Peroxisome Proliferator Response Element Research Articles

The acyl-CoA thioesterase I is regulated by PPARα and HNF4α via a distal response element in the promoter

The cytosolic acyl-coenzyme A thioesterase I (Acot1) is an enzyme that hydrolyzes long-chain acyl-CoAs of C(12)-C(20)-CoA in chain length to the free fatty acid and CoA. Acot1 was shown previously to be strongly upregulated at the mRNA and protein level in rodents by fibrates. In this study, we show that Acot1 mRNA levels were increased by 90-fold in liver by treatment with Wy-14,643 and that Acot1 mRNA was also increased by 15-fold in the liver of hepatocyte nuclear factor 4alpha (HNF4alpha) knockout animals. Our study identified a direct repeat 1 (DR1) located in the Acot1 gene promoter in mouse, which binds the peroxisome proliferator-activated receptor alpha (PPARalpha) and HNF4alpha. Chromatin immunoprecipitation (ChIP) assay showed that the identified DR1 bound PPARalpha/retinoid X receptor alpha (RXRalpha) and HNF4alpha, whereas the binding in ChIP was abrogated in the PPARalpha and HNF4alpha knockout mouse models. Reporter gene assays showed activation of the Acot1 promoter in cells by the PPARalpha agonist Wy-14,643 after cotransfection with PPARalpha/RXRalpha. However, transfection with a plasmid containing HNF4alpha also resulted in an increase in promoter activity. Together, these data show that Acot1 is under regulation by an interplay between HNF4alpha and PPARalpha.

Ciglitazone mediates COX-2 dependent suppression of PGE2 in human non-small cell lung cancer cells

Background: Cyclooxygenase-2 (COX-2) over-expression and subsequent prostaglandin E2 (PGE2) production are frequently associated with human non-small-cell lung cancer (NSCLC) and are involved in tumor proliferation, invasion, angiogenesis, and resistance to apoptosis. Here, we report that ciglitazone downregulates PGE2 in NSCLC cells. Methods: PGE2 ELISA assay and COX-2 ELISA assay were performed for measuring PGE2 and COX-2, respectively, in NSCLC. The mRNA level of COX-2 was measured by semi-quantitative RT-PCR. The transient transfection experiments were performed to measure COX-2 and peroxisome proliferator-response element (PPRE) promoter activity in NSCLC. Western blots were unitized to measure PGE synthase (PGES) and 15-hydroxyprostaglandin dehydrogenase (15-PGDH) protein expression. Results: COX-2 ELISA assays suggested that ciglitazone-dependent inhibition of PGE2 occurs through the suppression of COX-2. Ciglitazone treatment suppressed COX-2 mRNA expression and COX-2 promoter activity while upregulating PPRE promoter activity. Ciglitazone did not modify the expression of enzymes downstream of COX-2 including PGES and 15-PGDH. Utilization of a dominant-negative PPAR γ showed that the suppression of COX-2 and PGE2 by ciglitazone is mediated via non-PPAR pathways. Conclusion: Taken together, our findings suggest that ciglitazone is a negative modulator of COX-2/PGE2 in NSCLC.

Synergistic effects of RXRα and PPARγ ligands to inhibit growth in human colon cancer cells—phosphorylated RXRα is a critical target for colon cancer management

Background and aims:The activation of the peroxisome proliferator-activated receptor γ (PPARγ) that forms heterodimers with retinoid X receptors (RXRs) elicits an antineoplastic effect on colorectal cancer. It was previously reported...

Peroxisome proliferator‐activated receptor γ agonists inhibit adipocyte expression of α1‐acid glycoprotein

alpha1-Acid glycoprotein (alpha1-AGP) is an acute phase protein that can potentiate cytokine secretion by mononuclear cells and may induce thrombosis by stabilizing the inhibitory activity of plasminogen activator inhibitor-1. Thus, alpha1-AGP may promote pathobiologies associated with type 2 diabetes mellitus (T2DM) including insulin resistance and cardiovascular disease. Here, we demonstrate that antidiabetic peroxisome proliferator-activated receptor gamma (PPARgamma) agonists inhibited expression of 3T3-L1 adipocyte alpha1-AGP in a concentration- and time-dependent manner via an apparent PPARgamma-mediated mechanism. As a result, synthesis and secretion of the glycoprotein was reduced. While PPARgamma agonist regulation of genes with functional peroxisome proliferator response elements in their promoter such as phosphoenolpyruvate carboxykinase were unaffected when cellular protein synthesis was inhibited, downregulation of alpha1-AGP mRNA was ablated thereby supporting the proposition that PPARgamma activation inhibits alpha1-AGP expression indirectly. These results suggest a potential novel adipocytic mechanism by which PPARgamma agonists may ameliorate T2DM-associated insulin resistance and cardiovascular disease.

Transcriptional Regulation of Cidea, Mitochondrial Cell Death-inducing DNA Fragmentation Factor α-Like Effector A, in Mouse Liver by Peroxisome Proliferator-activated Receptor α and γ

Cidea (cell death-inducing DNA fragmentation factor alpha-like effector A), a member of a novel family of proapoptotic proteins, is expressed abundantly in the brown adipose tissue of the mouse. Although Cidea mRNA is not detectable in the mouse liver, we now show that peroxisome proliferator-activated receptor (PPAR) alpha ligands Wy-14,643 and ciprofibrate increase the Cidea mRNA level in a PPARalpha-dependent manner, whereas Cidea induction in liver by PPARgamma overexpression is PPARalpha independent. Increase in Cidea mRNA content in liver did not alter the expression of uncoupling protein 1 (Ucp1) gene, which regulates thermogenesis, lipolysis, and conservation of energy. Although Cidea is considered to be a proapoptotic factor, Cidea induction in liver did not result in increased apoptosis. To elucidate the mechanism by which PPARalpha and PPARgamma regulate Cidea gene expression in the liver, we analyzed the promoter region of the Cidea gene. Three putative peroxisome proliferator response elements (PPREs) are found in the Cidea gene promoter. Transactivation, gel-shift, and chromatin immunoprecipitation assays indicated that the proximal PPRE in Cidea gene (Cidea-PPRE1 at -680/-668) is functional for both PPARalpha and -gamma. We conclude that Cidea is a novel target gene for both PPARalpha and -gamma in the liver where these two transcription factors utilize the same PPRE region for dual regulation. The induction of Cidea in liver with these PPARalpha and -gamma agonists suggests a possible role for Cidea in energy metabolism and a less likely role in hepatocyte apoptosis.

Stimulation of Adenosine A2A Receptor Regulates Peroxisome Proliferator Activated Receptors in Macrophages

Adenosine signals via A2A receptor to inhibit many immune/inflammatory reactions. The mechanisms involved are not completely clear. Peroxisome proliferators-activated receptors (PPARs) are transcription factors that regulate lipid and glucose homeostasis and also are able to regulate the expression of inflammatory molecules in macrophages. In this study, we assessed whether adenosine and a selective A2A receptor agonists (ATL313) regulate the expression or/and activation of PPARs in macrophages. Adenosine and ATL313 increased expression of PPAR alpha, PPAR gamma and PPAR beta in THP-1 cells and mouse peritoneal macrophages. In mouse peritoneal macrophages adenosine as well as ATL313 were able to suppress LPS-induced PPAR gamma downregulation. EMSA showed that the binding of PPARs to their peroxisome proliferator response element (PPRE) was enhanced by adenosine. Thus, A2A receptor activation increase the expression and activation of PPARs and this may be an important mechanism by which adenosine inhibits inflammation. This work was supported by NIH HL078679, HL080569 and AHA 0430151N to Y. Huo.

Prostaglandin J2 causes dedifferentiation via PPAR‐gamma‐dependent pathway, but not COX‐2 expression in articular chondrocyte.

The peroxisome proliferator-activated receptors(PPARs) represent a family of nuclear receprtor ligand activated transcription factors that have been implicated in maintenance of lipid and glucose homeostasis, control of cell growth and differentiation, and regulation of inflammation. PPARs bind to specific response elements as heterodimers with retinoid X receptor (RXR) and activate transcription in response to a variety of endogenouse and exogenouse ligands, whereas its physiological role is unclear. Therefore we investigated the role of PPAR-gamma in dedifferantiation and COX-2 expression in articular chondrocyte with 15d-PGJ2, the natural receptor ligand for PPAR-gamma. Our experiments demonstrated that 15d-PGJ2 dose- and time-dependently induced dedifferantiation and COX-2 expression. The inductive effect on dedifferentiation of chondrocyte seems to be dependent on PPAR activation, as the peroxisome proliferator response element(PPRE) luciferase activity increased and PPAR antagonist, BADGE abolished it. Also, ectopic expression of PPAR-gamma was sufficient to cause dedifferentiation, whereas dominant negative PPAR-gamma, S112A, suppressed 15d-PGJ2-induced dedifferentiation. In the other hands, COX-2 expression was not affected by treatment of PPAR-gamma antagonist(BADGE) as well as transfection of PPAR-gamma dominant negative mutant(S112A). These results indicate that COX-2 regulation by 15d-PGJ2 is independent on PPAR-gamma signaling pathway. Additionally, these data suggest that targeted modulation of the PPAR-gamma pathway may offer a novel approach for therapeutic inhibition of joint tissue.

International Union of Pharmacology. LXI. Peroxisome Proliferator-Activated Receptors

The three peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors of the nuclear hormone receptor superfamily. They share a high degree of structural homology with all members of the superfamily, particularly in the DNA-binding domain and ligand- and cofactor-binding domain. Many cellular and systemic roles have been attributed to these receptors, reaching far beyond the stimulation of peroxisome proliferation in rodents after which they were initially named. PPARs exhibit broad, isotype-specific tissue expression patterns. PPARalpha is expressed at high levels in organs with significant catabolism of fatty acids. PPARbeta/delta has the broadest expression pattern, and the levels of expression in certain tissues depend on the extent of cell proliferation and differentiation. PPARgamma is expressed as two isoforms, of which PPARgamma2 is found at high levels in the adipose tissues, whereas PPARgamma1 has a broader expression pattern. Transcriptional regulation by PPARs requires heterodimerization with the retinoid X receptor (RXR). When activated by a ligand, the dimer modulates transcription via binding to a specific DNA sequence element called a peroxisome proliferator response element (PPRE) in the promoter region of target genes. A wide variety of natural or synthetic compounds was identified as PPAR ligands. Among the synthetic ligands, the lipid-lowering drugs, fibrates, and the insulin sensitizers, thiazolidinediones, are PPARalpha and PPARgamma agonists, respectively, which underscores the important role of PPARs as therapeutic targets. Transcriptional control by PPAR/RXR heterodimers also requires interaction with coregulator complexes. Thus, selective action of PPARs in vivo results from the interplay at a given time point between expression levels of each of the three PPAR and RXR isotypes, affinity for a specific promoter PPRE, and ligand and cofactor availabilities.

Identification of peroxisome-proliferator responsive element in the mouse HSL gene

Hormone-sensitive lipase (HSL) catalyzes the rate-limiting step of lipolysis in adipose tissue. Several studies suggest that protein phosphorylation regulates the HSL enzymatic activity. On the other hand, the precise mechanism of the transcriptional regulation of the HSL gene remains to be elucidated. Here, we identified a functional peroxisome-proliferator responsive element (PPRE) in the mouse HSL promoter by reporter assay in CV-1 cells using serial deletion and point mutants of the 5′-flanking region. Chromatin immunoprecipitation (ChIP) analysis revealed that both peroxisome-proliferator activated receptor (PPARγ) and retinoid X receptor (RXRα) interacted with the region. Binding of the PPARγ/RXRα heterodimer to the PPRE sequence was also confirmed by electrophoretic mobility shift assay. These results indicate that the HSL gene is transcriptionally regulated by PPARγ/RXRα heterodimer, and suggest that a cis-acting element regulates the HSL gene expression.

Peroxisome proliferator-activated receptor γ (PPARγ) regulates trefoil factor family 2 (TFF2) expression in gastric epithelial cells

Although trefoil factor family 2 (TFF2) plays a critical role in the defense and repair of gastric mucosa, the regulatory mechanism of TFF2 expression is not fully understood. In this study, we investigated the regulation of TFF2 expression by peroxisome proliferator-activated receptor γ (PPARγ) in gastric epithelial cells. MKN45 gastric cells were used. TFF2 mRNA expression was analyzed by real-time quantitative RT-PCR. The promoter sequence of the human TFF2 gene was cloned into pGL3-basic vector for reporter gene assays. Ciglitazone was mainly used as a specific PPARγ ligand. MKN45 cells expressed functional PPARγ proteins. Endogenous TFF2 mRNA expression and TFF2 reporter gene transcription was significantly up-regulated by ciglitazone in a dose-dependent manner. Reporter gene assays showed that two distinct cis-elements are involved in the response to PAPRγ activation. Within one of these element (nucleotides −558 to −507), we identified a functional peroxisome proliferator responsive element (PPRE) at −522 (5′-GGGACAAAGGGCA-3′). Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay confirmed the binding of PPARγ to this sequence. Another element (nucleotides −407 to −358) appeared to be a composite enhancer element indirectly regulated by PPARγ and a combination of these two cis-elements was required for the full response of the human TFF2 gene expression to PPARγ. These data demonstrate that human TFF2 gene is a direct target of PPARγ in gastric epithelial cells. Since TFF2 is a critical gastroprotective agent, PPARγ may be involved in the gastric mucosal defense through regulating TFF2 expression in humans.

Molecular cloning and functional expression of human cytosolic acetyl-CoA hydrolase.

A cDNA encoding human cytosolic acetyl-CoA hydrolase (CACH) was isolated from a human liver cDNA library, sequenced and functionally expressed in insect cells. The human CACH cDNA encodes a 555-amino-acid sequence that is 81.4%/78.7% identical to those of the mouse/rat homologue, suggesting a conserved role for this enzyme in the human and rodent livers. Bioinformatical study further reveals a high degree of similarity among the human and rodent CACHs as follows: First, the gene is composed of 15 exons ranging in size from 56 to 157 bp. Second, the protein consists of two thioesterase regions and a C-terminal steroidogenic acute regulatory protein-related lipid transfer (START) domain. Third, the promoter region is GC-rich and contains GC boxes, but lacks both TATA and CCAAT boxes, the typical criteria of housekeeping genes. A consensus peroxisome proliferator responsive element (PPRE) present in the rodent CACH promoter regions supports marked CACH induction in rat liver by peroxisome proliferator (PP).

Identification of a peroxisome proliferator responsive element (PPRE)-like cis-element in mouse plasminogen activator inhibitor-1 gene promoter

PAI-1 is expressed and secreted by adipose tissue which may mediate the pathogenesis of obesity-associated cardiovascular complications. Evidence is presented in this report that PAI-1 is not expressed by preadipocyte, but significantly induced during 3T3-L1 adipocyte differentiation and the PAI-1 expression correlates with the induction of peroxisome proliferator-activated receptor γ (PPARγ). A peroxisome proliferator responsive element (PPRE)-like cis-element (-206TCCCCCATGCCCT-194) is identified in the mouse PAI-1 gene promoter by electrophoretic mobility shift assay (EMSA) combined with transient transfection experiments; the PPRE-like cis-element forms a specific DNA–protein complex only with adipocyte nuclear extracts, not with preadipocyte nuclear extracts; the DNA–protein complex can be totally competed away by non-labeled consensus PPRE, and can be supershifted with PPARγ antibody. Mutation of this PPRE-like cis-element can abolish the transactivation of mouse PAI-1 promoter mediated by PPARγ. Specific PPARγ ligand Pioglitazone can significantly induce the PAI-1 expression, and stimulate the secretion of PAI-1 into medium.

Genome-wide identification of peroxisome proliferator response elements using integrated computational genomics

Peroxisome proliferator-activated receptor (PPAR) agonists are currently used therapeutically in humans, even though many of their direct gene targets are unknown. Because PPARs can directly regulate gene expression through peroxisome proliferator response elements (PPREs), we pursued the computational prediction of PPREs on a genome-wide scale. Contrary to current hypotheses, PPREs are not isotype-specific, nor do flanking nucleotides confer additional information. However, a position weight matrix-based search for PPREs within upstream conserved elements yielded sufficient selectivity for a genome-wide search. Additionally, a novel motif occurring with greater prevalence than PPREs was revealed. Microarray and gene ontology analyses further validated our search technique and provided new functional clusters of genes that were not previously known to be directly regulated by PPARs (e.g., chromatin remodeling, DNA damage response, Wnt, and mitogen-activated protein kinase signaling). This first genome-wide library of high-confidence predicted PPAR target genes will be a valuable resource to PPAR biologists.

PPARα is involved in triiodothyronine-induced cardiac hypertrophy and high-energy phosphate metabolism: A 31P MRS study of the PPARα-null mouse

Triiodothyronine (T3) acts via nuclear receptors to cause cardiac hypertrophy and alter cardiac energy metabolism. Peroxisome proliferator-activated receptor alpha (PPARα) is a nuclear receptor that plays an important role in cardiac fatty acid metabolism. Both the T3 receptor and PPARα form heterodimers with the retinoid × receptor (RXR), the heterodimers bind to T3 and peroxisome proliferator response elements, respectively, to stimulate the transcription of T3-responsive genes or genes involved in fatty acid metabolism.

Differential regulation of the cynomolgus, human, and rat acyl-CoA oxidase promoters by PPARα

Peroxisome proliferator-activated receptor α (PPARα) is a member of the nuclear receptor family of transcription factors and is recognized as the molecular target of the hypolipidemic fibrate drugs. Fibrates promote lipid catabolism by inducing genes involved in fatty acid β-oxidation. In rodents this is accompanied by peroxisome proliferation, and after chronic dosing hepatocarcinoma, whereas epidemiological studies suggest these adverse events are lacking in humans. Rodents such as rats and mice appear particularly sensitive to PPARα-induced peroxisome proliferation while humans are resistant. These findings question the utility of rodent models for safety monitoring of experimental PPARα agonists and highlight the need for additional preclinical models that display greater physiological relevance for human response. Thus we have focused on elucidating the molecular mechanism of species-dependent peroxisome proliferation by understanding the PPARα-dependent regulation of the acyl-CoA oxidase (AOX) promoter, the rate-limiting step of peroxisomal β-oxidation. We have chosen the cynomolgus monkey as a model that is modestly responsive to peroxisome proliferation and functionally characterized it against the highly responsive rat and non-responsive human species. We report the identification of a putative peroxisome proliferator response element (PPRE) within the 2.3 kb proximal promoter of the cynomolgus monkey AOX gene. Characterization of these promoters using a series of constitutively active, PPARα constructs demonstrate that the PPREs within the proximal cynomolgus and human AOX promoters are non-responsive to PPARα whereas the rat PPRE is highly responsive. These findings were verified in vivo using a small molecule PPARα agonist. Taken together, we demonstrate concordant regulation of the AOX promoter by PPARα in cynomolgus monkeys and humans and suggest that this model is superior to rodent models with respect to preclinical evaluation of PPARα agonists.

Transcriptional Regulation of Nephrin Gene by Peroxisome Proliferator–Activated Receptor-γ Agonist

The renoprotective potential of the peroxisome proliferator-activated receptor-gamma (PPAR-gamma) agonist pioglitazone was explored in an immune model of progressive nephropathy, passive Heymann nephritis (PHN), compared with that of an angiotensin II receptor antagonist, taken as standard therapy for renoprotection. PHN rats received orally vehicle, pioglitazone (10 mg/kg twice daily), or candesartan (1 mg/kg twice daily) from months 2 to 8. Pioglitazone reduced proteinuria as effectively as candesartan and limited renal functional and structural changes. Kidneys from untreated PHN rats showed lower nephrin mRNA and protein than controls, both restored by pioglitazone. The effect was seen both early and late during the course of the disease. Whether the antiproteinuric effect of pioglitazone could be due to its effect on nephrin gene transcription also was investigated. HK-2 cells were transfected with plasmids that harbor the luciferase gene under portions (2-kb or 325-bp) of human nephrin gene promoter that contain putative peroxisome proliferator-responsive elements (PPRE) and incubated with pioglitazone (10 muM). Transcriptional activity of luciferase gene was highly increased by pioglitazone, with the strongest expression achieved with the 325-bp fragment. Increase in luciferase activity was prevented by bisphenol A diglycidyl ether, a PPAR-gamma synthetic antagonist. Electrophoretic mobility shift assay experiments showed a direct interaction of PPAR/retinoid X receptor heterodimers to PPRE present in the enhancer region of the nephrin promoter. In conclusion, pioglitazone exerts an antiproteinuric effect in immune-mediated glomerulonephritis as angiotensin II receptor antagonist does. Enhancement of nephrin gene transcription through specific PPRE in its promoter discloses a novel mechanism of renoprotection for PPAR-gamma agonists.

Cloning and molecular dissection of the 8.8 kb pig uroplakin II promoter using transgenic mice and RT4 cells

Uroplakin II (UPII) gene expression is highly tissue and cell specific, with mRNA present in the suprabasal cell layers of the bladder and urethra. Previous reports described the mouse UPII (mUPII) promoter as primarily urothelium selective. However, ectopic expression of a transgene under the 3.6 kb mUPII promoter was also detected in brain, kidney, and testis in some transgenic mouse lines. Here, we have cloned an 8.8 kb pig UPII (pUPII) promoter region and investigated which cells within the bladder and urethra express a transgene consisting of the pUPII promoter fused to human erythropoietin (hEPO) or a luciferase gene. pUPII-luciferase expression vectors with various deletions of the promoter region were introduced into mouse fibroblast (NIH3T3), Chinese hamster ovary (CHO), and human bladder transitional carcinoma (RT4). A 2.1 kb pUPII promoter fragment displayed high levels of luciferase activity in transiently transfected RT4 cells, whereas the 8.8 kb pUPII promoter region displayed only low levels of activity. The pUPII-hEPO expression vector was injected into the pronucleus of zygotes to make transgenic mice. To elucidate the in vivo molecular mechanisms controlling the tissue- and cell-specific expression of the pUPII promoter gene, transgenic mice containing 2.1 and 8.8 kb pUPII promoter fragments linked to the genomic hEPO gene were generated. An erythropoietin (EPO) assay showed that all nine transgenic lines carrying the 8.8 kb construct expressed recombinant human erythropoietin (rhEPO) only in their urethra and bladder, whereas two transgenic lines carrying the 2.1 kb pUPII promoter displayed hEPO expression in several organs including bladder, kidney, spleen, heart, and brain. These studies demonstrate that the 2.1 kb promoter contains the DNA elements necessary for high levels of expression, but lacks critical sequences necessary for tissue-specific expression. We compared binding sites in the 2.1 and 8.8 kb promoter sequences and found five peroxisome proliferator responsive elements (PPREs) in the 8.8 kb promoter. Our data demonstrated that proliferator-activated receptor (PPAR)-gamma activator treatment in RT4 cells induced the elevated expression of hEPO mRNA under the control of the 8.8 kb pUPII promoter, but not the 2.1 kb promoter. Collectively, our data suggested that all the major trans-regulatory elements required for bladder- and urethra-specific transcription are located in the 8.8 kb upstream region and that it may enhance tissue-specific protein production and be of interest to clinicians who are searching for therapeutic modalities with high efficacy and low toxicity.

Peroxisome Proliferator–Activated Receptor Subtypes Differentially Cooperate with Other Transcription Factors in Selective Transactivation of the Perilipin/PEX11α Gene Pair

Perilipin is an adipocyte-specific protein associated with lipid droplets that is crucial for the regulation of storage and mobilization of lipids. We earlier reported that the mouse perilipin gene is regulated by peroxisome proliferator-activated receptor (PPAR) gamma through a peroxisome proliferator-response element (PPRE) positioned upstream of the perilipin promoter. Moreover, we showed that this PPRE also controls expression of the PEX11alpha gene, which is located further upstream. We show here that three elements, A, B, and C, in close proximity downstream of the PPRE, are essential for transactivation of the perilipin gene by PPARgamma. Electrophoretic gel-mobility shift assays demonstrated that nuclear factor (NF)-1 subtypes bind specifically to element B. Furthermore, chromatin immunoprecipitation using 3T3-L1 cells revealed that NF-1A and NF-1B bind to element B in a differentiation-dependent fashion, whereas binding is constitutive with NF-1C and NF-1X. Element C is likely to be a binding motif for nuclear receptors. With PPARalpha, elements A-C do not appear to be required for transactivation of the PEX11alpha gene, so that cooperation with other transcription factors may be differentially involved in selective transactivation of the PEX11alpha and perilipin genes by different PPAR subtypes.

15d‐PGJ2‐induced dedifferentiation and cyclooxygenase‐2 expression in articular chondrocytes via the ERK‐1/‐2 pathway

Peroxisome proliferators-activated receptors (PPARs) are members of the ligand-activated nuclear receptor superfamily. PPARs bind to specific response elements as heterodimers with retinoid X receptor (RXR) and activate transcription in response to a variety of endogenous and exogenous ligands, whereas its physiological role is unclear. Therefore, we investigated the role of PPAR-gamma in COX-2 expression in articular chondrocytes with 15d-PGJ2, the natural receptor ligand for PPAR-gamma, Our experiments demonstrated that 15d-PGJ2 dose- and time-dependently induced dedifferentiation and COX-2 expression, but suppressed proliferation. The inductive effect seems to be dependent on PPAR-gamma activation, as the peroxisome proliferator response element (PPRE) luciferase activaty increased and PPAR antagonist, BADGE abolished it. Also, ectopic expression of PPAR-gamma was sufficient to cause dedifferentiation and COX-2 expression, wheareas dominant negative PPAR-gamma, S112A, suppressed 15d-PGJ2-induced dedifferentiation and COX-2 expression. We found that 15d-PGJ2 treatment stimulated activation of ERK-1/-2. Inhibition of ERK-1/-2 with PD98059 rescued 15d-PGJ2-induced dedifferentiation and blocked COX-2 expression. Our findings collectively suggest that PPAR-gamma-dependent regulation of dedifferentiation and COX-2 expression by 15d-PGJ2 via ERK-1/-2 in articular chondrocytes.

Peroxisome proliferator-activated receptor gamma regulates expression of signal transducer and activator of transcription 5A

Signal transducer and activator of transcription 5A (STAT5A) has been shown to be important for terminal differentiation of mammary epithelial cells. In order to understand regulation of expression of STAT5A, the 5′ end of the mouse Stat5a gene was isolated. Putative regulatory elements was searched for and several peroxisome proliferator response elements (PPREs) were found, one with high (12/13 nucleotides) and three with less (8–10/13) similarity to the reported consensus sequence. Mouse mammary epithelial HC11 cells were treated with peroxisome proliferator-activated receptor γ (PPARγ) ligand, the thiazolidinedione (TZD) troglitazone, and an increase in STAT5A protein expression was seen. The 5′ flank of Stat5a gene was cloned in a luciferase reporter vector. A concentration dependent activation of the STAT5A-luciferase reporter was detected, when transiently transfected HC11 cells were treated with TZD. The activation could be inhibited by treatment with a PPARγ antagonist. It has earlier been shown that epidermal growth factor (EGF) induces MAPK phosphorylation of PPARγ resulting in a less transcriptionally active receptor. In HC11 cells, EGF inhibited TZD induced STAT5A-reporter activity suggesting that our previously reported EGF-mediated suppression of STAT5A expression is mediated in all or partly through inhibition of PPARγ activity. Furthermore, the MEK inhibitor PD98059 inhibited the EGF effect. All together, data presented suggest that PPARγ participates in regulation of STAT5A expression.