Glucocorticoids Repress Transcription of Phosphoenolpyruvate Carboxykinase (GTP) Gene in Adipocytes by Inhibiting Its C/EBP-mediated Activation*

Yael OlswangDagger §, Barak BlumDagger §, Hanoch CassutoDagger §, Hannah CohenDagger , Yael BibermanDagger , Richard W. Hanson, and Lea ReshefDagger ||

From the Dagger  Department of Developmental Biochemistry, Hebrew University-Hadassah Medical School, Jerusalem, Israel 91120 and the  Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4935

Received for publication, January 9, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The cytosolic form of the phosphoenolpyruvate carboxykinase (PEPCK-C) gene is selectively expressed in several tissues, primarily in the liver, kidney, and adipose tissue. The transcription of the gene is reciprocally regulated by glucocorticoids in these tissues. It is induced in the liver and kidney but repressed in the white adipose tissue. To elucidate which adipocyte-specific transcription factors participate in the repression of the gene, DNase I footprinting analyses of nuclear proteins from 3T3-F442A adipocytes and transient transfection experiments in NIH3T3 cells were utilized. Glucocorticoid treatment slightly reduced the nuclear C/EBPalpha concentration but prominently diminished the binding of adipocyte-derived nuclear proteins to CCAAT/enhancer-binding protein (C/EBP) recognition sites, without affecting the binding to nuclear receptor sites in the PEPCK-C gene promoter. Of members of the C/EBP family of transcription factors, C/EBPalpha was the strongest trans-activator of the PEPCK-C gene promoter in the NIH3T3 cell line. The glucocorticoid receptor (GR), in the presence of its hormone ligand, inhibited the activation of the PEPCK-C gene promoter by C/EBPalpha or C/EBPbeta but not by the adipocyte-specific peroxisome proliferator-activated receptor gamma 2. This inhibition effect was similar using the wild type or mutant GR and did not depend on GR binding to the DNA. The glucocorticoid response unit (GRU) in the PEPCK-C gene promoter (-2000 to +73) restrained C/EBPalpha -mediated trans-activation, because mutation of each single GRU element increased this activation by 3-4-fold. This series of GRU mutations were repressed by wild type GR to the same percent as was the nonmutated PEPCK-C gene promoter. In contrast, the repression by mutant GR depended on the intact AF1 site in the gene promoter, whereby mutation of the AF1 element abolished the repression.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Glucocorticoids play a fundamental role in the maintenance of homeostasis in mammals. Removal of the adrenals severely compromises the ability of animals to withstand fasting (for reviews see Refs. 1 and 2). Glucocorticoids exert their effects via the glucocorticoid receptor (GR),1 predominantly by modulating gene transcription (3-5). An attractive mode of regulation, especially in light of the coordinated effects of glucocorticoids in maintaining homeostasis, is the opposing control of the same gene in different tissues by GR. PEPCK-C gene provides an optimal model for studying this mode of regulation. The transcription of this gene is stimulated by glucocorticoids in the liver and kidney (6, 7) but is repressed in the adipose tissue (8).

PEPCK-C catalyzes a key reaction that determines the rates of gluconeogenesis in the liver and kidney and glyceroneogenesis in the adipose tissue and liver (9). Glyceroneogenesis, the de novo synthesis of 3-glycerophosphate from pyruvate and amino acids (via an abbreviated version of gluconeogenesis), provides this precursor for the synthesis of triglycerides (10, 11). Recently, we have performed a targeted mutation in the adipose tissue-specific enhancer of the PEPCK-C gene in embryonic stem cells. The mutation ablated PEPCK-C gene expression in white adipose tissue of mice homozygous for this mutation and caused a decrease in the storage of triglycerides, which in some mice developed into lipodystrophy (12). This mutation therefore established the importance of PEPCK-C and glyceroneogenesis in the homeostasis of triglycerides in the adipose tissue.

Because PEPCK-C is encoded by a unique copy gene, and is transcribed from a single promoter, it is likely that tissue-specific factors are involved in the reciprocal regulation that leads to stimulation (liver and kidney) or repression (adipose tissue) of the gene transcription in the presence of glucocorticoids. Yamamoto and colleagues (13) proposed the term composite GRE to describe a nonconsensus sequence that binds the GR with low affinity and, in turn, is capable of mediating either repression or activation of genes.

The GRE identified in the PEPCK-C gene (14) is a nonconsensus sequence that binds GR at a very low affinity and is not able by itself to transmit a transcriptional response to glucocorticoids. In fact, PEPCK-C gene promoter harbors a GR unit (GRU) containing two low affinity, nonconsensus GR-binding sites and two accessory elements, AF1 and AF2. These elements do not bind steroid receptors, but their occupancy is required for the response of the PEPCK-C gene to glucocorticoids (see scheme in Fig. 2). The factors binding to the AF1 element are all nonsteroid nuclear receptors; these include hepatocyte nuclear factor (HNF) 4, chicken ovalbumin upstream transcription factor (COUPTF), retinoic acid receptor, retinoid X receptor (RXR), and members of the peroxisome proliferator-activated receptor (PPAR) family. The AF2 site (15) binds HNF3beta (16, 17) and has been proposed to comprise an insulin-response element as well (18).

To identify tissue-specific factors that are involved in the glucocorticoid-mediated repression of PEPCK-C gene transcription in adipocytes, we have employed a systematic DNase I footprinting analysis of the PEPCK-C gene promoter, using adipocyte nuclear proteins. Their functional participation in the repression has been assessed using transient transfection experiments in PEPCK-nonexpressing NIH3T3 cells. The results from these two independent experimental systems consistently identified the involvement of members of the C/EBP family, but not those of PPAR, in the GR repression of the PEPCK-C gene promoter activity. Furthermore, experiments in NIH3T3 cells revealed a hierarchical constraint of PEPCK-C gene promoter trans-activation by the separate GRU elements. Both wild type and mutant GR (incapable of binding the DNA) repress the C/EBP-mediated trans-activation by 50-60%, regardless of whether the trans-activation is low or high. However, the repression by mutant GR critically requires an intact AF1 site in the PEPCK-C gene.

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ABSTRACT
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Materials-- Dulbecco's modified Eagle's medium (DMEM), F-12, and fetal and newborn calf serum were purchased from Biological Industries, Kibutz Beit Haemek, Israel. Biosynthetic human insulin was obtained from Novo Nordisk (Denmark). Dexamethasone, the synthetic glucocorticoid hormone, was purchased from Teva, Israel Pharmaceutical Industry. Ultraspec, the commercial reagent for the preparation of tissue RNA, was purchased from Biotecx Laboratories, Inc. (Austin, TX). Radioactive signals were quantified using a PhosphorImager (Fujix BAS 1000, Fuji, Japan). Reverse transcriptase was obtained from Invitrogen. Random hexanucleotide pd(N)6 was purchased from Amersham Biosciences, and the ribonuclease inhibitor, RNasin, was purchased from Promega (Madison, WI). The enzyme-linked immunosorbent assay kit for the determination of human somatotropin was purchased from Roche Molecular Biochemicals.

Differentiation of Adipocytes-- 3T3-F442A cells, obtained from Dr. Howard Green (19), were grown to confluence in DMEM and supplemented with 10% newborn calf serum. For differentiation of the cells to adipocytes, newborn calf serum was replaced by 10% fetal calf serum; isobutylmethylxanthine was added at a final concentration of 0.2 mM, and the cells were incubated for 3 days. The cells were further incubated for at least 8 days in a medium containing 4 milliunits/ml insulin, until ~80-90% of cells contained fat droplets.

Dexamethasone Treatment-- Dexamethasone (10-7 M) was added to NIH3T3 cells no later than 20 h after transfection, when expression of the reporter gene was barely detected. Cells were harvested no later than 24 h after addition of dexamethasone to prevent cell lysis. 3T3-F442A adipocytes were similarly treated with 10-7 M dexamethasone for 16-18 h.

RNA Isolation and RT-PCR Analysis-- Total RNA was isolated from a single 100-mm cultured plate of 3T3-F442A adipocytes or from 30 mg of mouse liver using the commercial reagent Ultraspec according to the manufacturer's instructions. One µg of total RNA was reverse-transcribed according to manufacturer's instructions, using the Invitrogen reverse transcriptase kit in the presence of 7.5 units/ml random hexanucleotide pd(N)6 as primer and 1 unit/µl RNasin ribonuclease inhibitor, except that the incubation was for 30 min at 42 °C.

PCR was performed using the PEPCK-C primers 5'-CTTGTCTACGAAGCTCTCAG from exon 9 and 3'-CGTCCGAACATCCACTC from exon 10. Primers for the aP2 gene were 5'-CCTGGAAGCTTGTCTCCAG from exon 1 and 3'-CTCTTGTGGAAGTCACGCC from exon 4. Primers for beta -actin were the same as published previously (20). PCR was performed in the presence of a trace of [32P]dCTP (0.5 × 106 dpm) to allow semi-quantification (20). The PCR program included denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min, and elongation at 72 °C for 2 min, 20 cycles each consisting of denaturation for 30 s, annealing for 1 min, and elongation for 1 min. The PCR product was separated by electrophoresis on 8% polyacrylamide gel and quantified using a PhosphorImager apparatus and visualized by its exposure to autoradiographic film.

Preparation of Nuclear Proteins-- Nuclear proteins were extracted from rat liver according to Gorski et al. (21) as modified (22). Nuclear protein extracts from adipocytes were prepared essentially as described previously (22), except that the sucrose gradient step to further purify the nuclei was omitted. DNase I footprinting assays were performed as described previously (22). The autoradiographic density signals of specific bands in the exposed film (see Figs. 2-4) were quantified using Fluor-STM MultiImager with Multianalyst version 1.1 (Bio-Rad).

Western Blot Analysis-- 5 µg of nuclear proteins from adipocytes treated or untreated with dexamethasone (a synthetic glucocorticoid) were separated on 15% SDS-PAGE and transferred to nitrocellulose membrane (Protran BA 85, Schleicher & Schuell). C/EBPalpha was probed with rabbit polyclonal anti-C/EBPalpha antibody c100 diluted 1:1000 (a gift from Dr. Steven McKnight) and was detected using horseradish peroxidase-conjugated goat anti-rabbit antibody diluted 1:4000. Nuclear Y12 protein was probed with mouse anti-sm monoclonal antibody Y12 (23) (a gift from Dr. Ruth Sperling), diluted 1:10, and detected using horseradish peroxidase conjugated to affinity-purified goat anti-mouse IgG F(ab)' fragment diluted 1:3000 (23). Secondary antibodies were visualized with SuperSignal West Pico chemiluminescent substrate (Pierce).

Cell Culture, Transfection Conditions, and CAT Assays-- NIH3T3 cells were grown on 100-mm plates in DMEM containing 10% fetal calf serum. For transfection, cells were transferred to DMEM containing 10% newborn calf serum. Transfection was performed essentially according to Chen and Okayama (24), as described previously (25), 1 or 2 days after the cells reached confluency. Supercoiled PEPCK-CAT plasmid (2 µg) and additional carrier pBlueScript DNA (Stratagene), to make a total of 12 µg, were used, and the transfection efficiency was monitored as described previously (25). Where indicated, 1 µg each of the expression vectors for C/EBPalpha and PPARgamma 2 together with RXRalpha or GR was used. The optimal quantity of C/EBPbeta or C/EBPdelta expression vectors added to the transfection mixture was 0.5 µg each. In all cases, titration test of various concentrations of the expression vectors allowed us to choose the amounts that yielded optimal effects. Assays for chloramphenicol acetyltransferase (CAT) activity were determined 44 h after transfection, as described (25), and quantified using a PhosphorImager apparatus. The S.E. was calculated as the S.D. divided by the square root of the number of experiments.

Plasmids Used in the Transfection Studies-- The previously described plasmid 597-pck-CAT (PCK (600)-CAT) contains 597 bp of the rat PEPCK-C gene promoter region fused to the CAT reporter gene (26). The derived 2000-pck-CAT plasmid (PCK(2000)-CAT) contains 2000 bp of the PEPCK-C gene promoter (27). The mutated AF1 (AF1-mut), the combined mutation of GRE1 and GRE2 (mGRE1-2) (28), and the mutated AF2 (AF2-mut) (29) plasmids contain 2000 bp of the rat PEPCK-C gene promoter mutated at these sites. The mouse promoters PCK(840)-CAT and PCK(1500)-CAT contain 840 or 1500 bp upstream from the transcription start site of PEPCK-C gene promoter, derived from a mouse genomic clone, and fused to the CAT reporter gene as described previously (26). The PCK(1500-mut)-CAT plasmid contains a site-specific mutation of the PPARE sequence. The mutation was generated by inserting the restriction sites XhoI and SmaI 3' and 5', respectively, to the PPARE site. This enabled us to replace the PPARE site with 47 bp of the pBlueScript polylinker residing between XhoI and SmaI, except that the EcoRI site of the polylinker insert was deleted.

The DNA concentration of the constructs containing the longer PEPCK-C gene promoters was corrected to achieve the same number of molecules as that of the shorter PEPCK-C gene promoters. Expression vectors encoding adipocyte-enriched transcription factors used in this work included the following: C/EBPalpha (30) obtained from Dr. Steven McKnight; C/EBPbeta (31) from Dr. David Ron; C/EBPdelta (32) from Dr. Daniel Lane; PPARgamma 2 (33) from Dr. Bruce Spiegelman; RXRalpha (34) and human GR (35) from the laboratory of Dr. Ron Evans; and rat wild type and mutant GR were a gift from Dr. Keith Yamamoto (13).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Repression of PEPCK-C Expression by Glucocorticoids in Vivo-- The addition of glucocorticoids to fully differentiated 3T3-F442A adipocytes for 16-18 h caused a strong repression of PEPCK-C gene expression as shown by the absence of its RT-PCR product (Fig. 1). This repression was distinct because glucocorticoids failed to inhibit the expression of the adipocyte-specific aP2 gene (Fig. 1).


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Fig. 1.   Effect of the glucocorticoids on PEPCK-C gene expression in adipocytes. cDNA made from 1 µg of total RNA, using random hexamer primers, was amplified by PCR (1 µl out of the total 30 µl of RT reaction mix), using PEPCK-C and beta -actin primers. The PCR products from adipocytes treated with dexamethasone (A + Dex) compared with the nontreated cells (- Dex), were identified by size separation as indicated on the right-hand side of the figure. The RT-PCR products of liver PEPCK-C RNA (L) and of the adipocyte-specific aP2 gene are shown as controls.

DNase I Footprinting Assays-- In order to assess whether GR affects the binding of nuclear proteins to specific sites in the PEPCK-C gene promoter, we systematically footprinted the gene promoter, using nuclear proteins extracted from 3T3-F442A adipocyte cells that had been incubated without or with glucocorticoids. A scheme of PEPCK-C gene promoter depicting the binding sites of transcription factors is shown (Fig. 2a). The figure also includes the binding sites for nuclear proteins present in the liver, fetal liver (where the gene for PEPCK-C is not actively transcribed (22, 36)), kidney, and adipocytes (Fig. 2b). Note that nuclear proteins from the liver and adipocytes bind to most sites included within positions -70 to -1200 of the transcription start site of the PEPCK-C gene promoter. Adipocyte nuclear proteins do not bind to the P2 site (HNF-1 recognition motif) in the PEPCK-C gene promoter (37), whereas nuclear proteins from the liver bind poorly to PPARE (the PPAR recognition site (see Fig. 3b)). Unlike the kidney and fetal liver, nuclear proteins from adipocytes and liver bind all C/EBP recognition sites. These include the CRE-1 (cAMP-response element), P3I, P4, and CRE-2 sites (CRE-2 is a CRE-like sequence). Of these, P3I binds exclusively isoforms of the C/EBP family (38). CRE-1 and P4 sites bind AP1 as well, and CRE-1 also binds cAMP-response element-binding protein, ATF-2 (39), and ATF-3 (40). CRE-2 binds C/EBPalpha with very low affinity but binds a nonidentified temperature-labile protein in the liver nuclear proteins (22).


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Fig. 2.   DNase I footprint analysis of the PEPCK-C gene promoter. a, scheme of the gene promoter. The entire GRU in the proximal region of the gene promoter includes two GR-binding sites (GR) and two accessory sites, out of which the more 5' is AF1 and the more 3' is AF2 (IRS, insulin response signal) (14). A distal element (PPARE) is homologous to the AF1 element except that it binds only PPAR and RXR (33). Note that four structures (CRE-1, CRE-2, P3, and P4) contain C/EBP recognition sequences (22, 41). b, binding (+) and absence of binding (-) of nuclear proteins from the indicated tissues. DNase I footprinting data of adult liver (61), fetal liver (22), kidney (61), and adipocytes (37) are indicated. c, a 390-bp DNA fragment, spanning positions -8 to -390 of the rat PEPCK-C gene promoter was 32P-end-labeled at the 3' site (position -8) of the fragment. 50,000 cpm per reaction of the labeled fragment were incubated without proteins (0') or with 15 µg of nuclear proteins from 3T3-F442A adipocytes not treated (- Dex) or treated overnight with Dexamethasone (A + Dex). The lane on the left (seq) designates sequencing of A + G, which enabled the identification of the regions protected by protein binding to CRE-2 and P1/CRE-1 sites. The binding regions are marked by the positions 5' to the transcription start site. Bands used for quantification of the signals (see Fig. 3c), a band at position -88 (within the CRE-1 protected area) and at position -160 (outside the protected area) (b), are marked by arrows on the right. d, the same probe as described in c was used, except that a longer separation enabled the identification of regions protected by protein binding to the C/EBP recognition sites P3 and P4 indicated in the figure by their positions 5' to the transcription start site.


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Fig. 3.   DNase I footprint analysis of the nuclear receptor-binding sites in the PEPCK-C gene promoter. a, a 128-bp DNA fragment, spanning positions -490 to -362 of the rat PEPCK-C gene, was 32P-end-labeled at the 5' site (position -490) of the fragment. 50,000 cpm of the labeled probe was added per reaction. Incubation without (0'), with 15 µg of nuclear proteins from 3T3-F442A adipocytes not treated (- Dex), or treated overnight (A + Dex) with dexamethasone or with 10 µg of rat liver nuclear proteins (L) are indicated. The lane on the right (seq) designates sequencing of A + G, which enabled the identification of the region protected by proteins binding to the AF1 site. The protected region is marked by the positions 5' to the transcription start site. b, a 160-bp DNA fragment, spanning positions -950 to -1110 of the rat PEPCK-C promoter, was 32P-end-labeled at the 3' site (position -950) of the fragment. 50,000 cpm per reaction of the labeled fragment were incubated without proteins (0') or with 15 µg of nuclear proteins from 3T3-F442A adipocytes not treated (- Dex) or treated overnight (A + Dex) with dexamethasone or with 10 µg of rat liver nuclear proteins (L). The lane on the right (seq) designates sequencing of A + G, which enabled the identification of the region protected by proteins binding to PPARE site, indicated by the positions 5' to the transcription start site. c, the ratio between the density signal of a distinct band within the protected CRE-1 site (position -88) and a band outside (position -160) (marked by arrows in Fig. 2b) are shown in the histogram. The ratio in the absence of proteins was set at 10. d, the ratio between the density signals of two distinct bands within the AF1 site-protected region (positions -445 and -446) and a band outside (position -457) (marked by arrows in a) is shown by the histogram. The ratio in the absence of proteins was set at 10.

Our footprinting analysis revealed that nuclear proteins extracted from glucocorticoid-treated adipocytes failed to bind to all C/EBP recognition sites in the PEPCK-C gene promoter: CRE-1 (Fig. 2c), P3 and P4 (Fig. 2d), and the single C/EBP-like CRE-2 site (Fig. 2c). The prominent inhibition of binding to all these sites occurred despite the wide spectrum of affinities of the C/EBP recognition sites to C/EBPalpha , which gradually decreases from the highest affinity (CRE-1 site) to the lowest (CRE-2 site) (22, 41). In addition, binding to the P1 site (nuclear factor 1 recognition site) in the PEPCK-C gene promoter was also inhibited (Fig. 2c), although this is not a C/EBP recognition site. In previous studies (38) the binding of C/EBPbeta to CRE-1 has been shown to cooperate with the binding to the P1 site. Therefore, a reduced binding to CRE-1 site might have led to a secondary effect on the binding to the P1 site.

The binding of adipocyte nuclear proteins to the recognition sites of nuclear receptors in the PEPCK-C gene promoter has not been affected by the glucocorticoid treatment. Thus, binding to AF1 site (also termed P6) remained similar whether using nuclear proteins from adipocytes not treated or treated with glucocorticoids (Fig. 3a). AF1 site is a nuclear receptor recognition site that interacts with a variety of nonsteroid nuclear receptors including HNF4, COUPTF, retinoic acid receptor, RXR, and members of the PPAR family. Similar to adipocytes, hepatic nuclear proteins also interact well with the AF1 site (Fig. 3a).

Another non-C/EBP-binding site, which likewise has not been affected by the glucocorticoid treatment, is PPARE (the recognition site of the heterodimer nuclear receptors PPARgamma 2/RXR) (Fig. 3b). In contrast to the adipocyte nuclear proteins, binding of hepatic nuclear proteins to the PPARE site could barely be detected (Fig. 3b), unlike their efficient binding to the AF1 site (Fig. 3a). Furthermore, the hypersensitive site (position -985) at the 3' end of the protected region appeared only in the presence of adipocyte nuclear proteins. It was undetectable both in the absence of nuclear proteins and in the presence of liver nuclear proteins (Fig. 3b). Therefore, PPAR isoforms seem less enriched in the liver (from nonfasted rat) than they are in adipocyte nuclear proteins. To gain quantitative estimation of the glucocorticoid effect, we measured the density signals of specific bands inside and outside the protected regions of the CRE-1 (Fig. 2a) and AF1 (Fig. 3a) sites. The footprinting intensity of the CRE-1 site was quantified from the autoradiographic films by measuring the density signal of a band within the CRE-1 site (position -88 from the transcription start site of the PEPCK-C gene) and a band outside, at position -160 (both marked by arrows). Likewise, the footprinting intensity of the AF1 site was quantified by measuring the density signals of two bands inside (positions -445 and -446) and a band outside (position -457) the AF1 site. The ratios between the density signals inside and outside the protected region were computed. The ratios obtained from the footprinting done without nuclear proteins were arbitrarily set at 10 and used to normalize other ratios of the footprinting done in the presence of nuclear proteins. These measurements have clearly assessed that dexamethasone treatment interfered with the adipocyte nuclear protein footprinting of the CRE-1 site (Fig. 3c) but not with the footprinting of the AF1 site (Fig. 3d).

Transcription of the gene for C/EBPalpha is also repressed by glucocorticoids but only for a few hours (32). Yet this repression might lead to a longer lasting reduction of C/EBPalpha protein concentration in adipocyte nuclei (32), resulting in an apparent, rather than real, interference of binding to its recognition sites. Therefore, we determined whether the concentration of C/EBPalpha in nuclei corresponded to the observed diminished footprinting of the PEPCK-C gene promoter.

C/EBPalpha Level in Extracted Adipocyte Nuclear Proteins-- The concentration of C/EBPalpha in the adipocyte extracts of nuclear proteins used for footprinting was determined by Western blot assay. The hormonal treatment diminished the nuclear concentration of C/EBPalpha by 30% when normalized to the level of the Y12 nuclear protein (Fig. 4a). Whether this reduced concentration of C/EBPalpha accounted for the inhibited footprinting of nuclear proteins from glucocorticoid-treated adipocytes was assessed. Thus, we used lower amounts of nuclear proteins extracted from untreated adipocytes (10 (2/3) and 7.5 µg (1/2)), compared with the whole amount (15 µg), to footprint the gene promoter region containing the CRE-1, P1, and CRE-2 sites. This region enabled us to assay the site with the highest affinity for C/EBP binding (CRE-1) and the site with the lowest affinity for C/EBP binding (CRE-2). The analysis showed that 10 µg of nuclear proteins footprinted all three sites (Fig. 4b). Footprinting of the CRE-1 site was quantified as detailed above for Fig. 2c. Thus, the ratio in the absence of proteins was set at 10, relative to a ratio of 2.5 obtained with 10 µg of protein and a ratio of 8.5 obtained with 7.5 µg of protein (Fig. 4c). The dexamethasone effect on CRE-1 footprinting (Fig. 2c) that was likewise quantified yielded a ratio of 1.7 in the presence of 15 µg of nuclear proteins from untreated adipocytes, and 15.8 in the presence of 15 µg of nuclear proteins from dexamethasone-treated adipocytes (Fig. 3c). In contrast, footprinting the AF1 site yielded similar ratios (2.7 and 3.3, respectively) using 15 µg of nuclear proteins from untreated or dexamethasone-treated adipocytes (Fig. 3d). We therefore conclude that in addition to lowering the level of C/EBPalpha in the nucleus, the hormonal treatment interfered with the binding to their recognition sites in the PEPCK-C gene promoter.


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Fig. 4.   The glucocorticoid inhibition of the binding to C/EBP recognition sites of the PEPCK-C gene promoter cannot be accounted for by a simple reduction of C/EBPalpha nuclear level. a, Western blot analysis of C/EBPalpha concentration in adipocyte nuclear proteins: 5 µg of nuclear proteins extracted from adipocytes untreated (control) or treated (+Dex) with 10-7 M dexamethasone. The equally blotted amounts of proteins were verified after stripping the membrane and re-probing with anti-Y12 antibody. The percent reduction of C/EBPalpha nuclear level by dexamethasone treatment is indicated above. b, footprinting analysis of the same probe as described in Fig. 2c. The probe was incubated without proteins (0') or with the indicated amounts (15, 10, or 7.5 µg) of nuclear proteins extracted from adipocytes not treated with dexamethasone. c, the ratio between the density signal of a band at position -88 (within the CRE-1 protected region) and a band at position -160 outside the protected area (marked by arrows on the right of the footprinting in b) was plotted against the amounts of nuclear protein used. The ratio in the absence of proteins was set at 10.

Transient Transfection Experiments in NIH3T3 Cells-- The involvement of adipocyte-enriched transcription factors in the repression of PEPCK-C gene transcription by glucocorticoids was further assessed using transient transfection assays in NIH3T3 cells. Although these cells do not express PEPCK-C or the adipocyte-specific transcription factors PPARgamma 2 or C/EBPalpha (42), co-transfecting expression vectors coding for PPARgamma 2 and RXRalpha (PPARgamma 2/RXR) were reported to stimulate transcription from the PEPCK-C gene promoter in these cells (33). We have compared the stimulation of the PEPCK-C gene promoter activity by PPARgamma 2/RXR and by C/EBPalpha expression vectors using PEPCK-CAT chimeric genes driven either by 600 (PCK(600)-CAT) or 2000 bp (PCK(2000)-CAT) of the PEPCK-C gene promoter (27). PPARgamma 2/RXR preferentially stimulated the transcription from PCK(2000)-CAT PEPCK-C gene promoter, whereas C/EBPalpha preferentially and markedly stimulated transcription from the PCK(600)-CAT gene promoter (Fig. 5a). The latter is most likely because of the localization of a cluster of C/EBP recognition sites in the proximal region of the PEPCK-C gene promoter (9). For orientation see the scheme of the PEPCK-C gene promoter (Fig. 2a). In addition to C/EBPalpha , other members of the C/EBP family also trans-activated the PEPCK-C gene promoter but to a lesser extent. C/EBPalpha was the most effective; C/EBPbeta was half as effective, and C/EBPdelta was the least effective. There was a 5-fold difference in the magnitude of stimulation between C/EBPalpha and C/EBPdelta (Fig. 5b). We then determined the effect of GR and its hormone (dexamethasone) on the level of trans-activation of the PCK(600)-CAT gene promoter by co-transfected C/EBPalpha , C/EBPbeta , or PPARgamma 2/RXR. Co-transfection of GR expression vector and the addition of dexamethasone for the last 24 h after transfection inhibited (by about 60%) the activation of transcription from the PEPCK-C gene promoter either by C/EBPalpha or by C/EBPbeta . In contrast, there was no effect of GR and dexamethasone on the trans-activation by PPARgamma 2/RXR of either PCK(600)-CAT (Fig. 5c) or PCK(2000)-CAT (Fig. 5d) gene promoters.


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Fig. 5.   Effect of PPARgamma 2 and C/EBPalpha on transcription from the PEPCK-C gene promoter in NIH3T3 cells. a, trans-activation by PPARgamma 2/RXR (dark bars) or C/EBPalpha (hatched bars) over basal activity (open bars) of PCK(600)-CAT (600) and PCK(2000)-CAT (2000) rat PEPCK-C gene promoters is shown. The fold stimulation by the transcription factors over basal transcription activity of the PCK(600)-CAT gene promoter, taken as one, represents the mean ± S.E. for at least five independent experiments. b, comparison of the fold stimulation by isoforms of C/EBP. Expression vectors encoding C/EBPalpha (1 µg), C/EBPbeta , or C/EBPdelta (0.5 µg each) were co-transfected with the PCK(600)-CAT rat PEPCK-C gene promoter as described under "Experimental Procedures." The fold stimulation by members of the C/EBP family, over basal transcription activity of the PCK(600)-CAT promoter, taken as one, represents the mean ± S.E. for at least six independent experiments. c, effect of GR and dexamethasone on the activation of transcription from the PEPCK-C gene promoter by C/EBPalpha (alpha ), C/EBPbeta (beta ), and PPARgamma 2/RXR (PPARgamma 2). Details as described in a, without GR (-GR, open bars), except that 1 µg of the human GR expression vector was included in the transfection mix and dexamethasone (10-7 M, final concentration) was added 19 h later for 24 h (+GR, dark bars). The fold stimulation over basal transcription activity of the PCK(600)-CAT promoter, taken as one, represents the mean ± S.E. for six independent experiments. d, effect of GR and dexamethasone on the activation of transcription from the PCK(2000)-CAT gene promoter by PPARgamma 2/RXR (PPARgamma 2). Details as described in c are as follows: without GR (-GR, open bars) and with GR (+GR, dark bars). The fold stimulation over basal transcription activity, taken as one, represents the mean ± S.E. for four independent experiments.

The preferential PPARgamma 2/RXR trans-activation of the longer region of the PEPCK-C gene promoter, either PCK(2000)-CAT (rat) or PCK(1500)-CAT (mouse) gene promoters compared with PCK(600)-CAT (rat) and PCK(840)-CAT (mouse) (Figs. 5a and 6a), is due to the two PPARgamma 2/RXR recognition sites (a proximal AF1 side and a distal PPARE site). Moreover, mutation of the PPARE sequence in PCK(1500)-CAT gene promoter (mouse PCK(1500-mut)-CAT) abolished its response to PPARgamma 2/RXR, although it contained the proximal AF1-binding site (Fig. 6a). Note that the sequence of PEPCK-C gene promoter is highly preserved between the rat (43) and the mouse (44). Furthermore, mutation of the PPARE sequence specifically ablated PEPCK-C gene expression in the adipose tissue in vivo as shown with the mutated rat PEPCK-C transgene in transgenic mice (45) and the targeted mutation in the mouse endogenous PEPCK-C gene (12). In contrast, mutation of the proximal PPARgamma 2/RXR-binding site (AF1 site) in the context of PCK(2000)-CAT gene promoter markedly increased the PPARgamma 2/RXR trans-activation of the gene promoter (Fig. 6b). The constraining features of the wild type AF1 site of the PEPCK-C gene promoter in NIH3T3 cells have been studied recently in detail by Eubank et al. (46). These authors showed that the AF1 site tightly binds the nuclear receptor COUPTF II whose expression vector inhibited the PPARgamma 2/RXR-mediated trans-activation of the PEPCK-C gene promoter via the AF1-binding site (46). These features of AF1 site are not shared by other GRU elements, because mutation of the AF2 site had no effect on the trans-activation of PCK(2000)-CAT by PPARgamma 2/RXR (Fig. 6b).


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Fig. 6.   Effects of site-specific mutations on the trans-activation of PCK(2000)-CAT rat PEPCK-C gene promoter. a, fold trans-activation by PPARgamma 2/RXR (dark bars) over basal activity (open bars) of the mouse PCK(840)-CAT (840) and PCK(1500)-CAT (1500) PEPCK-C gene promoters and of the PCK(1500)-CAT PEPCK-C gene promoter containing a mutation of the PPARE site (1500-mut) is shown. The fold stimulation by PPARgamma 2/RXR over basal transcription activity of the PCK(840)-CAT gene promoter, taken as one, represents the mean ± S.E. for at least nine independent experiments. b, effect of mutations of the AF1 (AF1-mut) and AF2 (AF2-mut) sites in the context of the PCK(2000)-CAT rat PEPCK-C gene promoter on its trans-activation by PPARgamma 2/RXR (dark bars) over basal activity (open bars). The fold stimulation by PPARgamma 2/RXR over basal transcription activity of the PCK(2000)-CAT gene promoter (2000), taken as one, represents the mean ± S.E. for at least six independent experiments. c, effect of mutations of GRE1 and GRE2 (mGRE1-2), AF1 (AF1-mut), and AF2 (AF2-mut) sites in the context of the PCK(2000)-CAT rat PEPCK-C gene promoter on its trans-activation by C/EBPalpha (hatched bars) over basal activity (open bars). The fold stimulation by C/EBPalpha over basal transcription activity from the PCK(2000)-CAT chimeric gene (2000), taken as one, represents the mean ± S.E. for at least four independent experiments.

C/EBPalpha -mediated trans-activation of the PCK(2000)-CAT chimeric gene is also constrained (Fig. 6c). However, unlike the trans-activation by PPARgamma 2/RXR, in this case mutations of any single element of the GRU (14), AF1, AF2, and GRE1-2 of PCK(2000)-CAT chimeric gene, enhanced the C/EBPalpha -mediated trans-activation of the gene promoter by 3-4-fold (Fig. 6c). Therefore, unlike PPARgamma 2/RXR, not only the AF1 site but the entire GRU domain constrains the C/EBPalpha trans-activation of the PCK(2000)-CAT chimeric gene. Because each of the elements comprising the GRU constrained the C/EBPalpha trans-activation of the PCK(2000)-CAT chimeric gene, we asked whether-binding of GR to the PEPCK-C gene promoter is required for its mediated repression. To this end, the capacity of the wild type rat GR was compared with its counterpart rat mutant GR that is incapable of binding to the DNA (13, 47) to inhibit the C/EBPalpha -mediated trans-activation of PCK(2000)-CAT. The data showed that either wild type or mutant rat GR equally repressed the stimulation of transcription from the PEPCK-C gene promoter by C/EBPalpha (Fig. 7a).


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Fig. 7.   Effect of rat wild type and mutant GR on the trans-activation of the PCK(2000)-CAT rat PEPCK-C gene promoter by C/EBPalpha . a, effect of wild type rat GR (wt GR) and rat mutant GR (mut GR) in the presence of dexamethasone (as described in the legend of Fig. 5c) on the trans-activation of the PCK(2000)-CAT rat PEPCK-C gene promoter by C/EBPalpha . The fold stimulation by C/EBPalpha over basal transcription activity of the PCK(2000)-CAT promoter, taken as one, represents the mean ± S.E. for at least eight independent experiments. b, effect of wild type GR (wt GR) and mutant GR (mut GR) on the trans-activation of the PCK(2000)-CAT rat PEPCK-C gene promoter and its derived series of the GRU mutants described in Fig. 6c. The values are expressed as percent repression by GR from the C/EBPalpha -stimulated activity of each gene promoter. These represent the mean ± S.E. of at least four independent experiments.

Finally, we have assessed whether the enhanced C/EBPalpha -mediated transcriptional stimulation of the GRU-mutated series of PCK(2000)-CAT could be repressed by GR and whether the mutant GR was also effective. The wild type GR repressed the C/EBPalpha -mediated stimulation of the GRU series of PCK(2000)-CAT mutants to a similar extent as that of the wild type (about 50%) (Fig. 7b). The mutant GR repressed the stimulation of the wild type and GRE1-2 mutant of PCK(2000)-CAT to a similar extent, less so the AF2 mutant, but completely failed to repress the stimulation of PCK(2000)-CAT-AF1 mutant (Fig. 7b). Therefore, the AF1 site is required for the repression by mutant GR but not by the wild type GR (Fig. 7b). In fact, the AF1 site emerges as an inherent constraining element on transcription from the PEPCK-C gene promoter because it markedly restrains the trans-activation of PCK(2000)-CAT gene promoter activity either by PPARgamma 2 or by C/EBPalpha (Fig. 6, b and c). Moreover, intact AF1 is also required for the mutant GR-mediated repression of C/EBPalpha trans-activation of the PEPCK-C gene promoter. When AF1 is mutated, the repression by mutant GR is abolished (Fig. 7b).

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Our results shed light on the involvement of transcription activators from the C/EBP family, but not those from the PPAR family, in the glucocorticoid-mediated repression of PEPCK-C gene transcription in adipocytes. Glucocorticoid treatment of 3T3-F442A adipocytes led to a reduced nuclear concentration of C/EBPalpha , in addition to a markedly diminished binding of nuclear proteins to the C/EBP recognition sites (but not to the PPAR/RXR recognition sites) in the PEPCK-C gene promoter. Previous studies from our laboratory (26) documented a requirement for the C/EBP recognition sites of the PEPCK-C gene promoter that were crucial for its basal activity in adipocytes. Transient transfection in NIH3T3 cells revealed that GR together with glucocorticoids partially inhibited the C/EBP-mediated, but not PPARgamma 2-mediated, trans-activation of PEPCK-C gene promoter. We have thus described the involvement of members of the C/EBP family in the adipocyte-specific glucocorticoid repression of PEPCK-C gene expression. This occurs in at least two ways: (a) by reducing the concentration of C/EBPalpha in adipocyte nuclei, and (b) interfering with the binding of nuclear proteins to the C/EBP recognition sites in the DNA and as a consequence of, or in addition to, inhibiting the trans-activation of PEPCK-C gene promoter by C/EBP isoforms. Clearly, using nuclear proteins from the PEPCK-expressing adipocytes and transient transfection in the PEPCK-nonexpressing NIH3T3 cells comprised two independent experimental approaches and systems, both of which consistently revealed the involvement of C/EBP but not PPAR activators in the glucocorticoid repression of PEPCK-C gene transcription. However, because GR succeeded to inhibit the C/EBP trans-activation only by 60%, it is likely that additional factors beside members of C/EBP family participate in PEPCK-C gene transcription in the adipocytes.

Experiments using NIH3T3 cells have disclosed a hierarchical regulation of PEPCK-C gene transcription, in particular the constraint on the gene promoter response to C/EBP-mediated activation by the GRU element in the PEPCK-C gene promoter. Mutating any single element within the GRU relieves this constraint, and the trans-activation by C/EBPalpha is markedly elevated. This is different from trans-activation by PPARgamma 2/RXR, which is also constrained, but in this case, it is not exerted via each element of the GRU but exclusively by the AF1 site. In that sense, the AF1 site has emerged as a unique element whose mutation elevates by 4-fold the response of PCK(2000)-CAT to either PPARgamma 2/RXR or C/EBPalpha . These constraining features of AF1 are accentuated by the very modest response of this site to PPARgamma 2/RXR activation of transcription from the PEPCK-C gene promoter (Fig. 6a). The main PPARgamma 2/RXR-responsive element is PPARE (Fig. 6a) that comprises an adipose tissue-specific enhancer, as has been shown previously in transgenic mice (45). Furthermore, a targeted mutation of PPARE in embryonic stem cells resulted in ablation of PEPCK-C gene expression in the white adipose tissue of offspring mice homozygous for the mutation (12).

Is the AF1 site involved in the GR repression of the C/EBPalpha -mediated trans-activation of PCK(2000)-CAT? The present data seem to support such involvement. Initially, our results have established that the GR repression does not require binding to the DNA. A GR mutated in the zinc finger (13), making it incapable of binding the DNA, was as active as wild type in repressing the C/EBPalpha stimulation. Subsequently, we have assessed that the wild type GR equally repressed the wild type PCK(2000)-CAT and derived mutated GRU series, whereas the mutant GR repressed the mutated GRE1-2 sites and, to a lesser extent, the mutated AF2 site of PCK(2000)-CAT. However, mutation of the AF1 site abolished the capability of the mutant GR to repress the C/EBP-mediated trans-activation. Because the mutated AF1 site did not hinder the repression by wild type GR, we suggest that the AF1 site undertakes a docking role to facilitate the mutant GR-mediated repression of the PCK(2000)-CAT gene promoter. Thus, these observations expand the hierarchy of regulation to strongly suggest that, in addition to its constraint features, the wild type AF1 site is crucial for the hormonal repression.

Because the repression by GR of the C/EBP-mediated trans-activation does not require DNA binding of the receptor, the GR probably inhibits trans-activation of PEPCK-C gene transcription via protein-protein interactions. This notion is supported by the fact that the percent repression by GR of the C/EBPalpha -mediated activation of transcription remained 50-60% whether the activation was low (wild type PCK(2000)-CAT gene promoter) or elevated by 3-4-fold (GRU-mutated series of PCK(2000)-CAT gene promoter).

Evidence of cross-talk between members of the C/EBP family and GR has been documented in numerous systems. Members of the C/EBP family have been shown to bind directly to the ligand binding domain of a number of nuclear receptors, including GR (see Refs. 48 and 49 and for review see Refs. 5 and 50). These interactions resulted either in induction or inhibition of the target genes and did not necessarily involve binding of GR to the DNA (48, 49). Alternatively, binding of C/EBPbeta to the DNA binding domain of GR has been implicated recently in the GR repression of the vitellogenin gene transcription in hepatocytes from the rainbow trout. The vitellogenin gene promoter lacks a GR-binding site (51).

Beyond the molecular aspects of the glucocorticoid repression of PEPCK-C gene transcription in adipose tissue, its physiological significance has gained new relevance. Metabolic studies have recently documented very substantial rates of glyceroneogenesis in the liver of both rats and humans during fasting (52, 53) or after ingestion of a diet high in protein but devoid of carbohydrate (52). These findings shed new light on the metabolic significance of the reciprocal control of PEPCK-C gene transcription by glucocorticoids. PEPCK-C activity catalyzes the rate-limiting step of both gluconeogenesis and glyceroneogenesis, hence regulating both pathways. This has been verified by deletion of the PEPCK-C gene in mice, resulting in neonatal lethality from hypoglycemia (54). In addition, a targeted mutation of the adipose tissue-specific PPARgamma 2-binding site of the PEPCK-C gene promoter, which selectively ablates gene expression in white adipose tissue, caused a marked diminution of glyceroneogenesis in this tissue. Mice homozygous for this mutation lost lipid from adipose tissue even to the extent of lipodystrophy, attesting to the metabolic significance of PEPCK-C and glyceroneogenesis in the adipose tissue (12). Moreover, a recent adipocyte-specific knockout of glucose transporter 4, required for glucose metabolism in the adipose tissue, generated mice exhibiting features of noninsulin-dependent diabetes mellitus without a loss of triglycerides from adipose tissue (55). Finally, Franckhauser et al. (56) overexpressed a chimeric gene containing the PEPCK-C structural gene linked to the aP2 promoter in mice resulting in obesity in adult mice. Taken together, these results further emphasize the crucial role of PEPCK-C and glyceroneogenesis in maintaining lipid homeostasis in the adipose tissue. Therefore, it is conceivable that hormonally mediated alterations of PEPCK-C gene expression ultimately regulate glyceroneogenesis in both liver and adipose tissue.

Lipid is released from the adipose tissue as free fatty acids and from the liver as triglycerides. Thus, glyceroneogenesis affects lipid metabolism in opposite ways in the two tissues; it restrains fat release from adipose tissue (57) and enhances it from the liver (58). It has been shown in rats that adrenalectomy enhances glyceroneogenesis and diminishes free fatty acid release from incubated epididymal fat pads (59, 60). In a reciprocal experiment, the addition of dexamethasone to cultured hepatocytes stimulated the synthesis of triglycerides and apolipoproteins E and B, as well as stimulating the release of very low density lipoproteins to the medium (58). How then is lipid homeostasis coordinated between the two tissues? We propose that the reciprocal regulation of PEPCK-C gene transcription by glucocorticoids provides a mechanism for such coordination because it represses PEPCK-C gene transcription in the adipose tissue and simultaneously enhances it in the liver. Experiments to further prove this hypothesis in vivo are under way.

    ACKNOWLEDGEMENTS

We are grateful to Dr. Keith Yamamoto for the wild type and mutant rat GR, to Dr. Bruce Spiegelman for supplying the PPARgamma expression vector, to Dr. Daniel Lane for the C/EBPdelta expression vector, and to Dr. Ron Evans for the human GR and mouse RXRalpha expression vectors. We especially appreciate the gift from Dr. Steven L. McKnight of the anti-C/EBPalpha antiserum, the valuable advice during the transfection experiments in confluent NIH3T3 given by Dr. Peter Tontonoz, and the many fruitful discussions with Dr. Oded Meyuhas.

    FOOTNOTES

* This work was supported by Grants 1999346 and 9600117 from the United States-Israel Binational Science Foundation, Grant 540197-19 from the Israel Science Foundation, a grant from the Israel Ministry of Health, and by Grant DK22541 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ These authors contributed equally to this work.

|| To whom correspondence should be addressed: Dept. of Developmental Biochemistry, Hebrew University-Hadassah Medical School, Jerusalem, Israel 91120. Fax: 972-2-675-7379; E-mail: reshef@cc.huji.ac.il.

Published, JBC Papers in Press, January 30, 2003, DOI 10.1074/jbc.M300263200

    ABBREVIATIONS

The abbreviations used are: GR, glucocorticoid receptor; PEPCK-C, phosphoenolpyruvate carboxykinase-C; C/EBP, CCAAT/enhancer-binding protein; GRE, glucocorticoid-response element; RXR, retinoid X receptor; PPAR, peroxisome proliferator-activated receptor; CRE, cyclic AMP-response element; PPARE, PPAR-response element; RT, reverse transcriptase; CAT, chloramphenicol acetyltransferase; GRU, glucocorticoid response unit; DMEM, Dulbecco's modified Eagle's medium; HNF, hepatocyte nuclear factor; COUPTF, chicken ovalbumin upstream transcription factor.

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