Down-Regulation of STAT5b Transcriptional Activity by Ligand- Activated Peroxisome Proliferator-Activated Receptor (PPAR) and PPAR

The nuclear receptor peroxisome proliferator-activated receptor (PPAR) is activated by a diverse group of acidic ligands, including many peroxisome proliferator chemicals present in the environment. Janus tyrosine kinase-signal transducer and activator of transcription (JAK-STAT) signaling is activated by multiple cytokines and hormones and leads to the translocation of dimerized STAT proteins to the nucleus where they activate transcription of target genes. Previous studies have shown that growth hormone (GH)-activated STAT5b can inhibit PPAR-regulated transcription. Here, we show that this inhibitory crosstalk is mutual, and that GH-induced, STAT5b-dependent -casein promoter-luciferase reporter gene transcription can be inhibited up to 80% by ligand-activated PPAR or PPAR . Dose-response experiments showed a direct relationship between the extent of PPAR activation and the degree of inhibition of STAT5-regulated transcription. PPAR did not block STAT5b tyrosine phosphorylation or inhibit DNA-binding activity. Both PPARs inhibited the transcriptional activity of a constitutively active STAT5b mutant, indicating that inhibition occurs downstream of the GH-stimulated STAT5 activation step. Transcriptionally inactive, dominant-negative PPAR mutants did not block STAT5b inhibition by wild-type PPAR, indicating that PPAR target gene transcription is not required. PPAR retained its STAT5b inhibitory activity in the presence of the histone deacetylase inhibitor trichostatin, indicating that enhanced histone deacetylase recruitment does not contribute to STAT5b inhibition. PPAR lacking the ligand-independent AF-1 trans-activation domain failed to inhibit STAT5b, highlighting the importance of the AF-1 region in STAT5-PPAR inhibitory cross-talk. These findings demonstrate the bidirectionality of cross-talk between the PPAR and STAT pathways and provide a mechanism whereby exposure to environmental chemical activators of PPAR can suppress expression of GH target genes. Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors that control a variety of cellular processes in response to a diverse group of natural and synthetic ligands (Escher and Wahli, 2000). Like other nuclear receptors, PPARs have a conserved protein structure containing distinct DNA-binding, trans-activation, and ligand-binding domains. After ligand binding, PPAR heterodimerizes with the retinoid X receptor and binds upstream of, and activates target genes. The three identified mammalian PPAR subtypes ( , , and ) have unique functions, tissue localizations, and ligand selectivities. PPAR regulates expression of genes involved in lipid metabolism, such as those encoding the peroxisomal enzymes acyl-CoA oxidase, bifunctional enzyme, and thiolase. PPAR has been implicated in rodent hepatocarcinogenesis (Corton et al., 2000), which reflects in part the inhibition of hepatocyte apoptosis (Roberts et al., 1998). Humans and several other species are resistant to the peroxisome proliferative and hepatocarcinogenic effects of PPAR activators, in part because of the significantly lower PPAR expression level in human liver (Palmer et al., 1998). In contrast, PPAR is expressed at high levels in multiple human tissues, including adipose tissue, where it plays a key role in adipocyte differentiation (Tontonoz et al., 1994). PPAR is activated by hypolipidemic compounds of the fibrate class, such as clofibrate and Wy-14,643, and by naturally occurring long-chain fatty acids. Specific ligands and activators of PPAR include antidiabetic thiazolidinedione drugs (Lehmann et al., 1995) and the prostaglandin metabolite 15-deoxy12,14 prostaglandin J2 (Escher and Wahli, This research was supported in part by National Institutes of Health grant 5-P42-ES07381 and the Superfund Basic Research Center at Boston University. ABBREVIATIONS: PPAR, peroxisome proliferator-activated receptor; STAT, signal transducer and activator of transcription; JAK, Janus tyrosine kinase; GH, growth hormone; GHR, growth hormone receptor; ER, estrogen receptor; PPC, peroxisome proliferator chemical; h, human; m, mouse; DMEM, Dulbecco’s modified Eagle’s medium; EMSA, electrophoretic mobility shift assay; HDAC, histone deacetylase; TSA, trichostatin A; CARM, coactivator-associated arginine methyltransferase; GRIP, glucocorticoid receptor interacting protein; Wy-14,643, pirinixic acid; CMV, cytomegalovirus. 0026-895X/03/6402-355–364$7.00 MOLECULAR PHARMACOLOGY Vol. 64, No. 2 Copyright © 2003 The American Society for Pharmacology and Experimental Therapeutics 2464/1078763 Mol Pharmacol 64:355–364, 2003 Printed in U.S.A. 355 at A PE T Jornals on N ovem er 8, 2017 m oharm .aspeurnals.org D ow nladed from 2000). Less is known about PPAR , which is thought to play a role in development (Peters et al., 2000). Previous studies have demonstrated the potential for cross-talk between STAT transcription factors and nuclear receptors such as PPARs. STATs are latent cytoplasmic signaling molecules activated by tyrosine-phosphorylation catalyzed by JAKs, tyrosine kinases associated with many cytokine and growth factor receptors, including growth hormone (GH) receptor (GHR) (Darnell, 1997). The tyrosine phosphorylated STATs form homoand heterodimeric complexes that translocate to the nucleus where they bind to specific DNA response elements and stimulate target gene transcription (Kisseleva et al., 2002). Inhibition of STAT1-regulated transcription by PPAR occurs in HeLa cells (Ricote et al., 1998), although not in COS-1 cells (this report). STAT1 can decrease PPAR -regulated gene transcription indirectly, by binding upstream of, and repressing transcription of the PPAR gene, leading to decreased PPAR protein expression (Hogan and Stephens, 2001). STAT5 transcriptional activity is strongly inhibited by the estrogen receptor (ER) via mechanisms that involve a direct interaction between the receptor and STAT5 (Faulds et al., 2001) and via an indirect inhibitory effect of ER on STAT5 activation and nuclear localization (Sueyoshi et al., 1999). STAT5 inhibits transcription stimulated by glucocorticoid receptor, mineralocorticoid receptor, and progesterone receptor, but, conversely, these three steroid receptors synergize with STAT5 to enhance STAT5 target gene transcription (Stoecklin et al., 1999). STAT5 can also inhibit PPAR and PPAR -regulated transcription, by a mechanism that involves the AF-1 ligandindependent trans-activation domain of PPAR (Zhou and Waxman, 1999a,b). The possibility that PPAR may, in turn, inhibit STAT5 transcriptional activity is suggested by the finding that ligand activation of PPAR leads to down-regulation of several GH-regulated, sexually dimorphic liver genes (Corton et al., 1998), which are regulated, in part, by STAT5b (Udy et al., 1997; Park et al., 1999; Park and Waxman, 2001). PPAR inhibition of STAT5 transcriptional activity would provide a mechanism whereby peroxisome proliferator chemicals (PPCs) may down-regulate such GHregulated genes. STAT5 is coexpressed with PPAR in many tissues, including hepatocytes (PPAR ) and preadipocytes (PPAR ). STAT5 increases in expression early during the course of adipogenesis (Stephens et al., 1999), becomes activated during differentiation, and contributes to the enhanced expression of proadipogenic transcription factors, including PPAR (Nanbu-Wakao et al., 2002). PPAR , as well as PPAR , can be activated by a broad range of environmental chemicals (Maloney and Waxman, 1999; Hurst and Waxman, 2003), and cross-talk with STATs is potentially an important route through which foreign chemical exposure may impact on endogenous pathways of metabolism and differentiation. The STAT and PPAR pathways are tightly regulated by an overlapping set of nuclear regulatory proteins, including coactivators (Chen and Li, 1998), and by post-translational modification, e.g., inhibitory serine phosphorylation of the NH2terminal AF-1 domain (A/B domain) of PPAR (Adams et al., 1997) and phosphorylation of several STATs, including STAT5a and STAT5b, at a conserved COOH-terminal serine, in some cases leading to stimulation and in other cases inhibition of transcriptional activity (Yamashita et al., 1998; Park et al., 2001). Given the multiple regulatory mechanisms controlling STAT and PPAR signaling pathways, there may be multiple mechanisms by which the activation of one pathway can lead to cross-talk with the other. In the present study, we investigate the effects that ligandactivated PPAR and PPAR have on STAT5b-regulated reporter gene transcription in GH-stimulated cells. PPAR and PPAR are shown to inhibit the transcriptional activity of STAT5b at a step downstream of GH activation, providing a mechanistic explanation for the previously observed downregulation of GH-activated genes by PPCs (Corton et al., 1998). We evaluate the mechanism that underlies this inhibitory cross-talk and highlight the importance of the NH2-terminal AF-1 trans-activation domain of PPAR, a protein domain that was previously found to be a target of the inhibitory effects of STAT5b on PPAR (Zhou and Waxman, 1999b). Materials and Methods 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 peroxisome proliferator response element from the rat enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase gene upstream of a minimal promoter cloned into the plasmid pCPS-luc. The reporter plasmid pZZ1, provided by Dr. B. Groner (Institute for Experimental Cancer Research, Freiburg, Germany), contains the -casein milk protein gene promoter upstream of the firefly luciferase gene. Mouse PPAR cloned into the expression plasmid pCMV5 was obtained from Dr. E. Johnson (Scripps Research Institute, La Jolla, CA). The STAT5 reporter plasmid pT109-4Xntcp-Luc, which contains four copies of a STAT5 response element from the rat ntcp gene, was provided by Dr. M. Vore (University of Kentucky, Lexington, KY). STAT5b1*6 cDNA was excised from the pMX-puro-STAT5b1*6 plasmid, provided by Dr. Toshio Kitamura (University of Tokyo, Tokyo, Japan), and the EcoRI-NotI fragment was subcloned into the expression vector pCI (Promega, Madison, WI) by Dr. S. H. Park of this laboratory. The PPAR expression plasmid pSV-Sport-mPPAR was obtained from Dr. J. Reddy (Northwestern University, Chicago, IL). Rat GHR cloned into the expression plasmid pcDNAI was provided by Dr. N. Billestrup (Hagedorn Research Institute, Gentofte, Denmark). pME18S expression plasmid encoding mouse STAT5b was obtained from Dr. A. Mui (DNAX Research Institute of Molecular and Cellular Biology, Inc.). An expression plasmid encoding hPPAR -6/29, a naturally occurring dominant-negative inhibitory variant of human liver PPAR , was provided by Dr. Ruth Roberts (Zeneca Central Toxicology Lab, Brixham, UK) (Roberts et al., 1998). FLAG epitope-tagged wild-type human PPAR and a dominantnegative human PPAR , PPAR -L466A/E469A, both subcloned into pcDNA, were provided by Dr. V.K.K. Chatterjee (University of Cambridge, Cambridge, UK) (Barroso et al., 1999). pNCMV-PPAR and PPAR lacking the A/B domain, pNCMV-PPAR / , were gifts of Dr. T. Osumi (Himeji Institute of Technology, Kamigori Hyogo, Japan) (Hi et al., 1999). The STAT1 luciferase reporter p36-8GAS-Luc, containing eight interferon -activated sites cloned upstream of a p36 minimal promoter, was provided by Dr. C. K. Glass (University of California San Diego, La Jolla, CA) (Ricote et al., 1998). Renilla reniformis luciferase expression plasmid pRL-CMV was purchased