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J Biol Chem, Vol. 274, Issue 42, 29874-29882, October 15, 1999


STAT5b Down-regulates Peroxisome Proliferator-activated Receptor alpha  Transcription by Inhibition of Ligand-independent Activation Function Region-1 trans-Activation Domain*

Yuan-Chun Zhou and David J. WaxmanDagger

From the Division of Cell and Molecular Biology, Department of Biology, Boston University, Boston, Massachusetts 02215

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Growth hormone-activated STAT5b inhibits by up to 80% the transcriptional activity of peroxisome proliferator-activated receptor (PPAR) alpha , a nuclear receptor activated by diverse environmental chemicals and hypolipidemic drugs classified as peroxisome proliferators. This inhibitory cross-talk between STAT5b and PPAR is now reported for PPAR forms gamma  and delta  and for thyroid hormone receptor, indicating a more general potential for inhibitory cross-talk between JAK/STAT and nuclear receptor signaling pathways. Further investigations revealed that SOCS-3, a growth hormone-inducible negative regulator of cytokine signaling to STAT5b, abolished the STAT5b inhibitory response. A constitutively active STAT5b mutant failed to inhibit PPARalpha activity, indicating that STAT5b does not induce synthesis of a more proximal PPARalpha inhibitor. STAT5b inhibition was not reversed by overexpression of the heterodimerization partner of PPAR (retinoid X receptor) or the nuclear receptor coactivators P300 and SRC-1, suggesting that STAT5b does not inhibit PPARalpha by competing for these limiting cellular cofactors. STAT5b did not inhibit a chimeric receptor comprised of yeast GAL4 DNA-binding domain linked to the ligand binding/AF-2 trans-activation domain of PPARalpha , indicating that the COOH-terminal AF-2 domain of PPAR is not the target of STAT5b inhibition. Rather, STAT5b inhibited transcription driven by the NH2-terminal ligand-independent AF-1 trans-activation domain of PPARalpha in a GAL4-linked chimera by ~80%. The conservation of this AF-1 trans-activation function in many nuclear receptors suggests that AF-1 may serve as an important target for inhibitory cross-talk between STAT transcription factors and nuclear receptors in a variety of signaling pathways.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

STATs1 and PPARs are distinct families of transcription factors that mediate cellular responses to diverse endogenous and environmental stimuli. STATs are activated by multiple cytokines and peptide hormones via receptor-associated JAK tyrosine kinases by a process that involves tyrosine phosphorylation and dimerization of the SH2 domain-containing STAT proteins, followed by STAT translocation to the nucleus, binding to the specific DNA enhancer elements, and activation of target gene expression (1). Two closely related STAT forms, STAT5a and STAT5b, share striking amino acid sequence homology (~90% identity) and can be activated by the same set of hormones and cytokines, which includes GH, prolactin, interleukins 2, 3, and 5, and erythropoietin (2). STAT5a and STAT5b both activate target gene expression via STAT5 response elements, albeit with some differences in binding specificity (3, 4). Mouse gene knock out studies demonstrate, however, that STAT5a and STAT5b have distinct and largely nonredundant tissue-specific functions respectively related to mammary gland development and liver metabolic function (5-7).

PPARs are transcription factors that belong to the nuclear receptor superfamily, and, like other nuclear receptors, they contain conserved domains that function in DNA binding, ligand binding, transcriptional activation, and dimerization (reviewed in Refs. 8 and 9). PPARs form high affinity DNA-binding complexes with the heterodimerization partner RXR, which facilitates their binding to specific PPAR response elements upstream of target genes. Mammalian PPARs include three subtypes (alpha , gamma , and delta ), which are characterized by unique functions, ligand specificities, and tissue distributions (10). A wide variety of structurally distinct PPAR ligands have been identified. These include certain environmental chemicals, fibrate hypolipidemic drugs, fatty acids, and eicosanoids for PPARalpha ; anti-diabetic thiazolidinediones and 15-deoxy-Delta 12,14 prostaglandin J2 for PPARgamma ; and the fatty acid-like compound L631033 for PPARdelta (11, 12). This diverse spectrum of PPAR ligands suggests that these receptors mediate a wide variety of biological functions. Although the function of PPARdelta is unknown, PPARalpha and PPARgamma regulate diverse biological processes, including lipid metabolism, cell differentiation, carcinogenesis, and apoptosis (13-15).

Extensive investigations have advanced our understanding of JAK/STAT and PPAR signaling pathways. However, the extent to which these two pathways interact remains unclear. The observation that hypophysectomy enhances hepatic peroxisome proliferation in female rats (16) suggests that the pituitary secretes an endocrine factor that inhibits this PPARalpha -dependent (17) liver response. GH and prolactin are two major pituitary hormones that directly activate JAK-STAT signaling pathways (18). GH treatment suppresses expression in rat liver in vivo of certain peroxisome proliferator-inducible liver P450 genes (19), and GH inhibits peroxisomal beta -oxidation induced by the PPARalpha activator clofibrate in rat primary hepatocyte culture (20, 21). We previously demonstrated that activation of STAT5b signaling by either GH or prolactin inhibits, by up to 80%, PPARalpha -dependent reporter gene transcription stimulated by both endogenous and foreign chemical peroxisome proliferators (22). This inhibition required both GH receptor (GHR) and STAT5b and was not seen with STAT1, a second GH-activated STAT. STAT5b-PPARalpha complexes could not be detected by anti-STAT5b supershift analysis of PPARalpha -DNA complexes, suggesting that the mechanism that underlies this inhibitory cross-talk between STAT5b and PPARalpha is distinct from the cross-talk between STAT5 and glucocorticoid receptor, where protein-protein complexes form (23, 24).

In the present study, we investigate the mechanism that underlies the inhibitory cross-talk between JAK/STAT and PPAR signaling pathways. We report that GH-activated STAT5b inhibits the transcriptional activity of PPARgamma , PPARdelta , and T3R in addition to PPARalpha and that the GH-dependent inhibition of PPARalpha -stimulated gene transcription is blocked by co-expression of SOCS3, a GH-inducible inhibitor (25) that belongs to a recently described family of cytokine signaling inhibitors (26-28). We evaluate the requirement for STAT5b-induced gene transcription, the role of transcriptional coactivators, and the role of the DNA-binding and ligand-binding and trans-activation domains of PPARalpha for STAT5b inhibition. Our findings suggest that STAT5b inhibition of PPAR transcriptional activity proceeds via a novel mechanism that involves the NH2-terminal AF-1 trans-activation domain of the nuclear receptor, which also serves as the target for insulin stimulation of PPARalpha activity (29), albeit by a distinct mechanism.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Plasmids-- The PPAR-activated firefly luciferase reporter pHD(X3)Luc, obtained from Dr. J. Capone (McMaster University, Toronto, ON, Canada), contains three tandem copies of the PPRE from the rat enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase gene upstream of a minimal promoter (30) cloned into the plasmid pCPS-luc (31). Mouse PPARalpha cloned into the expression plasmid pCMV5 was obtained from Dr. E. Johnson (Scripps Research Institute, La Jolla, CA) (32). pSG5-GAL4-mPPARalpha and (UAS)5-tk-CAT reporter construct were provided by Dr. Steven Kliewer (Glaxo Research Institute, Research Triangle Park, NC) (33). pSG5-GAL4-mPPARalpha encodes a chimeric receptor, herein designated GAL4-PPARalpha (167-468), that contains the initiation sequence and amino acids 1-76 of the glucocorticoid receptor fused to amino acids 1-147 of the yeast transcription factor GAL4 in the pSG5 expression vector (Stratagene), followed by amino acid 167-468 of mouse PPARalpha . pM-GAL4-BD-hPPARalpha (1-92) (herein designated GAL4-PPARalpha (1-92)), pM-GAL4-BD-hPPARalpha (1-92)-S12A, and pM-GAL4-BD-hPPARalpha (1-92)-S21A were provided by Dr. Christoph Meier (University Hospital, Geneva, Switzerland) (29). (UAS)5-tk-CAT is a reporter construct that contains five copies of a GAL4 DNA-binding site upstream of the thymidine kinase promoter driving the reporter gene chloramphenicol acetyl transferase (CAT). Human PPAARdelta /Nuc1 cloned into the expression plasmid pJ3 was obtained from Dr. A. Schmidt (Merck Research Labs, West Point, PA) (34). Mouse PPARgamma cloned in pSV-Sport1 expression plasmid was provided by Dr. J. Reddy (Northwestern University Medical School, Chicago, IL) (35). T3R (TRbeta ) expression plasmid and T3R reporter plasmid (TRE-CAT) were provided by Dr. D. Moore (Baylor College of Medicine, Houston, TX). Expression plasmids encoding mouse RXRalpha (Dr. R. Evans, Salk Institute, San Diego, CA) (36), CIS-1 (also known as CIS), CIS-4 (=SOCS-6), SOCS-2, and SOCS-3 (Dr. A. Yoshimura, Kurume University, Kurume, Japan) (37) and mouse SRC-1 and P300 (Dr. M. Brown, Dana Farbar Cancer Institute, Boston, MA) were each obtained from the sources indicated. Renilla luciferase expression plasmid pRL-CMV was purchased from Promega (Madison, WI). STAT5a1*6 and STAT5b1*6 cDNAs were excised from the corresponding pMX-puro-STAT5-1*6 plasmids, provided by Dr. Toshio Kitamura (University of Tokyo) (38), and the EcoRI-NotI fragments were subcloned into the expression vector pCI (Promega) by Dr. S. H. Park of this laboratory. The STAT5 reporter plasmid 4X-pT109-Luc, which contains four copies of a STAT5 response element from the rat ntcp gene (39), was provided by Dr. M. Vore (University of Kentucky, Lexington, KY).

Cell Culture and Transfection-- COS-1 cells were grown in Dulbecco's modified Eagles medium with 10% fetal calf serum. Transfection of COS-1 cells grown in 48-well tissue culture plates was carried out using FuGENE 6 transfection reagent (Roche Molecular Biochemicals; catalog number 1814443). FuGENE 6 (0.5 µl) was used for each well in 48-well plates. 24 h after addition of the DNA-FuGENE 6 mixture to the cells, the medium was changed to nonserum Dulbecco's modified Eagles medium. PPAR activators (e.g. Wy-14,643 at 1-20 µM) were then added to the culture medium in combination with GH at concentrations specified in the figure legends. Cell were lysed 24 h later, and firefly luciferase and Renilla luciferase activity were measured using a dual luciferase assay kit (Promega, Madison, WI; catalog number E1910). CAT activity was determined by thin layer chromatographic analysis of the incorporation of [14C]chloramphenicol (NEN Life Science Products) into acetyl CoA. CAT activity was normalized to the protein concentration of each extract. Transfections were performed using the following amounts of plasmid DNA/well (0.75 cm2) of a 48-well tissue culture plate: 90 ng of pHD(X3)Luc, 5 ng of PPARalpha , PPARgamma , or PPARdelta , 5 ng of pM-GAL4BD-hPPARalpha (1-92) and its corresponding serine to alanine mutants, 50 ng of 4X-pT109-Luc, 50 ng of STAT5b, 2 ng of JAK2, 50 ng of GHR, 200 ng of STAT5b1*6 or STAT5a1*6, 125 ng of SOCS or CIS, 50 ng of RXRalpha , 5 ng of GAL4-PPARalpha (167-468), 50 ng of (UAS)5-tk-CAT, 50-100 ng of P300, 10 ng of SRC-1, 1 ng of pRL-CMV, 50 ng of TRbeta , and 90 ng of TRE-CAT.

EMSA and Western Blot Analysis-- EMSA analysis using probes for STAT5 and PPAR DNA binding activity was performed as described previously (22). For Western blotting, whole cell lysates from transfected COS-1 cells were subjected to 10% SDS/polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes. Western blotting was performed using STAT5b-specific antibody (Santa Cruz Biotechnology, sc-835), as described elsewhere (22).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

STAT5b Inhibits PPARgamma , PPARdelta , and T3R Transcriptional Activity-- GH-activated STAT5b inhibits PPARalpha -dependent gene transcription by ~80% when evaluated in a reporter gene trans-activation assay (22). To determine whether this inhibition is specific to PPARalpha , we examined the other two mammalian PPAR forms, PPARgamma and PPARdelta , as well as T3R, all of which belong to family NR1 of the nuclear receptor superfamily (40). GH signaling and PPAR or T3R transcriptional activation pathways were reconstituted in COS-1 cells by co-transfection of key components: GHR, JAK2 kinase, and STAT5b for GH signal transduction; PPARgamma or PPARdelta and the reporter plasmid pHD(X3)Luc for PPAR signaling; and T3R and the reporter plasmid TRE-CAT for thyroid hormone signaling. The transfected COS-1 cells were stimulated for 24 h with the nuclear receptor form-specific ligands troglitazone (1 µM), L631033 (5 µM) or T3 (74 nM) to activate PPARgamma , PPARdelta , and T3R, respectively, either in the presence or the absence of GH. Fig. 1 shows that activation of PPARgamma , PPARdelta , and T3R transcriptional activity by the respective ligand of each receptor is substantially decreased in the presence of GH. The extent of STAT5b inhibition of these three nuclear receptors is similar to that seen previously for PPARalpha (80% inhibition for PPARalpha (22) compared with 89, 60, and 73% inhibition for PPARgamma , PPARdelta , and T3R, respectively). This finding suggests that these nuclear receptors share a common target for STAT5b inhibition.


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  		Fig. 1.   Transcriptional activities of PPARgamma (A), PPARdelta (B), and T3R (C) are inhibited by GH-activated STAT5b. COS-1 cells were cotransfected with expression plasmids encoding GHR, JAK2, STAT5b, reporter plasmids pHD(X3)Luc (A and B) or TRE-CAT (C) together with expression plasmids for each indicated nuclear receptors. A Renilla luciferase expression plasmid (pRL-CMV) was included to normalize samples for transfection efficiencies. Cells were stimulated for 24 h with GH and each of the indicated nuclear receptor ligands. Cell lysates were then prepared and assayed for normalized luciferase (A and B) or CAT enzyme activities (C). Data shown are firefly luciferase reporter activities normalized for the level of Renilla luciferase activities. A, GH (25, 100 ng/ml) inhibits troglitazone-stimulated (Tro; 1 µM) PPARgamma induction of luciferase reporter activity. B, GH inhibits PPARdelta activation by L631033 (L6; 5 µM). C, GH inhibits triiodothyronine-stimulated (T3; 74 nM) CAT reporter activity.

STAT5b Does Not Inhibit Ligand-dependent AF-2 Transcriptional Activation Domain of PPARalpha -- PPAR and other nuclear receptors contain a central DNA-binding domain and a COOH-terminal ligand-binding domain that displays ligand-dependent trans-activation activity, termed AF-2. The hydrophobicity of this region is conserved in all nuclear receptors, and mutational analysis suggests that AF-2 contains an interaction surface for coactivators, such as SRC-1 (41). To determine whether STAT5b exerts its inhibitory effect on nuclear receptor activity by inhibiting the COOH-terminal AF-2 region, we evaluated the effect of STAT5b on the chimeric receptor GAL4-PPARalpha (167-468), where the COOH-terminal region of mouse PPARalpha (amino acids 167-468) is fused to the DNA-binding domain of the yeast transcription factor GAL4 (33). Activation of this chimeric receptor by peroxisome proliferators was monitored in transfected COS-1 cells using the reporter gene (UAS)5-tk-CAT, which contains five copies of the DNA response element of GAL4 upstream of the thymidine kinase promoter driving a CAT reporter gene. Stimulation of the cells with the PPARalpha activator and potent peroxisome proliferator Wy-14,643 strongly activated the GAL4-PPARalpha chimera; however, GH-activated STAT5b failed to inhibit receptor activity (Fig. 2). Thus, the presence of the COOH-terminal AF-2 trans-activation/ligand-binding domain of PPARalpha is not sufficient for STAT5b to inhibit receptor activity, suggesting that STAT5b does not directly target this region of PPARalpha .


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  		Fig. 2.   GH-activated STAT5b fails to inhibit activation of GAL4-PPARalpha ligand-binding domain chimeric receptor by Wy-14,643. COS-1 cells were cotransfected for 24 h with expression plasmids encoding GHR, JAK2, STAT5b, GAL4-PPARalpha (167-468), and the reporter plasmid (UAS)5-tk-CAT. Transfected cells were stimulated for 24 h with Wy-14,643 (20 µM) with or without GH (25 and 100 ng/ml, as indicated). Cell extracts were assayed for CAT activity as described under "Materials and Methods." In the diagram shown, the GAL4-PPARalpha chimera is represented by a GAL4 DNA-binding domain (DBD) fused to the COOH-terminal trans-activation domain (TAD) of PPARalpha .

STAT5b Inhibits the NH2-terminal Ligand-independent AF-1 trans-Activation Domain of PPARalpha via a MAP Kinase-independent Mechanism-- A ligand-independent trans-activation domain, designated AF-1, has recently been identified within the NH2-terminal 92 amino acids (A/B region) of PPARalpha (29). We therefore investigated whether GH-activated STAT5b can inhibit the AF-1 function of PPARalpha , which was assayed using a fusion construct linking the GAL4 DNA-binding domain to the first 92 amino acids of hPPARalpha , designated GAL4-PPARalpha (1-92). Fig. 3A shows that the AF-1 region of hPPARalpha exhibits strong ligand-independent transcriptional activity (vehicle control versus GAL4 DNA-binding domain alone; compare the first two bars), in agreement with recent studies by Meier and co-workers (29). Moreover, GH activation of STAT5b inhibited this trans-activation activity in a dose-dependent manner. The extent of inhibition, ~80%, is consistent with the extent of STAT5b inhibition of full-length PPARalpha that we reported previously (22).


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  		Fig. 3.   STAT5b inhibits NH2-terminal AF-1 domain of PPARalpha in a manner that is independent of serine 12 and serine 21. COS-1 cells were transfected with GAL4-PPARalpha (1-92) (A), GAL4-PPARalpha (1-92)-S12A (B), or GAL4-PPARalpha (1-92)-S21A (C) together with expression plasmids encoding GHR, JAK2, STAT5b, and the reporter plasmid (UAS)5-tk-CAT. Cells were either untreated (vehicle control) or were treated for 24 h with GH at 5-500 ng/ml beginning 24 h after transfection. CAT reporter activity is expressed as the percentage of [14C]chloramphenicol incorporated into acetylated metabolites. CAT activity was normalized by protein concentration. A also presents the basal activity measured with an expression plasmid encoding GAL4 DNA-binding domain (GAL4DB) alone, in the absence of PPARalpha residues 1-92.

Serine residues 12 and 21 within the AF-1 region of PPARalpha have been identified as targets of MAP kinase, and phosphorylation of both of these serines is required for insulin stimulation of PPARalpha activity (29). To examine whether these serines participate in STAT5b inhibition of the AF-1 activity of PPARalpha , we tested the corresponding serine to alanine mutants of the GAL4-PPARalpha (1-92) chimera. Fig. 3 (B and C) show that mutation of these serine residues does not prevent STAT5b inhibition, indicating that the GH-dependent inhibition of the AF-1 activity of PPARalpha does not involve changes in the phosphorylation of these residues.

Coactivators P300 and SRC-1 Do Not Reverse STAT5b Inhibition of PPARalpha Activity-- The observation that GH-activated STAT5b inhibits activation of all three PPAR isoforms as well as T3R suggests that STAT5b may compete with a common regulatory factor of the PPARs, perhaps a nuclear receptor coactivator (42). This possibility is further supported by our identification of PPARalpha 's AF-1 region, which in the case of PPARgamma interacts with coactivators (43, 44), as a target of STAT5b. P300 and SRC-1 are widely expressed nuclear receptor coactivators that interact directly with PPARalpha (45). P300 can interact with both the AF-1 and AF-2 regions of PPAR (44) and can also coactivate STAT5 (46). To investigate whether these coactivators become limiting for PPARalpha transcriptional activity in cells that simultaneously signal via STAT5b, we examined the effects of P300 and SRC overexpression on the STAT5b-dependent inhibition of PPARalpha activity. Fig. 4A shows that cotransfection of P300 or SRC-1 substantially enhances both basal and Wy-14,643-inducible PPARalpha activity. This confirms that P300 and SRC-1 can both coactivate PPARalpha and indicates that their endogenous expression levels in transfected COS-1 cells are indeed limiting. Fig. 4B shows, however, that overexpression of these coactivators does not block the GH-induced inhibition of PPARalpha activity. Therefore, STAT5b does not inhibit PPARalpha by competing for the limiting endogenous P300 or SRC-1. Of note, STAT5b expression alone, in the absence of GH treatment, reversed the stimulation of PPARalpha activity by co-expressed P300 or SRC-1; whereas P300 and SRC-1 alone enhanced Wy-14,643-stimulated PPARalpha activity by 5- and 8-fold, respectively (Fig. 4A), these enhancing effects were abolished in cells cotransfected with GHR, JAK2, and STAT5b but not treated with GH (cf. data points on y axis of Fig. 4B). This result is consistent with the report that P300 directly interacts with the trans-activation domain of STAT5 (46) and may be explained by STAT5b competing with PPARalpha for the transfected P300 and perhaps also SRC-1.


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  		Fig. 4.   Coactivators P300 and SRC-1 enhance PPARalpha activity, but their overexpression does not reverse STAT5b inhibition. A, PPARalpha transcriptional activity is enhanced by cotransfection of P300 or SRC-1. COS-1 cells were transfected with reporter plasmid pHD(X3)Luc and expression plasmids encoding PPARalpha (5 ng) and P300 (100 ng) or SRC-1 (10 ng). Transfected cells were treated with Wy-14,643 (1 µM). 24 h after Wy-14,643 treatment (1 µM), cells were lysed and assayed for firefly luciferase activity normalized to a Renilla luciferase control. B, overexpression of P300 or SRC-1 fails to reverse GH inhibition of PPARalpha activity. COS-1 cells were cotransfected with expression plasmids encoding GHR, JAK2, STAT5b, and PPARalpha in the presence or absence of P300 or SRC-1, as described in A. After 24 h of treatment with Wy-14,643 (1 µM) and GH at the concentrations indicated, firefly luciferase activity was measured and normalized to a Renilla luciferase control.

Effects of STAT5b on PPARalpha DNA Binding Activity-- We next investigated whether PPARalpha DNA binding activity is affected by STAT5b activation. Fig. 5A shows that Wy-14,643 stimulation of PPARalpha leads to a 3.3-fold increase in PPARalpha DNA binding activity, assayed by EMSA using a PPRE probe derived from the rabbit CYP4A6 gene (lane 2 versus lane 1). In the absence of GHR, JAK2, and STAT5b, GH had no effect on Wy-14,643-induced PPARalpha DNA-binding (Fig. 5B, lanes 3 and 4 versus lane 2). In contrast, under conditions where GHR, JAK2, and STAT5b are cotransfected with PPARalpha , GH treatment led to a small (~25%) decrease in the abundance of the PPARalpha -DNA binding complex (Fig. 5A, lanes 3 and 4 versus lane 2). Because the DNA binding activity of PPARalpha is obligatorily dependent on the presence of its heterodimerization partner RXRalpha (47), we investigated whether GH activation of STAT5b might decrease PPARalpha DNA binding activity by interfering with PPAR/RXR heterodimerization. To test this possibility, the cells were co-transfected with RXR to augment the limited amount of endogenous RXR in COS-1 cells. RXR overexpression significantly increased basal PPARalpha DNA binding activity, which, as expected (47), is no longer further increased by Wy-14,643 treatment (Fig. 5, A, lanes 6-8, and B, lanes 5-7). Nevertheless, GH still decreased PPAR DNA binding activity by ~30% (Fig. 5A, lane 8 versus lane 7), despite the co-expression of RXR. This suppression varied from 24 to 50% in different experiments and was not seen in the absence of GHR, JAK2 and STAT5b (Fig. 5B, lanes 7 versus lane 6). Overexpression of RXRalpha significantly increased both basal and Wy-14,643-induced luciferase reporter gene activity; however, it did not reverse STAT5b inhibition of PPARalpha activity (Fig. 5C). We conclude that STAT5b does not inhibit PPARalpha by decreasing cellular RXR levels or the availability of RXR for PPARalpha heterodimerization.


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  		Fig. 5.   GH partially inhibits Wy-14,643-stimulated PPARalpha DNA binding activity in an RXRalpha -independent manner. A and B, GH partially inhibits PPARalpha DNA binding activity, both with (lanes 1-4) and without (lanes 6-8) RXRalpha coexpression. COS-1 cells were transfected with expression plasmids as indicated. 24 h after transfection, cells were treated with Wy-14,643 (10 µM) and GH (100 or 500 ng/ml) for 24 h. Whole cell lysates (10 µg) were assayed for EMSA activity using a CYP4A6 PPRE probe. Lanes 5 and 9, untransfected Cos-1 cell control. The experiment shown in lanes 1-4 of A and B used three times more PPARalpha expression plasmid/well than in the standard methods (i.e. 15 versus 5 ng PPARalpha /well of a 48-well plate). This was required to provide sufficient PPARalpha DNA binding activity to visualize the EMSA complexes, which are weak in the absence of cotransfected RXRalpha (cf. left-hand panels versus right-hand panels of A and B). C, overexpression of RXRalpha does not reverse STAT5b inhibition of PPARalpha transcriptional activity. COS-1 cells were transfected with pHD(X3)Luc reporter plasmid and expression plasmids encoding GHR, JAK2, STAT5b, and PPAR in the presence or absence of RXRalpha expression plasmid. 24 h after transfection, cells were treated with Wy-14,643 (20 µM) and GH (25 ng/ml). Cell lysates were prepared and assayed for luciferase activity 24 h later. Activities are expressed as firefly luciferase normalized by the reporter activity of a Renilla luciferase internal standard.

Constitutively Active STAT5 Mutants Do Not Inhibit PPARalpha -- STAT5a and STAT5b site-specific mutants with amino acid substitutions H299R and S711F are constitutively phosphorylated on tyrosine and undergo nuclear translocation and transcriptional activation in the absence of hormone or cytokine stimulation (38). These constitutively active STAT5 mutants, designated STAT5a1*6 and STAT5b1*6, were used to investigate whether STAT5b transcriptional activity is sufficient for PPARalpha inhibition. STAT5a1*6 and STAT5b1*6 were confirmed to be constitutively active when expressed in COS-1 cells, as shown by their strong activation of a luciferase reporter gene containing four copies of a STAT5 response element from the ntcp gene (39) in the absence of GHR expression and GH stimulation (37-fold activation by STAT5b1*6 and 7-fold by STAT5a1*6) (Fig. 6A). Cotransfection of GHR with STAT5b1*6 followed by GH stimulation did not result in further activation of the reporter plasmid (data not shown), suggesting that STAT5b1*6 is already fully active in COS-1 cells without GH stimulation. We next examined whether these constitutively active STAT5 mutants inhibit PPARalpha activity. Fig. 6B shows that neither STAT5a1*6 nor STAT5b1*6 blocked PPARalpha activity stimulated by Wy-14,643. STAT5b1*6 also failed to inhibit PPARalpha activity in GHR- and JAK2-transfected cells stimulated with GH (data not shown). Because these constitutively active STAT5 mutants failed to affect the inhibition of PPARalpha activity observed with wild-type STAT5b, we conclude that STAT5b does not inhibit PPARalpha transcriptional activity by stimulating transcription of a more proximal inhibitory factor.


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  		Fig. 6.   Effect of constitutively active STAT5 mutants on Wy-14,643 activation of PPARalpha . A, constitutively active STAT5a1*6 and STAT5b1*6 induce transcription from the ntcp promoter in the absence of GH stimulation. COS-1 cells were transfected with 4X-pT109-Luc reporter plasmid (39) and expression plasmids encoding STAT5a1*6 or STAT5b1*6. pCI empty vector was used as a DNA control. 24 h after transfection, cells were lysed and assayed for firefly luciferase activity normalized to a Rellina luciferase control. B, constitutively active STAT5 mutants do not inhibit PPARalpha activation by Wy-14,643. COS-1 cells were transfected with pHD(X3)Luc reporter plasmid and expression plasmids encoding PPARalpha , STAT5a1*6 or STAT5b1*6. Transfected cells were treated with vehicle or Wy-14,643 (10 µM) for 24 h. Transcriptional activity is expressed as relative firefly luciferase activity normalized to Renilla luciferase control. Values shown are the means ± S.E. (n = 3).

SOCS-3 Blocks GH-stimulated STAT5b Inhibition of PPARalpha -- SOCS/CIS proteins constitute a novel family of cytokine-inducible suppressors that negatively regulate the JAK/STAT signaling pathways (26-28). One of these proteins, SOCS-3, may, in part, inhibit GH signaling by binding to the tyrosine phosphorylated cytoplasmic domain of GHR (48). To investigate whether the STAT5b-dependent inhibition of PPARalpha is subjected to SOCS protein negative regulation, we first examined whether SOCS protein expression blocks GH-stimulated STAT5b activation. COS-1 cells were transiently transfected with GHR, STAT5b, and SOCS expression plasmids. 24 h later, the cells were stimulated with GH for 30 min, and cell lysates were assayed for STAT5b DNA binding activity by EMSA. Fig. 7A shows that SOCS-3 fully blocks GH activation of STAT5b EMSA activity (lanes 9 and 10 versus lanes 3 and 4). Partial or no inhibition was observed with three other SOCS/CIS proteins (CIS, SOCS-2, and SOCS-6; Fig. 7A). Western blot analysis (Fig. 7B) confirmed that STAT5b protein expression levels were unaffected by SOCS transfection, although in the case of SOCS-3 a slight increase in STAT5b protein mobility, consistent with the inhibition of STAT tyrosine phosphorylation, could be discerned (lane 6). To investigate the impact of SOCS-3 on PPARalpha signaling, GHR/JAK2/STAT5b and PPARalpha signaling were reconstituted in COS-1 cells in the absence or presence of SOCS-3. As seen in Fig. 7C, the inhibition of PPARalpha transcriptional activity by STAT5b was fully blocked by SOCS-3. These findings confirm that STAT5b must be activated to its DNA-binding form (Fig. 7A) to exert its inhibitory effect on PPARalpha and suggest that STAT5b inhibition of PPARalpha activity is subjected to negative regulation by SOCS-3.


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  		Fig. 7.   SOCS-3 abolishes the GH inhibition of PPARalpha by blocking GH activation of STAT5b. A, expression of SOCS-3 blocks GH activation of STAT5b DNA binding activity in transfected COS-1 cells. COS-1 cells were transfected with expression plasmids encoding GHR, STAT5b and each of the indicated SOCS/CIS proteins. 24 h later, cells were treated with GH (200 ng/ml) for 30 min followed by preparation of whole cell extracts. EMSA assays for STAT5 DNA binding activity were carried out using a 32P-labeled beta -casein promoter DNA probe, which contains a STAT5b binding site. The specific STAT5b-DNA complex is marked STAT5b EMSA, and the nonspecific protein binding to the beta -casein probe is labeled n.s. B, STAT5b protein expression level is not affected by overexpression of SOCS-3 protein. The same cell lysates assayed in A were analyzed for STAT5b protein expression by anti-STAT5 Western blotting (Santa Cruz antibody sc-835). C, SOCS-3 blocks STAT5b-dependent GH inhibition of PPARalpha activity. COS-1 cells were cotransfected with expression plasmids encoding GHR, JAK2, STAT5b, PPARalpha , SOCS-3, and the reporter plasmid pHD(X3)Luc as described above. Transfected cells were treated with Wy-14,643 (Wy, 20 µM), either alone or in the presence of GH (5, 25, or 100 ng/ml) for 24 h. Cell extracts were then prepared and assayed for firefly luciferase activities normalized to a Renilla luciferase transfection control.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

GH-activated STAT5b inhibits the transcriptional activation of PPARalpha by structurally diverse peroxisome proliferators (22), suggesting a mechanism whereby hormones and cytokines that activate STAT5b (2) may inhibit the pleiotropic responses of liver and perhaps other tissues to the numerous environmental chemicals, hypolipidemic agents, and other structurally diverse chemicals that are classified as peroxisome proliferators (13). The present study reports that GH also exerts inhibitory effect on PPARgamma , PPARdelta , and T3R transcriptional activity. STAT5b transcriptional activity alone (in the form of the constitutively active STAT5b1*6) was found to be insufficient for inhibition of PPARalpha , excluding the possibility that STAT5b acts by stimulating transcription of a more proximal PPARalpha inhibitor. Chimeric GAL4-PPARalpha chimeras were utilized to establish that the NH2-terminal AF-1 trans-activation domain of PPARalpha , which is active in a ligand-independent fashion (29), is the target of STAT5b inhibition, whereas the COOH-terminal, ligand-dependent AF-2 trans-activation domain is not. EMSA analysis suggested that PPARalpha DNA binding activity may be partially inhibited in cells containing GH-activated STAT5b, although it is difficult to exclude the possibility that experimental variation in protein yield and transfection efficiency lead to variation in the amount of PPARalpha protein available for DNA complex formation. GH-activated STAT5b may inhibit these processes by competing for a common factor required for nuclear receptor DNA binding and/or trans-activation, such as a nuclear receptor coactivator (42), although our data indicate that the coactivators P300 and SRC-1 are not the targets of STAT5b inhibition.

Ligand-dependent transcriptional activation by nuclear receptors is mediated by a large, complex COOH-terminal domain that integrates several critical functions: ligand binding, dimerization, trans-activation, and interaction with transcriptional coactivators (49). The three mammalian PPAR isoforms share high homology in the DNA-binding domain and weaker homology in the ligand-binding domain. The AF-2 domain of the receptor, a highly conserved hydrophobic region within the distal COOH-terminal portion of the ligand binding domain, is required for transcriptional activation. Mutations in AF-2 abolish trans-activation function, whereas DNA binding activity is preserved (41). AF-2 is likely to serve as an adapter surface for interaction with coactivators such as SRC-1 (41). Indeed, amino acids within the COOH terminus of PPARalpha as well as residues within the hinge region of the receptor are required for ligand-dependent interaction with P300 (45). However, our data show that, unlike full-length PPARalpha , the isolated PPARalpha COOH-terminal region, when fused to a GAL4 DNA-binding domain, is not subjected to inhibition by STAT5b. The AF-2 domain of PPARalpha is thus not a direct target of STAT5b inhibition, making it unlikely that STAT5b competes for coactivators that interact selectively with the AF-2 domain. AF-2-independent inhibition has also been reported for T3R and the transcription factors Jun and Fos at an AP-1 site, and mutation of the AF-2 domain of that receptor does not affect the inhibition (50).

Interdomain communication has been described as a novel mechanism to regulate PPARgamma activity (43). PPARgamma ligand binding activity can be modulated by interactions between the NH2-terminal A/B domain (AF-1 domain) of this nuclear receptor and its COOH-terminal ligand-binding domain. Modification of the A/B domain by MAP kinase phosphorylation reduces PPARgamma ligand-binding affinity and inhibits the transcriptional activity of that receptor. A strong ligand-independent trans-activating domain (AF-1-like domain) was recently localized to human PPARalpha amino acids 1-92 (29). In contrast to the inhibition that characterizes MAP kinase phosphorylation of PPARgamma , insulin-induced, MAP kinase-dependent phosphorylation of two serine residues within the AF-1 region stimulates PPARalpha activity, leading to the suggestion that the strong AF-1 region of PPARalpha is partially silenced by corepressor proteins (29). In the present study, we show that the AF-1 function of PPARalpha is inhibited by GH-activated STAT5b to an extent that is similar to the STAT5b inhibition of full-length PPARalpha . This inhibition of the AF-1 domain of PPARalpha is not dependent on the serine residues at positions 12 and 21, and thus the corepressor proteins that are proposed to dissociate upon MAP kinase phosphorylation of PPARalpha (29) are probably not the target of STAT5b inhibition. These findings suggest that the inhibition of PPARalpha transcriptional activity by GH-activated STAT5b occurs by a mechanism that is distinct from the MAP kinase-dependent regulation of PPARalpha activity by insulin, even though both hormones target the AF-1 region of PPARalpha . This latter conclusion is supported by our earlier observation that the MAP kinase inhibitor PD98059 does not block the inhibition of PPARalpha activity by GH-activated STAT5b (22).

STAT5b may directly inhibit the AF-1 activity of PPARalpha , or STAT5b may inhibit PPARalpha transcription by modulating the binding of coactivators or corepressors that interact with the AF-1 region in a manner that is independent of MAP kinase phosphorylation. PPARalpha ligand binding and DNA binding activity could conceivably also be affected by such an interaction. Our finding that STAT5b may partially inhibit PPARalpha DNA binding activity (Fig. 5) may be explained by two possibilities. First, STAT5b interaction with PPARalpha AF-1 region may modulate the interdomain communication of the receptor, as suggested for PPARgamma (43), such that PPARalpha ligand binding and DNA binding activity are inhibited. Alternatively, STAT5b may alter the binding of PPARalpha to its consensus PPRE, in addition to its inhibition of the NH2-terminal AF-1 function of the receptor. The PPRE recognized by PPAR is composed of a direct repeat of two AGGTCA half-site separated by a single nucleotide (DR-1 sequence motif). PPAR obligatorily binds to DNA as a heterodimer with RXR (47), which is also a heterodimerization partner for many other nuclear receptors. RXR can thus define a point for convergence and cross-talk between PPAR and other intracellular signaling pathways. In fact, competition for RXR is the basis for the cross-talk between PPAR and T3R (51). In our study, however, overexpression of RXRalpha did not block the STAT5b inhibition of PPARalpha activity.

Cells respond to GH through a signal transduction cascade that is initiated at the cell surface GHR. GHR dimerizes and is activated upon GH binding, leading to activation of the receptor-associated JAK2 kinase, tyrosine phosphorylation, and activation of STAT proteins, which translocate to the nucleus and ultimately stimulate target gene expression. Although protein tyrosine phosphatases play a role in terminating the GH response, the recently identified family of SOCS/CIS proteins (26-28) sheds new light on the mechanism of negative regulatory control. GH induces a rapid and transient expression of several SOCS/CIS genes, including SOCS-3 (25), which appears to establish a classic negative feedback loop that down-regulates GH signaling. In our studies, SOCS-3 expression fully blocked the STAT5b-dependent inhibition of PPARalpha activity in GH-treated cells by preventing the activation of STAT5b. This suggests that the cross-talk between PPARalpha and STAT5b is subjected to negative feedback regulation by SOCS-3. Mechanistically, SOCS-3 appears to inhibit JAK2 kinase signaling to STAT5b by binding via its SH2 domain to tyrosine phosphorylated residues on GHR (25, 48). This may, in turn, prevent the recruitment of STAT5b or facilitate ubiquitination of the GHR-JAK2 kinase complex, as suggested in the case of the erythropoietin receptor and the SOCS/CIS family member CIS-1 (52).

Functional interactions between STAT5a and glucocorticoid receptor, another nuclear receptor family member, have been described (23, 24). STAT5a and glucocorticoid receptor form a molecular complex that inhibits transcription from a glucocorticoid response element. Although the coactivator P300 enhances STAT5a-dependent, prolactin-stimulated transcription through a direct interaction with STAT5a, P300 cannot reverse the STAT5a-dependent inhibition of the glucocorticoid response (46), a finding that is analogous to that reported here for STAT5b and PPARalpha . Direct protein-protein interactions between STAT5a and glucocorticoid receptor have been proposed as the mechanism that underlies the STAT5a inhibitory effect of that nuclear receptor. However, in contrast to the STAT5a-glucocorticoid receptor interaction, no STAT5b-PPARalpha complex was detected on a PPRE element by supershift assay using STAT5b-specific antibody (22). This suggests that an indirect mechanism, one that does not involve direct STAT5b-PPARalpha interactions, may account for STAT5b inhibition of PPARalpha activity. One possibility is that STAT5b induces transcription of a secondary gene that yields a negative regulatory protein that inhibits PPARalpha activation. This model is unlikely, however, in view of our finding (Fig. 6) that a constitutively active STAT5b mutant (STAT5b1*6) (38) failed to inhibit PPARalpha activity, even though this mutant STAT strongly induced reporter gene expression driven by STAT5 response elements, even in the absence of GH stimulation. However, given that a single amino acid exchange between STAT5a and STAT5b can interconvert their distinguishable DNA binding activities on STAT5 form-specific promoter sequences (3), the possibility remains that, in combination, the two amino acid changes that confer constitutive activity to STAT5b (38) alter the DNA binding specificity of STAT, such that induction of the hypothetical inhibitory factor is lost.

An alternative model for STAT5b inhibition is that STAT5b competes for an essential coactivator or other interacting protein of PPARalpha , which may interact with the AF-1 domain of PPARalpha , leading to loss of PPARalpha ligand-independent transcriptional activity. This model is supported by our finding that STAT5b can inhibit trans-activation by all three PPAR isoforms and by T3R, which suggests that activated STAT5b interferes with the action of a common regulatory factor required for full activity of each of these nuclear receptors. (Of note, however, STAT5b did not inhibit an endogenous PPAR-like activity in the same COS-1 cell model (22).) The coactivators P300 and SRC-1 were both considered as candidate factors, because both proteins can interact with and coactivate PPARalpha (Fig. 4) (45, 53). Interaction with the nuclear receptor A/B domain has been described for the coactivator SRC-1 (54); however, overexpression of SRC-1 did not block STAT5b inhibition of PPARalpha . Moreover, P300 has been demonstrated to interact with STAT1 (55), STAT2 (56), and STAT5a (46). P300 interacts with transcriptional machinery and RNA polymerase II, and it also interacts with the nuclear factor P/CAF, which exhibits histone acetylase activity. Histone acetylation induces conformational changes in nucleosome structure and facilitates the entry of core transcription factors. Thus, P300 serves as an integrator of multiple signaling pathways. Indeed, P300 has been implicated in the antagonism between STAT and Ap-1 signaling (57). P300 plays an essential coactivator role for an expanding number of regulated transcription factors, including CREB, Jun, and Fos, in addition to STATs (46, 56). However, the present study shows that P300 cannot reverse STAT5b inhibition of PPARalpha activity. Although it is thus unlikely that STAT5b inhibits PPARalpha by competing for P300 or SRC-1, STAT5b and PPARalpha may compete for other nuclear receptor coactivators and/or other interacting factors. Further study will be needed to fully elucidate the mechanism for this inhibitory cross-talk between STAT transcription factors and nuclear receptor and to better understand its consequences in terms of the impact of hormonal factors on PPAR signaling initiated by diverse pharmacological agents and environmental chemicals.

    ACKNOWLEDGEMENTS

We thank Drs. J. Capone, E. Johnson, S. Kliewer, C. Meier, A. Schmidt, J. Reddy, D. Moore, R. Evans, A. Yoshimura, M. Brown, T. Kitamura, and M. Vore for provision of plasmid DNAs used in this study.

    FOOTNOTES

* This work supported in part by National Institutes of Health Grant ES07381 (to D. J. W.) funded via the Environmental Protection Agency-sponsored Superfund Basic Research Program at Boston University.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Biology, Boston University, 5 Cummington St., Boston, MA 02215. Tel.: 617-353-7401; Fax: 617-353-7404; E-mail: djw@bio.bu.edu.

    ABBREVIATIONS

The abbreviations used are: STAT, signal transducer and activator of transcription; PPAR, peroxisome proliferator-activated receptor; PPRE, peroxisome proliferator response element; GH, growth hormone; GHR, growth hormone receptor; JAK2, Janus kinase 2; RXR, retinoid X receptor; MAP, mitogen-activated protein; AF-1, activation function region-1; AF-2, activation function region-2; SRC-1, steroid receptor co-activator 1; T3R, thyroid hormone receptor; CAT, chloramphenicol acetyltransferase; EMSA, electrophoretic nobility shift assay; CIS, cytokine-inducible SH2-containing protein; SOCS, suppressor of cytokine signaling.

    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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