Cross-talk between Janus Kinase-Signal Transducer and Activator of Transcription (JAK-STAT) and Peroxisome Proliferator-activated Receptor-alpha (PPARalpha ) Signaling Pathways
GROWTH HORMONE INHIBITION OF PPARalpha TRANSCRIPTIONAL ACTIVITY MEDIATED BY STAT5b*

Yuan-Chun Zhou and David J. WaxmanDagger

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

    ABSTRACT
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Abstract
Introduction
References

Hepatic peroxisome proliferation induced by structurally diverse non-genotoxic carcinogens is mediated by the nuclear receptor peroxisome proliferator-activated receptor (PPARalpha ) and can be inhibited by growth hormone (GH). GH-stimulated Janus kinase-signal transducer and activator of transcription 5b (JAK2/STAT5b) signaling and the PPAR activation pathway were reconstituted in COS-1 cells to investigate the mechanism for this GH inhibitory effect. Activation of STAT5b signaling by either GH or prolactin inhibited, by up to 80-85%, ligand-induced, PPARalpha -dependent reporter gene transcription. GH failed to inhibit 15-deoxy-Delta 12,14-prostaglandin-J2-stimulated gene transcription mediated by an endogenous COS-1 PPAR-related receptor. GH inhibition of PPARalpha activity required GH receptor and STAT5b and was not observed using GH-activated STAT1 in place of STAT5b. GH inhibition was not blocked by the mitogen-activated protein kinase pathway inhibitor PD98059. STAT5b-PPARalpha protein-protein interactions could not be detected by anti-STAT5b supershift analysis of PPARalpha -DNA complexes. The GH inhibitory effect required the tyrosine phosphorylation site (Tyr-699) of STAT5b, an intact STAT5b DNA binding domain, and the presence of a COOH-terminal trans-activation domain. Moreover, GH inhibition was reversed by a COOH-terminal-truncated, dominant-negative STAT5b mutant. STAT5b must thus be nuclear and transcriptionally active to mediate GH inhibition of PPARalpha activity, suggesting an indirect inhibition mechanism, such as competition for an essential PPARalpha coactivator or STAT5b-dependent synthesis of a more proximal PPARalpha inhibitor. The cross-talk between STAT5b and PPARalpha signaling pathways established by these findings provides new insight into the mechanisms of hormonal and cytokine regulation of hepatic peroxisome proliferation.

    INTRODUCTION
Top
Abstract
Introduction
References

The peroxisome proliferator-activated receptors (PPARs)1 comprise a family of three nuclear receptors characterized by unique functions, ligand specificities, and tissue distributions (1-3). Ligands for PPARalpha include fibrate hypolipidemic drugs, specific fatty acids and eicosanoids, and leukotriene B4 (4-7), whereas anti-diabetic thiazolidinediones (8) and 15-deoxy-Delta 12,14-prostaglandin J2 (15d-PGJ2) (9, 10) are high affinity ligands for PPARgamma . Like many other nuclear receptors, PPARs are transcription factors that bind to specific DNA response elements (PPREs) upstream of target genes in heterodimeric complexes with the 9-cis-retinoid acid receptor RXR, leading to the activation of target gene transcription (11).

PPARalpha and PPARgamma play a key role in lipid homeostasis, adipocyte differentiation, and inflammatory responses (3, 12). PPARalpha target genes include liver-expressed enzymes involved in fatty acid beta -oxidation and microsomal omega -hydroxylation (13-15). PPARalpha thus plays a direct role in liver homeostasis by regulating lipid storage and by modulating the metabolism of important lipid signaling molecules, including prostaglandins and leukotrienes. PPARalpha gene knock-out mice do not exhibit hepatic (16) or renal (17) peroxisome proliferative responses induced by fibrate drugs and other PPAR activators, demonstrating the key role played by PPARalpha in these processes. PPARalpha also mediates the carcinogenicity of foreign peroxisome proliferators (18), which are non-genotoxic hepatocarcinogens when administered chronically to rodents (19).

PPARalpha gene expression and PPARalpha -stimulated transcriptional activity are tightly controlled by a variety of hormones that act at multiple levels and via different mechanisms. Glucocorticoids induce PPARalpha protein expression at the transcriptional level (20), which may account for the expression of PPARalpha diurnally and in a stress-inducible manner (21), whereas insulin treatment decreases PPARalpha mRNA levels (22). Peroxisome proliferative responses in rodents are suppressed by the thyroid hormone triiodothyronine (23), whose receptor may compete with PPAR for heterodimerization with the retinoid X receptor RXR and for trans-activation of PPAR DNA response elements (PPREs) (24). Hepatic peroxisome proliferation can also be modulated by sex hormones, with female rats being less responsive than males to clofibrate and other peroxisome proliferators (25) and testosterone treatment abolishing this sex difference (26).

The observation that hypophysectomy enhances peroxisome proliferation in female rats (26) suggests that a pituitary factor(s) serves as a negative regulator of peroxisome proliferation. In rats, the continuous plasma growth hormone (GH) profile characteristic of adult females fully suppresses liver expression of the clofibrate-inducible P450 4A2 fatty acid omega -hydroxylase (27). The same suppressive effect is observed in primary rat hepatocyte cultures, where GH inhibits peroxisomal beta -oxidation induced by clofibrate (28, 29). GH has diverse effects on metabolism and growth, some of which are indirectly mediated by insulin-like growth factor-1 but many of which reflect the direct effects that GH has on gene expression. In particular, GH, like many cytokines and growth factors, directly activates JAK-STAT signaling pathways. GH binds to and thereby dimerizes its plasma membrane receptor (GHR) in a process that leads to JAK2 kinase-catalyzed tyrosine phosphorylation of STAT proteins, which are latent, cytoplasmic transcription factors (30). The tyrosine-phosphorylated STAT proteins dimerize and translocate into the nucleus, where they bind to specific DNA response element and thereby activate target gene transcription (31). Among the seven mammalian STATs, four forms (STATs 1, 3, 5a, and 5b) can be activated by GH (32-35).

In the present study, we investigate the mechanism that underlies the inhibitory effect of GH on peroxisome proliferation using COS-1 cells transfected to express both the GHR/JAK/STAT signaling pathway and the peroxisome proliferator-activated PPAR pathway. We find that GH inhibits PPARalpha -stimulated reporter gene transcription in a process that is mediated by STAT5b but not by STAT1. We further demonstrate that STAT5b tyrosine phosphorylation, DNA binding, and transcriptional activation are each essential for GH to mediate its inhibitory effects on PPARalpha activity. These findings are discussed in the context of the implications of this cross-talk between STAT5b and PPARalpha for the regulation of PPARalpha -dependent responses by hormones and cytokines.

    MATERIALS AND METHODS

Plasmids-- PPRE-firefly luciferase reporter plasmid derived from the rabbit CYP4A6 gene, pLUCA6-880, and mouse PPARalpha cloned into the expression plasmid pCMV5 (pCMV-mPPARalpha ) were provided by Dr. E. Johnson (Scripps Research Institute, La Jolla, CA) (13). The PPRE-firefly luciferase reporter plasmid pHD(X3)Luc, obtained from Dr. J. Capone (McMaster University, Ontario, Canada), contains three tandem copies of the PPRE from the rat enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase gene upstream of a minimal promoter (15) cloned into the plasmid pCPS-luc (36). The PPARgamma expression plasmid pSV-Sport-mPPARgamma was obtained from Dr. J. Reddy (Northwestern University, Chicago) (37). Mouse RXRalpha expression plasmid pCMX-mRXRalpha was obtained from Dr. R. Evans (Salk Institute, San Diego) (38). Rat GHR cloned into the expression plasmid pcDNAI and mouse JAK2 cloned into the expression plasmid pRK5 were respectively provided by Dr. N. Billestrup (Hagedorn Research Institute, Denmark) (39) and Dr. J. Ihle (St. Jude Children's Research Hospital, Memphis, TN) (40). pME18S expression plasmids encoding mouse STAT1, STAT3, STAT5a, and STAT5b were obtained from Dr. A. Mui (DNAX Research Institute of Molecular and Cellular Biology, Inc., Palo Alto, CA) (41). Mouse prolactin receptor long form cloned into the expression plasmid pcDNAI (42), and COOH-terminal truncated forms of mouse STAT5a and STAT5b (designated STAT5aDelta 749 and STAT5bDelta 754, respectively) and cloned into the expression plasmid pXM were provided by Dr. B. Groner (Institute for Experimental Cancer Research, Freiburg, Germany) (43). Human STAT5a, STAT5b, STAT5a-Y694F, and STAT5b-Y699F cloned into the expression plasmid pSX were provided by Dr. W. Leonard (NHLBI, National Institutes of Health) (44). pRc/CMV expression plasmids encoding rat STAT5b and the DNA-binding region mutants STAT5b-VVVI-(466-469) and STAT5b-EE-(437-438), where each of the indicated residues is replaced by alanine, were provided by Dr. L. Yu-Lee (Baylor College of Medicine) (45).

Cell Culture and Transfections-- COS-1 cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. Transfection of COS-1 cells grown in 12-well tissue culture plates was carried out by calcium phosphate precipitation (46). At 9 h after addition of the DNA-calcium phosphate precipitate, cells were washed and incubated in Dulbecco's modified Eagle's medium without serum for 12 h. Peroxisome proliferators were then added to the culture medium in combination with GH at concentrations specified in each figure. Cells were lysed 24 h later, and firefly luciferase and beta -galactosidase (pCMV-beta gal; internal control) reporter activities were measured using a Galacto-light chemiluminescent reporter kit (Tropix, Bedford, MA). In some experiments, Renilla luciferase expression plasmid (pRL-TK; Promega, Madison, WI) was used in place of the pCMV-beta gal internal control plasmid, as indicated in the figure legends. Firefly and Renilla luciferase activities were measured using a dual-luciferase assay kit (Promega, Madison, WI). Transfections were performed using the following amounts of plasmid DNA/well (3.8 cm2) of a 12-well tissue culture plate: 0.36 µg of reporter construct (pHD(X3)Luc or pLUCA6-880), 14 ng of pCMV-mPPARalpha , 0.2 µg of GHR expression plasmid, 0.12 µg of JAK2 expression plasmid, and 0.2 µg of STAT expression plasmid. pCMV-beta gal (0.16 µg) or pRL-TK (30 ng) were included as internal controls in each cell transfection. The total amount of DNA was adjusted to 0.96 µg/well using salmon sperm DNA (Stratagene, La Jolla, CA). Data shown in each figure are mean values ± S.D. (for n = 3 replicates) or mean values ± half the range (for duplicates) and are representative of at least three such independent experiments.

EMSA Analysis-- Whole cell extracts were prepared by lysing transfected COS-1 cells in lysis buffer (Tropix, Inc.) containing 1 mM dithiothreitol added prior to use. 10 µg of cell extract was added to 2 µl of 5× gel-shift buffer (20% glycerol, 5 mM MgCl2, 2.5 mM EDTA, 2.5 mM dithiothreitol, 50 mM Tris-HCl), plus 1 µl of 2 µg/µl poly(deoxyinosinic-deoxycytidylic) acid (Boehringer Mannhein), with water added to adjust the total volume to 15 µl. Samples were incubated for 10 min at room temperature. 32P-Labeled double-stranded DNA probe (1 µl; 10 fmol) was then added, and the incubation was continued for 20 min at room temperature and then 10 min on ice. Loading dye (2 µl of 30% glycerol, 0.25% bromphenol blue, 0.25% xylene cyanol) was then added before the mixture was loaded onto an acrylamide gel (5.5% acrylamide, 0.7% bisacrylamide in 0.5× TBE). The gel was electrophoresed at 4 °C for 40 min at 100 V before loading. The gel was first electrophoresed for 20 min at 100 V at 4 °C at which point the dye entered the gel; electrophoresis was then continued at room temperature for 5 h. This procedure minimizes formation of nonspecific protein-DNA complexes (47). For STAT5b supershift assays, 3 µl of anti-STAT5b antibody (Santa Cruz Biotechnology, Santa Cruz, CA; antibody sc-835) was added 10 min after the labeled DNA probe, followed by a 10-min incubation at room temperature and 10-min incubation on ice before samples were loaded on the gel. The STAT5 binding site of the rat beta -casein promoter (5'-GGA-CTT-CTT-GGA-ATT-AAG-GGA-3') was used as gel-shift probe for GH-activated STAT5a and STAT5b, and the sis-inducible element (SIE)-binding site (5'-gtc-gaC-ATT-TCC-CGT-AAA-TCg-tcga-3') was used as gel-shift probe for analyzing GH-activated STAT1 and STAT3 (34). 32P-Labeled DNA probe corresponding to the Z element of the CYP4A6 gene (5'-g-CGC-AAA-CAC-TGA-ACT-AGG-GCA-AAG-TTG-AGG-GCA-G-3') was used as probe for PPARalpha binding (48).

Western Blotting-- Whole cell extracts from COS-1 cells (15 µg; see above) were resolved on 10% SDS-polyacrylamide gels, electrotransferred to nitrocellulose, and then probed with anti-STAT antibodies. STAT5b-specific antibody sc-835 is a rabbit polyclonal antibody raised to mouse STAT5b amino acid 711-727 (Santa Cruz Biotechnology). Mouse anti-STAT antibodies (Transduction Laboratories, Lexington, KY) were as follows: anti-STAT5 (antibody S21520) was raised to sheep STAT5 amino acids 451-649; anti-STAT3 (antibody S21320) was raised to rat STAT3 amino acids 1-175; anti-STAT1 (antibody S21120) was raised to human STAT1 amino acids 592-731.

    RESULTS

GH Activation of STAT5b Inhibits PPARalpha Transcriptional Activity-- To investigate the mechanism by which GH inhibits PPARalpha -dependent liver peroxisome proliferative responses, we reconstituted GH signaling and PPARalpha -dependent peroxisome proliferation pathways by cotransfection of key components into COS-1 cells. GH signal transduction was reconstituted by cotransfection of expression plasmids encoding GHR, JAK2 kinase, and STAT5b, the major GH-responsive STAT form in liver (34). The PPARalpha pathway was reconstituted by cotransfection of a mouse PPARalpha expression plasmid, together with reporter plasmid containing 880 nucleotides of 5'-flanking DNA of the rabbit CYP4A6 gene (13) fused to a firefly luciferase reporter gene. Transfected cells were stimulated with the peroxisome proliferator Wy-14,643 at 20 µM for 24 h in the presence or absence of GH (200 ng/ml). Fig. 1A shows that Wy-14,643 activation of the CYP4A6 promoter is significantly decreased in the presence of GH. Wy-14,643 activation of the CYP4A6 promoter is mediated by PPARalpha , which binds to one strong and two weaker PPREs within the 5'-flanking 880 nucleotides (13, 49). This GH inhibitory effect may be due to GH suppression of PPARalpha -dependent transcription via the CYP4A6 PPREs; alternatively, GH may interfere with other transcription factors required for either basal or Wy-14,643-inducible CYP4A6 promoter transcription, independent of PPRE. To distinguish these possibilities, we examined the effect of GH on PPARalpha -activation of the reporter construct pHD(X3)Luc, which contains three tandem repeats of a PPRE from the gene that encodes enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase, cloned upstream of a minimal promoter and luciferase reporter gene (15). Fig. 1B shows that GH inhibits, by up to 80-85%, PPARalpha induction mediated by this isolated PPRE. Maximal GH inhibition was achieved at 25 ng/ml, well within the physiological range of GH concentrations and consistent with the reported Kd of GHR for GH of ~2 ng/ml (50). The inhibitory effects of GH on PPARalpha activation were also apparent with several other PPARalpha activators, including both foreign chemicals (nafenopin, a fibrate hypolipidemic drug) and the endogenous PPARalpha activators (8S)-hydroxyeicosatetraenoic acid and leukotriene B4 (Fig. 1C).


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Fig. 1.   PPARalpha activation of CYP4A6 promoter (A) and an isolated PPRE reporter (B) is inhibited by GH-activated STAT5b when using multiple PPARalpha activators (C). COS-1 cells were cotransfected with expression plasmids encoding PPARalpha , GHR, JAK2, STAT5b, and the indicated reporter plasmid. A beta -galactosidase expression plasmid (pSV-beta gal) was included to normalize samples for transfection efficiency. Cells were stimulated with GH together with the indicated peroxisome proliferators 24 h before preparation of cell extracts and measurement of luciferase activity. Data shown are firefly luciferase reporter activities normalized for the level of beta -galactosidase activity in each extract. A, GH (200 ng/ml) suppresses Wy-14,643-stimulated (Wy; 20 µM) PPARalpha induction of CYP4A6 promoter activity (nucleotides -880 to -1). B, GH suppresses PPARalpha activation of an isolated PPRE linked to a firefly luciferase reporter, pHD(X3)Luc. Shown are the effects of 1-100 ng/ml GH on Wy-14,643-stimulated (20 µM) reporter gene activity. C, GH suppresses PPARalpha activation stimulated by a range of foreign chemicals and endogenous PPARalpha activators, as monitored with the reporter plasmid pHD(X3)Luc. Luciferase activity was measured after the cells were treated for 24 h with Wy-14,643 (20 µM), (8S)HETE (8(S)H) (5 µM), leukotriene B4 (LTB)(10 µM), or nafenopin (Naf) (100 µM) in the absence and presence of GH (100 ng/ml), as indicated. Data shown are relative luciferase activities (firefly luciferase/beta -galactosidase) for cells treated with PPAR activators compared with vehicle controls (fold induction).

Requirement of GHR, JAK2, and STAT5b for GH Suppression of PPARalpha Activity-- Since some, but not all GH intracellular events involve JAK/STAT signaling pathways (30), we examined whether GHR, JAK2, and STAT5b are each required for the inhibitory effect of GH on PPARalpha activity. Fig. 2A shows that inhibition of PPARalpha activity by 25 ng/ml GH (lanes 3 versus lane 2) was not observed in the absence of cotransfected GHR (lane 4) or STAT5b (lane 5). A similar dependence on GHR and STAT5b was observed at higher GH concentrations (100-500 ng/ml GH; data not shown). In the absence of transfected JAK2, GH could still activate STAT5b and inhibit PPARalpha activity using the low levels of JAK2 expressed endogenously in COS-1 cells, although a higher GH concentration (500 ng/ml) was required for maximal PPARalpha inhibition (data not shown). Interestingly, cotransfection of GHR, JAK2, and STAT5b significantly reduced the moderate PPARalpha -dependent but Wy-14,643-independent basal luciferase reporter activity, even in the absence of GH (Fig. 2A, lanes 6 versus 1). This Wy-14,643-independent activity has been associated with the presence of endogenous PPAR activators, such as fatty acids (4, 5). Inhibition of this basal PPAR activity likely results from the constitutive activation of STAT5b upon overexpression of JAK2, as shown in the gel-shift studies described below (Fig. 2B). GH stimulation further decreased the Wy-14,643-independent PPARalpha activity up to 4-fold (data not shown).


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Fig. 2.   GH activation of STAT5b is required for inhibition of PPARalpha activity. A, GHR and STAT5b are both required for GH inhibition of PPARalpha activity. COS-1 cells were transiently transfected with expression plasmids encoding GHR, JAK2, and STAT5b, as indicated in each lane, in combination with PPARalpha expression plasmid and the PPRE-luciferase reporter gene pHD(X3)Luc. Cells were stimulated with Wy-14,643 (Wy) (20 µM) and GH (25 ng/ml), as indicated. Luciferase activities relative to a beta -galactosidase transfection control were measured in whole cell lysates. B, GH activates STAT5b DNA binding activity in transfected COS-1 cells. COS-1 cells were transfected with expression plasmids encoding GHR, JAK2, STAT5b, PPARalpha , and the pHD(X3)Luc reporter gene. Cells were treated with Wy-14,643 (20 µM) and GH (100 ng/ml) for 24 h, as indicated, followed by preparation of whole cell extracts. EMSA assays were carried out with a 32P-labeled beta -casein promoter DNA probe, which contains a STAT5b-binding site. The specific STAT5b-DNA complex is marked 5b. Anti-STAT5b-specific antibody was used to supershift STAT5b-containing DNA-protein complexes (STAT5b SShift), as indicated. Nonspecific protein binding to the beta -casein probe is labeled ns.

To confirm the reconstitution of STAT5b activity in the GH-stimulated COS-1 cells, EMSA assays were carried out using a beta -casein promoter probe, which contains a single STAT5b-binding site. No STAT5b DNA binding was detected upon GH stimulation of untransfected COS-1 cells (Fig. 2B, lanes 1 and 2). This indicates that COS-1 cells have at most low levels of GHR and/or STAT5b and are thus suitable for use as recipient cells in these transfection studies. In cells cotransfected with GHR, JAK2, and STAT5b, a low basal STAT5b DNA binding was observed in the absence of GH; this STAT5b activity was not affected by Wy-14,643 treatment (Fig. 2B, lanes 3 versus 5). Thus, overexpression of JAK2 and STAT5b results in some constitutive activation of STAT5b DNA binding activity. Stimulation with GH yielded a much higher level of STAT5b DNA binding activity (lane 7 versus 5). The beta -casein DNA-binding complexes obtained in these experiments were confirmed to contain STAT5b, as shown by supershift analysis using anti-STAT5b antibody (lanes 4, 6, and 8).

Inhibition of PPARalpha Activity by GH-activated STAT3 but Not STAT1-- In addition to STAT5b, GH can activate three other STAT proteins, STATs 1, 3, and 5a (32-35). Fig. 3 shows, however, that in COS-1 cells transfected with STAT1, GH did not decrease PPARalpha -induced luciferase reporter activity, even at a high GH concentration (500 ng/ml). Partial GH inhibition of PPARalpha activity was seen in COS-1 cells transfected with STAT3 or with the closely related (41, 51) STAT5a. In control experiments, all four STATs were shown by Western blotting to be highly expressed in the transfected COS-1 cells compared with untransfected controls (Fig. 3B; lanes 2-4 versus lane 1). The lower effectiveness of STAT5a compared with STAT5b is not because of its inefficient activation by GH, as shown by EMSA using 32P-labeled beta -casein probe (Fig. 3C, lanes 3 and 4 versus lanes 6 and 7). STAT1 and STAT4 expressed in GH-treated cells were also shown to be functionally active in DNA binding using an SIE (sis-inducible element) EMSA probe (see "Materials and Methods") (data not shown).


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Fig. 3.   Comparison of the capability of GH-activated STATs 1, 3, 5a, and 5b to inhibit PPARalpha activity. A, COS-1 cells were transiently transfected with the reporter plasmid pHD(X3)Luc and with expression plasmids encoding PPARalpha , GHR, JAK2 kinase and the indicated STAT protein. Cells were stimulated for 24 h with Wy-14,643 (Wy) (20 µM) and GH at the indicated concentrations. Whole cell extracts were prepared, and luciferase activities relative to a beta -galactosidase transfection control were determined. B, expression of STAT proteins in transiently transfected COS-1 cells. The same cell lysates assayed in A were analyzed for STAT protein expression by Western blot using each of the indicated STAT form-specific antibodies. Small amounts of STAT1 and STAT5a are seen to be present in untransfected COS-1 cell extracts (lane 1). C, GH activates STAT5a and STAT5b DNA binding activity in transfected COS-1 cells. COS-1 cells were transfected with expression plasmids encoding GHR, STAT5a or STAT5b, and PPARalpha . Cells were treated with GH (500 ng/ml) for 30 min, and cell extracts were assayed for EMSA activity using the beta -casein probe. Supershift analysis was carried out using anti-STAT5a (Santa Cruz, sc-1081x) or anti-STAT5b antibody (Santa Cruz sc-835), as indicated. GH-activated STAT5a (lane 3) migrates more slowly than STAT5b (lane 6). ns, nonspecific protein-DNA complex.

Prolactin Inhibition of PPARalpha Activity-- Prolactin is a GH-related pituitary polypeptide hormone that also signals via its cell-surface receptor through a JAK2/STAT5 pathway (42). STAT5a, originally identified as a prolactin-activated mammary gland factor, and STAT5b can both confer a prolactin response to the mammary beta -casein gene promoter (42, 51). Fig. 4 shows that in the presence of prolactin, STAT5b, but not STAT5a, inhibits PPARalpha activation in cells cotransfected with prolactin receptor and JAK2. This inhibition by prolactin is less extensive (~40%) than seen for GH-activated STAT5b in the same system (~80%; cf., Fig. 3A) and also required a higher concentration of hormone to achieve its maximum effect (100 ng/ml prolactin versus 25 ng/ml GH). STAT5b activation by prolactin was confirmed by gel-shift assay using a beta -casein probe (data not shown).


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Fig. 4.   Prolactin inhibition of PPARalpha activity mediated by STAT5b. COS-1 cells were cotransfected with expression plasmids encoding prolactin receptor, JAK2 kinase, STAT5a or STAT5b, PPARalpha , and the reporter plasmid pHD(X3)Luc. Cells were treated with Wy-14,643 (Wy) (20 µM) and prolactin (PRL) (0, 10 or 100 ng/ml, as indicated) for 24 h. Cell extracts were then prepared and assayed for luciferase activity relative to a beta -galactosidase transfection control. In some experiments, prolactin-activated STAT5a inhibited PPARalpha activity by ~20%.

GH-activated STAT5b Fails to Inhibit PPRE-dependent Transcription Induced by 15-Deoxy-Delta 12,14,-PGJ2 (15d-PGJ2)-- 15d-PGJ2 is a naturally occurring PPAR ligand that activates PPARgamma and other PPARs (52). Treatment of COS-1 cells with 5 µM 15d-PGJ2 stimulated PPRE-dependent luciferase reporter activity by ~20-fold, even in the absence of PPARgamma transfection (Fig. 5). This induction was not further enhanced by co-transfection of a PPARgamma expression plasmid (data not shown), suggesting that the 15d-PGJ2-stimulated PPRE response observed in this experiment involves an endogenous COS-1 PPAR that is distinct from PPARgamma or PPARalpha . GH did not, however, inhibit 15d-PGJ2 -induced, PPRE-dependent reporter gene transcription (Fig. 5), demonstrating that the GH suppressive response has specificity for the PPARalpha activation pathway.


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Fig. 5.   GH activated STAT5b fails to inhibit activation of endogenous COS-1 cell PPAR activity stimulated by 15d-PGJ2. COS-1 cells were cotransfected with expression plasmids encoding GHR, JAK2, STAT5b, and the reporter plasmid pHD(X3)Luc. Transfected cells were stimulated with 15d-PGJ2 (5 µM) with or without GH (100 ng/ml). Cell extracts were prepared 24 h later and assayed for luciferase activities relative to beta -galactosidase transfection controls.

STAT5b Inhibition of PPARalpha Activity Is Not Mediated by the MAP Kinases ERK1 and ERK2-- We next investigated whether MAP kinase plays a role in the GH-induced down-regulation of PPARalpha activity. GH can activate the MAP kinase kinase MEK1, which phosphorylates and thereby activates the MAP kinases ERK1 and ERK2 (53). MAP kinases are actively involved in growth factor and cytokine receptor signaling (54, 55) and catalyze phosphorylation on serine and/or threonine of several transcription factors, including PPARgamma (56). By analogy to the MAP kinase regulation of PPARgamma , PPARalpha , which is also a phosphoprotein (57), might be regulated by MAP kinase-catalyzed serine phosphorylation. To investigate whether MAP kinase activity is required for GH inhibition of PPARalpha activity, we used the MEK1 and MEK2 inhibitor PD98059 (58) to block MAP kinase activation. Stimulation of transfected COS-1 cells with Wy-14,643 in combination with PD98059 increased PPRE-dependent luciferase activity compared with Wy-14,643 treatment alone (Fig. 6). The enhancement by PD98059 of PPRE luciferase activity varied from ~1.5- to 4-fold in different experiments. However, GH treatment inhibited Wy-14,643-induced reporter activity to a similar extent in the presence and in the absence of PD98059 (Fig. 6). Since PD98059 did not block the GH inhibitory effect, the MAP kinases ERK1 and ERK2 are not likely to mediate GH inhibition of PPARalpha activity. PD98059 treatment also enhanced Wy-14,643-induced PPARalpha activity in the absence of GHR, JAK2, and STAT5b (data not shown). This suggests that basal MAP kinase activity in these cells exerts a negative effect on PPARalpha activity but in a manner that is independent of GHR or STAT5b expression or GH treatment.


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Fig. 6.   Effect of MAP kinase pathway inhibitor PD98059 on GH inhibition of PPARalpha activity. COS-1 cells were transfected with expression plasmids encoding GHR, JAK2, STAT5b, PPARalpha , and the reporter plasmid pHD(X3)Luc. Transfected cells were treated with Wy-14,643 (Wy) (20 µM) and GH (100 ng/ml) in the presence or absence of PD98059 (PD) (20 µM). Luciferase activities relative to beta -galactosidase transfection controls were measured in whole cell extracts prepared 24 h after Wy-14,643 and GH treatment. In the experiment shown, GH inhibited PPARalpha activity by ~90% in the absence of PD98059 and by ~70% in its presence. Similar results were obtained in an experiment carried out at 1 µM Wy-14,643 (73 and 84% inhibition by GH in the absence and presence of 10 µM PD98059, respectively), except that PD98059 induced an ~4-fold increase in PPARalpha activity in Wy-14,643-treated cells (data not shown).

Tyr-699 Phosphorylation of STAT5b Is Required for GH Inhibition of PPARalpha Activity-- STAT5b is activated by JAK2 kinase-dependent phosphorylation on tyrosine 699. Mutation of this tyrosine to phenylalanine (STAT5b-Y699F) results in a loss of STAT5b dimerization, DNA binding, and transcriptional activation (44). Fig. 7 shows that, when activated by GH, wild-type human STAT5b inhibited PPARalpha activity to a similar extent as did mouse STAT5b. By contrast, the Y699F substitution abrogated GH inhibition of PPARalpha activity. Accordingly, STAT5b tyrosine phosphorylation and/or its downstream activities (STAT5b dimerization, nuclear translocation, DNA binding, or transcriptional activation) are obligatory for STAT5b to mediate GH inhibition of PPARalpha . When equal amounts of human STAT5a expression plasmid were transfected, it failed to inhibit PPARalpha activity (Fig. 7). Partial inhibition (~40%) was observed, however, at 4-fold higher human STAT5a plasmid levels (data not shown).


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Fig. 7.   Mutation of STAT5b Tyr-699 abolishes GH inhibition of PPARalpha activity. Expression plasmids encoding human STAT5a, human STAT5b and their site-specific mutants (STAT5b-Y699F and STAT5a-Y694F) were transiently transfected into COS-1 cells together with GHR, JAK2, PPARalpha expression plasmids and pHD(X3)Luc reporter plasmid. Transfected cells were treated with Wy-14,643 (Wy) (20 µM) and GH (0, 25 or 100 ng/ml) for 24 h. Cell extracts were then prepared and assayed for luciferase activity relative to a beta -galactosidase transfection control.

Requirement of STAT5b COOH-terminal trans-Activation Domain and Effects of Dominant-negative STAT5b Mutant-- Deletions of the COOH-terminal trans-activation domain of mouse STAT5a and STAT5b (constructs STAT5aDelta 749 and STAT5bDelta 754, respectively) result in a loss of transcriptional activity and yield truncated STAT5 proteins that exert dominant-negative effects on wild-type STAT5-induced gene transcription (43). These COOH-terminal truncated STAT5 proteins undergo hormone-induced tyrosine phosphorylation and retain DNA binding activity but show delayed tyrosine dephosphorylation (43). These mutants were used to examine whether the transcriptional activity of STAT5b is necessary for GH inhibition of PPARalpha activity. Fig. 8A shows that, in contrast to wild-type STAT5b, STAT5bDelta 754 cannot mediate GH-induced PPARalpha inhibition, suggesting that the trans-activation domain of STAT5b is required for GH inhibitory effects. In contrast, STAT5aDelta 749 inhibited PPARalpha activity by ~40% following GH stimulation.


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Fig. 8.   Dominant-negative STAT5b blocks GH inhibition of PPARalpha activity by wild-type STAT5b. A, effects of COOH-terminal truncated STAT5b and STAT5a on PPARalpha activity. COS-1 cells were cotransfected with expression plasmids encoding GHR, JAK2, and the STAT5b COOH-terminal deletion constructs STAT5bDelta 754 (Delta STAT5b) or STAT5aDelta 749 (Delta STAT5a). Transfected cells were treated with Wy-14,643 (Wy) (20 µM) alone or in the presence of GH (100 ng/ml) for 24 h. Cell extracts were then prepared and assayed for firefly luciferase activity relative to a Renilla luciferase transfection control. Delta STAT5a, but not Delta STAT5b, partially inhibited Wy-14,643-induced reporter gene activity in GH-treated cells. B, STAT5bDelta 754 blocks wild-type STAT5b-dependent inhibition of PPARalpha activity. COS-1 cells were transfected with pHD(X3)Luc reporter and PPARalpha , GHR, JAK2, and mouse STAT5b expression plasmids together with increasing amounts of Delta STAT5b or Delta STAT5a expression plasmids (0-, 0.5-, 1-, and 5-fold relative to the amount of cotransfected wild-type STAT5b, calculated on a per microgram plasmid DNA basis). Cells were stimulated with Wy-14,643 (20 µM) and GH (100 ng/ml) for 24 h. Luciferase activities relative to a beta -galactosidase transfection control were then assayed in whole cell extracts. C, expression of STAT5a, STAT5b, and their COOH-terminal truncated mutants. The same cell lysates analyzed in B were analyzed for STAT5 protein expression by anti-STAT5 Western blotting (Transduction Laboratories, antibody S21520). The COOH-terminal truncated Delta STAT5b and Delta STAT5a bands were not detectable by a carboxyl-terminal targeted STAT5b antibody (Santa Cruz antibody sc-835; data not shown). D, EMSA analysis of STAT5b, Delta STAT5a, and Delta STAT5b DNA binding activity. The same cell lysates shown in B and C were analyzed for STAT5 DNA binding activity using the beta -casein promoter probe, as described under "Materials and Methods." STAT5b COOH-terminal targeted antibody (Santa Cruz antibody sc-835) was used for supershift (SS) where indicated (anti-5b).

To test for dominant-negative activity, COS-1 cells were transfected with wild-type STAT5b in the presence of increasing amounts of STAT5aDelta 749 or STAT5bDelta 754. Fig. 8B shows that STAT5bDelta 754 could fully block the STAT5b-dependent GH inhibition of PPARalpha activity in a dose-dependent manner (lane 2 versus lanes 3 and 4). Interestingly, while STAT5aDelta 749 has dominant-negative activity toward wild-type STAT5b and can block its transcriptional activity (43), this mutant had only a modest effect on STAT5b-dependent PPARalpha inhibition (lane 2 versus lanes 5 and 6). However, interpretation of this result is complicated by the fact that STAT5aDelta 749 itself confers partial inhibition of PPARalpha in response to GH stimulation (Fig. 8A). Fig. 8C confirms the expression of STAT5aDelta 749, STAT5bDelta 754, and wild-type STAT5 proteins in the transfected COS-1 cells, detected with an antibody against the STAT5 SH2 and SH3 domains, which are upstream of the deleted sequences. As expected, the STAT5bDelta 754 and STAT5aDelta 749 bands seen on this blot were of lower molecular weight (lanes 4 and 7) and could not be detected using an antibody that specifically recognizes the extreme COOH-terminal region of STAT5b (Santa Cruz antibody sc-835; data not shown). When equal amounts of expression plasmid were transfected, STAT5aDelta 749 protein was expressed at a level similar to wild-type STAT5b (lane 7 versus lanes 2 and 3). The level of STAT5bDelta 754 protein (lane 4) was much lower (Fig. 8C), highlighting the potency of its dominant-negative effects (Fig. 8B). EMSA analysis showed that STAT5aDelta 749 and STAT5bDelta 754 both bind to a beta -casein DNA probe much more efficiently than wild-type STAT5b (Fig. 8D; lanes 2, 3, 8, 9, 11, and 12 versus lanes 5 and 6), in agreement with earlier studies (43). This suggests that these mutants achieve their dominant-negative effect, at least in part, by interfering with wild-type STAT5b DNA binding activity. As expected, antibody specific for the STAT5b COOH-terminal peptide can completely supershift wild-type STAT5b but not the much more intense STAT5bDelta 754 binding to the beta -casein probe (Fig. 8D, lanes 7 versus 13).

STAT5b DNA Binding Activity Is Required for Inhibition of PPARalpha Activity-- STAT5a interacts with glucocorticoid receptor through formation of a protein-protein complex that inhibits glucocorticoid-induced gene expression (59, 60). STAT5b inhibition of the stimulatory effects of prolactin at the IRF-1 promoter is also proposed to involve direct protein-protein interactions (45). We therefore considered whether a direct interaction between GH-activated STAT5b and PPARalpha can be detected using an EMSA assay for PPARalpha -PPRE protein-DNA complexes. Fig. 9 shows that anti-STAT5b antibody does not alter the mobility of a PPARalpha -containing DNA complex formed on a PPRE probe from the CYP4A6 gene (lanes 3 versus 2) under conditions where the antibody fully supershifts STAT5b-containing DNA complexes (Figs. 2B and 3C). These data indicate that STAT5b does not bind directly to a PPARalpha -DNA complex. In control samples, the PPAR-PPRE complex seen in lane 2 was either disrupted or supershifted by various anti-PPARalpha antibodies (lanes 4 and 5), competed by an excess of unlabeled PPRE probe (lane 6) and was fully dependent on transfected PPARalpha for its formation (lane 7).


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Fig. 9.   STAT5B does not bind directly to PPARalpha -DNA complex. COS-1 cells were transfected with expression plasmids encoding GHR, JAK2, STAT5b and RXRalpha in combination with (lanes 2-6) or in the absence of PPARalpha expression plasmid (lane 7). Cell were stimulated with Wy-14,643 (20 µM) and GH (100 ng/ml) for 24 h before cell extracts were prepared. EMSA assays were carried out with a 32P-labeled CYP4A6 Z-element EMSA probe, which contains a single PPARalpha binding site. Lane 1, untransfected COS-1 cell extract. Anti-STAT5b-specific antibody (2 µl; Santa Cruz, sc-835) was included in lane 3. Anti-PPARalpha antibodies were used to verify the presence of PPARalpha in the DNA-binding complex: lane 4, `a', 4 µl of anti-PPARalpha antibody which specifically inhibits complex formation (Affinity Bioreagents, Inc., cat# PA1-822); lane 5, `b', 2 µl of a supershifting anti-PPARalpha antibody provided by Dr. E. Johnson (Scripps Research Institute, La Jolla, CA).

We next investigated whether STAT5b DNA binding activity is required to inhibit PPARalpha activity. The effects of two rat STAT5b mutants, STAT5b-EE and STAT5b-VVVI (45), were compared with that of wild-type rat STAT5b. In these mutants, amino acid residues EE (437-438) and VVVI (466-469) within the VTEE and SLPVVVI sequences of the DNA binding domain of STAT5b are replaced by alanine. The STAT5b-VVVI mutant is devoid of DNA binding activity and is transcriptionally inactive following prolactin stimulation, whereas the STAT5b-EE mutation does not abolish STAT5b DNA binding activity or prolactin-induced transcription from the beta -casein promoter (45). STAT5b-EE and STAT5b-VVVI were therefore tested for their ability to inhibit PPARalpha activity in GH-treated COS-1 cells. Fig. 10 shows that wild-type rat STAT5b and rat STAT5b-EE both inhibit PPARalpha activity in GH-treated cells in a manner similar to the effects of mouse and human STAT5b shown above. By contrast, much less inhibition is seen with STAT5b-VVVI. Thus, the VVVI residues in the DNA binding domain of STAT5b are critical for GH-activated STAT5b to efficiently inhibit PPARalpha activity, strongly suggesting that this inhibition requires STAT5b DNA binding and transcriptional activation activity.


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Fig. 10.   DNA-binding activity is required for rat STAT5b to mediate GH inhibition of PPARalpha activity. COS-1 cells were transiently transfected with pHD(X3)Luc reporter and expression plasmids encoding PPARalpha , GHR, JAK2 and rat STAT5b or either of two rat STAT5b site-specific mutants localized to STAT5b's DNA binding domain. STAT5b-EE retains DNA-binding activity, while STAT5b-VVVI is devoid of DNA-binding activity (45). Transfected cells were treated with Wy-14,643 (20 µM) and GH (25 and 100 ng/ml) for 24 h. Cell extracts were assayed for firefly luciferase activity relative to renilla luciferase activity.


    DISCUSSION

GH and several other hormones, including thyroid hormone (triiodothyronine) and glucocorticoids, modulate the pleiotropic responses of rodent liver to structurally diverse peroxisome proliferators mediated by the nuclear receptor PPARalpha . The present studies demonstrate that the GH-activated transcription factor STAT5b, and to a lesser extent STAT3, can mediate the inhibitory effects of GH on PPARalpha transcriptional activity and that these effects occur at a physiological GH level. This specific inhibitory effect on PPARalpha activity was seen both with rodent and human STAT5b proteins and occurred when STAT5b was activated by either GH or prolactin. Given that STAT5b can also be activated by a large number of cytokines and growth factors, and is widely expressed in mammalian tissues (41, 51), the potential for inhibitory interactions between PPARalpha and JAK-STAT5b signaling pathways is widespread. Although STAT5a (mammary gland factor) and STAT5b show ~90% amino acid sequence identity, and both bind to and trans-activate target genes via STAT5 response elements such as that found in the beta -casein promoter, they have distinct tissue-specific functions that cannot be compensated by each other in vivo, as shown in the case of mouse STAT5 gene knock-out studies (61, 62). The present study provides further evidence for differences between the two STAT5 forms, insofar as STAT5a was less effective at inhibiting PPARalpha activity, particularly when activated by prolactin (Fig. 4).

Functional interactions involving the binding of STAT factors and another nuclear receptor family member, glucocorticoid receptor, have been described (59, 60, 63). STAT5a and glucocorticoid receptor form a molecular complex that enhances STAT5a-induced transcription from the beta -casein promoter and inhibits glucocorticoid-stimulated transcription from a glucocorticoid response element. Specific DNA binding by STAT5a is required for cooperation with glucocorticoid receptor on the beta -casein promoter, and although the receptor does not bind the STAT5a DNA-binding element directly, it associates with the STAT5a-DNA complex. A potential glucocorticoid receptor-binding site is present within the beta -casein promoter, and this site is required for the synergism between STAT5a and glucocorticoid receptor (64). These studies establish a model in which STAT proteins cross-talk with nuclear receptors by direct protein-protein interactions that modulate gene expression. By contrast, we were unable to detect molecular complexes involving both STAT5b and PPARalpha in the present study, as judged by EMSA supershift analysis. Transcriptional inhibitory effects of prolactin-activated STAT5b have also been observed in studies of the full-length IRF-1 (interferon regulatory factor-1) promoter but not with an isolated IRF-1 STAT response element (45). In contrast to our findings with PPARalpha , the prolactin inhibitory effects seen on the IRF-1 promoter are conferred equally well by STAT5a or STAT5b. Moreover, unlike our findings in the present study, the effects of STAT5b on the IRF-1 promoter do not require the DNA binding activity of STAT and are proposed to involve STAT5b in protein-protein interactions with other transcription factors (45).

GH activates the MAP kinase pathway in many cell types, including liver cells (32, 53, 65). MAP kinase can, in turn, phosphorylate and thereby modulate the activity of a variety of transcription factors, including STAT proteins, which may undergo MAP kinase-catalyzed serine phosphorylation required for full transcriptional activity (66, 67). Growth factor-activated MAP kinase phosphorylates PPARgamma , resulting in an inhibition of the transcriptional activity of that nuclear receptor (56, 68). In our experiments, the MAP kinase kinase inhibitor PD98059 increased the responsiveness of COS-1 cells to PPARalpha activators, suggesting that MAP kinase phosphorylates and thereby inhibits PPARalpha by a mechanism similar to that described for PPARgamma (56, 68). However, PD98059 did not alter the extent to which GH-activated STAT5b inhibited PPARalpha activity.

Several mechanisms may account for the inhibition of PPARalpha by GH-activated STAT5b described in the present study. First, activated STAT5b could inhibit PPARalpha protein expression and thereby block PPAR activation of PPRE. However, this is considered unlikely, since GH-activated STAT5b had no effect on expression of the internal control beta -galactosidase expression plasmid, which utilizes the same cytomegalovirus promoter as does the PPARalpha expression plasmid used in our experiments. Second, STAT5b might compete with PPARalpha for binding to the PPAR reporter PPRE elements of plasmid. This is also unlikely, since the pHD(X3)Luc reporter used in this study does not contain STAT5b-binding sites. Moreover, gel-shift assays revealed that STAT5b does not bind to an isolated PPRE (Fig. 9, lane 7). Third, by analogy to the interaction of STAT5a with glucocorticoid receptor discussed above, STAT5b may form a protein-protein complex with PPARalpha and thereby prevent PPARalpha from trans-activating its target genes. However, the inability of a STAT5b-specific antibody to supershift activated PPARalpha when bound on a PPRE element (Fig. 9) argues against a direct association of STAT5b with PPARalpha , indicating a mechanism distinct from the previously described STAT5a-glucocorticoid receptor association. We cannot rule out the possibility, however, that STAT5b might form a complex with PPARalpha that is independent of its binding to PPRE and thus not detected in our experiments.

An alternative possibility is that STAT5b inhibits PPARalpha activity by an indirect mechanism. For example, when present in the nucleus in a transcriptionally active state, STAT5b may compete for an essential coactivator of PPAR, such as SRC-1, CBP/p300, or PBP (69-71), or perhaps modulate the binding of PPAR to other interacting proteins, such as RXRalpha or RIP140 (72), leading to inhibition of transcriptional activity of PPARalpha . Indeed, CBP and/or p300 have been implicated in the antagonism between STAT and AP-1 signaling pathways (73). Alternatively, given the requirement for intact, functional STAT5b DNA binding and transcriptional activation domains, STAT5b may activate transcription of a second gene, leading to production of a distinct protein factor that serves as a more proximal inhibitor of PPARalpha activity. Both of these possibilities are consistent with our observation that a dominant-negative STAT5b mutant (STAT5bDelta 754 (43)) blocks wild-type PPARalpha inhibitory activity of STAT5b (Fig. 8). Of note, some caution about the interpretation of the effects of the STAT5b mutants employed in this study is required. For example, if mutation of the STAT5b DNA binding domain residues VVVI (45) were to interfere with STAT5b tyrosine phosphorylation and/or nuclear translocation, then a failure to translocate into the nucleus rather than the loss of DNA binding activity per se could account for the observed lack of PPARalpha inhibition (Fig. 10). The precise mechanism underlying the dominant-negative effect of the COOH-terminal truncated STAT5b also needs to be elucidated. Further study will be required to test and evaluate these and other possible inhibitory mechanisms.

The inhibition of PPARalpha activity by activated STAT5b described in this study may have diverse physiological consequences. PPARalpha is an important intracellular messenger that transmits pharmacological as well as nutritional and immunological stimuli to cells. PPARalpha target genes are involved in fatty acid oxidation, transport, and synthesis in liver and other tissues, and PPARalpha activators include certain steroids, fatty acids, and metabolites of arachidonic acid (12). STAT5b can be activated by many cytokines and growth factors in addition to GH and prolactin, including erythropoietin and interleukins 2, 3, and 5 (41, 44). If, as seems likely, STAT5b inhibits PPARalpha activity when activated by these other cytokines and growth factors, additional cytokine and hormonal effects on the regulation of lipid metabolism and the degradation of lipid signaling molecules are also possible.

Leukotriene B4, a mediator of certain inflammatory and immunological reactions, has been identified as an endogenous ligand for PPARalpha (7). Peroxisome proliferators and PPARalpha activators, such as clofibrate, are reported to have both inductive (74, 75) and suppressive effects (76) on the enzymes involved in leukotriene B4 omega -hydroxylation, a reaction that deactivates this potent chemotactic agent and thereby shortens the duration of an inflammatory response. The inhibition of PPARalpha activity by STAT5b activators may therefore provide a mechanism through which STAT5b-activating cytokines modulate leukotriene B4-induced inflammatory responses. The pituitary hormones GH and prolactin can have a marked influence on immune cell types (77, 78). Leukocytes express receptors for GH and prolactin (77) and have an intact JAK/STAT pathway (79), indicating that these polypeptide hormones have the potential to modulate inflammatory responses by suppression of PPARalpha -regulated leukotriene B4 degradation. Inflammatory cells also have the capacity to synthesize and secrete GH (80), which could provide for fine adjustment of the leukotriene B4 response in immune cells.

The present demonstration that GH-activated STAT5b can inhibit PPARalpha -dependent transcriptional responses provides a mechanistic basis for the observed inhibitory effects of GH on peroxisomal enzyme induction (28, 29). Additional mechanisms may also be operative, however, as suggested by the longer term down-regulation of PPARalpha mRNA levels seen in GH-treated liver cells (81). Foreign chemical peroxisome proliferators, including chlorinated hydrocarbons and their metabolites, induce hepatocarcinogenesis via PPARalpha -dependent non-genotoxic mechanisms (18, 19). Increased oxidative stress and proto-oncogene induction (82) are both postulated to contribute to the carcinogenicity of these peroxisome proliferators; however, the precise mechanism remains unclear. PPARalpha plays a critical role in tumor development in mice in response to the foreign peroxisome proliferator Wy-14,643, and targeted disruption of the PPARalpha gene results in loss of this carcinogenic response (18). GH suppression of PPARalpha activity may therefore inhibit tumor development, suggesting a novel mechanism whereby endogenous hormones beneficially modulate cellular responses to non-genotoxic chemical carcinogens.

    ACKNOWLEDGEMENTS

We thank Drs. E. Johnson, J. Capone, J. Reddy, R. Evans, N. Billestrup, J. Ihle, A. Mui, B. Groner, W. Leonard, and L. Yu-Lee for provision of plasmid DNAs used in this study.

    FOOTNOTES

* This work was supported in part by the Environmental Protection Agency via National Institutes of Health Grant ES07381 (to D. J. W.).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{at}bio.bu.edu.

The abbreviations used are: PPAR, peroxisome proliferator-activated receptor; GH, growth hormone; RXR, retinoid X receptor; PPRE, peroxisome proliferator response element; GHR, GH receptor; JAK2, Janus kinase 2; STAT, signal transducer and activator of transcription; CYP, cytochrome P450; EMSA, electrophoretic mobility shift assay; 15d-PGJ2, 15-deoxy-Delta 12,14 prostaglandin J2; MAP kinase, mitogen-activated protein kinase; MEK, MAP kinase kinase; IRF-1, interferon regulatory factor-1.
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