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Originally published In Press as doi:10.1074/jbc.M200585200 on March 7, 2002

J. Biol. Chem., Vol. 277, Issue 19, 16906-16912, May 10, 2002
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Overexpression and Ribozyme-mediated Targeting of Transcriptional Coactivators CREB-binding Protein and p300 Revealed Their Indispensable Roles in Adipocyte Differentiation through the Regulation of Peroxisome Proliferator-activated Receptor gamma *

Nobuyuki TakahashiDagger §, Teruo KawadaDagger §, Takayuki YamamotoDagger , Tsuyoshi GotoDagger , Aki TaimatsuDagger , Naohito Aoki||, Hiroaki Kawasaki**Dagger Dagger , Kazunari Taira**Dagger Dagger , Kazunari K. Yokoyama§§, Yasutomi Kamei¶¶, and Tohru FushikiDagger

From the Dagger  Laboratory of Nutrition Chemistry, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan, § Project for Obesity and Lipid Metabolism Regulation, Bio-oriented Research Advancement Institute, Tokyo 105-0001, Japan, || Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan, ** Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Tokyo 113-8656, Japan, Dagger Dagger  Gene Discovery Research Center, National Institute of Advanced Industrial Science and Technology, Tsukuba Science City 305-8562, Japan, §§ Tsukuba Life Science Center, The Institute of Physical and Chemical Research, Tsukuba Science City 305-0074, Japan, and ¶¶ Precursory Research for Embryonic Science and Technology, Japan Science and Technology Corp., Tokyo 162-8636, Japan

Received for publication, January 18, 2002, and in revised form, February 25, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The cAMP-response element-binding protein-binding protein (CBP) and p300 are common coactivators for several transcriptional factors. It has been reported that both CBP and p300 are significant for the activation of peroxisome proliferator-activated receptor gamma  (PPARgamma ), which is a crucial nuclear receptor in adipogenesis. However, it remains unclear whether CBP and/or p300 is physiologically essential to the activation of PPARgamma in adipocytes and adipocyte differentiation. In this study, we investigated the physiological significance of CBP/p300 in NIH3T3 cells transiently expressing PPARgamma and CBP and in 3T3-L1 preadipocytes stably expressing CBP- or p300-specific ribozymes. In PPARgamma -transfected NIH3T3 cells, induction of expression of PPARgamma target genes such as adipocyte fatty acid-binding protein (aP2) and lipoprotein lipase (LPL) by adding thiazolidinedione was enhanced, depending on the amount of a CBP expression plasmid transfected. Expression of aP2 and LPL genes, as well as glycerol-3-phosphate dehydrogenase activity and triacylglyceride accumulation after adipogenic induction, was largely suppressed in 3T3-L1 adipocytes expressing either the CBP- or p300-specific active ribozyme, but not in inactive ribozyme-expressing cells. These data suggest that both CBP and p300 are indispensable for the full activation of PPARgamma and adipocyte differentiation and that CBP and p300 do not mutually complement in the process.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Adipose tissues are significant in regulation of common diseases such as obesity, type 2 diabetes, coronary artery disease, and hypertension (1). This is because, during their differentiation and maturation, adipocytes release many bioactive molecules (called "adipocytokines"), including adipsin, angiotensinogen, leptin, tumor necrosis factor alpha  (TNF-alpha ),1 and adiponectin (2). TNF-alpha is a negative factor released from mature adipocytes, that is, it suppresses glucose uptake into adipose tissues or skeletal muscles (3). On the other hand, adiponectin is a positive factor released from nonmature adipocytes, that is, it enhances insulin sensitivity (4, 5). Thus, understanding the mechanism underlying adipocyte differentiation is essential to management of common diseases.

Adipocyte differentiation is a complex process regulated by various factors. Upon induction of differentiation, a cascade of gene transcription events occurs, leading to the expression of adipocyte-specific genes (6). One of the essential genes involved in the cascade encodes peroxisome proliferator-activated receptor gamma  (PPARgamma ), a member of the ligand-activated nuclear receptor superfamily (7). PPARgamma binds to the retinoid X receptor (RXR) (8) and up-regulates the expression of adipocyte-specific genes to promote adipocyte differentiation (9). Exogenous expression of PPARgamma transforms NIH3T3 fibroblasts and G8 myoblasts into adipocyte-like cells (10, 11). Moreover, PPARgamma is activated by anti-diabetes drugs, such as thiazolidinediones (TZDs) (12). TZDs stimulate differentiation of preadipocytes and up-regulate glucose uptake into the adipose tissue by activating PPARgamma . TZDs also suppress the expression of TNF-alpha and enhance that of adiponectin in differentiated adipocytes (13, 14). Therefore, activation of PPARgamma is involved in the regulation of adipocyte differentiation as well as insulin activity in adipose tissues.

It has recently been reported that coactivators are necessary for the activation of nuclear receptors, including PPARgamma (15, 16). Coactivators interact with nuclear receptors in a ligand-dependent manner and recruit basal transcriptional factors such as RNA polymerases proximal to a nuclear receptor complex in a gene promoter region. Among the coactivators, the cAMP-response element-binding protein (CREB)-binding protein (CBP) and its highly related p300 protein have been rather well characterized to date. These coactivators are expressed ubiquitously, and they participate in many basic cellular events (17). PPARgamma interacts with CBP and p300 in a ligand-dependent manner, and p300, in turn, enhances the activity of PPARgamma (18, 19). However, it has not yet been clarified whether CBP and/or p300 can actually affect the expression of PPARgamma target genes in adipocytes or whether expression of endogenous CBP and/or p300 is indispensable for adipocyte differentiation.

The aim of this study was to elucidate the physiological role of CBP and p300 in PPARgamma -mediated gene expression in preadipocytes and adipocytes. Detailed analyses revealed that overexpression of CBP or p300 with PPARgamma enhanced the expression of PPARgamma target genes in NIH3T3 cells. Moreover, either CBP- or p300-specific ribozyme-mediated targeting resulted in suppressed gene expression of adipogenic markers such as adipocyte fatty acid-binding protein (aP2) and lipoprotein lipase (LPL), as well as reduction in glycerol-3-phosphate dehydrogenase activity and lipid accumulation in 3T3-L1 cells upon induction of adipocyte differentiation. This suggests that both CBP and p300 are indispensable for adipocyte differentiation and that CBP and p300 do not mutually complement in the process. To our knowledge, this is the first report of the physiological relevance of CBP and p300 in adipocytes.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- T174 TZD, a specific ligand for PPARgamma (18), was kindly provided by Tanabe Seiyaku Co., Ltd. (Osaka, Japan). All other chemicals were from Sigma or Nacalai Tesque (Kyoto, Japan) and were of guaranteed reagent grade or tissue culture grade.

Plasmid Construction and Preparation of Recombinant Retroviruses-- Expression plasmids for coactivators, pCMX-CBP and pCMX-p300, were kind gifts from Dr. R. H. Goodman (Oregon Health Sciences University) and Dr. D. M. Livingston (Harvard Medical School), respectively. pSK-CBP, which included CBP cDNA in pBlueScript-SK(+) (Stratagene), was used as a control plasmid to make the amounts of DNA transfected constant in transient expression assays. pSG5mPPARgamma for mouse PPARgamma and pE1A for adenoviral oncoprotein E1A were supplied by Dr. P. A. Grimaldi (INSERM U470) and Dr. T. Kouzarides (Wellcome/Cancer Research Campaign (CRC) Institute), respectively. A luciferase reporter plasmid containing four tandem repeats of the PPAR response element (PPRE) followed by a thymidine kinase promoter, p4xPPRE-tk-luc, was from Dr. K. Umesono (Kyoto University). pRL-CMV (Promega) was used as an internal control to normalize transfection efficiencies in luciferase assays. CBP- and p300-specific active ribozymes (RzCBP-wt and Rzp300-wt, respectively) have target sequences against nucleic acid sequences 484-502 in CBP cDNA and 364-382 in p300 cDNA, respectively (20). Inactive mutants of the CBP- and p300-specific ribozymes (RzCBP-mut and Rzp300-mut, respectively) have point mutations on each ribozyme active site, which cannot cleave target mRNAs (20).

Fragments of RzCBP and Rzp300 with EcoRI and SalI ends were inserted into a retrovirus expression vector, pMX-puro (a gift from Dr. T. Kitamura, University of Tokyo) (21) via the same sites, generating pMX-RzCBP and pMX-Rzp300, respectively. For the preparation of recombinant retroviruses, expression constructs were transiently transfected into Phoenix ecotroping packaging cells (a kind gift from Dr. G. Nolan, Stanford University) using LipofectAMINE Plus (Invitrogen) according to the manufacturer's protocol, and then the conditioned medium was recovered for subsequent infection.

Cell Culture-- Murine NIH3T3 fibroblasts and murine 3T3-L1 preadipocytes were purchased from American Type Culture Collection. All cell lines were maintained in a maintenance medium (10% fetal bovine serum, 200 µM ascorbic acid, and 10 mg/ml penicillin/streptomycin in Dulbecco's modified Eagle's medium) at 37 °C in 5% CO2/95% air under a humidified condition. For luciferase assays using NIH3T3 cells cultured on 24-well tissue culture plates, pSG5-mPPARgamma (0.4 µg/well), pCMX-CBP and/or pSK-CBP (0.4 µg/well), p4xPPRE-tk-luc (0.4 µg/well), and pRL-CMV (0.4 ng/well) were transfected into NIH3T3 cells. For quantification of PPARgamma target transcripts, pSG5-mPPARgamma (0.5 µg/well) and pCMX-CBP and/or pSK-CBP (0.5 µg/well) were transfected into NIH3T3 cells cultured on 6-well tissue culture plates. An expression vector for E1A, pE1A (0.5 µg/well), was included as indicated in Fig. 1B to inhibit CBP/p300 activity. The transfections were performed using LipofectAMINE (Invitrogen) according to the manufacturer's protocol. Twenty-four h after transfection, cells were supplemented with 10 µM TZD, cultured for another 24 h, and then lysed in the recommended lysis buffer for estimation of luciferase activity or harvested for mRNA preparation. Luciferase assays were performed using the dual luciferase assay system (Promega).

3T3-L1 cells expressing RzCBP-wt/-mut or Rzp300-wt/-mut were selected in a cell culture medium containing 2.0 µg/ml puromycin after infection with the corresponding retrovirus. To exclude the clonal variation in adipocyte differentiation, polyclonal cells were used directly for subsequent assays. The cells expressing ribozymes were cultured on 6-well tissue culture plates for immunoblotting and differentiation assays as described previously (22). Briefly, after 4 days, when confluence was reached, cells were incubated in a differentiation medium (DM), which is the maintenance medium supplemented with 0.25 µM dexamethazone, 10 µg/ml insulin, and 0.5 mM 3-isobutyl-1-methylxanthine. After 40 h, the cell culture medium was changed to post-DM, which is DM supplemented with 5 µg/ml insulin, and then the medium was replaced with fresh medium every 2 days. Eight days after differentiation induction, the cells were washed with phosphate-buffered saline, and total RNA was prepared using an RNeasy minikit (Qiagen, Hilden, Germany) according to the manufacturer's protocol.

Biochemical Assays, Immunoblots, and Oil-Red O Staining-- Samples for biochemical assays were prepared using cells cultured on 6-well tissue culture plates. The measurement of glycerol-3-phosphate dehydrogenase activity was performed as described previously (22). The content of cellular triacylglycerol was measured using a TG Test WAKO kit (Wako Pure Chemical Industry Ltd., Osaka, Japan). Protein concentrations of samples for immunoblotting were determined using a protein assay kit (Bio-Rad). Immunoblotting was carried out using an enhanced chemiluminescence system (PerkinElmer Life Sciences) as described previously (22). The anti-mouse PPARgamma antibody was obtained from Affinity Bioreagents, Inc., and antibodies against RXRalpha , C/EBPalpha , C/EBPbeta , C/EBPdelta , CBP, and p300 were from Santa Cruz Biotechnology Inc. Anti-beta -actin and horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgGs were purchased from Chemicon International Inc. and DAKO A/S (Copenhagen, Denmark), respectively. Oil-Red O staining was performed as follows: cells were washed with phosphate-buffered saline and then stained with 60% filtered Oil-Red O stock solution (0.15 g of Oil-Red O in 50 ml of isopropanol) for 30 min at 37 °C. Cells were washed with 60% isopropanol and then washed briefly with water and examined under a microscope.

mRNA Preparation and Quantification-- Aliquots of total RNA were reverse-transcribed using Moloney murine leukemia virus reverse transcriptase (Invitrogen) and a thermal cycler (Takara PCR Thermal Cycler SP; Takara Shuzo Co., Kyoto, Japan) according to the manufacturers' instructions. To quantify mRNA expression, PCR was performed using a fluorescence temperature cycler (LightCycler System; Roche Diagnostics). The oligonucleotide primer sets of mouse PPAR target genes were designed using a PCR primer selection program at the web site of the Virtual Genomic Center from the GenBankTM data base as follows: (a) mouse LPL (GenBankTM accession number J03302), forward primer 5'-ATCCATGGATGGACGGTAACG-3' and reverse primer 5'-CTGGATCCCAATACTTCGACCA-3'; (b) aP2 (GenBankTM accession number K02109), forward primer 5'-AAGACAGCTCCTCCTCGAAGGTT-3' and reverse primer 5'-TGACCAAATCCCCATTTACGC-3'); and (c) glyceraldehyde-3-phosphate dehydrogenase (GAPDH; GenBankTM accession number M32599), forward primer 5'-GAAGGTCGGTGTGAACGGATT-3' and reverse primer 5'-GAAGACACCAGTAGACTCCACGACATA-3'). Amplification was performed according to a published protocol (23). Briefly, the reaction solution (10 µl, final volume) contained 3 µM MgCl2, 2.0 µl of LightCycler DNA Master SYBR Green I dye, and 5 µM of each primer. The standard amplification program included 30 cycles of three steps each, which involved heating the product to 95 °C at 20 °C/s with a 30-s hold, annealing to 55 °C at 20 °C/s with a 5-s hold, and extension to 72 °C at 20 °C/s with a 10-s hold. The fluorescence at 530 nm was recorded on-line at the end of the extension phase. The amplified products were subcloned into the T-easy vector (Promega), sequenced, and used as PCR standards. The copy number of each standard plasmid was calculated from the absorbance at 260 nm and the molecular mass of each plasmid. The copy numbers of standards and samples were amplified simultaneously in the LightCycler. The first cycle number indicated specific fluorescence against noise, and the logarithm of the concentration of the PCR product standard, the external standard curve, was calculated using LightCycler software. To confirm the amplification of specific transcripts, melting curve profiles were generated at the end of each run. To compare the mRNA expression level among samples, the copy number of each transcript was divided by that of GAPDH, which showed a constant mRNA expression level. All data indicating the mRNA expression level were presented as a ratio with respect to that of the control in each experiment.

Statistical Analysis-- The data are presented as means ± S.E. and were analyzed statistically using the unpaired t test or the Welch t test when variances were heterogeneous. Differences were considered significant at p < 0.05.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Increase in Expression Level of CBP/p300 Proteins Enhances the Expression of PPARgamma Target Genes in Intact Cells-- It has been reported in detail that CBP and p300 interact with PPARgamma in a ligand-dependent manner, and increasing the amount of p300 enhanced PPARgamma activity (18, 19). However, it remained unknown whether an increase in the CBP protein expression level could promote PPARgamma transactivation in intact cells. To elucidate this, we performed luciferase assays by transfecting a reporter plasmid with the PPRE into PPARgamma -transfected NIH3T3 fibroblasts, which differentiate into adipocyte-like cells in a PPARgamma ligand-dependent manner (10). Increasing the amount of an expression plasmid for CBP enhanced luciferase activity in the presence of 10 µM TZD (Fig. 1A). Cotransfection of 0.2, 0.4, and 0.8 µg/well pCMX-CBP (a CBP expression vector) induced luciferase activities that were 1.2-, 1.9-, and 2.8-fold higher, respectively, than those of mock transfectants in the presence of TZD. These results suggest that the expression of CBP could enhance PPARgamma activity against ligand as much as expression of p300.


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  		Fig. 1.   CBP enhances PPARgamma activation in NIH3T3 cells. A, NIH3T3 cells were transfected with an expression plasmid for mouse PPARgamma (0.4 µg/well), a reporter plasmid with PPRE (0.4 µg/well), and increasing amounts of an expression plasmid for CBP. pRL-CMV (0.8 ng/well) was also included as an internal control to normalize transfection efficiency. Twenty-four h after transfection, cells were incubated in the presence or absence of 10 µM TZD (T174) for 24 h. Cells were lysed, and luciferase activity was assayed as described under "Experimental Procedures." Relative luciferase activity was presented as fold induction with respect to that of mock transfectants (without CBP) in the absence of TZD. The values are the means ± S.E. of four tests. *, p < 0.05 compared with mock transfectants. B, NIH3T3 cells were transfected with an expression vector for mouse PPARgamma (0.5 µg/well) and increasing amounts of an expression vector for CBP. An expression vector for E1A (0.5 µg/well) was included as indicated. Cells were cultured in the presence or absence of 10 µM TZD (T174) for 24 h after transfection, and then total RNA samples were prepared. The expression levels of aP2 (left panel) and LPL (right panel) were estimated using LightCycler and normalized with respect to the GAPDH expression level (each copy number of aP2 and LPL was divided by that of GAPDH). The relative gene expression is presented as the ratio of the expression level of a gene to that of the vehicle control without transfection of the CBP expression vector. The values are the means ± S.E. of six tests. *, p < 0.05 compared with a sample cotransfected without a CBP expression vector. **, p < 0.05 compared with a sample cotransfected without an E1A expression vector.

It was also investigated whether coexpression of CBP/p300 could regulate the gene promoter activity of endogenous promoters of PPARgamma target genes in cells. We used the PPARgamma -transfected NIH3T3 cells for luciferase assays. As shown in Fig. 1B, cotransfection of PPARgamma and CBP expression plasmids up-regulated the expression of endogenous aP2 (left panel) and LPL (right panel) by the addition of 10 µM TZD. PPARgamma regulates the expression of aP2 and LPL, which is parallel to adipocyte differentiation (24, 25). Therefore, aP2 and LPL have been used as well-characterized PPARgamma target genes and typical adipocyte differentiation markers. The expression of these endogenous genes depended on the amount of CBP plasmids transfected; cotransfection of 0.5 and 1.0 µg/well pCMX-CBP resulted in 2.3- and 4.1-fold increases in the up-regulation of aP2, respectively, compared with mock transfectants in the presence of 10 µM TZD (Fig. 1B, left panel). In a similar manner, cotransfection of 0.5 and 1.0 µg/well pCMX-CBP increased the expression level of LPL by 1.5- and 2.0-fold, respectively, compared with mock transfectants (Fig. 1B, right panel). Nearly the same results were obtained for p300 coexpression (data not shown). Moreover, the enhancement by CBP coexpression was significantly suppressed by coexpression of adenoviral oncoprotein E1A, which is known as a viral regulatory protein that specifically suppresses CBP/p300 activity in virus-infected cells (26) (Fig. 1B). Therefore, this result indicates that the increase in the CBP expression level was involved in the up-regulation of PPARgamma target genes in cells. These results strongly suggest that CBP and p300 proteins were involved in the up-regulation of PPARgamma target genes in intact cells.

CBP or p300 Targeting Specific Ribozymes Inhibits the Expression of PPARgamma Target Genes in 3T3-L1 Preadipocytes-- Next, to further deepen the understanding of the physiological relevance of CBP and p300, we established 3T3-L1 preadipocytes expressing a CBP- or p300-specific active ribozyme (RzCBP-wt or Rzp300-wt, respectively). RzCBP-wt and Rzp300-wt, which specifically cleave target mRNAs, can down-regulate the expression of CBP and p300 protein, respectively, in cells expressing the ribozyme (20). 3T3-L1 cells expressing a mutant of RzCBP-wt or Rzp300-wt (RzCBP-mut or Rzp300-mut, respectively), which is inactive on mRNA cleavage, were also established as controls. After puromycin selection, polyclonal cells for each ribozyme were used directly for subsequent analyses. Cells were lysed, and expression levels of CBP were determined by immunoblot analysis (Fig. 2, left panels). The expression of CBP was suppressed in 3T3-L1 cells expressing RzCBP-wt (3T3-L1-RzCBP-wt) to ~20% as compared with that in RzCBP-mut-expressing cells (3T3-L1-RzCBP-mut). However, there was no difference in the expression levels of p300 between 3T3-L1-RzCBP-wt and 3T3-L1-RzCBP-mut (Fig. 2). Conversely, the p300 expression level in 3T3-L1-Rzp300-wt decreased ~30% as compared with that in 3T3-L1-Rzp300-mut (Fig. 2, right panels). Steroid receptor coactivator 1 (SRC-1), another coactivator for PPARgamma (27), and beta -actin were expressed at nearly the same levels in any cells used. These results indicate that ribozymes specifically decreased the CBP or p300 mRNA expression level, resulting in a decreased expression level of each protein in 3T3-L1 preadipocytes.


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  		Fig. 2.   Ribozyme-mediated targeting of CBP or p300 in 3T3-L1 preadipocytes. Protein samples were prepared from 2-day confluent 3T3-L1 cells expressing RzCBP-mut (Inactive), RzCBP-wt (Active), Rzp300-mut (Inactive), or Rzp300-wt (Active). The same amounts of protein (30 µg) were loaded and blotted onto polyvinylidene fluoride membranes. The membranes were sequentially treated with primary antibodies as indicated and with secondary antibodies conjugated with horseradish peroxidase. The enhanced chemiluminescence system was used for visualization. The expression level of each protein in 3T3-L1 cells expressing either 3T3-L1-RzCBP-mut or 3T3-L1-Rzp300-mut was set at 100%, and the relative densitometric ratio values (estimated by NIH Image software) are indicated. The data are representative of three independent blots.

We next examined the expression levels of transcription factors involved in PPARgamma activation in 3T3-L1-RzCBP-wt/-mut. Confluent cells were lysed and separated by SDS-PAGE followed by immunoblotting. As shown in Fig. 3A, the expression level of PPARgamma 2 in 3T3-L1-RzCBP-wt was comparable to that in 3T3-L1-RzCBP-mut (Fig. 3A). Although PPARgamma 1, another isoform of PPARgamma in preadipocytes, was not detected, the total expression levels of PPARgamma 1 and PPARgamma 2 were shown to be comparable in 3T3-L1-RzCBP-wt and 3T3-L1-RzCBP-mut by quantitative reverse transcription-PCR analyses (data not shown). RXRs are heterodimer partners of PPARgamma and are essential to various functions of PPARgamma (28). In 3T3-L1 cells, RXRalpha is a functional subtype, and RXRbeta is expressed as well (8, 30). The expression levels of RXRalpha also did not differ in 3T3-L1-RzCBP-wt and 3T3-L1-RzCBP-mut (Fig. 3A). The absence of difference in the PPARgamma and RXRalpha expression levels in undifferentiated cells suggests that a decreased expression level of CBP does not affect the basal expression levels of PPARgamma and RXRalpha in 3T3-L1 preadipocytes.


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  		Fig. 3.   Ribozyme-mediated targeting of CBP resulted in suppressed expression of PPARgamma target genes in 3T3-L1 preadipocytes. A, expression levels of PPARgamma and its partner, RXRalpha , in 3T3-L1-RzCBP-mut (Inactive) and 3T3-L1-RzCBP-wt (Active). The same amounts of protein (15 µg) were subjected to immunoblotting. The results are shown on each lane as the percentage for the relative densitometric ratio (estimated by NIH Image software) compared with that of the band for 3T3-L1-RzCBP-mut (Inactive). The expression level of each protein in 3T3-L1-RzCBP-mut was set at 100%, and the relative densitometric ratios (estimated by NIH Image software) are indicated. The data are representative of three independent blots. B, the mRNA expression level of PPARgamma target genes in 3T3-L1-RzCBP-wt/-mut. Confluent 3T3-L1-RzCBP-mut (Inactive) and 3T3-L1-RzCBP-wt (Active) cells were cultured in the presence or absence of 10 µM TZD (T174) for 48 h. The expression levels of aP2 (left panel) and LPL (right panel) were estimated using LightCycler and normalized with respect to the GAPDH expression level. The relative gene expression was presented as fold induction with respect to vehicle controls. The values are the means ± S.E. of five tests. *, p < 0.05 compared with the inactive ribozyme controls.

3T3-L1-RzCBP-wt and 3T3-L1-RzCBP-mut were then stimulated with TZD, and the expression of PPARgamma target genes aP2 and LPL was investigated. The addition of 10 µM TZD resulted in a 53- and 5.4-fold increase in the expression level of aP2 and LPL mRNA, respectively, in 3T3-L1-RzCBP-mut, whereas only a 20- and 1.4-fold increase in aP2 and LPL mRNA, respectively, was observed in 3T3-L1-RzCBP-wt (Fig. 3B). Experiments using 3T3-L1-Rzp300-wt/-mut showed results similar to those using 3T3-L1-RzCBP-wt/-mut (data not shown). These data suggest that the decrease in expression levels of CBP/p300 resulted in the suppression of PPARgamma target gene expression in 3T3-L1 preadipocytes and that endogenous expression of CBP/p300 was essential to the induction of PPARgamma target genes in 3T3-L1 preadipocytes.

Targeting of CBP or p300 by Specific Ribozymes Inhibits Adipocyte Differentiation in 3T3-L1 Preadipocytes-- Finally, we investigated whether the decrease in the expression level of endogenous CBP or p300 in 3T3-L1 preadipocytes could affect their differentiation into adipocytes. 3T3-L1-RzCBP-wt/-mut and 3T3-L1-Rzp300-wt/-mut were cultured in DM for 40 h and then cultured in post-DM. Eight days after differentiation induction, 3T3-L1-RzCBP-mut accumulated fat droplets in cells (Fig. 4A, a). On the other hand, 3T3-L1-RzCBP-wt showed a low level of accumulation of fat droplets (Fig. 4A, b). Essentially the same results were obtained in Oil-Red O staining, by which triacylglycerides were stained red (Fig. 4A, c and d). To confirm the low level of accumulation of lipid droplets, we determined the triacylglyceride level in the cells. As shown in Fig. 4A, e, 8 days after differentiation induction, the triacylglyceride content in 3T3-L1-RzCBP-wt cells was significantly lower than that in 3T3-L1-RzCBP-mut cells. As observed in 3T3-L1-RzCBP-wt, the lipid levels in 3T3-L1-Rzp300-wt were also significantly lower than that in 3T3-L1-Rzp300-mut (Fig. 4B). Moreover, glycerol-3-phosphate dehydrogenase activity, which is one of the biochemical markers of adipocyte differentiation, was also significantly suppressed in 3T3-L1-RzCBP-wt and in 3T3-L1-Rzp300-wt throughout the course of adipocyte differentiation (Fig. 5). Therefore, it was shown that the decrease in the expression level of endogenous CBP or p300 in the presence of the active ribozymes resulted in the suppression of 3T3-L1 preadipocyte differentiation.


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  		Fig. 4.   Ribozyme-mediated targeting of CBP or p300 suppresses differentiation of 3T3-L1 preadipocytes into adipocytes. Confluent 3T3-L1 cells expressing the inactive and active ribozymes (A, CBP; B, p300) were treated by cultivation in DM for 40 h and then in post-DM for 8 days. Photomicrographs of representative 3T3-L1 cells expressing the inactive (a and c) or active (b and d) ribozymes are shown in reverse phase (a and b) and stained with Oil-Red O (c and d). Bars, 50 µm. At the same point, the cellular triacylglyceride content was estimated (e). Inactive and Active in this figure represent the results of 3T3-L1 adipocytes expressing inactive ribozymes (RzCBP-mut and Rzp300-mut) and active ones (RzCBP-wt and Rzp300-wt), respectively. The values are the means ± S.E. of six independent tests. *, p < 0.05 compared with the inactive ribozyme controls.


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  		Fig. 5.   Glycerol-3-phosphate dehydrogenase activities were suppressed in 3T3-L1 adipocytes expressing the active CBP- or p300-specific ribozyme. Glycerol-3-phosphate dehydrogenase activities in 3T3-L1 adipocytes expressing CBP-specific (A) or p300-specific (B) ribozyme were estimated after differentiation induction.  and black-square, glycerol-3-phosphate dehydrogenase activities of 3T3-L1 adipocytes expressing inactive (RzCBP-mut or Rzp300-mut) and active (RzCBP-wt and Rzp300-wt) ribozymes, respectively. Cells on 6-well plates were recovered at the indicated days after differentiation induction. Glycerol-3-phosphate dehydrogenase activities were measured as described under "Experimental Procedures." The values are the means ± S.E. of six tests. *, p < 0.05 compared with the inactive ribozyme controls.

Eight days after differentiation induction, the expression levels of aP2 and LPL genes, which are PPARgamma target genes and adipocyte differentiation markers, were estimated by the quantitative real-time reverse transcription-PCR method. As shown in Fig. 6A, the expression level of aP2 in 3T3-L1-RzCBP-wt was lower than that in 3T3-L1-RzCBP-mut (Fig. 6A, left panel). The expression of LPL was also significantly suppressed, but the extent of suppression was smaller (Fig. 6A, right panel). The suppressed expression of aP2 and LPL genes in 3T3-L1-Rzp300-wt was the same as that in 3T3-L1-RzpCBP-wt (data not shown).


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  		Fig. 6.   Expression of PPARgamma target genes as well as PPARgamma and C/EBPalpha is suppressed by decreased CBP expression level in differentiated 3T3-L1 adipocytes. 3T3-L1-RzCBP-wt/-mut adipocytes were induced to differentiate, and then their mRNA and proteins were subjected to reverse transcription-PCR analyses (A) and immunoblotting (B), respectively. Eight days after differentiation induction, total RNA samples were prepared from differentiated 3T3-L1 cells expressing either RzCBP-wt (Active) or RzCBP-mut (Inactive). The expression levels of aP2 (left panel) and LPL (right panel) were estimated as described in the Fig. 1 legend. The values are the means ± S.E. of five tests. *, p < 0.05 compared with the inactive ribozyme controls. For immunoblotting, cells were lysed at the indicated days after differentiation induction, and the same amounts of protein (20 µg) were immunoblotted with the indicated primary antibodies. The expression level of each protein in 3T3-L1 cells expressing RzCBP-mut (Inactive) was set at 100%, and the relative densitometric ratios (estimated by NIH Image software) are indicated. The data are representative of three independent blots.

Although the expression of aP2 and LPL is regulated mainly by PPARgamma with respect to the differentiation of 3T3-L1 cells, the C/EBP family, as well as PPARgamma , is also known to be involved in the regulation of adipocyte differentiation (31). Therefore, the expression of the C/EBP family, as well as PPARgamma , in the early and late phases of adipocyte differentiation was examined by immunoblotting. Eight days after the differentiation induction in 3T3-L1-RzCBP-mut, the PPARgamma 2 expression level was about 3.5-fold higher than that on day 0 (the start of differentiation induction) (Fig. 6B, top panels). However, the expression level of PPARgamma 2 in 3T3-L1-RzCBP-wt was lower than that in 3T3-L1-RzCBP-mut (~65%). PPARgamma 1 was not detected by the antibody used, but the expression of PPARgamma 1 mRNA was also suppressed in differentiated 3T3-L1-RzCBP-wt (data not shown). The expression levels of the full-length 42-kDa C/EBPalpha in 3T3-L1-RzCBP-wt and 3T3-L1-RzCBP-mut were nearly comparable 2 days after differentiation induction. Eight days after differentiation induction, an ~3-fold increase in the expression level of C/EBPalpha was induced in 3T3-L1-RzCBP-mut, whereas only about a 1.5-fold increase in the C/EBPalpha expression level was induced in 3T3-L1RzCBP-wt (Fig. 6, second panel from the top). On the other hand, there was no obvious difference in the expression level of C/EBPdelta and C/EBPbeta /liver activator protein (LAP) (32-kDa form), which were thought to be regulators of PPARgamma and C/EBPalpha induction (31) (Fig. 6B, third and fourth panels from the top). Other isoforms of C/EBPs, such as 30-kDa C/EBPalpha and 18-kDa C/EBPbeta /liver inhibitory protein (LIP), exhibited nearly the same expression profiles as 42-kDa C/EBPalpha and 18-kDa C/EBPbeta /LAP, respectively (data not shown). These results suggest that the inhibition of adipocyte differentiation by the decrease in CBP expression is due primarily to the suppression of PPARgamma expression and activity, but other transcriptional factors such as C/EBPalpha could be involved in the differentiation process.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this study, we showed that the increase in the expression level of CBP, a coactivator for PPARgamma , resulted in the activation of PPARgamma in NIH3T3 cells by targeting PPRE in luciferase reporter plasmid (Fig. 1A) and endogenous promoters of aP2 and LPL genes (Fig. 1B). NIH3T3 fibroblasts are not preadipocytes, but exogenous expression of PPARgamma transforms the transfected NIH3T3 cells into preadipocytes, which can differentiate into adipocytes by treatment with a combination of dexamethazone, 3-isobutyl-1-methylxanthine, and insulin or TZD (10). To our knowledge, our results first showed that PPARgamma ligand-dependent expression of PPARgamma target genes in intact cells, as in NIH3T3 fibroblasts, was induced by the ectopic expression of CBP or p300. This suggests that the expression of endogenous CBP/p300 could be a rate-limiting factor in PPARgamma activation in NIH3T3 cells.

The physiological significance of CBP/p300 in complete activation of PPARgamma was further examined in ribozyme-mediated targeting experiments. Decreasing the CBP or p300 expression level in 3T3-L1 preadipocytes using specific ribozymes suppressed PPARgamma ligand-dependent induction of aP2 and LPL (Fig. 3). This suggests that the expression levels of both CBP and p300 were indispensable for induction of PPARgamma target genes in 3T3-L1 preadipocytes. Moreover, the expression level of endogenous CBP or p300 was essential for differentiation of 3T3-L1 preadipocytes (Figs. 4 and 5). Although CBP and p300 share high sequence similarity throughout their entire structure (32), several differences in their functions have been reported (20, 33), suggesting that CBP and p300 might function at different points in the course of adipocyte differentiation. It is known that many nuclear transcriptional factors such as PPARs and C/EBPs are involved in adipocyte differentiation and that the activation of those transcriptional factors requires several coactivators (or a coactivator complex), including CBP/p300 (18, 19), steroid receptor coactivator 1 (27), and PPARgamma coactivators (34, 35). Inhibition of one or more steps of transcriptional regulation in adipocyte differentiation by a decrease in either CBP or p300 expression could totally suppress the transcriptional cascade, leading to the inhibition of adipocyte differentiation. Another possibility is that CBP and p300 might function equally, and the total expression level of CBP and p300 is essential to the complete activation of PPARgamma and adipocyte differentiation. In this sense, it might be interesting to examine whether ectopic expression of CBP or p300 in 3T3-L1-Rzp300-wt or 3T3-L1-RzCBP-wt could complement the suppression of PPARgamma activity and adipocyte differentiation.

Although our experiments focused mainly on PPARgamma in preadipocytes and adipocytes, the C/EBP family is also important in adipocyte differentiation (36). The following model is widely accepted. C/EBPbeta and C/EBPdelta are induced early and temporally in adipocyte differentiation, and then they stimulate PPARgamma and C/EBPalpha expression. Finally, PPARgamma and C/EBPalpha induce their mutual expressions under the control of CBP and p300 coactivators (Refs. 37 and 38; namely, there is a positive feedback loop between PPARgamma and C/EBPalpha (39)) and synergistically promote adipocyte differentiation. Targeting of CBP by RzCBP-wt resulted in suppression of PPARgamma and C/EBPalpha expression, whereas C/EBPbeta and C/EBPdelta were expressed at comparable levels (Fig. 6). These data suggest that CBP/p300 are necessary for induction of PPARgamma and C/EBPalpha expression, but not for that of C/EBPbeta or C/EBPdelta . The decrease in the PPARgamma and C/EBPalpha expression might be due to decreased activities of C/EBPbeta and C/EBPdelta because CBP can act as a coactivator for the transcriptional factors (40, 41). Alternatively, there might be a mechanism independent of C/EBPbeta and C/EBPdelta activation that regulates PPARgamma and C/EBPalpha expression, as Akira and co-workers reported previously (42). They proposed an alternative mechanism that regulates PPARgamma and C/EBPalpha expression because PPARgamma and C/EBPalpha expression was normal in mice lacking C/EBPbeta and C/EBPdelta . Thus, it is suggested that CBP and p300 function sequentially in both activation and expression of transcriptional factors involved in the adipocyte differentiation process.

The expression levels of PPARgamma in undifferentiated 3T3-L1-RzCBP-wt and 3T3-L1-RzCBP-mut cells were apparently the same (Fig. 3), suggesting that CBP and/or p300 is not necessary in the basal expression of PPARgamma , although they were essential in the differentiation-dependent induction of PPARgamma . This might be because other coactivators compensated for the function of CBP/p300 in basal expression of PPARgamma or because the basal expression was regulated by a possible mechanism that is independent of CBP/p300.

Our data showing that the expression of CBP and p300 was indispensable for complete activation of PPARgamma and adipocyte differentiation suggest that dysfunction of CBP and/or p300 might be associated with common diseases such as obesity and diabetes. A recent study showed that the interaction of PPARgamma with distinct coactivators was ligand type-specific (43), suggesting that PPARgamma target genes could be regulated by various combinations of coactivators and PPARgamma ligands. In the present study, a decrease in the CBP expression level suppressed gene expression of aP2 more strongly than that of LPL in differentiated 3T3-L1 cells (Fig. 6), and this was somehow consistent with the observation that the overexpression of CBP in NIH3T3 cells up-regulated aP2 expression more strongly than LPL expression (Fig. 2). Thus, CBP/p300 might be more significant in aP2 gene expression in PPARgamma -transfected NIH3T3 cells and in 3T3-L1 adipocytes. The expression of aP2 in adipose tissues links obesity to insulin resistance. Obese aP2 knockout mice did not develop insulin resistance and diabetes due to failure in TNF-alpha expression in adipose tissues (44). Expression of aP2 is central to the pathway that links obesity to insulin resistance by linking fatty acid metabolism to TNF-alpha expression. With respect to the significance of CBP in aP2 expression, we propose that CBP might be a good candidate for treatment of obesity and diabetes. This is supported by our preliminary data showing that the CBP expression level in adipose tissues of KK-Ay strain mice (obesity and diabetes model mice) was higher than that of A/J strain mice (obesity resistance model mice), although the expression levels of other coactivators such as SRC-1 were almost the same.2 In this sense, it is interesting to elucidate how CBP expression is regulated in adipocytes. Abnormality in CBP expression will result in critical damage to many tissues as well as adipose tissues because CBP is ubiquitously expressed and is essential to basic cellular events (17), and it has been shown that disruption of the mouse CBP gene was lethal to the embryo (45). Furthermore, CBP dysfunction caused Rubinstein-Taybi syndrome (46). Recently, such diseases have been called "coactivator diseases" (29), which are characterized by severe generalized dysfunctions. Thus, we again emphasize the physiological relevance of CBP/p300 in adipocyte differentiation and lipid metabolism.

PPARgamma plays a central role in adipocyte differentiation and lipid metabolism by adipocytes. Understanding the mechanisms by which PPARgamma is activated leads to effective management of common diseases including obesity, diabetes, and atherosclerosis. PPARgamma activation is regulated mainly in a ligand-dependent manner. However, because the interaction of PPARgamma with coactivators is also important in adipocyte differentiation and is regulated in a ligand type-specific manner as discussed above, the regulation of PPARgamma activation and its own expression by coactivators may be the primary system. In this regard, our study is very important not only in investigating adipocyte differentiation but also in clarifying the relationship between coactivators and common diseases such as obesity and diabetes.

    ACKNOWLEDGEMENTS

We thank Dr. R. H. Goodman for the gift of CBP DNAs, Dr. D. M. Livingston for the p300 DNAs, Dr. P. A. Grimaldi for the gift of the mouse PPARgamma expression vector, Dr. T. Kouzarides for the E1A expression vector, Dr. K. Umesono for the PPRE reporter plasmid, Dr. T. Kitamura for the retrovirus expression vector, and Dr. J. Ohkawa for valuable discussion about ribozymes.

    FOOTNOTES

* This work was supported by the PROBRAIN Project of the Bio-oriented Technology Research Advancement Institution, Japan and by Grant-in-aid for Scientific Research 13460058 from the Ministry of Education, Culture, Sports, Science and Technology of Japan.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.

To whom correspondence should be addressed: Laboratory of Nutrition Chemistry, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan. Fax: 81-75-753-6264; E-mail: fat@kais.kyoto-u.ac.jp.

Published, JBC Papers in Press, March 7, 2002, DOI 10.1074/jbc.M200585200

2 T. Kawada and N. Takahashi, manuscript in preparation.

    ABBREVIATIONS

The abbreviations used are: TNF-alpha , tumor necrosis factor alpha ; PPAR, peroxisome proliferator-activated receptor; PPRE, PPAR response element; TZD, thiazolidinedione; DM, differentiation medium; CREB, cAMP-response element-binding protein; CBP, CREB-binding protein; RXR, retinoid-X receptor; C/EBP, CCAAT/enhancer-binding protein; LPL, lipoprotein lipase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

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