Frederick M. Stanley, Ph.D.

Insulin acts through FOXO3a to activate transcription of plasminogen activator inhibitor type 1
Jag, Ushma R; Zavadil, Jiri; Stanley, Frederick M
2009 Oct;23(10):1587-1602, Molecular endocrinology
Plasminogen activator inhibitor-1 (PAI-1) is an important regulator of fibrinolysis. PAI-1 levels are elevated in type 2 diabetes, and this elevation correlates with macro- and microvascular complications of diabetes. However, the mechanistic link between insulin and up-regulation of PAI-1 is unclear. Here we demonstrate that overexpression of Forkhead-related transcription factor (Fox)O1, FoxO3a, and FoxC1 augment insulin's ability to activate the PAI-1 promoter. In addition, insulin treatment promotes the phosphorylation of nuclear and cytoplasmic Fox03a and an increase of cytoplasmic Fox03a. In contrast, insulin treatment led to the accumulation of phospho-Fox01 only in the cytoplasm. Furthermore, insulin also increased the ability of chimeric LexA-FoxO1, LexA-FoxO3a, and LexA-FoxC1 proteins to increase the activity of a LexA reporter, suggesting that the effect of insulin on FoxO3a was direct. Using small interfering RNA to specifically deplete each of the Fox transcription factors tested, we demonstrate that only reduction of FoxO3a inhibits insulin-increased PAI-1-Luc expression and PAI-1 mRNA accumulation. Finally, chromatin immunoprecipitation assays confirm the presence of FoxO3a on the PAI-1 promoter. These results suggest that FoxO3a mediates insulin-increased PAI-1 gene expression
— id: 102930, year: 2009, vol: 23, page: 1587, stat: Journal Article,

Insulin-increased prolactin gene expression requires actin treadmilling: potential role for p21 activated kinase
Stanley, Frederick M
2007 Dec;148(12):5874-5883, Endocrinology
Insulin-increased prolactin gene transcription in GH4 cells was enhanced by binding on fibronectin. This was mediated by receptor-like protein tyrosine phosphatase alpha, which activated Src, Rho, and phosphatidylinositol 3-kinase. It suggested that insulin signaling to gene transcription was partly dependent on actin rearrangement. This was confirmed through studies using inhibitors of actin treadmilling. Cytochalasin D, jasplakinolide, latrunculin B, and swinholide A altered the actin cytoskeleton of GH4 cells, as assessed by Alexa Fluor phalloidin staining, and inhibited insulin-increased prolactin gene transcription. These reagents did not affect the controls. Nor was it due to a gross defect of insulin signaling because activation/translocation of glycogen synthase kinase 3beta and mammalian target of rapamycin were not affected. Expression of wild-type and mutant actin treadmilling agents, Cdc42, TC10, neuronal Wiskott-Aldrich syndrome protein, and Nck, indicated that they were essential to insulin-increased prolactin gene expression, and suggested that activation of p21 associated kinase (PAK) might also be essential to this process. PAK expression also increased and PAK mutants decreased prolactin promoter activity in insulin-treated cells. The activation of PAK in the presence of inhibitors was also consistent with a role in activation of insulin-increased prolactin gene expression. Finally, small interfering RNA-mediated reduction of PAK decreased the effect of insulin on prolactin gene expression. Thus, it is likely that insulin activation of actin treadmilling through Cdc42/TC10 and neuronal Wiskott-Aldrich syndrome protein activates PAK and prolactin gene transcription
— id: 79290, year: 2007, vol: 148, page: 5874, stat: Journal Article,

Integrin activates receptor-like protein tyrosine phosphatase {alpha}, Src, and Rho to increase prolactin gene expression through a final PI 3-kinase/cytoskeletal pathway that is additive with insulin
Vulin, Anthony I; Jacob, Kirsten K; Stanley, Frederick M
2005 Aug;146(8):3535-3546, Endocrinology
We previously showed that receptor-like protein tyrosine phosphatase alpha (RPTPalpha) inhibited insulin-increased prolactin gene transcription. Others suggested that RPTPalpha was a key intermediary between integrins and activation of Src. We present evidence that inhibition of insulin-increased prolactin gene transcription was secondary to RPTPalpha activation of Src reflecting its role as mediator of integrin responses. Src kinase activity was increased in GH4 cells transiently or stably expressing RPTPalpha and in cells plated on the integrin alpha5beta1 ligand fibronectin. C-terminal Src Kinase inactivated Src and blocked RPTPalpha inhibition of insulin-increased prolactin gene transcription. Expression of dominant negative Src also prevented the RPTPalpha mediated inhibition of insulin-increased prolactin gene expression. Low levels of a constitutively active Src mutant (SrcY/F) stimulated while higher expression levels of Src Y/F inhibited prolactin gene expression. Src-increased prolactin gene transcription was inhibited by expression of a blocking rho mutant (RhoN19) suggesting that Src acted through or required active rho. Experiments with an activated rho mutant (RhoL63) demonstrated a biphasic activation/repression of prolactin gene transcription that was similar to the effect of Src. The effects of both Src and rho were phosphatidylinositol 3-kinase dependent. Expression of SrcY/F or RhoL63 altered the actin cytoskeleton and morphology of GH4 cells. Taken together, these data suggest a physiological pathway from the cell matrix to increased prolactin gene transcription mediated by RPTPalpha/Src/rho/PI 3-kinase and cytoskeletal change that is additive with effects of insulin. Over activation of this pathway, however, caused extreme alteration of the cytoskeleton that blocked activation of the prolactin gene
— id: 56165, year: 2005, vol: 146, page: 3535, stat: Journal Article,

Oxidative stress activates the plasminogen activator inhibitor type 1 (PAI-1) promoter through an AP-1 response element and cooperates with insulin for additive effects on PAI-1 transcription
Vulin, Anthony I; Stanley, Frederick M
2004 Jun 11;279(24):25172-25178, Journal of biological chemistry
Oxidative stress is one of the characteristics of diabetes and is thought to be responsible for many of the pathophysiological changes caused by the disease. We previously identified an insulin response element in the promoter of plasminogen activator inhibitor 1 (PAI-1) that was activated by an unidentified member of the forkhead/winged helix (Fox) family of transcription factors. This element mediated a 5-7-fold increase in PAI-1 transcription because of insulin. Here we report that oxidative stress also caused a 3-fold increase in PAI-1 transcription and that the effect was additive with that of insulin. Antioxidants prevent this response. Mutational analysis of the PAI-1 promoter revealed that oxidative stress acted at an AP-1 site at -60/52 of the promoter. Gel mobility shift analysis demonstrated that binding to an AP-1 oligonucleotide was increased 4-fold by oxidative stress. Jun levels were increased by oxidants as assessed by reverse transcriptase-PCR. Western blotting demonstrated that a rapid and prolonged nuclear accumulation of phospho-c-Jun followed oxidant stimulation. The nuclear c-Jun phosphorylation was not observed in cells treated with reduced glutathione. Finally, JNK/SAPK activity was found to increase in response to oxidants, and inhibition of JNK/SAP blocked TBHQ-increased PAI-1-luciferase expression. Thus, oxidative stress stimulated AP-1 and activated the PAI-1 promoter
— id: 45320, year: 2004, vol: 279, page: 25172, stat: Journal Article,

Insulin receptor tyrosine kinase activity and phosphorylation of tyrosines 1162 and 1163 are required for insulin-increased prolactin gene expression
Jacob, Kirsten K; Whittaker, Jonathan; Stanley, Frederick M
2002 Jan 15;186(1):7-16, Molecular & cellular endocrinology
Insulin treatment increased prolactin gene expression in GH4 cells, a rat pituitary tumor cell line, through the endogenous insulin receptor. However, insulin regulation of transfected plasmids required the expression of cotransfected insulin receptor. Prolactin-CAT expression was increased 12-fold in cells transfected with wild type insulin receptor, but insulin did not increase prolactin gene expression when a kinase negative mutant of the ATP binding site (K1030R) was expressed. Thus, receptor kinase activity was required for signaling to gene transcription. Mutation of tyrosine 1158 did not reduce insulin-increased prolactin-CAT expression while individual mutations of tyrosine 1162 and tyrosine 1163 each reduced insulin-increased prolactin-CAT expression by 50% and a triple mutant of tyrosines 1158/1162/1163 was inactive. Thus, mutation of tyrosine 1162 and 1163 was also sufficient to inactivate signaling by the insulin receptor. Insulin-stimulated auto phosphorylation occurred in all mutants in vitro except the ATP binding site mutant. However, the ability of mutant insulin receptors to mediate insulin-increased prolactin-CAT expression correlated with the substrate-specific catalytic activity of the receptors. This suggested that phosphorylation of these tyrosines was important for substrate access to the catalytic domain of the receptor
— id: 32124, year: 2002, vol: 186, page: 7, stat: Journal Article,

A Forkhead/winged helix-related transcription factor mediates insulin-increased plasminogen activator inhibitor-1 gene transcription
Vulin, Anthony Igor; Stanley, Frederick M
2002 Jun 7;277(23):20169-20176, Journal of biological chemistry
Plasminogen activator inhibitor-1 (PAI-1) is an important regulator of fibrinolysis by its inhibition of both tissue-type and urokinase plasminogen activators. PAI-1 levels are elevated in type II diabetes and this elevation correlates with macro- and microvascular complications of diabetes. Insulin increases PAI-1 production in several experimental systems, but the mechanism of insulin-activated PAI-1 transcription remains to be determined. Deletion analysis of the PAI-1 promoter revealed that the insulin response element is between -117 and -7. Mutation of the AT-rich site at -52/-45 abolished the insulin responsiveness of the PAI-1 promoter. This sequence is similar to the inhibitory sequence found in the phosphoenolpyruvate carboxylkinase/insulin-like growth factor-I-binding protein I promoters. Gel-mobility shift assays demonstrated that the forkhead bound to the PAI-1 promoter insulin response element. Expression of the DNA-binding domain of FKHR acted as a dominant negative to block insulin-increased PAI-1-CAT expression. A LexA-FKHR construct was also insulin responsive. These data suggested that a member of the Forkhead/winged helix family of transcription factors mediated the effect of insulin on PAI-1 transcription. Inhibition of phosphatidylinositol 3-kinase reduced the effect of insulin on PAI-1 gene expression, a result consistent with activation through FKHR. However, it was likely that a different member of the FKHR family (not FKHR) mediated this effect since FKHR was present in both insulin-responsive and non-responsive cell lines
— id: 39692, year: 2002, vol: 277, page: 20169, stat: Journal Article,

Elk-1, C/EBPalpha, and Pit-1 confer an insulin-responsive phenotype on prolactin promoter expression in Chinese hamster ovary cells and define the factors required for insulin-increased transcription
Jacob KK; Stanley FM
2001 Jul 6;276(27):24931-24936, Journal of biological chemistry
The transcription factor(s) that mediate insulin-increased gene transcription are not well defined. These studies use phenotypic conversion of Rat2 and Chinese hamster ovary (CHO) cells with transcription factors to identify components required for regulation of prolactin promoter activity and its control by insulin. The pituitary-derived GH4 cells contain all of the transcription factors required for insulin-increased prolactin-chloramphenicol acetyltransferase (CAT) expression while HeLa cells require only Pit-1, a pituitary-specific factor. However, Rat2 and CHO cells require additional factors. We had determined previously that the transcription factor that mediates insulin-increased prolactin gene expression was likely an Ets-related protein. Elk-1 and Sap-1 were the only Ets-related transcription factors tested as chimeras with LexA DNA-binding domain that were able to mediate insulin-increased expression of a LexA-CAT reporter plasmid. Elk-1 and Sap-1 are expressed in GH4 and HeLa cells but Rat2 and CHO cells express Sap-1, but not Elk-1. Expression of Elk-1 made Rat2 cells (but not CHO cells) insulin responsive. C/EBPalpha also binds to the prolactin promoter at a sequence overlapping the binding site for Elk-1. Expression of both C/EBPalpha and Pit-1 in CHO cells is required for high basal transcription of prolactin-CAT. Expression of Elk-1 converts CHO cells into a phenotype in which prolactin gene expression is increased by insulin treatment. Finally, antisense mediated reduction of Elk-1 in GH4 cells decreased insulin-increased prolactin gene expression and confirmed the requirement for Elk-1 for insulin-increased prolactin gene expression. Thus, both C/EBPalpha and Pit-1 were required for high basal transcription while insulin sensitivity required Elk-1
— id: 48162, year: 2001, vol: 276, page: 24931, stat: Journal Article,

Embryonic dopamine cell transplantation for Parkinson's disease: Voxel-based analysis of data from a double-blind placebo controlled PET study
Ma, Y; Dhawan, V; Mentis, MJ; Greene, P; Stanley, F; Freed, C; Eidelberg, D
2000 ;54(7):A114-A115, Neurology
— id: 104831, year: 2000, vol: 54, page: A114, stat: Journal Article,

CCAAT/enhancer-binding protein alpha is a physiological regulator of prolactin gene expression
Jacob KK; Stanley FM
1999 Oct;140(10):4542-4550, Endocrinology
The sequence -101/-92 of the PRL promoter has been shown to be essential for both basal and hormone-increased PRL gene transcription. It is important to identify transcription factors that bind to this sequence if we are to understand the regulation of the PRL gene. Nuclear proteins, metabolically labeled with 35S were used in gel mobility shift experiments to examine which protein(s) binds to this region of the PRL promoter. An abundant 43-kDa protein binds to the PRL promoter at -106/-87. Two 43-kDa transcription factors were identified in cytosolic extracts of GH4 cells, CCAAT enhancer-binding protein alpha (C/EBP alpha) and cAMP response element-binding protein. Both of these bind to the PRL promoter, and both were present in GH4 cell nuclear extract, but only C/EBP alpha was definitively identified in complexes with PRL promoter DNA. Expression of C/EBP alpha increased basal PRL gene expression almost 6-fold, whereas expression of Chop10 that can act as an inhibitor of C/EBP alpha reduced the basal activity of the PRL promoter 60-75%. Mutational analysis demonstrated that the ability of C/EBP alpha to increase basal expression of the PRL promoter was dependent on the sequence -101/-92. These data suggest that C/EBP alpha is an important transcription factor that regulates PRL gene expression
— id: 8482, year: 1999, vol: 140, page: 4542, stat: Journal Article,

The EGF response element in the prolactin promoter
Jacob KK; Wininger E; DiMinni K; Stanley FM
1999 Jun 25;152(1-2):137-145, Molecular & cellular endocrinology
Epidermal growth factor (EGF) increases prolactin gene expression in GH4 cells, but the promoter element(s) required for this response has not been clearly defined. We identified a bipartite element - 96/ - 87, - 76/ - 67 in the rat proximal promoter that is essential for EGF signaling using deletion and linker-scanning mutants of the prolactin promoter. This element was active in either normal or inverted orientation when transferred to a heterologous promoter (mammary-tumor virus). We had previously identified this element as the cAMP/insulin response element of the prolactin promoter. However, the effects of EGF are additive with the responses to insulin or cAMP implying that EGF activated prolactin gene transcription by a mechanism different from insulin or cAMP. The EGF response element of the prolactin promoter is a recognition sequence for the Ets-related family of transcription factors and Ets-related factors have been shown to bind this element. Expression of the DNA-binding domain of c-Ets-1, which acts as a dominant negative inhibitor of Ets-related transcription factors, reduces EGF-increased prolactin-CAT expression 65% in GH4 cells. Thus, both EGF and insulin may signal through Ets-related transcription factors to activate prolactin gene transcription at the same response element in the prolactin proximal promoter
— id: 6170, year: 1999, vol: 152, page: 137, stat: Journal Article,

Receptor-like protein-tyrosine phosphatase alpha specifically inhibits insulin-increased prolactin gene expression
Jacob KK; Sap J; Stanley FM
1998 Feb 20;273(8):4800-4809, Journal of biological chemistry
A physiologically relevant response to insulin, stimulation of prolactin promoter activity in GH4 pituitary cells, was used as an assay to study the specificity of protein-tyrosine phosphatase function. Receptor-like protein-tyrosine phosphatase alpha (RPTPalpha) blocks the effect of insulin to increase prolactin gene expression but potentiates the effects of epidermal growth factor and cAMP on prolactin promoter activity. RPTPalpha was the only protein-tyrosine phosphatase tested that did this. Thus, the effect of RPTPalpha on prolactin-chloramphenicol acetyltransferase (CAT) promoter activity is specific by two criteria. A number of potential RPTPalpha targets were ruled out by finding (a) that they are not affected or (b) that they are not on the pathway to insulin-increased prolactin-CAT activity. The negative effect of RPTPalpha on insulin activation of the prolactin promoter is not due to reduced phosphorylation or kinase activity of the insulin receptor or to reduced phosphorylation of insulin receptor substrate-1 or Shc. Inhibitor studies suggest that insulin-increased prolactin gene expression is mediated by a Ras-like GTPase but is not mitogen-activated protein kinase dependent. Experiments with inhibitors of phosphatidylinositol 3-kinase suggest that insulin-increased prolactin-CAT expression is phosphatidylinositol 3-kinase-independent. These results suggest that RPTPalpha may be a physiological regulator of insulin action
— id: 7617, year: 1998, vol: 273, page: 4800, stat: Journal Article,

GABP mediates insulin-increased prolactin gene transcription
Ouyang L; Jacob KK; Stanley FM
1996 May 3;271(18):10425-10428, Journal of biological chemistry
The insulin-response element from the prolactin gene is identical to the Ets-binding site, and dominant-negative Ets protein inhibits insulin-increased prolactin gene expression. Immunoblotting identified the Ets-related transcription factor GABP in nuclear extracts from GH cells. Expression of GABP alpha and GABP beta 1 squelches insulin-increased prolactin gene expression. GABP alpha and GABP beta 1 bind the insulin-response element of the prolactin promoter, and anti-GABP alpha and anti-GABP beta 1 antibodies supershift a species seen with nuclear extracts from GH cells. GABP alpha immunoprecipitated from insulin-treated, 32P-labeled GH cells was phosphorylated 3-fold more than GABP alpha from control cells. There was no increase in phosphorylation of GABP beta in response to insulin. Mitogen-activated protein (MAP) kinase activity is increased 10-fold in insulin-treated GH4 cells. MAP kinase immunoprecipitated from control cells does not phosphorylate GABP alpha while MAP kinase immunoprecipitated from insulin-treated cells shows substantial phosphorylation of GABP alpha. These studies suggest that GABP mediates insulin-increased transcription of the prolactin gene. GABP may be regulated by MAP kinase phosphorylation
— id: 7029, year: 1996, vol: 271, page: 10425, stat: Journal Article,

A consensus insulin response element is activated by an Ets-related transcription factor
Jacob KK; Ouyang L; Stanley FM
1995 Nov 17;270(46):27773-27779, Journal of biological chemistry
Insulin increases expression of somatostatin-chloramphenicol acetyltransferase (CAT) constructs 10-fold and thymidine kinase-CAT constructs 5-fold in GH4 cells. These responses are similar to our previously reported data on insulin-increased prolactin-CAT expression. They are also observed in HeLa cells and are thus not cell type specific. The evidence suggests that the insulin responsiveness of these genes is mediated by an Ets-related transcription factor. First, linker-scanning mutations and/or deletions of the prolactin, somatostatin, and thymidine kinase promoters suggest that their insulin responsiveness is mediated by the sequence CGGA. This sequence is identical with the response element of the Ets-related transcription factors. Second, CGGA-containing sequences placed at -88 in the delta MTV-CAT reporter plasmid conferred insulin responsiveness to the mammary tumor virus promoter. Third, expression of the DNA-binding domain of c-Ets-2, which acts by blocking effects mediated by Ets-related transcription factors, inhibits the response of these promoters to insulin. Finally, the Ets-related proteins Sap and Elk-1 bind to the prolactin, somatostatin, and thymidine kinase insulin-response elements. An Ets-like element was found in all insulin-sensitive promoters examined and may serve a similar function in those promoters
— id: 12713, year: 1995, vol: 270, page: 27773, stat: Journal Article,

Insulin and cyclic adenosine monophosphate increase prolactin gene expression through different response pathways
Jacob KK; Stanley FM
1995 Apr 1;109(2):175-181, Molecular & cellular endocrinology
Insulin and cAMP stimulate prolactin gene transcription and prolactin-CAT expression in rat pituitary tumor GH cells. Expression of prolactin-CAT construct, pPrl(-173/+75)CAT, is stimulated 10- to 30-fold by either insulin or cAMP. Addition of both insulin and cAMP resulted in an additive 20- to 60-fold stimulation. Although the regulatory sequences have not been defined precisely, both insulin and cAMP appear to stimulate transcription of prolactin-CAT expression through possibly identical sequences in the -106/-87 region of the promoter. Insulin mediated increases in prolactin-CAT expression are not ras-dependent in GH4 cells. Thus, a number of experiments were performed to determine that the effects of insulin and cAMP are independent. First, insulin does not stimulate cAMP levels in GH4 cells. Second, cAMP action was inhibited by expression of a mutant regulatory subunit of cAMP-dependent protein kinase A that does not bind cAMP and by expression of an inhibitor of cAMP-dependent protein kinase A, while insulin action was not affected by expression of these proteins. Thus, although the regulatory sequences for insulin and cAMP may be identical, the effects of insulin and cAMP on the prolactin gene are clearly mediated through distinct response pathway
— id: 6650, year: 1995, vol: 109, page: 175, stat: Journal Article,

The insulin and cAMP response elements of the prolactin gene are overlapping sequences
Jacob KK; Stanley FM
1994 Oct 14;269(41):25515-25520, Journal of biological chemistry
Prolactin gene expression is affected by numerous signals, but many of the promoter elements required for these responses have not been clearly identified. This report identifies sequences within the prolactin gene promoter that are required for the responses to cAMP and insulin. The cAMP response element, -101 to -92 shares a 6 of 8 base pair homology with previously identified cAMP response elements. Mutation of this element also results in a > 100-fold decrease in basal prolactin gene expression. This is characteristic of cAMP response elements, but the importance of this element to basal prolactin gene transcription was previously unrecognized. The insulin response element, -97 to -67, is not homologous to previously reported insulin response elements and mediates the 10-fold increases in prolactin gene expression due to insulin observed in GH cells. These elements also function to mediate insulin and cAMP responses from the heterologous delta MTV-CAT reporter plasmid. Together, insulin and cAMP increase prolactin gene expression additively. The clustering of these elements may provide clues to the independent and possible coordinate regulation by these effectors
— id: 6649, year: 1994, vol: 269, page: 25515, stat: Journal Article,

Chicken insulin discriminates between receptors for insulin and insulin-like growth factor I on centrally- and peripherally-derived glial cells
Stanley FM
1993 ;13(7):1009-1030, Journal of receptor research
Insulin and IGF-I receptors in G26-20 cells, derived from a mouse oligodendroglioma, and in RN-2 cells, derived from a rat Schwannoma, were characterized by specific binding to [125I]insulin and [125I]IGF-I respectively. In both cell lines, the Kd for insulin was 1.5 nM. Insulin receptor number was 33,000/cell for RN-2 cells and 17,000 receptors/cell for G26-20 cells. RN-2 cells have 700,000 IGF-I receptors/cell with a Kd of 2 nM while G26-20 cells have 60,000 receptors/cell with an affinity of 4.9 nM. However, the independence of these two receptor populations in each cell type was equivocal since the subunit structure of these receptors appears identical by electrophoresis. In both cell lines, competition with insulin analogs for [125I]insulin binding demonstrated chicken insulin > insulin > IGF-I. Competition for [125I]IGF-I binding showed that IGF-I was approximately 85-fold more potent than insulin. Chicken insulin was ineffective at all concentrations. Thus, chicken insulin can be used as a specific ligand to unequivocally discriminate between IGF-I and insulin receptors and effects
— id: 13319, year: 1993, vol: 13, page: 1009, stat: Journal Article,

An element in the prolactin promoter mediates the stimulatory effect of insulin on transcription of the prolactin gene
Stanley FM
1992 Aug 15;267(23):16719-16726, Journal of biological chemistry
An insulin response element (IRE) has been identified in the prolactin gene using chimeric plasmids in which prolactin promoter DNA directs expression of the bacterial chloramphenicol acetyltransferase gene. A series of 5'-deletion constructs starting between positions -173 and -106 and extending through position +75 of the prolactin gene were all stimulated greater than 10-fold by physiological concentrations of insulin in rat pituitary tumor GH4 cells. However, insulin did not stimulate constructs starting at positions -96 and -46, suggesting that the IRE of the prolactin gene may be located in region -106/-96. Insulin stimulation of prolactin-chloramphenicol acetyltransferase constructs requires cotransfection with a human insulin receptor expression vector. Estimation of insulin receptor levels by beta-subunit phosphorylation indicates that receptor levels are increased approximately 50-fold following transfection with the human insulin receptor expression vector. This requirement for cotransfection suggests that the endogenous receptor levels may not be adequate to couple the response of transfected genes to insulin. Gel mobility shift experiments reveal a nuclear factor from GH4 cells that specifically associates with prolactin DNA fragment -106/-87. The amount or binding activity of this factor is increased following insulin treatment of cells. The concordance between functional and binding analyses of the prolactin promoter confirms the presence of an IRE in region -106/-87. The insulin-sensitive DNA-binding factor may mediate effects of insulin on prolactin gene transcription
— id: 13476, year: 1992, vol: 267, page: 16719, stat: Journal Article,

The homeodomain protein, Pit-1/GHF-1, is capable of binding to and activating cell-specific elements of both the growth hormone and prolactin gene promoters
Fox SR; Jong MT; Casanova J; Ye ZS; Stanley F; Samuels HH
1990 Jul;4(7):1069-1080, Molecular endocrinology
Studies were conducted to determine whether the trans-acting protein Pit-1/GHF-1 can bind to and activate promoter elements in both the GH and PRL genes that are necessary for cell-specific expression. Four pituitary cell lines that differentially express the endogenous GH and PRL genes were examined for their ability to activate GH and PRL promoter constructs containing sequences necessary for cell-specific expression (CSEs). Plasmids containing one CSE, -96 PRL and -104 GH, were similarly expressed in each of the four cell lines. Of the plasmids containing two CSEs, -173 PRL was always activated to a greater extent than -145 GH, with this relative activation being stronger in GC and GH1 cells than in 235-1 and GH4C1 cells. Protein-DNA binding assays were used to show that the GH and PRL CSEs specifically bound two highly abundant nuclear proteins (31 and 33 kDa). The two proteins were present at similar levels in all four pituitary cell lines and were recognized by a Pit-1/GHF-1 antibody. In contrast, HeLa and Rat2 cells did not activate transfected GH or PRL plasmids and did not contain nuclear proteins that specifically bound to the GH and PRL CSEs. However, cotransfection of these cells with the expression vector RSV-Pit-1/GHF-1 resulted in the activation of -173 PRL and -145 GH (PRL greater than GH). HeLa cells transfected with RSV-Pit-1/GHF-1 also contained 31- and 33-kDa nuclear proteins that bound to the GH and PRL CSEs. These results show that Pit-1/GHF-1 is present at levels in pituitary cell lines that are sufficient to activate the minimal elements in both the GH and PRL promoters necessary for cell-specific expression of these genes
— id: 63083, year: 1990, vol: 4, page: 1069, stat: Journal Article,

Transcriptional regulation of prolactin gene expression by thyroid hormone--alternate suppression and stimulation in different GH cell lines
Stanley F
1989 Oct;3(10):1627-1633, Molecular endocrinology
Transient expression experiments, using chimeric plasmids containing 3000 base pairs of PRL 5'-flanking sequences linked to the bacterial chloramphenicol acetyl transferase structural gene, demonstrate that L-T3 can inhibit (GH1 cells) or stimulate (GH4C1 cells) chloramphenicol acetyl transferase activity. Deletion experiments have defined the region necessary for these effects to sequences between -176 and -11 of the PRL gene. This region seems to contain the sequences necessary both for basal expression and for L-T3 regulation. Gel mobility shift experiments revealed that proteins extracted from GH1 and GH4C1 cell nuclei but not rat-2 fibroblasts interact with the PRL gene from -176 to +75. DNase I footprinting studies reveal two footprints which are the same in all pituitary derived cells tested. These footprints are not seen in rat-2 fibroblasts. Neither of these footprints likely represents binding of the L-T3-receptor since extracts from cells containing very low levels of receptor form footprints identical to those from cells with an abundance of receptors. These results suggest that different trans-acting factors, not identifiable by conventional footprinting techniques, are present in these cell lines which account for their opposite responses to L-T3. The regulation of PRL gene expression by L-T3 is unique in that both stimulation and suppression can be demonstrated using a single hormone-gene system. This should allow us to answer fundamental questions regarding the molecular switch between stimulation and suppression of gene expression by hormones
— id: 10478, year: 1989, vol: 3, page: 1627, stat: Journal Article,

c-erbA protooncogenes mediate thyroid hormone-dependent and independent regulation of the rat growth hormone and prolactin genes
Forman BM; Yang CR; Stanley F; Casanova J; Samuels HH
1988 Oct;2(10):902-911, Molecular endocrinology
Regulation of gene expression by the thyroid hormones is thought to be mediated by a nuclear-associated receptor found in a wide variety of cells and tissues. Cellular homologues of the avian erythroblastosis virus oncogene, v-erbA, encode proteins which bind thyroid hormone with similar affinities as thyroid hormone receptors. However, it has not been shown that any of the c-erbA proteins can function as receptor and modulate thyroid hormone responsive genes. In this study, using transient expression of chimeric reporter constructs, we document that the chick fibroblast c-erbA-alpha and the human placental c-erbA-beta modulate cis-acting regulatory sequences of two thyroid hormone responsive genes; rat GH and PRL. From these results we conclude: 1) in a receptor deficient cell line (235-1) both c-erbA subtypes act as hormone-dependent modulators of PRL gene expression and hence function as thyroid hormone receptors, 2) in two different receptor containing cell lines (GH4C1 and GH1), both c-erbA proteins act in a hormone-independent fashion to regulate PRL and GH expression. This suggests that events other than ligand binding can result in formation of a c-erbA protein that modulates transcription of thyroid hormone responsive genes, 3) no qualitative functional differences were detected between alpha- and beta-c-erbA subtypes, and 4) depending on the cell-type, L-T3 acts through its endogenous receptor to stimulate (GH4C1) or suppress (GH1) expression of a chimeric PRL construct. In these cells, c-erbA expression results in the same positive or negative response as the endogenous receptor except that the response occurs in the absence of hormone. These results suggest that the endogenous receptor and the c-erbAs act by augmenting the effect of transcription factors which can positively or negatively control gene expression
— id: 10956, year: 1988, vol: 2, page: 902, stat: Journal Article,

Stimulation of prolactin gene expression by insulin
Stanley F
1988 Sep 15;263(26):13444-13448, Journal of biological chemistry
GH3 cells are a rat pituitary-derived cell line in which the expression of the growth hormone and prolactin genes is controlled by a variety of hormones including thyroid hormone. Since these cells contain insulin receptors, we explored whether these cells can be used to analyze the mechanisms involved in the regulation of gene expression by insulin. When GH3 cells were incubated with serum-free media, insulin stimulated prolactin production rates about 3-fold after 72 h and 10-fold after 96 h of incubation. Insulin stimulated prolactin mRNA levels and gene transcription rates to the same extent as the prolactin production rates. In contrast, insulin did not stimulate the rate of growth hormone production or growth hormone mRNA levels. As previously reported, thyroid hormone stimulated growth hormone production and mRNA levels. In these cells, thyroid hormone also stimulated prolactin synthesis and prolactin mRNA levels. Prolactin production and mRNA levels in cells incubated with both insulin and thyroid hormone showed a synergistic response which was about 5- to 10-fold greater than with either hormone alone. In contrast, growth hormone production rates in cells cultured with insulin and thyroid hormone was only slightly greater than cells incubated with only thyroid hormone. Half-maximal stimulation of prolactin production in cells incubated with insulin or with both insulin and thyroid hormone occurred between 2 and 5 nM insulin, suggesting that the response is mediated by the insulin receptor. These results indicate that GH3 cells should be useful in analyzing the mechanisms involved in regulation of gene expression by insulin
— id: 10960, year: 1988, vol: 263, page: 13444, stat: Journal Article,

Stimulation of facilitated [3H]uridine transport by thyroid hormone in GH1 cells. Evidence for regulation by the thyroid hormone nuclear receptor
Stanley F; Tsai JS; Samuels HH
1986 Jul 15;261(20):9400-9404, Journal of biological chemistry
We have previously shown that 3,5,3'-triiodo-L-thyronine (L-T3) stimulates cell growth and a 4- to 8-fold increase in growth hormone mRNA in GH1 cells. These effects appear to be mediated by a thyroid hormone nuclear receptor with an equilibrium dissociation constant for L-T3 of 0.2 nM and an abundance of about 10,000 receptors per cell nucleus. In this report, we show that L-T3 exerts a pleiotypic effect on GH1 cells to rapidly (within 2 h) stimulate [3H]uridine uptake to a maximal value of 2.5- to 3-fold after 24 h. This results from an increase in the number of functional uridine 'transport sites' as shown by studies documenting an increase in the apparent Vmax with no change in the Km, 17 microM. Although the labeling of the cellular uridine pool and pools of all phosphorylated uridine derivatives was increased by L-T3, there was no change in the relative amounts of the individual pools in cells incubated with or without hormone. The intracellular concentration of [3H]uridine was estimated to be similar to that of the medium, suggesting that facilitated transport mediates [3H]uridine uptake. That this increase in [3H]uridine transport was nuclear receptor-mediated is supported by the excellent correspondence of the L-T3 dose-response curve for [3H]uridine uptake and that for L-T3 binding to receptor. Finally, inhibition of protein synthesis by cycloheximide and RNA synthesis by actinomycin D demonstrated that the L-T3 effect required continuing protein and RNA synthesis. These results are consistent with an effect of the L-T3-nuclear receptor complex to increase uridine uptake in GH1 cells by altering the expression of gene(s) essential for the transport process
— id: 62194, year: 1986, vol: 261, page: 9400, stat: Journal Article,

n-Butyrate effects thyroid hormone stimulation of prolactin production and mRNA levels in GH1 cells
Stanley F; Samuels HH
1984 Aug 10;259(15):9768-9775, Journal of biological chemistry
Using cultured GH1 cells, a growth hormone and prolactin-producing rat pituitary cell line, we have shown that n-butyrate and other short chain carboxylic acids stimulate histone acetylation and elicit a reduction of thyroid hormone nuclear receptor which is inversely related to the extent of acetylation (Samuels, H. H., Stanley, F., Casanova, J., and Shao, T. C. (1980) J. Biol. Chem. 255, 2499-2508). In this study, we compared the n-butyrate and propionate modulation of receptor levels to regulation of the growth hormone and prolactin response by 3,5,3'-triiodo-L-thyronine (L-T3). n-Butyrate (0.1-10 mM) did not stimulate growth hormone production. L-T3 stimulated the growth hormone response 4- to 5-fold and n-butyrate (0.5-1 mM) increased L-T3 stimulation of growth hormone production 1.5- to 2-fold compared to L-T3 alone. L-T3 stimulation of growth hormone production at higher n-butyrate concentrations decreased in parallel with the n-butyrate-mediated reduction of receptor levels. In contrast with the growth hormone response, n-butyrate (0.5 mM) increased basal prolactin production about 5-fold. Prolactin production, which is inhibited 25 to 50% by L-T3, was stimulated between 20- and 70-fold by L-T3 + n-butyrate (0.5-1 mM) and this decreased at higher n-butyrate levels. Prolactin mRNA and growth hormone mRNA levels paralleled the changes in prolactin and growth hormone production rates. These effects of L-T3, n-butyrate, or L-T3 + n-butyrate appeared unrelated to changes in cAMP levels or global changes in DNA methylation of the growth hormone or prolactin genes. Propionate elicited the same effects as n-butyrate but at a 5- to 10-fold higher concentration consistent with their relative effect on stimulating acetylation of chromatin proteins. These results suggest that prolactin gene expression is under partial regulatory repression which is reversed by a carboxylic acid-mediated postsynthetic modification event which allows for stimulation of the prolactin gene by thyroid hormone
— id: 63093, year: 1984, vol: 259, page: 9768, stat: Journal Article,

Thyroid hormone nuclear receptor. Evidence for multimeric organization in chromatin
Perlman AJ; Stanley F; Samuels HH
1982 Jan 25;257(2):930-938, Journal of biological chemistry
— id: 62192, year: 1982, vol: 257, page: 930, stat: Journal Article,

Organization of the thyroid hormone receptor in chromatin
Samuels HH; Perlman AJ; Raaka BM; Stanley F
1982 ;38:557-599, Recent progress in hormone research
— id: 62267, year: 1982, vol: 38, page: 557, stat: Journal Article,

Thyroid hormone nuclear receptor levels are influenced by the acetylation of chromatin-associated proteins
Samuels HH; Stanley F; Casanova J; Shao TC
1980 Mar 25;255(6):2499-2508, Journal of biological chemistry
The thyroid hormone receptor is a chromatin-associated protein which appears to mediate the actions of the thyroid hormones in mammalian cells. Unlike steroid hormone receptors, a cytoplasmic form of the receptor has not been identified, and the factors which govern the nuclear concentrations of the receptor are poorly understood. Using cultured GH1 cells, a rat pituitary cell line, we having previously demonstrated that thyroid hormones reduces the concentration of its receptor by a mechanism which involves the association of the ligand with the receptor binding site (Samuels, H.H., Stanley, F., and Shapiro, L.E. (1977) J. Biol. Chem. 252, 6052-6060). In this study, we demonstrate that n-butyrate and other aliphatic carboxylic acids elicit a reduction of thyroid hormone nuclear receptor levels without altering total cell protein synthetic rates. In contrast, the nuclear association and total cell level of the glucocorticoid receptor is not altered by n-butyrate. Evidence is presented that the aliphatic carboxylic acid-mediated reduction of thyroid hormone nuclear receptor levels is secondary to the inhibitory effect of these compounds on chromatin-associated deacetylases which is reflected as an increase in the acetylation of the nucleosome core histones. Isokinetic gradient centrifugation of chromatin solubilized from GH1 cell nuclei by micrococcal nuclease indicates that the receptor exists as a form associated with high molecular weight chromatin, as a 12.5 S form that sediments slightly faster than the bulk of the mononucleosomes, and as a 6.5 S form which appears to remain associated with low molecular weight chromatin components. Exclusive of the receptor associated with the high molecular weight chromatin, the 6.5 S form represents 80% and the 12.5 S form 10% of the receptor resolved in the gradient. n-Butyrate decreases both forms to the same degree suggesting that they are generated from the same 'entity' of chromatin structure. Studies on the reappearance of receptor after restoration of the chromatin to the 'normal' acetylated state are consistent with a model in which the affinity of chromatin for newly synthesized receptor is diminished in the 'hyperacetylated' state
— id: 62182, year: 1980, vol: 255, page: 2499, stat: Journal Article,

Depletion of L-3,5,3'-triiodothyronine and L-thyroxine in euthyroid calf serum for use in cell culture studies of the action of thyroid hormone
Samuels HH; Stanley F; Casanova J
1979 Jul;105(1):80-85, Endocrinology
GH1 cells are a clonal strain of rat pituitary tumor cells which synthesize GH and PRL. We have previously demonstrated that these cells respond to physiological concentrations of L-T3 and L-T4 when cultured with medium supplemented with thyroidectomized calf serum to achieve a thyroid hormone-depleted state under cell culture conditions. In this study, we describe a method to deplete euthyroid calf serum of L-T3 and L-T4 using an anion exchange resin. We demonstrate that the procedure only minimally alters the low molecular weight anion components of the serum and does not change the total protein content or the electrophoretic pattern of serum proteins. Moreover, we show that euthyroid calf serum depleted of L-T3 and L-T4 by this procedure yields serum which, when used as a medium supplement, results in biological responses identical to those obtained with media supplemented with thyroidectomized calf serum. In addition, resin treatment does not alter the growth-promoting properties of the serum if the thyroid hormone concentration is restored. This procedure should be useful in preparing thyroid hormone-depleted serum for cell culture studies in situations where thyroidectomy is not feasible or would require surgical procedures on a large number of small animals
— id: 63095, year: 1979, vol: 105, page: 80, stat: Journal Article,

Relationship of receptor affinity to the modulation of thyroid hormone nuclear receptor levels and growth hormone synthesis by L-triiodothyronine and iodothyronine analogues in cultured GH1 cells
Samuels HH; Stanley F; Casanova J
1979 Jun;63(6):1229-1240, Journal of clinical investigation
We have previously demonstrated that L-triiodothyronine (L-T3) induces an increase in growth hormone synthesis and messenger RNA in cultured GH1 cells, a rat pituitary cell line. In addition to regulating the growth hormone response, L-T3 elicits a time- and dose-dependent reduction in the level of its nuclear receptor, which is a direct function of the occupancy of the receptor binding site. In this study we have compared the relative affinity of L-T3, triiodothyroacetic acid, D-triiodothyronine (D-T3), and L-thyroxine (L-T4) for the receptor with the induction of the growth hormone synthesis and the ability of these compounds to elicit a reduction in thyroid hormone nuclear receptor levels. Triiodothyroacetic acid and D-T3 were specifically examined because the biologic effect of these compounds in the intact rat is significantly lower than predicted by their affinity for the receptor using isolated rat liver nuclei in vitro. In intact cells each compound demonstrated an excellent relationship between the relative receptor affinity, the induction of growth hormone production, and the concentration-dependent reduction in nuclear receptor levels. With the exception of D-T3, the relative affinity of iodothyronine was identical for the receptor using intact cells in serum-free media, or isolated GH1 cell nuclei in vitro. The apparent receptor affinity of D-T3 with intact cells was 5.5-fold lower than with isolated nuclei, which suggests a decrease in cell entry of D-T3 relative to the other iodothyronines. Quantitation of the [125I]iodothyronine associated with the receptor in GH1 cells after a 36-h incubation with L-125I-T4 was 90% L-T4 and 10% L-T3, which indicates that the major effect of L-T4 in GH1 cells is a result of intrinsic L-T4 activity. Studies with dispersed rat anterior pituitary cells demonstrated that L-T3 induces growth hormone synthesis and elicits a reduction in nuclear receptor levels in the same fashion as GH1 cells. The observation that thyroid hormone influences dispersed rat pituitary cells in a fashion qualitatively similar to GH1 cells may have implications for the growth hormone response of the somatotroph cell in vivo to different thyroidal states
— id: 62208, year: 1979, vol: 63, page: 1229, stat: Journal Article,

Control of growth hormone synthesis in cultured GH1 cells by 3,5,3'-triiodo-L-thyronine and glucocorticoid agonists and antagonists: studies on the independent and synergistic regulation of the growth hormone response
Samuels HH; Stanley F; Shapiro LE
1979 Feb 20;18(4):715-721, Biochemistry
— id: 63096, year: 1979, vol: 18, page: 715, stat: Journal Article,

Evidence for thyroid hormone-dependent and independent glucocorticoid actions in cultured cells. Studies on the induction of growth hormone and glutamine synthetase in GH1 cells and tyrosine aminotransferase in Reuber H-35 cells
Samuels HH; Klein D; Stanley F; Casanova J
1978 Sep 10;253(17):5895-5898, Journal of biological chemistry
The induction of growth hormone synthesis and mRNA by thyroid hormone in cultured GH1 cells is mediated by the thyroid hormone nuclear receptor. In addition, the regulation of the growth hormone response by glucocorticoid is highly dependent on the action of thyroid hormone. To clarify whether thyroid hormone has a general influence on glucocorticoid action in GH1 cells, the glucocorticoid induction of growth hormone and glutamine synthetase was simultaneously examined. In contrast to the growth hormone response, the induction of glutamine synthetase by glucocorticoid was not influenced by thyroid hormone. Both responses appear to be modulated by the glucocorticoid receptor, and thyroid hormone had no influence on nuclear-associated glucocorticoid receptor levels. These results suggest that the thyroid hormone control of glucocorticoid induction of growth hormone may be a selective process, and the nuclear associated receptors for both thyroid and glucocorticoid hormones interrelate to control the growth hormone response
— id: 63097, year: 1978, vol: 253, page: 5895, stat: Journal Article,

Thyroid hormone controls glucocorticoid action in cultured GH1 cells
Samuels HH; Horwitz ZD; Stanley F; Casanova J; Shapiro LE
1977 Jul 21;268(5617):254-257, Nature
— id: 63099, year: 1977, vol: 268, page: 254, stat: Journal Article,

Modulation of thyroid hormone nuclear receptor levels by 3,5,3'-triiodo-L-thyronine in GH1 cells. Evidence for two functional components of nuclear-bound receptor and relationship to the induction of growth hormone synthesis
Samuels HH; Stanley F; Shapiro LE
1977 Sep 10;252(17):6052-6060, Journal of biological chemistry
— id: 62189, year: 1977, vol: 252, page: 6052, stat: Journal Article,

Dose-dependent depletion of nuclear receptors by L-triiodothyronine: evidence for a role in induction of growth hormone synthesis in cultured GH1 cells
Samuels HH; Stanley F; Shapiro LE
1976 Nov;73(11):3877-3881, Proceedings of the National Academy of Sciences of the United States of America
The relationship between the binding of L-triiodothyronine (T3) to nuclear receptors and the induction of growth hormone synthesis was examined in cultured GH1 cells, a rat pituitary cell line. After 24 hr, T3 induced a maximal 4-fold increase in the rate of growth hormone wynthesis; the T3 concentration that induced a half-maximal increase was 0.22 nM. The biologic dose-response curve was shifted to the left of the receptor occupancy curve (Kd = 0.5 nM) by a factor of approximately 2 when receptor binding was examined for 4 hr but showed a significantly closer agreement when examined for the same 24-hr period as the biologic response. This shift in the fractional occupancy curve is not due to further equilibration but occurs as a result of a time- and dose-dependent depletion of the nuclear receptor by T3
— id: 62244, year: 1976, vol: 73, page: 3877, stat: Journal Article,

Thyroid hormone action: in vitro characterization of solubilized nuclear receptors from rat liver and cultured GH1 cells
Samuels HH; Tsai JS; Casanova J; Stanley F
1974 Oct;54(4):853-865, Journal of clinical investigation
— id: 62207, year: 1974, vol: 54, page: 853, stat: Journal Article,