The peroxisome proliferator-activated receptor ␣ (PPAR␣) is a ligand-inducible transcription factor, which belongs to the nuclear receptor superfamily. PPAR␣ mediates the carcinogenic effects of peroxisome proliferators in rodents. In humans, PPAR␣ plays a fundamental role in regulating energy homeostasis via control of lipid metabolism. To study the possible role of chaperone proteins in the regulation of PPAR␣ activity, a monoclonal antibody (mAb) was made against PPAR␣ and designated as 3B6/PPAR. The specificity of mAb 3B6/PPAR in recognizing PPAR␣ was tested in immunoprecipitations using in vitro translated PPAR subtypes. The mAb 3B6/PPAR recognized PPAR␣, failed to bind to PPAR␤ or PPAR␥, and is efficient in both immunoprecipitating and visualizing the receptor on protein blots. The immunoprecipitation of PPAR␣ in mouse liver cytosol using mAb 3B6/PPAR has resulted in the detection of two co-immunoprecipitated proteins, which are heat shock protein 90 (hsp90) and the hepatitis B virus X-associated protein 2 (XAP2). The concomitant depletion of PPAR␣ in hsp90-depleted mouse liver cytosol was also detected. Complex formation between XAP2 and PPAR␣/FLAG was also demonstrated in an in vitro translation binding assay. hsp90 interacts with PPAR␣ in a mammalian two-hybrid assay and binds to the E/F domain. Transient expression of XAP2 co-expressed with PPAR␣ resulted in down-regulation of a peroxisome proliferator response element-driven reporter gene activity. Taken together, these results indicate that PPAR␣ is in a complex with hsp90 and XAP2, and XAP2 appears to function as a repressor. This is the first demonstration that PPAR␣ is stably associated with other proteins in tissue extracts and the first nuclear receptor shown to functionally interact with XAP2.The ability of peroxisome proliferators to activate a receptor in the steroid receptor superfamily was first discovered in 1990, and the cognate protein was designated as PPAR 1 (1). ThePPARs are soluble transcription factors that are activated by a diverse class of lipophilic compounds (2). With the activation of PPAR, a concomitant induction of a number of genes that code for peroxisomal fatty acid metabolizing enzymes was observed in mouse liver. Among these, the peroxisomal enzyme AOx is the most broadly used indicator of peroxisome proliferator action. Transcription of the AOx gene is increased by exposure to the hypolipidemic peroxisome proliferator WY-14,643, and this effect is mediated by a PPRE located 570 base pairs upstream of the transcriptional start site (3). This PPRE contains a direct repeat of the sequence motifs TGACCT and TGTCCT, which is separated by a single nucleotide. A heterodimer of PPAR and RXR binds to PPREs located in upstream regulatory regions of various target genes and the RXR ligand, 9-cis-retinoic acid, increases PPAR/RXR transcriptional activity (4). There are three PPAR subtypes, designated as PPAR␣ (NR1C1), -␤ (NR1C2), and -␥ (NR1C3); and each subtype is capable of binding to DNA after heterodimerizing with RXR (NR2B1) ...

The nuclear receptor (NR) peroxisome proliferator-activated receptor-alpha (PPARalpha) mediates the effects of several hypolipidemic drugs, endogenous fatty acids, and peroxisome proliferators. Despite belonging to a class of NR not known to interact with cytosolic chaperone complexes, we have recently shown that PPARalpha interacts with heat shock protein 90 (Hsp90), although the biological consequence of this association was unknown. In the present study, PPARalpha directly associated with Hsp90 in vitro to a much greater extent than either PPARbeta or PPARgamma. This interaction is similar to other NR-Hsp90 complexes with association occurring between the middle of Hsp90 and the hinge (D) and ligand binding domain (EF) of PPARalpha. Using several different approaches to disrupt Hsp90 complexes within the cell, we demonstrate that Hsp90 is a repressor of both PPARalpha and PPARbeta activity. Treatment with geldanamycin (GA) increased the activity of PPARalpha and in the presence of ligand in transient transfection assays. PPARalpha-response element (PPRE)-reporter assays in a stable cell line treated with GA resulted in enhanced expression of a known target gene, acyl-CoA oxidase. Similarly, overexpression of the tetratricopeptide repeat (TPR) of protein phosphatase 5 (PP5) increased PPARalpha or PPARbeta activity in a PPRE-reporter assay and decreased the interaction between PPARalpha or PPARbeta and Hsp90 in a mammalian two-hybrid assay. Finally, cotransfection with the C-terminal hsp-interacting protein (CHIP) construct, a TPR-containing ubiquitin ligase that interacts with hsp90, increased PPARalpha's and decreased PPARbeta's ability to regulate PPRE-reporter activity upon ligand activation. All three methods to disrupt Hsp90 function (GA, PP5-TPR, CHIP) resulted in an alteration in PPARalpha or PPARbeta activity to a much greater extent than PPARgamma. While FKBP52 had no effect on PPARalpha activity, p23 greatly enhanced constitutive and Wy14 643 induced PPRE-reporter activity. Thus, we describe the chaperone complex as being a regulator of PPARalpha and PPARbeta activity and have identified a novel, subtype-specific, inhibitory role for Hsp90.