Peroxisome Proliferator-activated Receptor α Inhibits Hepatic S14 Gene Transcription

The peroxisome proliferator-activated receptor (PPARα) has been implicated in fatty acid regulation of gene transcription. Lipogenic gene transcription is inhibited by polyunsaturated fatty acids (PUFA). We have used the PUFA-sensitive rat liver S14 gene as a model to examine the role PPARα plays in fatty acid regulation of hepatic lipogenic gene transcription. Both PPARα and the potent peroxisome proliferator, WY14643, inhibit S14CAT activity in transfected primary hepatocytes. WY14643 and PPARα target the S14 T3 regulatory region (TRR, −2.8 to −2.5 kilobases), a region containing 3 T3 response elements (TRE). Transfer of the TRR to the thymidine kinase (TK) promoter conferred negative control to the TKCAT gene following WY14643 and PPARα treatment. Gel shift analysis showed that PPARα, either alone or with RXRα, did not bind the S14TRR. However, PPARα interfered with TRβ/RXRα binding to a TRE (DR+4). Functional studies showed that co-transfected RXRα, but not T3 receptor β1 (TRβ1), abrogated the inhibitory effect of PPARα on S14 gene transcription. These results suggest that WY14643 and PPARα functionally interfere with T3 regulation of S14 gene transcription by inhibiting TRβ1/RXR binding to S14 TREs. Previous studies had established that the cis-regulatory targets of PUFA control were located within the proximal promoter region of the S14 gene, i.e. between −220 and −80 bp. Finding that the cis-regulatory elements for WY14643/PPARα and PUFA are functionally and spatially distinct argues against PPARα as the mediator of PUFA suppression of S14 gene transcription.

The S14 gene has been used as a model for PUFA control of lipogenic gene transcription because the developmental, tissuespecific, hormonal, and nutritional control of S14 is similar to that found for several lipogenic enzymes (2,3). For example, transcription of both fatty acid synthase (FAS) and S14 genes is induced during postnatal development and by insulin, high carbohydrate feeding, and triiodothyronine (T 3 ) and suppressed by diabetes, starvation, and hormones that elevate hepatic cAMP levels. Feeding rats diets enriched in n Ϫ 3 PUFA rapidly and coordinately inhibits transcription of both hepatic genes (1,3). PUFA administration to primary hepatocytes suppresses mRNA FAS and mRNA S14 levels (ED 50 Յ 100 M), as well as inhibiting transcription of S14CAT fusion genes. Transfection analysis has localized PUFA-response elements (PUFA-RE) to the proximal promoter region of the S14 gene, i.e. between Ϫ220 and Ϫ80 bp upstream from the transcription start site. The targets for PUFA control of S14 are distinct from the cis-regulatory elements involved in T 3 (between Ϫ2.8 and 2.5 kb) and insulin/glucose (between Ϫ1.6 and Ϫ1.4 kb) control of S14 gene transcription (3).
Since our goal is to define the molecular basis of PUFAmediated control of gene transcription, we became interested in the peroxisome proliferator-activated receptor (PPAR) as a prospective candidate for a PUFA-regulated factor (PUFA-RF) affecting lipogenic gene transcription. PPAR belongs to the steroid/thyroid supergene family and is involved in lipid metabolism (10 -12). PPAR activates gene transcription by binding peroxisome proliferator response elements (PPRE) in association with retinoid X receptor (RXR) (10 -19). Several PPAR subtypes have been identified, some showing tissue-specific distribution and ligand-dependent activation (10 -12, 19 -26). PPAR␣ appears to be the predominant PPAR subtype in liver (27), and PPAR␣ can be activated by fatty acids as well as peroxisome proliferators to induce enzymes involved in peroxisomal and mitochondrial ␤-oxidation and cholesterol metabolism (12, 20, 21, 24, 29 -41).
Three lines of evidence suggest that PPAR␣ might be involved in PUFA control of lipogenic gene expression: (a) feeding rats high fat diets induces peroxisomal enzymes (37)(38)(39)(40), (b) in vitro transfection studies show that fatty acids activate PPAR␣ (12, 20, 21, 24, 26, 28, 34 -37), and (c) the potent peroxisome proliferator, WY14643, suppressed both mRNA S14 level and S14CAT activity in cultured primary rat hepatocytes (42). In this report, we characterize the peroxisome proliferator regulation of S14 gene transcription. In contrast to our expectation, the cis-regulatory targets for WY14643 and PPAR␣ action mapped to a region of the S14 promoter containing TREs and not to the proximal promoter region containing the negative PUFA-response elements (nPUFA-RE). Based on these studies, PPAR␣ may not be the mediator of PUFA regulation of hepatic S14 gene expression.
Transfections-Primary rat hepatocytes were prepared by the collagenase perfusion method, cultured on Primaria tissue culture plates, and transfected with Lipofectin (Life Technologies) as described previously (3). Hepatocytes were treated with 100 M WY14643 (Chemsyn Science Laboratories, Lenexa, KS). WY14643 was dissolved in Me 2 SO, and Me 2 SO was used as a control for WY14643 treatment. Media were changed after 24 h, and cells were harvested for protein assay and CAT activity assay after 48 h of treatment (3). CAT activity: CAT units ϭ counts/min of 14 C-butylated chloramphenicol/h/100 g of protein.

RESULTS
Effect of WY14643 and mPPAR␣ on S14 Gene Promoter Activity-In primary rat hepatocytes, the potent peroxisome proliferator WY14643 suppressed hepatic mRNA S14 and S14CAT activity in a dose-dependent manner with ED 50 Յ 50 M (42). In the current study, the effects of WY14643 and PPAR␣ on S14 gene transcription were examined using the S14CAT124 fusion gene containing a 5Ј-flanking region extending from Ϫ4315 to ϩ19 bp of the S14 gene. Primary hepatocytes were co-transfected with S14CAT124 and a rat TR␤1 expression vector (MLVTR␤1) in the presence and absence of co-transfected mPPAR␣. Hepatocytes were treated with T 3 to induce (Ն50-fold) CAT activity (3). WY14643 was added to cells without and with co-transfected PPAR␣. WY14643 treatment and mPPAR␣ co-transfection alone suppressed T 3 -induced CAT activity by 45% and 60%, respectively (Fig. 1). In the presence of its activator (WY14643), mPPAR␣ inhibited S14CAT activity by 84%.
Titration studies (data not shown) demonstrated that over a range of 0.1 to 1 g of pSG5-PPAR␣/30-mm culture well, PPAR␣ suppressed S14CAT activity (ED 50 ϭ 0.25 g of PPAR␣/culture). Therefore, ϳ0.2 g/well pSG5-PPAR␣ was used for most co-transfection studies. Substitution of an equivalent amount of the empty vector, i.e. pSG5, or another nuclear receptor, pSG5-RXR␣, for pSG5-PPAR␣ was not inhibitory to S14CAT activity (data not shown). Finally, WY14643 only marginally affected RSV promoter activity, and PPAR␣ had no effect on RSVCAT activity (Fig. 1). More importantly, the WY14643 effect on RSVCAT was not enhanced by PPAR␣ co-transfection. These studies show that: 1) WY14643 suppression of S14CAT activity is not due to generalized effects on hepatocytes, 2) elevating hepatocellular PPAR␣ levels through co-transfection inhibits S14 in a promoter-and receptor-specific fashion, and 3) WY14643 augments the inhibitory effect of PPAR␣ on S14CAT activity. This information coupled with the finding that WY14643 suppressed mRNA S14 in primary hepatocytes (42) suggests that PPAR␣ inhibits transcription of both the endogenous and transfected S14 genes.
WY14643/PPAR␣ cis-Regulatory Elements Localize to the FIG. 1. Effects of PPAR␣ and WY14643 on hepatic S14 gene promoter activity. Primary rat hepatocytes prepared by the collagenase perfusion method (described under "Experimental Procedures") were plated into 6-well Primaria tissue culture dishes. Hepatocytes were transfected with 1 g of T 3 receptor expression vector (MLVTR␤1) and 2 g of S14CAT124 per well. After transfection, cells were treated with 1 M T 3 and Me 2 SO (open bars (DMSO)) or T 3 and 100 M WY14643 (solid bars), respectively. A second group of cells were cotransfected with 0.2 g of pSG5-PPAR␣, a mouse PPAR␣ expression vector. PPAR␣-transfected cells were also treated with T 3 and Me 2 SO (hatched bars) or T 3 and WY14643 (horizontally striped bars). After 48 h, cells were harvested and assayed for CAT activity. CAT activity (mean Ϯ S.E.) for Me 2 SO-treated cells transfected with S14CAT124 and RSVCAT was 3207 Ϯ 625 units, n ϭ 27, and 53972 Ϯ 3270 units, n ϭ 6, respectively. The data are represented as Relative CAT Activity (mean Ϯ S.E.), which is the change in CAT activity induced by WY14643, mPPAR␣, or the combination of treatments relative to the Me 2 SO-treated cells. The structure of S14CAT124 is shown in Fig. 2A. Statistical analysis was by analysis of variance. S14CAT124: a, WY14643 versus Me 2 SO, p Ͻ 0.001; b, PPAR versus Me 2 SO, p Ͻ 0.001; c, PPAR versus PPAR ϩ WY14643, p Ͻ 0.001; RSVCAT: d, WY14643 versus Me 2 SO, p Յ 0.02. S14TRR-The cis-regulatory elements targeted by PPAR␣ and WY14643 were localized by deletion analysis using a strategy similar to the one used to localize the nPUFA-RE within the S14 proximal promoter region (3). Fig. 2A illustrates the organization of functional elements controlling S14 gene transcription and the promoter deletion constructs used in this study. The S14 gene has three major functional cis-regulatory regions. The proximal promoter (Ϫ290/ϩ19 bp) contains the basal elements required to initiate gene transcription and to confer tissue specificity. This region also contains targets for PUFAmediated suppression of transcription (3,44). The pluripotent response region (PRR, Ϫ1.6/Ϫ1.4 kb) contains cis-acting regulatory elements required for tissue-specific, insulin/carbohydrate, and glucocorticoid control of transcription (2). The thyroid hormone response region (TRR, Ϫ2.9/Ϫ2.5 kb) contains three functional thyroid hormone response elements (TRE) that bind TR␤1/RXR heterodimers and are required for the T 3 -mediated transactivation of the S14 gene (45).
A series of promoter deletions was prepared in which the S14TRR region was retained to ensure high transcriptional activity and to allow for an examination of an inhibitory effect of WY14643 and PPAR␣ on S14CAT activity (Fig. 2B). As   FIG. 2. Deletion analysis of the S14 promoter. A, schematic representation of the 5Ј regulatory region controlling the S14 gene transcription. TRR, thyroid hormone response region (at Ϫ2.8 to Ϫ2.5 kb); PRR, pluripotent response region (at Ϫ1.6 to Ϫ1.4 kb); PIC, preinitiation complex binding at the TATA-box; NF1, B and C represent tissue-specific transcription factor binding sites (44); PUFA-RR, PUFA response region (at Ϫ220/Ϫ80 bp). The deletion constructs are shown numbered, 124, 149, 155, 156, and 158. Each construct contains the same S14TRR upstream from the S14 promoter element. The 3Ј end point (at ϩ19 bp) is common to all constructs; the 5Ј end points vary (at Ϫ290, Ϫ220, Ϫ120, and Ϫ80 bp, respectively). B, primary hepatocytes were co-transfected with various promoter constructs and MLVTR␤1 as described under "Experimental Procedures." These cells were treated with T 3 and Me 2 SO (open bars) or T 3 and 100 M WY14643 (solid bars). Half of the hepatocytes also received pSG5-PPAR␣ (0.2 g/culture) and were treated with either Me 2 SO (hatched bars) or WY14643 (horizontally striped bars). T 3 -stimulated CAT activity for control (Me 2 SO)-treated cells transfected with S14CAT124, -149, -155, -156, or -158 was 2293 Ϯ 220, 1799 Ϯ 255, 547 Ϯ 17, 633 Ϯ 76, and 69 Ϯ 8.2 CAT units, respectively. Results are expressed as Relative CAT Activity (see Fig. 1) (mean Ϯ S.E., N Ն 6). An analysis of variance test for statistical significance of the effect of WY14643, PPAR␣, or the combination of these treatments, all were p Ͻ 0.001. shown in Fig. 1, both WY14643 and PPAR␣ inhibited S14CAT124 by ϳ50%, and the combination inhibited S14CAT124 activity by 85%. This same pattern of control was seen with all S14 deletion constructs suggesting that the PRR (at Ϫ1.6/Ϫ1.4 kb) or the PUFA-RE (at Ϫ220/Ϫ80 bp) was not involved in WY14643/PPAR␣-mediated control of transcription.
The minimal elements required for the S14 gene to be responsive to WY14643 and PPAR␣ were the elements within the proximal promoter region (Ϫ80/ϩ19 bp) containing a TATA box and an NF-1 site and the upstream TRR. The S14TRR was tested as the prospective target of WY14643/PPAR␣ control by fusing this element (Ϫ2.9/Ϫ2.5 kb) to the heterologous thymidine kinase promoter (TKCAT222). The specificity of WY14643/PPAR␣ effects on transcription was examined by comparing the CAT activity of TKCAT222 with that of: 1) an enhancer-less TKCAT fusion gene (TKCAT202); 2) a S14CAT fusion gene containing the S14TRR fused upstream from the Ϫ290/ϩ19-bp region of the S14 promoter (S14CAT149); and 3) a TKCAT fusion gene containing the AOX-PPRE, TKCAT223 (Fig. 3A). TKCAT202 was not significantly affected by WY14643 or co-transfected PPAR␣ (Fig. 3B). Inserting the S14TRR upstream from the TK promoter conferred high levels of T 3 -induction (Ͼ50-fold) of CAT activity (see legend for Fig. 3 and Ref. 3). Both WY14643 and PPAR␣ and the combination of these treatments inhibited TKCAT222 by ϳ50% and Ͼ90%, respectively. This pattern of control is identical with that seen with S14CAT149 and suggests that the S14TRR is the target of WY14643/PPAR␣ action.
In contrast to TKCAT222, TKCAT223, which contains the AOX-PPRE upstream from the TK promoter, was only marginally induced by WY14643 (2-fold), but significantly induced by co-transfected PPAR␣ (56-fold). The combination of mPPAR␣ co-transfection and WY14643 enhanced CAT activity by 140fold (Fig. 3C). These results indicate that the direction of control and sensitivity to WY14643 and PPAR␣ regulation is enhancer-dependent. While the S14TRR confers negative control, the AOX-PPRE confers positive control to the TKCAT fusion gene following WY14643/PPAR␣ treatment. These studies confirm and extend the deletion studies (Fig. 2) by showing that the S14TRR is sufficient and necessary for the negative effect of WY14643/PPAR␣ on the S14 or TK promoter activity. We previously established that the nPUFA-RE was localized to the Ϫ220/Ϫ80-bp region (Ref. 3 and Fig. 2A). The current studies show that the cis-regulatory elements for WY14643/ PPAR␣ and PUFA control of S14 gene transcription are functionally and spatially distinct.
mPPAR␣ Does Not Bind the S14TRR Directly-Gel shift analysis was used to determine if PPAR␣-mediated effects on S14 gene transcription were due to direct binding to the S14TRR. Using the AOX-PPRE as a positive control for binding, neither PPAR␣ nor RXR␣ alone binds the AOX-PPRE (Fig.  4). However, the combination of these receptors bind as a heterodimer. This observation is consistent with previous reports (13,16,19,42). Addition of a 100-fold molar excess of unlabeled AOX-PPRE effectively competes for the formation of the PPAR␣⅐RXR complex. In contrast, a 100-fold molar excess of the S14TRR failed to compete for binding (Fig. 4). No competition was seen with a 500-fold molar excess of TRR (not shown). The S14TRR region contains 3 TREs, which consist of direct repeats of AGGTCA-related motifs separated by 4 nucleotides (45). These elements, also known as far upstream regulatory elements (FUR 10, 11, and 12) did not compete for PPAR␣/RXR␣ binding (data not shown). PPAR␣/RXR␣ also did not bind directly to a canonical DRϩ4 (gatcctcAGGTCAcag-gAGGTCAgag, see Fig. 6). These studies show that PPAR␣, either alone or with RXR␣, does not bind the S14 FUR elements or other DNA elements within the Ϫ2.9to Ϫ2.5-kb S14TRR.
PPAR␣ Suppresses Hepatic S14 Gene Expression by Functionally Interfering with TR/RXR Action-Since PPAR␣ did not interact directly with the TRR, we speculated that PPAR␣ might affect T 3 action indirectly. A recent report showed that co-transfected PPAR effects on T 3 -dependent gene transcription were eliminated by elevating cellular RXR levels (46). To determine if PPAR␣ action on S14CAT activity was affected by hepatocellular levels of other receptors, additional TR␤1 (as MLVTR␤1) or RXR␣ (as pSG5-RXR␣) was co-transfected with a constant amount of pSG-PPAR␣ (0.5 g/well) and TKCAT222 FIG. 3. The S14TRR is the target for WY14643 and PPAR␣ negative control of S14 gene transcription. A, schematic representation of the different CAT fusion genes. TK, thymidine kinase proximal promoter; AOX-PPRR, the peroxisome proliferator response region of the rat acyl-CoA oxidase gene. B and C, hepatocytes were co-transfected with reporter genes (2 g) and the pMLVTR␤1 (1 g) and were treated with T 3

and either Me 2 SO (open bars) or 100 M WY14643 (solid bars).
Half of the hepatocyte cultures were also co-transfected with 0.2 g of pSG5-PPAR␣, then treated with T 3 and either Me 2 SO (hatched bars) or WY14643 (horizontally striped bars). CAT activity for Me 2 SO-treated cells transfected with TKCAT202, TKCAT222, and S14CAT149 (B) or TKCAT223 (C) was 45 Ϯ 11, 4732 Ϯ 720, 1910 Ϯ 94, and 41 Ϯ 8.8 units, respectively. Relative CAT activity was calculated as described in Fig.  1 (mean Ϯ S.E., N Ն 6). Statistical analysis was by analysis of variance. There was no significant difference between treatments in cells transfected with TKCAT202. For cells transfected with TKCAT222 and S14CAT149: a, WY14643 versus Me 2 SO; b, PPAR␣ versus Me 2 SO; and c, WY14643 ϩ PPAR␣ versus PPAR␣, p Ͻ 0.001. For cells transfected with TKCAT223: a, WY14643 versus Me 2 SO was not significant; b, PPAR␣ versus Me 2 SO, p Ͻ 0.001; and c, PPAR␣ ϩ WY14643 versus PPAR␣ p Ͻ 0.001.
(1 g/well). While co-transfected TR␤1 is required for T 3 control of transfected TKCAT222, increasing hepatocellular levels above the 1 g/well level did not enhance T 3 activation or affect PPAR␣-mediated inhibition of TKCAT222 activity (Fig. 5).
Co-transfected RXR␣ is not required for T 3 -mediated control of TKCAT222 activity in hepatocytes co-transfected with MLVTR␤1. However, increasing hepatocellular RXR␣ levels by co-transfecting pSG5-RXR␣ (at 1 g/well) was sufficient to override the inhibitory effect of PPAR␣ on TKCAT222 activity (Fig. 5). This pattern of control suggests that RXR might be limiting in primary hepatocytes.
To determine how this interference might occur, gel shift analysis was used to examine the effect of PPAR␣ on TR␤ binding to a DRϩ4. While RXR␣ fails to bind a DRϩ4, TR␤ binds as a monomer (Fig. 6). Addition of both TR␤ and RXR␣ yields a heterodimer (Fig. 6). Addition of PPAR␣ inhibited heterodimer formation and favored TR monomer formation. Addition of unprogrammed reticulolysate lysate had no effect on binding of TR␤ as monomers or TR␤/RXR␣ heterodimers. Thus, PPAR␣ interferes with TR/RXR binding to DRϩ4. This observation essentially confirms and extends the report by Juge-Aubry et al. (46) by showing that PPAR␣ inhibits TR␤/ RXR␣ binding to a DRϩ4 (Fig. 6) or TRE-PAL (46). DISCUSSION Dietary polyunsaturated fatty acids (PUFA) suppress transcription of genes encoding hepatic lipogenic enzymes (1)(2)(3)(4) and induce expression of genes encoding peroxisomal enzymes (38 -41). The fact that PPARs are activated by long chain fatty acids as well as peroxisome proliferators suggests PPAR might regulate both pathways (11, 12, 21, 24, 26, 34 -37, 47-49). We initiated this study with two goals in mind; first, to determine whether PPAR␣ was the mediator of PUFA regulation of hepatic lipogenic gene transcription, and second, to define the molecular basis of WY14643 and PPAR inhibition of S14 gene transcription.
For PPAR␣ to be a mediator of PUFA action, two criteria must be satisfied: 1) both PPAR␣ and its activators must inhibit S14 gene transcription, and 2) the cis-regulatory targets for PPAR␣ and its activators must map to the PUFA-RE within the S14 promoter. Mapping the nPPRE to the nPUFA-RE would provide strong evidence for PPAR␣ serving as the mediator of PUFA action. We found that the nPPRE and nPUFA-RE within the S14 gene are functionally and spatially distinct.
This observation makes it difficult to envision how PPAR␣ can function as the common mediator for both PUFA and peroxisome proliferator control of hepatic lipogenic gene expression.
Recent homologous recombination studies show that PPAR␣ is the predominant subtype expressed in rodent liver, and this subtype accounts for the peroxisome proliferator regulation of several enzymes involved in lipid metabolism (27). Fatty acids appear to be activators of PPAR␣ only under conditions of lipid overload, which occurs following peroxisome proliferator treatment, high fat feeding, diabetes mellitus, starvation, or pathophysiological states when hepatic mitochondrial ␤-oxidation is suppressed, i.e. alcoholic liver disease (27,35,36). PUFA me- FIG. 5. Co-transfection of RXR␣ eliminates PPAR␣ inhibitory effect on S14TRR activity. Hepatocytes were co-transfected with TKCAT222 (2 g) and MLVTR␤ (ranging from 1 to 2 g/well). Cells were also co-transfected without (open bars) or with pSG5-PPAR␣ (0.5 g/well) (solid bars). All cells received T 3 to induce TKCAT222 activity. The results were pooled from 3 separate experiments and are expressed as CAT Activity, Units (mean Ϯ S.D., n ϭ 9). Statistical analysis of the data used Student's t test: a, PPAR␣ versus control, p Ͻ 0.001. diated suppression of lipogenic gene transcription is rapid, occurs within hours of PUFA administration (1)(2)(3)(4), and precedes changes in acyl-CoA oxidase mRNA levels. 2 Other arguments against PPAR␣ as the common mediator for PUFA and peroxisome proliferator action include the finding that PPARs are activated by monounsaturated and polyunsaturated fatty acids and to a lesser extent by saturated fatty acids (12,20,41). Lipogenic enzyme gene expression is suppressed by PUFA, but not affected by saturated or monounsaturated fatty acids (1)(2)(3)(4)50). Certain peroxisome proliferators, like nafenopin, bezafibrate, and MEDICA 16, actually stimulate lipogenic as well as peroxisomal enzyme gene expression (51). Taken together, these studies suggest that fatty acids might regulate two pathways. One involves PPAR␣ and may function in states of lipid overload. The other pathway involves ill-defined PUFA-regulatory factors (PUFA-RF) that are activated by PUFA ingestion. In contrast to PPAR␣, PUFA-RF do not target the S14TRR (3).
The second part of this study focused on defining the molecular basis of PPAR␣ inhibition of S14 gene transcription. The transfection and gel shift studies show that PPAR␣ and activators of PPAR␣, like WY14643, target the S14TRR. The mechanism of inhibition appears to be due to an interference of PPAR␣ with TR␤/RXR␣ function at the TREs. The gel shift studies indicated that PPAR␣ inhibited TR␤/RXR␣ binding and that co-transfected RXR␣ abrogated the PPAR␣-mediated inhibition of T 3 activation of S14 gene transcription (Figs. 5 and 6).
The interaction of PPAR␣ with other transcription factors has been reported previously (50 -55). For example, PPAR/RXR interact with Sp1 to synergistically induce AOX gene transcription (52). In contrast, competitive binding of PPAR/RXR heterodimers to estrogen response elements in the vitellogenin A2 promoter or to the HNF4 site in the apolipoprotein CIII promoter lead to inhibition of transcription (53,54). Both these examples require PPAR/RXR to bind PPREs to exert their effect on gene transcription. Other reports indicate that PPAR effects on T 3 -regulated gene transcription may not involve direct DNA binding. Bogazzi et al. (55) first reported that PPAR co-transfection interfered with T 3 control of malic enzyme and thyroid-stimulating hormone ␤1 gene transcription. Their studies suggested that PPAR␣ heterodimerized with TR␤ to form inactive complexes that prevent TR/RXR binding to DNA. In contrast, Juge-Aubry et al. (46) showed that PPAR␣ interfered with T 3 -regulated gene transcription by forming heterodimers with RXR␣ in solution. The interaction between PPAR and RXR was not dependent on PPAR/TR heterodimerization or competition for DNA binding. Under these circumstances, endogenous RXR or TR auxiliary proteins were limiting. We show that overexpression of RXR␣, but not TR␤1, overrides the negative effect of PPAR␣ on TKCAT reporter genes containing the S14TRR (Fig. 5), and that PPAR␣ interfered with the formation of TR/RXR heterodimer formation on S14TRE (FUR 10, 11, and 12) and a canonical DRϩ4 (Fig. 6). Our findings are consistent with the report by Juge-Aubry et al. (46) and suggest that, under the conditions of hepatocyte transfection, RXR is limiting. PPAR␣ inhibits S14 gene transcription by inhibiting TR/RXR heterodimer formation on the S14TREs. The finding that WY14643 acting through PPAR␣ affects T 3 receptor action suggests that under conditions of hepatic lipid overload, i.e. starvation and diabetes, PPAR␣ may play an important role in modulating T 3 regulation of hepatic lipogenic gene expression.