Regulation of Pyruvate Dehydrogenase Kinase 4 (PDK4) by CCAAT/Enhancer-binding Protein β (C/EBPβ)

The conversion of pyruvate to acetyl-CoA in mitochondria is catalyzed by the pyruvate dehydrogenase complex (PDC). Activity of PDC is inhibited by phosphorylation via the pyruvate dehydrogenase kinases (PDKs). Here, we examined the regulation of Pdk4 gene expression by the CCAAT/enhancer-binding protein β (C/EBPβ). C/EBPβ modulates the expression of multiple hepatic genes including those involved in metabolism, development, and inflammation. We found that C/EBPβ induced Pdk4 gene expression and decreased PDC activity. This transcriptional induction was mediated through two C/EBPβ binding sites in the Pdk4 promoter. C/EBPβ participates in the hormonal regulation of gluconeogenic genes. Previously, we reported that Pdk4 was induced by thyroid hormone (T3). Therefore, we investigated the role of C/EBPβ in the T3 regulation of Pdk4. T3 increased C/EBPβ abundance in primary rat hepatocytes. Knockdown of C/EBPβ with siRNA diminished the T3 induction of the Pdk4 and carnitine palmitoyltransferase (Cpt1a) genes. CPT1a is an initiating step in the mitochondrial oxidation of long chain fatty acids. Our results indicate that C/EBPβ stimulates Pdk4 expression and participates in the T3 induction of the Cpt1a and Pdk4 genes.

Regulation of gene expression by steroid hormones involves the interaction of transcription factors, nuclear receptors, and coactivators (1). In this study, we investigated the role of the CCAAT/enhancer-binding protein ␤ (C/EBP␤) 4 in the regulation of pyruvate dehydrogenase kinase (PDK) and carnitine palmitoyltransferase (CPT1a) genes. C/EBPs constitute a family of transcription factors with multiple members including C/EBP␣, C/EBP␤, and others (2,3). C/EBP isoforms share two highly conserved domains, the C-terminal basic DNA binding domain and the leucine zipper domain as well as the less homologous N-terminal activation domain (4). C/EBP␤ is highly expressed in liver, adipose tissue, intestine, lung, and others (3,4). In the liver, C/EBP␤ stimulates genes encoding gluconeogenic enzymes including phosphoenolpyruvate carboxykinase (PEPCK) (5-7) as well as genes involved in hepatic lipogenesis such as fatty acid synthase (FASN) and acetyl-CoA carboxylase (8,9). Insulin and glucocorticoids regulate transcription of C/EBP␤ and the binding of C/EBP␤ alternate translation proteins to gluconeogenic genes (10 -12).
Thyroid hormone (T 3 ) plays an important role in various aspects of metabolism and development (13). The genomic actions of T 3 are mediated through the binding of thyroid hormone receptors (TRs) to T 3 -response elements (TREs) (14). Two TR isoforms (TR␣ and TR␤) are encoded on separate genes with TR␤ being the more abundantly expressed isoform in the liver (14). Liganded TR stimulates transcription through the recruitment of various coactivators as well as via interactions with other transcription factors (1,15). T 3 increases the expression of genes involved in hepatic fatty acid oxidation, especially Cpt1a (16). T 3 elevates hepatic triglyceride production as well as a range of genes involved in hepatic lipogenesis and low density lipoprotein receptor expression (17)(18)(19). With respect to glucose metabolism, T 3 stimulates gluconeogenesis by elevating the transcription of the gluconeogenic enzymes, glucose-6-phosphatase and PEPCK (20 -22).
It was observed by Menéndez-Hurtado et al. (23) that hypothyroidism in pregnant rats led to a decrease in both C/EBP␣ and C/EBP␤ hepatic gene expression in the pups during postnatal development. In addition, the hypothyroid neonatal pups had diminished C/EBP␣ and C/EBP␤ protein levels. This reduction in C/EBP expression was confined to the liver because C/EBP mRNA levels in brown fat were unchanged. Injection of hypothyroid animals with T 3 resulted in the recovery of C/EBP␣ and C/EBP␤ mRNA levels in the liver. Studies from our laboratory showed that C/EBP␣ was needed for the induction of Pepck by T 3 (24). Other investigators have shown that C/EBP␣ participates in the actions of T 3 in liver, kidney, and brown adipose tissue (25)(26)(27). The C/EBP␤ isoform has not been examined with respect to its role in T 3 action. Therefore, we investigated the role of C/EBP␤ in induction of Pdk4 and Cpt1a by T 3 .
The pyruvate dehydrogenase complex (PDC) catalyzes the oxidative decarboxylation of pyruvate into acetyl-CoA. The phosphorylation of PDC on three serine residues of the E1␣ subunit by PDK inhibits PDC activity (28,29). The abundance of the PDK4 isoform is transcriptionally controlled (30). Expression of the Pdk4 gene is increased by T 3 , glucocorticoids, retinoic acid, and long chain fatty acids, whereas it is inhibited by insulin (30,31). We recently identified two TREs in the Pdk4 gene promoter and demonstrated that the peroxisome proliferator-activated receptor ␥ coactivator (PGC-1␣) enhances the T 3 induction of the Pdk4 expression (32). CPT1a catalyzes the transfer of fatty acids from long chain acyl-CoA to carnitine and is an initiating step in the translocation of long chain fatty acids across the mitochondrial membranes (33). PGC-1␣ also enhances the induction of Cpt1a by T 3 (34).
In this study, we characterized the role of C/EBP␤ in the regulation of Pdk4 gene expression. Our data indicate that C/EBP␤ strongly stimulates the expression of the Pdk4 gene through multiple sites in the promoter. In addition, C/EBP␤ participates in the induction of Pdk4 expression by T 3 , and the abundance of C/EBP␤ is elevated by T 3 . Moreover, C/EBP␤ is involved in the T 3 stimulation of the Cpt1a gene, suggesting that C/EBP␤ can affect the hormonal regulation of genes involved in glucose and fatty acid metabolism.

MATERIALS AND METHODS
Transient Transfection of Luciferase Vectors-Pdk4-luciferase constructs (Pdk4-luc) were transiently transfected into HepG2 cells by the calcium phosphate method as described previously (34). Pdk4-luc was transfected with the expression vectors SV40-TR␤, MSV-C/EBP␤, and TK-Renilla. Cells were transfected in DMEM containing 5% calf serum and 5% FCS. After overnight incubation, the medium was replaced by DMEM without serum, and the cells were treated with 100 nM T 3 for 24 h. Transfected cells were harvested in passive lysis buffer (Promega, Madison, WI). Luciferase and Renilla luciferase activity was measured with the Promega Dual-Luciferase reporter kit (E1980). Protein content in each lysate was determined by Pierce BCA protein assay kit (Thermo Scientific). Luciferase activity was corrected for both protein content and Renilla luciferase activity.
Western Analysis-Western analysis was performed on whole cell extracts from primary rat hepatocytes (37). Hepatocytes were lysed in radioimmune precipitation buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5% deoxycholate, 5 mM EDTA, pH 8.0, 0.1% SDS, and diluted protease inhibitor mixture). The cells were vortexed for 1 min, and cell membranes were removed by centrifugation for 25 min at 4°C. An equal amount of protein was loaded on a 12% SDS-PAGE gel and transferred to a 0.45-m nitrocellulose membrane (Bio-Rad). The membranes were immunoblotted with primary antibodies C/EBP␤ (sc-150, Santa Cruz Biotechnology, Santa Cruz, CA) in PBS containing 5% nonfat dry milk powder and were incubated with HRP-conjugated anti-rabbit secondary antibody. Immunoreactive proteins were detected using SuperSignal West Femto chemiluminescent substrate (Thermo Scientific). The ChemiDoc TM XRS gel documentation system (Bio-Rad) was used to quantify the immunoreactive proteins. ␤-Actin (sc-1615, Santa Cruz Biotechnology) was used as the loading control for each lane. Determination of PDC Activity-The PDC activity was measured with the Dipstick assay kit from MitoSciences (MSP30, Eugene OR). The validity of the assay has been demonstrated previously (38,39). Cells transduced with 50 pfu/cell Ad-C/ EBP␤ or Ad-GFP were scraped from plates in the provided sample buffer and pelleted by centrifugation. Following the kit protocol, equal amounts of protein were added to plate wells, and dipsticks were inserted into the wells. The intensity of the bands representing active PDC was quantified densitometrically using Alphaimager EP and AlphaView SA software (Cell Biosciences, Santa Clara, CA).
Real-time PCR-The cDNA for real-time PCR was prepared using RNA isolated from primary rat hepatocytes. RNA was isolated with RNA-Stat-60 (Tel-test, Friendswood, TX) (40). The RNA was treated with DNase I (2 units) at 37°C for 1 h followed by the addition of DNase inactivation reagent (Ambion, Austin, TX), and the concentration of each sample was measured using NanoDrop (Thermo Scientific). Two g of DNA-free RNA was converted to cDNA using SuperScript reverse transcriptase III and random hexamers (Invitrogen). The parameters for real-time PCR were as follows: 95°C for 10 min, 40 cycles of 95°C for 30 s and 60°C for 1 min. The final concentration of primers in each well in the PCR plates was 0.1 M. The target genes were normalized with the 18S gene. The following FPs and RPs were used for real-time PCR: PDK4 FP, 5Ј-ggattactgaccgcctctttagtt-3Ј; PDK4 RP, 5Ј-gcattccgtgaattgtccatc-3Ј; CPT1a FP, 5Ј-cggttcaagaatggcatcatc-3Ј; CPT1a RP, 5Ј-tcacacccaccaccacgat-3Ј; 18S FP, 5Ј-cggctaccacatccaaggaa-3Ј; 18S RP, 5Ј-ttttcgtcactacctccccg-3Ј. Primers for C/EBP␤ and FASN were obtained from Qiagen (41) (QT00366478 and QT00371210, respectively).
Chromatin Immunoprecipitation Assays-ChIP assays were conducted with modifications following the protocol given by the Millipore Magna ChIP kit (17-610) (42). Rat primary hepatocytes were maintained for 18 h in RPMI 1640 medium containing 5% fetal bovine serum and 5% calf serum. Cells were  A, HepG2 cells were transiently transfected with Ϫ1256/ϩ78 rat Pdk4-luciferase, MSV-C/EBP␤, and TK-Renilla. The potential C/EBP␤ binding sites were disrupted. All transfections were performed in duplicate and repeated 4 times. Results are expressed as the relative percentage of induction of Pdk4 by C/EBP␤ Ϯ S.E. Significance is calculated relative to the wild type rat Pdk4 Ϫ1256/ϩ78 luciferase (*, p ϭ 0.01-0.05, **, p ϭ 0.001-0.01). B, C/EBP␤ binding to the promoter sites was tested with gel shift mobility assays. Gel shift assays were conducted as described under "Materials and Methods." Sequences for the wild type C/EBP␤ binding sites (Ϫ96) and (Ϫ629) oligomer (wt oligo) or the mutated version (mut) are shown below the gel shift with the mutated nucleotides underlined. Ab, antibody; RLNE, rat liver nuclear extract. treated with 100 nM T 3 overnight in serum-free medium. After treatment, cross-linking was performed with 1% formaldehyde for 10 min at room temperature, and cells were sonicated as described previously (42). Chromatin preparations were diluted with dilution buffer and protease inhibitor mixture and designated as input samples (no antibody) or immunoprecipitated with the control antibody rabbit IgG (sc-2027, Santa Cruz Biotechnology), anti-C/EBP␤ (sc-150, Santa Cruz Biotechnology), or anti-TR␤ (MA1-216, Thermo Scientific). Samples were mixed overnight at 4°C with the antibody and magnetic protein A beads. The beads were washed, and the DNA was eluted. Eluted DNA was purified using the PCR purification kit (Qiagen 28104). DNA was subjected to 35 cycles of PCR using 3-6 l of DNA. PCR products were analyzed on 2% NuSieve 3:1 agarose (Lonza, Walkersville, MD) and visualized with Multi-Image light cabinet with Alphaimager EP software. The following primers were used to amplify portions of the Pdk4 promoter: the proximal Pdk4 promoter (Ϫ591/Ϫ338) FP 5Јtaaggctatttaggcagttt-3Ј and RP 5Ј-ccagacttgtccttgtttac-3Ј, the TRE (Ϫ1535/Ϫ1228) FP 5Ј-agtgtctccaccagattgt-3Ј and RP 5Јctaagagagctaacctagt-3Ј, and the upstream region (Ϫ6634/ Ϫ6377) FP 5Ј-tatgagaagtgctgcaataa-3Ј and RP 5Ј-atgttaccacaaaccttcat-3Ј (42).
Knockdown of C/EBP␤ in Hepatocytes-Adenoviruses encoding shRNA specific for C/EBP␤ (Ad-siC/EBP␤) and control adenoviruses encoding non-template shRNA were constructed as described previously (43). Hepatocytes were plated at a density of 3 ϫ 10 6 in a 60-mm dish in RPMI 1640 medium. The adenoviruses were added at a multiplicity of infection of 50. Media were changed 24 h after transduction, and the cells were treated with 100 nM T 3 for another 24 h in serum-free RPMI 1640 before harvesting the cells.

RESULTS
Induction of Pdk4 by C/EBP␤-Previous studies from our laboratory and others found that C/EBP␤ participates in both the basal expression and the hormonal regulation of gluconeogenic genes (6,10,44). To test whether C/EBP␤ would induce the Pdk4 gene, we overexpressed C/EBP␤ in McA-RH7777 hepatoma cells by adenoviral infection. C/EBP␤ increased the abundance of Pdk4 mRNA 9.8-fold (Fig. 1A). The activity of PDC was decreased 48%, suggesting that the increase in PDK4 was inhibiting PDC (Fig. 1B). In addition, we measured Cpt1a mRNA as fatty acid oxidation is often increased as glucose oxidation is diminished. Cpt1a mRNA abundance was increased slightly (Fig. 1A).

Identification of C/EBP␤-responsive Elements in the Rat Pdk4
Promoter-To investigate the ability of C/EBP␤ to induce the Pdk4 promoter, we cotransfected serial deletions of the rat Pdk4 promoter driving a luciferase reporter with or without MSV-C/EBP␤ in HepG2 hepatoma cells and measured luciferase activity. Deletion of 210 nucleotides between Ϫ788 and Ϫ578 of the rat Pdk4 promoter reduced C/EBP␤ responsiveness from 7.6 Ϯ 0.7-fold to 3.3 Ϯ 0.5-fold. In addition, deletion of the 255 nucleotides between Ϫ325 and Ϫ70 of the rat Pdk4 promoter diminished C/EBP␤ responsiveness from 4.4 Ϯ 0.8to 1.00 Ϯ 0.3-fold (Fig. 2). These data indicate that C/EBP␤ induced Pdk4 gene expression through two regions in the promoter.
To identify specific C/EBP␤-response elements, we conducted site-directed mutagenesis of putative C/EBP binding sites in the Ϫ1256/ϩ78 Pdk4-luciferase vector (Pdk4-luc). Mutation of two C/EBP␤ elements, localized between Ϫ634 to Ϫ626 and Ϫ96 to Ϫ87, decreased the ability of C/EBP␤ to induce the Pdk4 gene (Fig. 3A). Gel shift mobility assays were conducted to determine whether C/EBP␤ could bind these sites. Oligomers were incubated with rat liver nuclear extract and antibodies to C/EBP␤. In each case, a supershift was observed indicating that C/EBP␤ bound the site (Fig. 3B). Multiple proteins were able to bind to the Ϫ96 to Ϫ87 site as the C/EBP␤ antibody supershifted only a small amount of the DNA-protein complex.
T 3 Increases C/EBP␤ Abundance in Hepatocytes-We next investigated whether T 3 treatment could induce C/EBP␤ expression. Primary rat hepatocytes were treated with 100 nM T 3 for 24 h. C/EBP␤ mRNA abundance was elevated 2.2 Ϯ 0.4-fold (Fig. 4A). C/EBP␤ protein abundance was increased 1.8 Ϯ 0.3fold in response to T 3 treatment (Fig. 4B). This experiment demonstrated that the C/EBP␤ is a T 3 -responsive gene and raised the possibility that it might participate in the regulation of the Pdk4 gene by T 3 .

C/EBP␤ Enhances the T 3 Induction of the Pdk4 Gene-To investigate whether C/EBP␤ participated in the T 3 induction of
Pdk4, we cotransfected wild type Ϫ1256/ϩ78 Pdk4-luc and MSV-C/EBP␤ into HepG2 cells with or without T 3 . The addition of 100 nM T 3 stimulated wild type Ϫ1256/ϩ78 Pdk4-luc 4.7 Ϯ 1-fold. In these experiments, overexpression of C/EBP␤ induced the Pdk4-luc vector 5.3 Ϯ 1-fold. When T 3 was added in the presence of C/EBP␤, a synergistic 16.9 Ϯ 2-fold induction was observed (Fig. 5A). These data indicate that C/EBP␤ can activate the Pdk4 gene promoter and amplify the T 3 effect on the Pdk4 promoter. In addition, we tested the Pdk4-luc vector in which both C/EBP binding sites were disrupted (Fig. 5A). This vector was modestly induced by C/EBP␤, suggesting that an additional C/EBP␤ binding site is present in the promoter.
The T 3 induction was reduced in the vector with disrupted C/EBP␤ binding sites, indicating that C/EBP␤ participates in the T 3 action.
To further investigate the role of C/EBP␤ in the induction of Pdk4 transcription, we tested a set of dominant negative vectors. The dominant negative C/EBP vector, A-C/EBP, has the C/EBP leucine zipper attached to an acidic amphipathic helix. This helix interacts with the basic region of C/EBP proteins to form a non-DNA binding heterodimer. For controls, we used A-CREB, which is a dominant negative CREB protein (45), and the dominant negative Jun vector A-Fos (45). The Ϫ1256/ϩ78 Pdk4-luc vector was cotransfected with the dominant negative vectors into HepG2 cells, and luciferase activity was assessed. A-C/EBP reduced basal

Regulation of Pdk4 Gene Expression by C/EBP␤
expression of the Pdk4 gene by 32% (p ϭ 0.02), whereas the A-Fos did not affect the basal level of the Pdk4 gene. A-CREB increased the Pdk4-luciferase activity (p ϭ 0.0007) (Fig. 5B), although the reason for this increase is not clear. In addition, A-C/EBP abolished the ability of T 3 to induce Pdk4-luciferase (Fig. 5C). In previous studies, we demonstrated that C/EBP␣ bound to the intron of the Cpt1a gene (46). Cpt1a is a T 3 -responsive gene, so we examined the effect of A-C/EBP on the T 3 induction of Cpt1a. A-C/EBP reduced the T 3 induction of Cpt1a-luciferase, suggesting that C/EBP␤ is important in the T 3 responsiveness of multiple genes (Fig. 5D).
Knockdown of C/EBP␤ Reduces Pdk4 Gene Expression in Primary Rat Hepatocytes-To examine the role of the C/EBP␤ in the T 3 induction of the endogenous Pdk4 gene, we infected rat primary hepatocytes with adenoviruses encoding shRNA to silence C/EBP␤ (Ad-siC/EBP␤). The hepatocytes were infected for 16 h prior to the addition of T 3 . Adenovirus encoding control shRNA that does not silence any rat genes was used as a control (Ad-siControl) (43). Ad-siC/EBP␤ knocked down the C/EBP␤ mRNA abundance by 50%. In addition, the induction of C/EBP␤ by T 3 was reduced in hepatocytes infected with Ad-siC/EBP␤ in comparison with the 2.2 Ϯ 0.4-fold induction of the T 3 -treated control cells (Fig. 6A). Knockdown of C/EBP␤ in primary hepatocytes decreased the T 3 induction of PDK4 as T 3 induced Pdk4 only by 1.2 Ϯ 0.2-fold, whereas T 3 increased Pdk4 in hepatocytes infected with Ad-siControl by 2.4 Ϯ 0.2fold (Fig. 6B). This inhibition was also observed with the Cpt1a gene expression as T 3 failed to induce Cpt1a following C/EBP␤ knockdown (Fig. 6C). Although the T 3 induction of Fasn was decreased by C/EBP␤ knockdown, the decrease did not reach statistical significance. These data indicate that C/EBP␤ is an important coregulator in the T 3 induction of the Pdk4 and Cpt1a genes.
Next, we tested whether T 3 altered the binding of C/EBP␤ to the Pdk4 gene. ChIP assays were conducted on the PDK4 promoter following T 3 treatment. We observed C/EBP␤ association with the Pdk4 promoter, but C/EBP␤ binding was not elevated, suggesting that T 3 does not increase C/EBP␤ association with the promoter (Fig. 7B). As a control to test whether we could observe elevated C/EBP␤ binding, we overexpressed C/EBP␤ by adenoviral infection and conducted ChIP assays. Under these conditions, we observed a 2-fold enhancement of C/EBP␤ association with the Pdk4 promoter (supplemental Fig. 1). These data suggested that the 2-fold induction of C/EBP␤ protein by T 3 might not generate sufficient increase in binding to be quantified in our ChIP assays at least at the time point at which the crosslinking was conducted. We also tested whether the knockdown of C/EBP␤ would result in decreased TR␤ recruitment to the Pdk4 promoter. Hepatocytes were infected with Ad-siC/EBP␤. Our ChIP assay results indicated that the knockdown of C/EBP␤ decreased the binding of TR␤ to the Pdk4 promoter (Fig. 7C). These data suggest that C/EBP␤ assists in recruitment of TR␤ to the Pdk4 gene.

DISCUSSION
T 3 controls multiple aspects of hepatic metabolism including fatty acid oxidation, lipogenesis, and glucose oxidation (47). T 3 stimulates Pdk4 gene expression and decreases PDC activity in the heart, liver, and skeletal muscle (48 -50). We recently reported that T 3 induces Pdk4 expression through two TREs in the Pdk4 promoter located 1,150 bp upstream of the transcriptional start site (32). Here, we investigated the role of C/EBP␤ in the basal and T 3 -regulated expression of the Pdk4 gene. Our data demonstrate that C/EBP␤ stimulates Pdk4 expression. In addition, C/EBP␤ enhances the T 3 induction of Pdk4 and Cpt1a. Our results suggest that C/EBP␤ will contribute to the hormonal regulation of a subset of hepatic genes.
In addition to activating the TR, T 3 can affect transcription by altering the abundance and activity of transcription factors and coactivators. For example, we and others found that T 3 increased the levels of the transcriptional coactivator PGC-1␣ (21,34). We also observed elevated association of PGC-1␣ with the Pdk4 and Cpt1a genes following T 3 administration (32,34). To our knowledge, only one study has investigated the effect of T 3 on the abundance of C/EBP proteins. Menéndez-Hurtado et al. (23) reported that both C/EBP␣ gene expression and C/EBP␤ gene expression were decreased in livers of pups delivered from hypothyroid rats. Administration of T 3 to the hypothyroid animals increased the hepatic C/EBP␣ and C/EBP␤ mRNA and protein levels (23). In our studies, we observed that the addition of T 3 to hepatocytes induced C/EBP␤ gene expression and protein abundance. Surprisingly, T 3 did not increase the association of C/EBP␤ with the Pdk4 gene in the ChIP assays. It seems that the mechanism by which C/EBP␤ enhances T 3 responsiveness does not involve increased C/EBP␤ binding. C/EBP␤ participates in the glucocorticoid induction of the Pepck gene (12). However, increased C/EBP␤ binding was not observed on the Pepck gene following the addition of dexamethasone (10).
Overexpression of C/EBP␤ increased the expression of Pdk4, suggesting that C/EBP␤ regulates this gene. Previous studies indicated that C/EBP␤ knock-out mice had altered regulation of gluconeogenic and lipogenic genes following various dietary manipulations (8,52). However, knocking out C/EBP␤ did not reduce significantly the basal levels of Pdk4 and Cpt1a mRNA in fed animals (52). The expression of PGC-1␣, an important coactivator of Pdk4 and Cpt1a, was reduced in C/EBP␤ knock-out mice (52). We also found that Pdk4 and Cpt1a mRNA levels were not decreased in the FIGURE 7. C/EBP␤ is associated with the rat Pdk4 gene promoter in vivo. A, a model of the Pdk4 promoter with the locations of the ChIP primers is shown. B, chromatin immunoprecipitation assays were conducted on primary rat hepatocytes. Hepatocytes were treated with 100 nM T 3 for 24 h prior to cross-linking as described under "Materials and Methods." Antibodies to C/EBP␤ or immunoglobulin G (IgG) were used for immunoprecipitation. The amplified PCR products using primers for the proximal and upstream regions of the rat Pdk4 gene were resolved on agarose gels. The association of C/EBP␤ with Pdk4 proximal promoter was quantified using the Quantity One software. In the right panel, the data measure the relative association of C/EBP␤ with (ϩT 3 ) or without T 3 (ϪT 3 ). These data are the average Ϯ S.E. of four independent ChIP assays. C, ChIP assays were conducted on cells infected with adenovirus expressing Ad-NC (Ad-siControl) or Ad-siC/EBP␤. Antibodies to TR␤ or IgG were used for the immunoprecipitation. The amplified products were for the TRE region of the Pdk4 promoter. On the right, the data show the relative decrease in TR␤ binding and are the average of four independent ChIP assays. *, p ϭ 0.01-0.05. livers from C/EBP␤ knock-out mice (data not shown). These results are similar to previous reports showing that C/EBP␤ is not essential for the maintenance of basal Pepck mRNA levels in mice (6). However, C/EBP␤ deletion limited the full induction of Pepck and glucose-6-phosphatase genes by streptozotocin-induced diabetes (6). Our results and those of others suggest that C/EBP␤ is not essential for maintaining the basal levels of Pdk4 and Cpt1a but likely contributes to the hormonal regulation of these genes (6).
Earlier studies demonstrated that C/EBP␤ is extensively involved in the hormonal control of the Pepck gene by cAMP and glucocorticoids (12,51). In addition, the switching of the liver-enriched activator protein (LAP) and liver-enriched inhibitory protein (LIP) isoforms of C/EBP␤ contributes to the insulin inhibition of Pepck gene expression (10). We have shown that C/EBP␤ participates in the T 3 induction of the Pepck gene and that the transactivation domain of C/EBP␤ is needed (24). Here, we used multiple approaches to demonstrate that C/EBP␤ is required for the full T 3 induction of Pdk4 and Cpt1a. Overexpression of C/EBP␤ enhanced the T 3 induction. The dominant negative A-C/EBP diminished the T 3 induction of Pdk4 and Cpt1a. We used adenoviral-mediated silencing of the C/EBP␤. The knockdown of C/EBP␤ diminished the ability of T 3 to induce Pdk4, Cpt1a, and Pepck (data not shown) gene expression. C/EBP␤ knockdown modestly impacted the induction of Fasn. These data indicate that C/EBP␤ is an important coregulator for the T 3 induction of selected genes in primary hepatocytes. For both the Pdk4 and the Cpt1a genes, the C/EBP binding sites are not adjacent to the TRE. Our ChIP data suggest that the role of C/EBP␤ may be to enhance the association of TR␤ with the Pdk4 gene. In addition, the absence of C/EBP␤ may impair the recruitment or abundance of coactivators. In fact, it has been found that C/EBP␤ increases the expression of PGC-1␣, and we have shown that PGC-1␣ enhances the T 3 induction of Cpt1a and Pdk4.
Based on our studies, we conclude that C/EBP␤ induces the Pdk4 gene expression through two C/EBP␤-response elements in the Pdk4 promoter. T 3 increases the abundance of the C/EBP␤ but does not increase its association with the Pdk4 promoter. Our data suggest that C/EBP␤ is an important accessory factor for the T 3 activation of several hepatic genes.