Mazzieri R; Munger JS; Noguera I; Sung J
1999 Jan;107(1):80-85, Apmis
Most growth factors are released from cells in a form that does not permit immediate interaction with their high affinity receptors. An important mechanism for presentation of these released latent growth factors is activation by the plasminogen activator-plasmin system. The involvement of this system in the biology of Transforming Growth Factor-beta (TGF-beta) is reviewed
— id: 6074, year: 1999, vol: 107, page: 80, stat: Journal Article,

Interactions between growth factors and integrins: latent forms of transforming growth factor-beta are ligands for the integrin alphavbeta1
Munger JS; Harpel JG; Giancotti FG; Rifkin DB
1998 Sep;9(9):2627-2638, Molecular biology of the cell
The multipotential cytokine transforming growth factor-beta (TGF-beta) is secreted in a latent form. Latency results from the noncovalent association of TGF-beta with its processed propeptide dimer, called the latency-associated peptide (LAP); the complex of the two proteins is termed the small latent complex. Disulfide bonding between LAP and latent TGF-beta-binding protein (LTBP) produces the most common form of latent TGF-beta, the large latent complex. The extracellular matrix (ECM) modulates the activity of TGF-beta. LTBP and the LAP propeptides of TGF-beta (isoforms 1 and 3), like many ECM proteins, contain the common integrin-binding sequence RGD. To increase our understanding of latent TGF-beta function in the ECM, we determined whether latent TGF-beta1 interacts with integrins. A549 cells adhered and spread on plastic coated with LAP, small latent complex, and large latent complex but not on LTBP-coated plastic. Adhesion was blocked by an RGD peptide, and cells were unable to attach to a mutant form of recombinant LAP lacking the RGD sequence. Adhesion was also blocked by mAbs to integrin subunits alphav and beta1. We purified LAP-binding integrins from extracts of A549 cells using LAP bound to Sepharose. alphavbeta1 eluted with EDTA. After purification in the presence of Mn2+, a small amount of alphavbeta5 was also detected. A549 cells migrated equally on fibronectin- and LAP-coated surfaces; migration on LAP was alphavbeta1 dependent. These results establish alphavbeta1 as a LAP-beta1 receptor. Interactions between latent TGF-beta and alphavbeta1 may localize latent TGF-beta to the surface of specific cells and may allow the TGF-beta1 gene product to initiate signals by both TGF-beta receptor and integrin pathways
— id: 12078, year: 1998, vol: 9, page: 2627, stat: Journal Article,

TGF-beta latency: biological significance and mechanisms of activation
Gleizes PE; Munger JS; Nunes I; Harpel JG; Mazzieri R; Noguera I; Rifkin DB
1997 ;15(3):190-197, Stem cells
Transforming growth factor (TGF-) beta is secreted as a latent complex in which the mature growth factor remains associated with its propeptide. In order to elicit a biological response, the cytokine must be released from the latent complex, a process termed latent TGF-beta activation or TGF-beta formation. Although latent TGF-beta activation is a critical step in the regulation of its activity, little is known about the molecular mechanisms that lead to the production of active TGF-beta. In this article, we present an overview of the data available on this topic, and we propose a tentative model for the mechanism of TGF-beta formation based upon the observations with different cell systems and on recent findings on the structure of the latent TGF-beta complex
— id: 7154, year: 1997, vol: 15, page: 190, stat: Journal Article,

Latent transforming growth factor-beta: structural features and mechanisms of activation
Munger JS; Harpel JG; Gleizes PE; Mazzieri R; Nunes I; Rifkin DB
1997 May;51(5):1376-1382, Kidney international
Transforming growth factor-beta are cytokines with a wide range of biological effects. They play a pathologic role in inflammatory and fibrosing diseases such as nephrosclerosis. TGF-beta s are secreted in a latent form due to noncovalent association with latency associated peptide (LAP), which is a homodimer formed from the propeptide region of TGF-beta. LAP is disulfide linked to another protein, latent TGF-beta binding protein (LTBP). LTBP has features in common with extracellular matrix proteins, and targets latent TGF-beta to the matrix. Activation of latent TGF-beta can be accomplished in vitro by denaturing treatments, plasmin digestion, ionizing radiation and interaction with thrombospondin. The mechanisms by which latent TGF-beta is activated physiologically are not well understood. Results to date suggest an important role for proteases, particularly plasmin, although other mechanisms probably exist. A general model of activation is proposed in which latent TGF-beta is released from the extracellular matrix by proteases, localized to cell surfaces, and activated by cell-associated plasmin
— id: 35177, year: 1997, vol: 51, page: 1376, stat: Journal Article,

Plasminogen/plasminogen activator and growth factor activation
Rifkin DB; Gleizes PE; Harpel J; Nunes I; Munger J; Mazzieri R; Noguera I
1997 ;212:105-115, CIBA Foundation symposium
The plasminogen/plasminogen activator system is widely used in extracellular proteolysis. In this review the involvement of this system in tumour invasion, cell migration, growth factor presentation and inhibition of angiogenesis are discussed
— id: 12147, year: 1997, vol: 212, page: 105, stat: Journal Article,

Tissue destruction by proteases
Munger, John S; Chapman, Harold A
Tuberculosis Boston : Little Brown, 1996,
— id: 4835, year: 1996, vol: , page: ?, stat: Chapter,

Structure and activation of the large latent transforming growth factor-beta complex
Nunes I; Munger JS; Harpel JG; Nagano Y; Shapiro RL; Gleizes PE; Rifkin DB
1996 Mar;20 Suppl 3:S4-S8, International journal of obesity & related metabolic disorders
Most cell types express transforming growth factor-beta (TGF-beta) as a large latent TGF-beta complex that must be converted to an active form before TGF-beta can interact with cell surface TGF-beta receptors. This conversion involves the release of mature TGF-beta from the complex by disrupting noncovalent interactions between mature TGF-beta and its propeptide, latency associated peptide. A critical step in regulating TGF-beta effects may be the activation of the large latent TGF-beta complex. Activation of the complex can be achieved by chemical and enzymatic treatments, or by various cell systems. We have identified that coculturing bovine endothelial and smooth muscle cells generates active TGF-beta. Coculture activation of the large latent TGF-beta complex occurs through a plasmin-dependent mechanism that requires concentration of reactants on the cell surface and/or extracellular matrix. The mechanism of latent TGF-beta activation self-regulates through effectors of plasmin generation
— id: 12639, year: 1996, vol: 20 Suppl 3, page: S4, stat: Journal Article,

The lysosomal cysteine protease, cathepsin S, is increased in Alzheimer's disease and Down syndrome brain. An immunocytochemical study
Lemere CA; Munger JS; Shi GP; Natkin L; Haass C; Chapman HA; Selkoe DJ
1995 Apr;146(4):848-860, American journal of pathology
Expression of cathepsin (cat) S, a lysosomal cysteine protease, has recently been shown to cause an increase in production of amyloid beta-peptides in transfected human cells. In this study, we examined the presence and localization of cat S by immunocytochemistry in 21 control, 24 Alzheimer's disease (AD), and 10 Down syndrome (DS) postmortem brains. An antiserum to a human cat S fusion protein was affinity purified and its specificity confirmed by abolition of immunoreactivity after adsorption with cat S but not cat L fusion protein. A small minority of control cases showed light, focal staining of scattered cortical neurons. Many control cases, as well as most AD and DS cases, showed prominent staining of vascular smooth muscle cells, particularly in leptomeningeal vessels. Both AD and DS brain tissue showed increased immunoreactivity in a subset of neocortical and hippocampal neurons and glia. Cat S immunoreactivity occurred in a granular, cytoplasmic pattern in some neurons or in a more dense staining pattern in certain neurofibrillary tangle-bearing neurons. Cat S-positive neurons were also present in amygdala and basal forebrain in AD brains. A subset of astrocytes were immunoreactive with the cat S antibody in AD and DS but not in control brains. In rare AD cases, cat S immunostaining was observed in astrocytes in the periphery of amyloid-beta-containing plaques. These results suggest that cat S is up-regulated in AD and DS brain. The association of cat S immunoreactivity with tangle-bearing neurons, astrocytes, and rare senile plaques implies a role for altered cat S activity in the pathogenesis of AD
— id: 35179, year: 1995, vol: 146, page: 848, stat: Journal Article,

Lysosomal processing of amyloid precursor protein to A beta peptides: a distinct role for cathepsin S
Munger JS; Haass C; Lemere CA; Shi GP; Wong WS; Teplow DB; Selkoe DJ; Chapman HA
1995 Oct 1;311 ( Pt 1)(3):299-305, Biochemical journal
To investigate the potential contribution of the lysosomal compartment in the processing of amyloid precursor protein (APP) to amyloid beta-peptides (A beta s), we stably overexpressed a series of lysosomal proteases (the cysteine proteases, cathepsins B, L and S, and the aspartic protease, cathepsin D) in a human kidney epithelial cell line (293) transfected to express high levels of beta APP. Preliminary experiments indicated that 293 cells endogenously synthesize cathepsins B, L and D, but not cathepsin S. A beta secretion was assessed by immunoprecipitation and ELISA and found to be increased approximately 2-fold following cathepsin S expression, but to be unchanged (cathepsins B, L) or decreased (cathepsin D) in the other double transfectants. E-64d, an inhibitor of lysosomal cysteine proteases, significantly reduced A beta secretion by the cathepsin S transfectants, but had no effect on cells expressing the other proteases. Radiosequencing of A beta secreted by cathepsin S-expressing cells revealed that a previously unreported variant beginning at Met -1 (relative to the most common A beta N-terminus, Asp -1) accounted for most of the increase in A beta secretion. Immunostaining of human brain sections revealed cathepsin S in cortical neurons and glia in samples of brain from patients with Alzheimer's disease. These results provide evidence in living cells for a pathway in which cathepsin S generates A beta from amyloidogenic fragments of beta APP in the endosomal/lysosomal compartment. This pathway appears to be inducible, distinct from a constitutive pathway used by 293 and other cells to generate A beta, and may be relevant to the pathogenesis of Alzheimer's disease
— id: 35178, year: 1995, vol: 311 ( Pt 1), page: 299, stat: Journal Article,

The role of thiol proteases in tissue injury and remodeling
Chapman HA Jr; Munger JS; Shi GP
1994 Dec;150(6 Pt 2):S155-S159, American journal of respiratory & critical care medicine
Human lung macrophages express all four of the known lysosomal thiol proteases: cathepsins B, H, L, and S. These enzymes share a similar size and targeting mechanism for lysosomal accumulation and all have relatively indiscriminate substrate specificity in comparison with such highly selective serine proteases as urokinase or thrombin. These enzymes do have distinctive properties: only cathepsin B has C-terminal dipeptidase activity, only cathepsin H has potent aminopeptidase activity, and only cathepsin L and S are elastolytic. Cathepsin S is unique in that it is stable at neutral pH; indeed, at neutral pH it has elastolytic activity roughly comparable with that of neutrophil elastase. Recent studies of the differential expression of these cathepsins suggest they not only cooperate in terminal degradation of endocytized protein but also have specific functions such as proenzyme activation, antigen processing, and tissue remodeling, especially bone matrix resorption. Lysates of lung macrophages degrade elastin at neutral pH, suggesting that necrosis of macrophages at sites of macrophage accumulation, e.g., caseation necrosis, could contribute to tissue destruction. Tissue destruction and remodeling by thiol proteases expressed by live macrophages, however, is limited by tight compartmentalization of cathepsins to lysosomes. Nonetheless, macrophages accumulate at sites of known injury in cigarette smokers. Because these cells contain potent elastases, and because lysosomal enzyme release and cell surface acidification are regulated events, dysregulation of thiol protease expression in stimulated macrophages may contribute to the injury observed in cigarette smokers with non-alpha-1-protease inhibitor-type emphysema
— id: 35180, year: 1994, vol: 150, page: S155, stat: Journal Article,

Human cathepsin S: chromosomal localization, gene structure, and tissue distribution
Shi GP; Webb AC; Foster KE; Knoll JH; Lemere CA; Munger JS; Chapman HA
1994 Apr 15;269(15):11530-11536, Journal of biological chemistry
The human lysosomal cysteine proteinases, cathepsins H, L, and B, have been mapped to chromosomes 15, 9, and 8, respectively, and the genomic structures of cathepsins L and B have been determined. We report here the chromosomal localization and partial gene structure for a recently sequenced human cysteine proteinase, cathepsin S. A 20-kilobase pair genomic clone of the human cathepsin S gene was isolated from a human fibroblast genomic library and used to map the human cathepsin S gene to chromosome 1q21 by fluorescence in situ hybridization. This clone contains exons 1 through 5, introns 1 through 4, part of intron 5, and > 7 kilobase pairs of the 5'-flanking sequence. The gene structure of human cathepsin S is similar to that of cathepsin L through the first 5 exons, except that cathepsin S introns are substantially larger. Sequencing of the 5'-flanking region revealed, similar to human cathepsin B, no classical TATA or CAAT box. In contrast to cathepsin B, cathepsin S contains only two SP1 and at least 18 AP1 binding sites that potentially could be involved in regulation of the gene. This 5'-flanking region also contains CA microsatellites. The presence of AP1 sites and CA microsatellites suggest that cathepsin S can be specifically regulated. Results of Northern blotting using probes for human cathepsins B, L, and S are consistent with this hypothesis; only cathepsin S shows a restricted tissue distribution, with highest levels in spleen, heart, and lung. In addition, immunostaining of lung tissue demonstrated detectable cathepsin S only in lung macrophages. The high level of expression in the spleen and in phagocytes suggests that cathepsin S may have a specific function in immunity, perhaps related to antigen processing
— id: 35181, year: 1994, vol: 269, page: 11530, stat: Journal Article,

Molecular cloning and expression of human alveolar macrophage cathepsin S, an elastinolytic cysteine protease
Shi GP; Munger JS; Meara JP; Rich DH; Chapman HA
1992 Apr 15;267(11):7258-7262, Journal of biological chemistry
Human alveolar macrophages (HAM) express an elastase activity of acidic pH optimum inhibitable by cysteine protease inhibitors. Recent studies indicate that the only known eukaryotic elastinolytic cysteine protease, cathepsin L, cannot completely account for this activity. In order to search for additional cysteine proteases with elastinolytic activity, low degeneracy oligonucleotide primers based on regions of strong homology among the known cysteine proteases were used to screen reverse-transcribed HAM RNA for cysteine proteases by the polymerase chain reaction. Among the cDNA sequences generated was a 493-base pair product highly homologous to bovine cathepsin S. Screening of a HAM cDNA eukaryotic expression library with this cDNA yielded a 1.7-kilobase full-length cDNA highly homologous to bovine cathepsin S (approximately 85% identical). This cDNA predicts a 331-amino acid preprocathepsin. Expression of this cDNA in COS cells revealed the active enzyme to be a single chain 28-kDa protease, as judged by active site labeling with a novel iodinated analogue of N-(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucylamido-(4-gua nido)butane (E-64). The recombinant enzyme was found to be elastinolytic toward 3H-labeled elastin (bovine ligamentum nuchae) at pH 5.5 but with 25% of this activity retained at pH 7.0. Labeling of HAM with the active site probe revealed these cells express a 28-kDa cysteine protease, and Northern blot analysis revealed the presence of a approximately 1.7-kilobase cathepsin S mRNA. These data establish that human macrophages express at least two cysteine proteases with elastinolytic activity. The relatively broad pH range of human cathepsin S activity suggests this enzyme may contribute to the contact-dependent elastase activity of live human alveolar macrophages
— id: 35182, year: 1992, vol: 267, page: 7258, stat: Journal Article,

A serine esterase released by human alveolar macrophages is closely related to liver microsomal carboxylesterases
Munger JS; Shi GP; Mark EA; Chin DT; Gerard C; Chapman HA
1991 Oct 5;266(28):18832-18838, Journal of biological chemistry
We identified a 60-kDa diisopropylfluorophosphate-(DFP) reactive protein in human bronchoalveolar lavage fluid, at a yield of 50-100 pmol/lavage. The protein is associated with the cell-free lavage fluid sediment, which consists mainly of surfactant. [3H]DFP labeling is inhibited by heating to 56 degrees C, 2 mM phenylmethylsulfonylfluoride and 1 mM bis(4-nitrophenyl)-phosphate. An identical 60-kDa [3H]DFP-reactive protein is present in the insoluble fraction of alveolar macrophage-conditioned culture medium and in total membrane preparations of alveolar macrophages. The [3H]DFP-labeled protein was purified approximately 30-fold from lavage fluid sediment by size-exclusion (Sephacryl S-200) and ion-exchange (Mono-Q) chromatography. Cyanogen bromide treatment of the partially purified protein produced a major labeled peptide of 14 kDa with an NH2-terminal sequence 90% identical to a region of form 1 rabbit liver microsomal carboxylesterase. Esterase activity in unlabeled starting material, detected using p-nitrophenyl valerate as substrate, copurified with the [3H]DFP-labeled enzyme. Degenerate oligonucleotide primers were designed based on the partial amino acid sequence and on a highly conserved region of known liver carboxylesterase sequences. Polymerase chain reaction using these primers and reverse-transcribed human alveolar macrophage mRNA yielded a 354-base pair product which was then used to screen a human alveolar macrophage cDNA library. A complete esterase sequence was obtained from two incomplete, overlapping clones, and is virtually identical to human liver carboxylesterase partial sequences. Northern blot analysis demonstrated a single approximately 1.7-kilobase transcript in human monocytes and alveolar macrophages, with much higher levels in the latter. These data indicate that human alveolar macrophages both contain and release a serine esterase that is apparently identical to liver microsomal carboxylesterase. Its enzymatic profile suggests it is a major component of alveolar macrophage-nonspecific esterase activity. We hypothesize that it acts as a detoxication enzyme in the lung
— id: 35183, year: 1991, vol: 266, page: 18832, stat: Journal Article,