Transcription coactivator PBP, the peroxisome proliferator-activated receptor (PPAR)-binding protein, is required for PPARalpha-regulated gene expression in liver.

Nuclear receptor coactivator PBP (peroxisome proliferator-activated receptor (PPAR)-binding protein) functions as a coactivator for PPARs and other nuclear receptors. PBP serves as an anchor for TRAP (thyroid hormone receptor-associated proteins)/mediator multisubunit cofactor transcription complex. Disruption of the PBP/TRAP220 gene results in embryonic lethality around embryonic day 11.5 by affecting placental, cardiac, hepatic, and bone marrow development. Because PPAR isoforms alpha, gamma, and beta/delta function as important regulators of lipid homeostasis in mammals, it becomes important to assess the requirement of coactivator PBP in the regulation of PPAR functions in vivo. Sustained activation of PPARalpha by structurally diverse classes of chemicals of biological importance, designated peroxisome proliferators, leads to proliferation of peroxisomes in liver, induction of PPARalpha target genes including those involved in fatty acid oxidation, and the eventual development of liver tumors. Here, we show that targeted deletion of PBP in liver parenchymal cells, using the Cre-loxP system, results in the near abrogation of PPARalpha ligand-induced peroxisome proliferation and liver cell proliferation, as well as the induction of PPARalpha-regulated genes in PBP-deficient liver cells. In contrast, scattered PBP(+/+) hepatocytes in these livers showed DNA synthesis and were markedly hypertrophic with peroxisome proliferation in response to PPARalpha ligands. Chromatin immunoprecipitation data suggest that in PBP conditional null livers, there appears to be reduced association of cofactors, especially of CBP and TRAP150, to the mouse enoyl-CoA hydratase/l-3-hydroxyacyl-CoA dehydrogenase gene promoter. These observations suggest that PBP is required for the stabilization of multiprotein cofactor complexes. In essence, the absence of PBP in hepatocytes in vivo appears to mimic the absence of PPARalpha, indicating that coactivator PBP is essential for PPARalpha-regulated gene expression in liver parenchymal cells.

Identification of increasing number of coactivators and coactivator-binding proteins raises many questions about their functional role in gene-, cell-, and stage-specific transcription. Delineation of the in vivo functional roles of these transcriptional coactivators becomes an important challenge. Such information may prove crucial to our understanding of many biological processes including development and differentiation in cancer and also in tissue-and species-specific responses to xenobiotic agents. Evidence obtained from gene knockout studies has established that both PBP and PRIP null mutations lead to embryonic lethality between gestational day 11.5 (E11.5) and 12.5 (E12.5), indicating that PBP and PRIP are essential and nonredundant coactivators (23)(24)(25)(26)(27)(28)(29). In contrast, SRC-1, TIF2/GRIP1/SRC-2, and ACTR/pCIP/AIB1/SRC-3 null mice are viable but show varying degree of partial hormone resistance and growth retardation (30 -34). The embryonic lethality of PBP and PRIP null mutants necessitates the creation of conditional null mice using the Cre-loxP strategy for elucidating the cell-and gene-specific roles of these coactivators. We now report the generation of PBP liver conditional null mice to determine the role of PBP/TRAP220 in the PPAR␣-regulated response to peroxisome proliferators. The results show that targeted deletion of PBP in liver parenchymal cells resulted in the abrogation of peroxisome proliferation as well as the induction of PPAR␣-regulated genes in mouse liver in response to peroxisome proliferators. In essence, the absence of PBP in hepatocytes in vivo mimics the absence of PPAR␣ (35), indicating that both PPAR␣ and PBP are essential for PPAR␣-regulated gene expression in liver parenchymal cells.

Generation of PBP loxP Mice and Liver-specific PBP Gene Targeting-
The targeting vector was generated by introducing a single loxP site and a neomycin (neo) cassette flanked by two additional loxP sites into the introns upstream and downstream of exons 8 and 10 of PBP gene (Fig. 1A). Linearized targeting vector was electroporated into HM1 embryonic stem (ES) cells. G418-selected, homologously recombined ES cells were confirmed by Southern blotting, and correctly targeted ES cells were injected into C57BL/6 blastocysts. Highly chimeric male mice were bred with C57BL/6 females to generate heterozygous mice, which were then crossed with an EIIa-Cre transgenic line (28,36) to delete the neo cassette. The neomycin cassette deleted heterozygous mice carrying one floxed (fl, for flanked by loxP) and one wild-type allele were bred with a mouse containing albumin-Cre (AlbCre) transgene (37) to generate PBP liver-null (PBP Ϫ/Ϫ ) mice. PCR genotyping of mice was performed using the following primers flanking loxP site 2 in the floxed allele: 5Ј-TCCATCTGACCTGCTGGATGATAA-3Ј and 5Ј-GGGTGTGA-CCCCATAATT-3Ј. Cre-specific primers used included: 5Ј-AGGTGTAG-AGAAGGCACTCAGC-3Ј and 5Ј-CTAATCGCCATCTTCCAGCAGG-3Ј. The Northwestern University Animal Care and Use Committee approved all animal studies.
Treatment with Peroxisome Proliferators-Mice were maintained on a 12-h light/dark cycle and fed water and a pellet chow diet ad libitum. Wy-14,643 (0.125% weight/weight) or ciprofibrate (0.025% weight/ weight) was administered in powdered diet form for 2 weeks. For light microscopy, liver sections were fixed in 10% neutral buffered formalin or 4% paraformaldehyde and embedded in paraffin. Sections (4-m thick) were cut and stained with hematoxylin and eosin or immunohistochemically stained using antibodies against PBP (TRAP220 (C-19), lot B230, Santa Cruz Biotechnology) and enoyl-CoA hydratase/L-3-hydroxyacyl-CoA dehydrogenase (L-bifunctional enzyme (L-PBE)) (38). For cytochemical localization of peroxisomal catalase (CTL), liver sections were fixed in 1.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 4 h at 4°C, incubated in alkaline 3,3-diaminobenzidine reaction mixture, and processed for light and electron microscopy as described (31,39). For cell proliferation analysis, mice were fed a diet containing Wy-14,643 (0.125%) and were given bromodeoxyuridine (0.5 mg/ml) in drinking water at the same time. They were killed at the end of 4 days to obtain nuclear labeling indices by analyzing immunohistochemically stained liver sections as described (31). Histological analysis and image processing were carried out using a Leica DMRE microscope equipped with Spot digital camera.
Northern and Immunoblot Analyses-Total RNA was isolated from liver using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. For Northern blotting, 20 g of total RNA from each sample was glyoxylated, separated on 0.8% agarose gel, transferred to nylon membrane, and probed with selected cDNA as described previously (31,39). To detect PBP mRNA in wild-type and PBP conditional null livers, RT-PCR was carried out with primers 5Ј-TGTATCTG-GCTCTCCAATCC-3Ј and 5Ј-AGTGATGAGTTCATACAGGGG-3Ј (40). Total RNA (1 g) was used for each sample for RT-PCR analysis using a One-Step RT-PCR kit (Invitrogen). For immunoblot analysis, whole liver proteins were subjected to 7.5 or 10% SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted as described previously (31).
Chromatin Protein Association Assays-Wild-type, PBP liver conditional, and PPAR␣ Ϫ/Ϫ mice were on a control diet or fed a diet containing Wy-14,643 (0.125% w/w) for 4 days. Animals were killed and liver nuclei purified and processed for chromatin immunoprecipitation (ChIP) assays as described (41). Nuclei were suspended in phosphatebuffered saline and fixed in 1% formaldehyde in phosphate-buffered saline at room temperature to cross-link the DNA-binding proteins to cognate cis-acting elements. The nuclei were harvested after 30 min, washed with phosphate-buffered saline, and sonicated on ice in buffer I containing 50 mM Tris-Cl, pH 8.0, 1% SDS, 5 mM EDTA, 5 mM EGTA, 0.5 mM phenylmethylsulfonyl fluoride, and complete protease inhibitor mix (Roche Applied Science) to shear chromosomal DNA to an average length of 1000 bp (41). After centrifugation to remove insoluble material, the chromatin extract was stored at Ϫ80°C until used. Chromatin supernatant was diluted 5-fold with buffer B containing 20 mM Tris-Cl pH 8.0, 1% Triton X-100, 1.2 mM EDTA and 150 mM NaCl. The diluted chromatin was precleared by incubating for 3 h with 5 l of preimmune serum coupled to protein A-agarose beads saturated with bovine serum albumin (1 mg/ml) and salmon sperm DNA (0.4 mg/ml). Precleared chromatin was incubated with 10 l of individual antibodies overnight on a spinner in the presence and absence of ligand. The complexes were pulled down with 50 l of 50% protein A-agarose beads. The complexes were washed sequentially with buffer C (50 mM Tris-Cl pH 8.0, 2 mM EDTA, 0.2% sarkosyl) and buffer D (100 mM Tris-Cl pH9.0, 500 mM LiCl, 1% Nonidet P-40, 1% deoxycholic acid). Immune complexes were eluted from the protein A beads by vortexing in 150 l of buffer E (50 mM sodium bicarbonate, 1% SDS) twice for 15 min. The eluants were pooled, the protein-DNA cross-linking was reversed by adding 5 M NaCl to a final concentration of 200 mM, and the RNA was removed with the addition of 10 g of DNase-free RNaseA followed by incubation at 65°C for 4 h. Genomic DNA from the immune complexes was ethanol-precipitated, and the proteins in the complex were digested with 10 g of proteinase K. Finally, the DNA-binding protein-bound cognate cis-acting elements were purified with phenol-chloroform-isoamyl alcohol and further on Qiagen columns. Similarly purified DNA fragments from the chromatin extracts (input) were used as control for PCR reactions. The primers used for amplification of mouse L-PBE (AC130214) were 5Ј-TTACTTTTTTACTCATTCAGCA-3Ј and 5Ј-TGTCACAGGAAAAAGAG-TAAAA-3Ј (42).
Immunoprecipitation and Immunoblot Analysis-Nuclei isolated from wild-type and PBP conditional null livers 4 days after His-tagged adenovirus PIMT injection, were used for immunoprecipitation with anti-His tag and immunoblotting with anti-CBP, anti-PBP, anti-His (for PIMT), and anti-PRIP as described previously (21,22).

Generation of a Conditional Null Allele of the PBP Gene-A
targeting vector containing three loxP sites in the same orientation was constructed (Fig. 1A), with the first loxP site in the intron upstream of exon 8 and the two other loxP sites flanking neomycin (neo) cassette in the intron between exons 10 and 11. The construct was introduced into ES cells by electroporation, and the cells were screened for homologous recombinants. One recombinant clone was identified by Southern blot analysis of genomic DNA. A single targeted ES clone was injected into C57BL/6 blastocysts to generate chimeric mice, which were bred with C57BL/6 females to generate heterozygous loxPtargeted/wild-type (t/ϩ) mice. The t/ϩ mice were crossed into a transgenic line expressing Cre under the control of the adeno-virus EIIa promoter (EIIaCre) to delete the neo cassette (28,36). EIIaCre-mediated recombination occurs only in the early mouse embryo (2-8 cell stage), and it induces the recombination between the two loxP sites with the same orientation. The recombination events were confirmed by Southern blotting.
Liver-specific Disruption of PBP-To evaluate the role of PBP in maintaining hepatic gene expression and to assess the response to PPAR␣ ligands, mice heterozygous for the floxed PBP allele (PBP fl/ϩ ) were bred with a mouse containing the AlbCre transgene, and genotypes were assessed by PCR (Fig.  1B). AlbCre transgenic mice express Cre in the postpartum liver (37). The time course, extent, and liver specificity of Cre- mediated recombination at the PBP locus were evaluated by RT-PCR or Northern blots of total RNA from 2-, 4-, and 6-weekold mouse livers. A marked decrease in PBP mRNA concentration was seen in PBP conditional null livers at 4 -6 weeks of age (data not shown). More critically, the extent of disruption of the PBP gene in hepatocytes was evaluated by immunohistochemical localization of PBP in liver, as it provides a direct estimate of the extent of PBP gene deletion in hepatocytes and their location in liver lobule for assessment of response to PPAR␣-ligands (Fig. 1, C and D). In the wild-type mouse liver, PBP is localized in the nuclei of all hepatocytes and nonparenchymal cells (Fig. 1C, arrows). In contrast, in 6 -8-week-old PBP liver conditional nulls, PBP was absent in the nuclei of ϳ99% of hepatocytes (based on counting of 10,000 hepatocyte nuclei) (Fig. 1D, arrows). Few hepatocytes in the centrilobular region exhibited nuclear staining, suggesting that not all hepatocytes express albumin promoter to drive Cre (Fig. 1D). As expected, PBP nuclear staining is also evident in nonparenchymal cells in PBP Ϫ/Ϫ liver (Fig. 1D, arrowheads). The presence of PBP in few hepatocytes in PBP liver conditional nulls provides a remarkable opportunity for side-by-side comparison and contrast of the response of PBP Ϫ/Ϫ and PBP ϩ/ϩ hepatocytes to PPAR␣ ligands (see below). These PBP immunohistochemical localization findings also emphasize the importance of ascertaining the percentages of hepatocytes exhibiting the gene disruption in liver conditional null mouse models.
Liver-specific Deletion of PBP Leads to Near Abrogation of Peroxisome Proliferative Response Caused by PPAR␣ Ligands-To evaluate the role of coactivator PBP in PPAR␣regulated gene expression and peroxisome proliferation in hepatic parenchymal cells, we fed PBP liver conditional, PPAR␣ Ϫ/Ϫ (34), and wild-type mice a diet containing Wy-14,643 (0.125% weight/weight) or ciprofibrate (0.025% weight/weight) for 2 weeks. The histological architecture of control PBP liver conditional null mice (Fig 2B) was essentially similar to that of wild-type mice ( Fig. 2A) and PPAR␣ Ϫ/Ϫ mice (Fig. 2C). As expected, wild-type mice treated with a peroxisome proliferator for 2 weeks exhibited marked hypertrophy of hepatocytes with increased cytoplasmic eosinophilia (Fig. 2G), which is due to marked proliferation of peroxisomes as ascertained by the cytochemical staining for peroxisomal CTL (Fig.  2, D and J). In contrast, PBP liver conditional nulls revealed minimal or no observable hepatocyte hypertrophy (Fig. 2, H  and K), except for the presence of a few hepatocytes with enormous hypertrophy (Fig. 2H, boxed), which in 0.5-m thick sections stained histochemically for catalase revealed an extreme degree of peroxisome proliferation (Fig. 2K, arrows). Hepatocytes demonstrating positive response to peroxisome proliferators are located predominantly in the centrilobular regions (Fig. 3), with a few clusters scattered randomly in liver lobules (Fig. 3A). A dramatic contrast in the response between PBP Ϫ/Ϫ and PBP ϩ/ϩ hepatocytes to a PPAR␣ ligand is visualized in liver sections stained for immunohistochemical localization of L-PBE, the second enzyme of the peroxisomal ␤-oxidation system (Fig. 3, A and B), as well as for PBP (Fig. 3C). L-PBE immunostaining was not prominent in PBP Ϫ/Ϫ hepatocytes, which are generally smaller than normal hepatocytes, with a larger nuclear volume and less prominent cytoplasmic volume (Fig. 3, A and B). In contrast, PBP ϩ/ϩ hepatocytes are hypertrophic and highly prominent with intense immunostaining for L-PBE (Fig. 3B, arrows). Adjacent sections stained for PBP revealed that the nuclei of large hepatocytes with intense L-PBE staining are positive for the coactivator PBP (Fig. 3C, arrows). Electron microscopy confirmed the presence of peroxisome proliferation in a few PBP ϩ/ϩ cells (Fig. 4, A and  B), similar to those shown in Figs. 2K and 3, A and B. The attenuation or absence of peroxisome proliferation in PBP Ϫ/Ϫ hepatocytes (Figs. 2K and 3, A and B) was further confirmed by electron microscopy (Fig. 4, A and B). The absence of peroxisome proliferative response of PBP Ϫ/Ϫ hepatocytes following exposure to PPAR␣ ligands appeared essentially similar to that of PPAR␣ Ϫ/Ϫ hepatocytes (Fig. 2, C, F, I, and L). In this respect, the presence of either PPAR␣ or PBP alone is not sufficient to elicit PPAR␣ ligand-induced peroxisome proliferation in mouse hepatic parenchymal cells under in vivo conditions.
To further assess the role of PBP in PPAR␣-regulated gene expression in liver, RNA isolated from livers of untreated and Wy-14,643-treated wild-type, PBP liver-null, and PPAR␣ Ϫ/Ϫ mice was analyzed by Northern blotting. As expected, mRNA levels of fatty acyl-CoA oxidase (AOX), L-PBE, peroxisomal thiolase (PTL), CYP4A1 and CYP4A3 (which encode microsomal cytochrome P450 fatty acid -hydroxylase), PEX11␣ (peroxin-peroxisome membrane protein), PDK-4 (pyruvate dehydrogenase kinase-4), and Ly-6D (lymphocyte antigen 6 complex locus D) were increased in Wy-14,643-treated wild-type mouse livers, but such increases were modest at best in the livers of Wy-14,643-treated PBP liver conditional nulls (Fig. 5A). No increases in the mRNA levels of these genes were noted in PPAR␣ Ϫ/Ϫ mice treated with Wy-14,643. The modest increase in mRNA content in the livers of PBP liver conditionals after treatment with a peroxisome proliferator is because of the massive induction of peroxisome proliferation in the few hepatocytes in which PBP gene was not knocked out (see Figs. 2, H and K, and 3, A and B). It should also be noted that PBP ϩ/ϩ hepatocytes in these PBP liver conditional nulls divided in response to PPAR␣ ligands; with 2 weeks of treatment, a net gain of PBP ϩ/ϩ hepatocytes appeared with increased peroxisome proliferation. The differential induction of PPAR␣-responsive genes in PBP Ϫ/Ϫ and PBP ϩ/ϩ hepatocytes is visible in tissues processed for catalase and L-PBE localization (Figs. 2K  and 3, A and B). No changes in CTL, PPAR␣, and PPAR␥ mRNA levels were noted. The constitutive and inducible levels of fatty acid-metabolizing enzymes in livers of PBP liver conditional nulls were also evaluated by immunoblotting (Fig. 5B). Constitutive levels of expression of AOX, L-PBE, D-PBE, PTL, steroid carrier protein x, and catalase were similar in the livers of wild-type, PBP liver null and PPAR␣ Ϫ/Ϫ mice (Fig. 5B). The hepatic levels of AOX, L-PBE, D-PBE, PTL, and steroid carrier protein x increased in Wy-14,643-treated wild-type livers, but the increases were considerably less prominent in PBP livernull mice exposed to PPAR␣ ligand (Fig. 5B). No induction was seen in PPAR␣ Ϫ/Ϫ mouse liver. CTL and PPAR␣ levels did not differ in wild-type and PBP liver-null mice.
PPAR␣ Ligand-induced Hepatocellular Proliferation is Diminished in PBP Ϫ/Ϫ Hepatocytes-To determine the role of PBP, if any, in peroxisome proliferator-induced liver cell pro-liferation, we studied the effect of Wy-14,643 on liver cell proliferation by assessing bromodeoxyuridine labeling (Fig. 6). Wild-type and PBP liver-null mice were fed a diet containing Wy-14,643 (0.125%) for 4 days and allowed to drink water with bromodeoxyuridine (0.5 mg/ml). At the end of 4 days, livers were processed for immunohistochemical localization of bromodeoxyuridine. In untreated wild-type (Fig. 6A) and PBP conditional null livers (Fig. 5B), an occasional cell displayed nuclear labeling (0.3% in wild-type versus 1% in PBP livernull). Wy-14,643-treated wild-type liver displayed prominent labeling of many hepatocyte nuclei (11.5%) throughout the liver lobule (Fig. 6C), confirming the hepatocyte proliferative effect of PPAR␣ ligand (30). In contrast, bromodeoxyuridine labeling was seen mostly in the hepatocyte nuclei (3.4%), located in the centrilobular region (Fig. 6D). This cell proliferative response corresponded with the residual PBP ϩ/ϩ hepatocytes in the PBP liver-nulls (compare Figs. 1D and 3C with 6D). Bromodeoxyuridine labeling data clearly point to a net gain of PBP ϩ/ϩ hepatocytes in PBP liver conditional null mice with exposure to PPAR␣ ligand. In essence, PBP Ϫ/Ϫ hepatocytes failed to respond to the cell-proliferative effects of PPAR␣ ligand.
Reduction in Coactivator Recruitment to L-PBE Gene Promoter-Transcriptional activation by PPAR␣ requires recruitment of coactivators. A ChIP assay was used to examine the associ- ation of L-PBE gene promoter with coactivators in wild-type and PBP conditional null livers. Cross-linked protein-DNA extracts prepared from wild-type and PBP conditional livers were immunoprecipitated with antibodies against PPAR␣, PBP, TRAP150, PRIP, PIMT, CBP, and SRC-1 (Fig. 7A). The DNA fragments extracted from these immunoprecipitates were analyzed by PCR using primers to amplify the peroxisome proliferator response elements of the mouse L-PBE gene (42). Recruitment of PPAR␣ to the L-PBE promoter was increased in both wild-type and PBP liver conditionals following Wy-14,643 (Fig. 7A). The recruitment to the L-PBE promoter of all coactivators examined was increased in Wy-14,643-treated wild-type livers (Fig. 7A). In PBP conditional null livers, reductions in PBP, TRAP150, CBP, and SRC-1 appeared prominent (Fig.  7A), suggesting that PBP is needed for the recruitment of TRAP150 of TRAP complex and other coactivators. It should be noted that these results represent only qualitative alterations because of the chimeric nature of the PBP conditional null livers and might prove to be more dramatic if all hepatocytes were PBP null.
Previously we showed that the nuclear receptor coactivator PRIP-interacting protein, designated PIMT, bridges the CBP/ p300-anchored coactivator complex with the PBP/TRAP220anchored coactivator complex (20). Disruption of the PRIP gene affected the recruitment of PBP and PIMT to aP2 gene promoter, a PPAR␥ target gene, in PRIP Ϫ/Ϫ mouse embryonic fibroblasts (40). To examine these interactions in PBP conditional null livers, we injected adenovirus-expressing Histagged PIMT into the tail vein of Wy-14,643-treated and untreated wild-type and PBP liver conditional null mice. PIMT and its putative associated proteins in nuclear extracts were immunoprecipitated with anti-His antibodies, and the immunoprecipitates were analyzed for the presence of PRIP, CBP, PIMT, PPAR␣, and RXR by immunoblotting. Although PRIP and CBP were present in PIMT immunoprecipitates obtained from liver nuclear extracts of wild-type mice treated with Wy-14,643 (Fig. 7B), these proteins were barely detectable in liver nuclear extracts derived from Wy-14,643-treated PBP conditional null mice (Fig. 7B), suggesting that the absence or reduction of PBP affects the formation of coactivator subcomplex pulled down with PIMT.

DISCUSSION
This report provides evidence that PBP/TRAP220 is essential for the PPAR␣-regulated peroxisome proliferator-induced pleiotropic responses in liver in an intact animal model. These observations establish a dramatic link between nuclear receptor PPAR␣ and coactivator PBP/TRAP220, the proposed anchor of the TRAP/DRIP/ARC/PRIC complex (7)(8)(9)22), and signify a special dependence of this nuclear receptor for PBP in a liver cell-specific function. In PBP conditional null livers, hepatocytes devoid of PBP failed to show the characteristic peroxisome proliferation in response to peroxisome proliferators, whereas hepatocytes with intact PBP gene displayed massive peroxisome proliferative response to PPAR␣ ligands (Fig. 3, A  and B). Despite the diversity of cofactors identified as associated with nuclear receptor function (4 -10), the observation that deletion of one coactivator nearly abolishes the response of hepatocytes to peroxisome proliferators is of considerable interest in PPAR␣ signaling. Peroxisome proliferators are a structurally diverse group of chemicals and include industrial plasticizers such as di(2-ethylhexyl) phthalate, industrial solvents, herbicides, and hypolipidemic drugs such as clofibrate, ciprofibrate, fenofibrate, gemfibrozil, Wy-14,643, and others (43)(44)(45)(46). Chronic exposure to peroxisome proliferators vis´a vis sustained activation of PPAR␣, by either synthetic or natural ligands, leads to the development of liver cancer in rodents (39,(45)(46)(47). Based on the abrogation of peroxisome proliferatorinduced pleiotropic responses, including the development of hepatocellular carcinomas, in PPAR␣ null mice (35,46), it is generally assumed that PPAR␣ is a limiting factor for peroxisome proliferation and that of PPAR␣ target gene induction and accounts for cell/species-specific differences (48). The essentiality of coactivator PBP in PPAR␣ transcriptional activity raises an important mechanistic parameter in assessing species-specific differences in peroxisome proliferator-induced effects and levels of risk. These observations clearly establish that both PPAR␣ and PBP are required for the activation of PPAR␣ target genes in liver. Furthermore, decreased expression of PPAR␣ but increased expression of PPAR␣ target genes in the livers of HNF4␣ (hepatocyte nuclear factor 4␣) null livers imply that PPAR␣ may not be limiting for PPAR␣ target gene induction (49).
PPAR␣ activation in liver also leads to transient induction of hepatocellular proliferation in rat and mouse liver (50), a response that is abrogated in PPAR␣ Ϫ/Ϫ mice (35,51). In PBP conditional null livers, PPAR␣ ligands elicited hepatocellular proliferative responses in PBP ϩ/ϩ hepatocytes but not in PBP Ϫ/Ϫ hepatocytes, implying that PPAR␣ ligand-induced liver cell proliferation requires both PPAR␣ and PBP. The presence of hepatocytes containing both PPAR␣ and PBP adjacent to PBP null but receptor-containing hepatocytes did not induce liver cell proliferation in PBP negative hepatocytes that still retained PPAR␣ (not illustrated). Additional long-term studies are needed to ascertain whether hepatocytes expressing both PPAR␣ and PBP/TRAP220 exhibit growth advantage to chronic exposure to hepatocellular mitogens or partial hepatectomy and expand rapidly as compared with PBP null hepatocytes in these conditional null livers.
PBP was originally cloned as PPAR␥-binding protein and was identified as a nuclear receptor coactivator (13). PBP contains two LXXLL signature motifs considered necessary and sufficient for the binding of coactivators to the nuclear receptor (5,6,13). PBP/TRAP220 is widely expressed and found to interact with various nuclear receptors including PPAR␥, PPAR␣, RXR, TR␤1, estrogen receptor-␣, and others (13,52,53). PBP/TRAP220 has been shown to be a crucial molecule for anchoring estrogen receptor and PPAR␥ to the TRAP/Mediator complex (54,55). The importance of PBP/TRAP220 is underscored by the observations that: (i) the PBP gene is amplified in more than 25% of human breast cancers, implying that this coactivator plays a broader role in the transcriptional activation of many receptors and transcription factors (52); (ii) PBP gene disruption leads to embryonic lethality because of a multitude of developmental abnormalities and placental defects (23)(24)(25)(26); (iii) PBP interacts with GATA transcription factors, suggesting a broader role for PBP in transcriptional control (25); and (iv) it has been demonstrated that PBP/TRAP220 is necessary for PPAR␥-induced adipogenesis in cultured mouse embryonic fibroblasts (55). The results presented here provide in vivo evidence for the PBP in PPAR␣ transcriptional activity in liver, implying that PBP is a bona fide and essential coactivator for PPAR␣ function. Thus, PBP appears to be the central coactivator for the two members of the PPAR subfamily, namely PPAR␣ and PPAR␥. In this context, it is important to note that SRC-1, the prototype member of SRC-1/p160 family of coactivators, is not required for the transcriptional activation of PPAR␣ target genes and peroxisome proliferation (31). The redundancy of SRC-1 for PPAR␣-mediated effects is in direct contrast with the essential role of PBP in PPAR␣ target gene transcription.
The mechanism by which PBP/TRAP220 affects the PPAR␣ function in vivo requires further investigation. ChIP assays performed with liver nuclear extracts revealed a reduced association of other cofactors, especially TRAP150 of the TRAP/ Mediator complex (14), CBP, and SRC-1 at the L-PBE gene promoter. Immunoprecipitation and immunoblotting studies provided support for the diminished association of CBP. These ChIP and immunoprecipitation results would be more dramatic if the PBP gene disruption in liver were more universal. Additional studies using PBP Ϫ/Ϫ hepatocytes would be needed to systematically assess the assembly and disassembly of cofactors on PPAR␣ target gene promoter. Recently, the coactivator binding has been shown to promote specific interaction between ligand and the receptor and to influence receptor stabilization (56). It would be of interest to investigate the role of two LXXLL motifs in the PBP gene in its interaction with PPAR␣. FIG. 7. ChIP assays and immunoblotting of PIMT immunoprecipitates. A, ChIP analysis for PPAR␣-mediated recruitment of nuclear receptor cofactors to the L-PBE gene promoter. Nuclear chromatin from control and Wy-14,643-fed wild-type (WT), PBP liver conditional null (PBP Ϫ/Ϫ ), and PPAR␣ Ϫ/Ϫ mice was immunoprecipitated with specific antibodies. PCR was used to analyze the L-PBE promoter. Wy-14,643 treatment increased the recruitment of various cofactors to L-PBE gene promoter. In PBP conditional null livers, the recruitment of PBP, TRAP150, and CBP is reduced. B, immunoprecipitation and immunoblotting to identify PIMT-interacting proteins in wild-type and PBP conditional null livers from control (Ϫ) and Wy-14,643 (ϩ)-fed groups. Nuclear extracts were immunoprecipitated with anti-His to pull down PIMT and associated proteins. The immunoprecipitates were immunoblotted with anti-His to recognize PIMT, anti-PPAR, anti-RXR, anti-CBP, and anti-PRIP. In PBP liver conditionals, PIMT immunoprecipitate shows no detectable CBP and PRIP proteins.
In conclusion, we have developed a mouse line carrying a conditionally null allele of the PBP/TRAP220 gene. Disruption of the PBP gene specifically in hepatocytes indicates that PBP is an essential coactivator for in vivo PPAR␣ target gene transcription in response to PPAR␣ ligands. The differential response of PBP Ϫ/Ϫ and PBP ϩ/ϩ hepatocytes in the PBP conditional liver knockout mouse provides a visual view of the differential response to compare and contrast the contradictions. This model clearly establishes that neither PPAR␣ nor PBP is sufficient to elicit pleiotropic responses caused by peroxisome proliferators. The availability of PBP floxed mice should facilitate further studies on the role of this coactivator in cell-, tissue-, and gene-specific transcription.