Repression of cellular retinoic acid-binding protein II during adipocyte differentiation

Abstract

In preadipocytes, retinoic acid (RA) regulates gene expression by activating the nuclear RA receptor (RAR) and its cognate intracellular lipid-binding protein CRABP-II. It was previously reported that RA inhibits adipocyte differentiation but only when administered early during the differentiation program. The data presented here indicate that the diminished ability of RA to activate RAR following induction of differentiation stems from down-regulation of CRABP-II. The observations show that expression of CRABP-II in preadipocytes is repressed by all three components of the classical hormonal mixture that induces adipocyte differentiation, i.e. isobutylmethylxanthine, insulin, and dexamethasone. Isobutylmethylxanthine-dependent activation of protein kinase A triggered the phosphorylation of the transcription factor cAMP-response element-binding protein, which induced the expression of the cAMP-response element-binding protein family repressor cAMP-response element modulator. In turn, cAMP-response element modulator was found to associate with a cognate response element in the CRABP-II promoter and to repress CRABP-II expression. The data further show that CRABP-II is a direct target gene for the glucocorticoid receptor and that it is subjected to dexamethasone-induced glucocorticoid receptor-mediated repression during adipogenesis. Finally, the observations demonstrate that permanent repression of CRABP-II in mature adipocytes is exerted by the master regulator of adipocyte differentiation CCAAT/enhancer-binding protein alpha and is directly mediated through CCAAT/enhancer-binding protein alpha-response elements in the CRABP-II promoter. Taken together, the observations emphasize the important role of CRABP-II in regulating the transcriptional activity of RA through RAR, and they demonstrate that repression of this gene is critical for allowing adipogenesis to proceed.

FIGURE 1.

CRABP-II sensitizes NIH3T3-L1 cells to RA-induced inhibition of adipogenesis. a, NIH3T3-L1 preadipocytes were infected with an empty adenovirus (Ad0) or adenovirus harboring CRABP-II cDNA (Ad-II) at 80% confluence. Two days later, cells were induced to differentiate. CRABP-II level in resulting mature adipocytes was assessed by immunoblotting. b, preadipocytes were infected with Ad0 or Ad-II as described in a and induced to differentiate in the absence or presence of denoted concentrations of RA. Micrographs show lipid accumulation at the end of differentiation. c, cells were treated as described in b, and triglyceride content at the end of differentiation was measured (Zen-Bio, triglyceride assay kit). d, cells were treated as described in b, and levels of C/EBP-β mRNA were measured by Q-PCR for 24 h following induction. Error bars indicate S.D. e, cells were treated as described in b, and expression of FABP4 in mature adipocytes was measured by immunoblots.

FIGURE 2.

Adipocyte differentiation is accompanied by down-regulation of CRABP-II expression. a, preadipocytes were induced to differentiate, and CRABP-II expression at denoted time points was analyzed by Q-PCR. b, immunoblots of CRABP-II expression in preadipocytes at the denoted time points following differentiation induction. c, preadipocytes were treated with actinomycin D (5 μg/ml) for denoted time points, and levels of CRABP-II mRNA were analyzed by Q-PCR. d and e, preadipocytes were treated with insulin (10 μg/ml), dexamethasone (dex) (0.25 mm), or IBMX (0.5 mm) for 4 h. Levels of CRABP-II mRNA were analyzed by Q-PCR (d) and CRABP-II protein levels assessed by immunoblots (e). Error bars indicate S.D.

FIGURE 3.

Insulin, dexamethasone, and IBMX down-regulate CRABP-II expression. a and b, preadipocytes were pretreated with denoted concentrations of wortmannin (wort) (a) or with RU486 (b) and then treated with 10 μg/ml insulin (a) or 250 nm dexamethasone (b). CRABP-II mRNA expression was analyzed by Q-PCR. c, preadipocytes were treated with denoted concentrations of IBMX, and CRABP-II mRNA expression was analyzed by Q-PCR. d, preadipocytes were treated with either actinomycin D (act D) (5 μg/ml) or IBMX (0.5 mm) for the denoted times, and CRABP-II mRNA expression was analyzed by Q-PCR. Error bars indicate S.D.

FIGURE 4.

Repression of CRABP-II by IBMX is mediated by PKA. a and b, preadipocytes were treated with IBMX (0.5 mm), forskolin (1 μm), or the PKA inhibitor H89 (1 μm) for 4 h or pretreated with H89 prior to treatment with IBMX or forskolin (fors). n/t, not treated. Levels of CRABP-II mRNA (a) and protein (b) were measured by Q-PCR and immunoblots, respectively. c, primary human preadipocytes isolated from three individual patients were treated with IBMX (0.5 mm, 4 h), and CRABP-II expression was analyzed by Q-PCR. d, preadipocytes were pretreated with cycloheximide (CHX) (10 μg/ml, 15 min) and then treated with IBMX (0.5 mm, 4 h). CRABP-II mRNA expression was analyzed by Q-PCR. Error bars indicate S.D.

FIGURE 5.

Repression of CRABP-II by PKA is mediated by CREB and its target gene CREM. a, preadipocytes were treated with IBMX (0.5 mm) for 15 min, and phosphorylation of CREB was assessed by immunoblots. b, preadipocytes were infected with empty lentivirus (ev) or lentivirus harboring CREB shRNA. Decreased expression of CREB was assessed by immunoblots. c and d, preadipocytes infected with lentivirus as denoted were then treated with IBMX (0.5 mm) for 4 (c) or 12 h (d), and levels of CRABP-II mRNA (a) and protein (d) were assessed by Q-PCR and immunoblots, respectively. e, preadipocytes were treated with IBMX (0.5 mm) for the denoted times. Levels of mRNA for CREM and CRABP-II were measured by Q-PCR. f, preadipocytes were infected with denoted lentiviruses and treated with IBMX for 4 h. Levels of mRNA for CREM were measured by Q-PCR. g, immunoblots of CREM in preadipocytes were infected with empty lentivirus or with lentivirus harboring CREM shRNA. h, top, immunoblots of CRABP-II in preadipocytes were infected with denoted lentiviruses and treated with IBMX for 12 h. Bottom, quantification of immunoblots from three independent experiments. Data are mean ± S.E. i, location of the Cre element in the first intron of the CRABP-II gene. j, ChIP assays indicating recruitment of CREM and HDAC1 to the Cre element of the CRABP-II gene following treatment with IBMX (0.5 mm, 4 h.) (see under “Experimental Procedures” for assay details). IP, immunoprecipitation.

FIGURE 6.

CRABP-II is directly repressed by GR. a, preadipocytes were pretreated with cycloheximide (CHX) (10 μg/ml, 15 min) and then treated with dexamethasone (dex) for 4 h. CRABP-II expression at the denoted time was assessed by Q-PCR. b, immunoblot of GR in preadipocytes infected with empty lentivirus (ev) or with lentivirus harboring GR shRNA. c and d, preadipocytes were infected with lentoviruses and treated with dexamethasone for 4 h (c) or 12 h (d). Expression levels of CRABP-II mRNA (c) and protein (d) were assessed by Q-PCR and immunoblots, respectively. e, GR half-site in the CRABP-II promoter. Error bars indicate S.D. f, ChIP assays indicating recruitment of GR to the GRE of the CRABP-II gene following treatment with dexamethasone (see “Experimental Procedures” for assay details). IP, immunoprecipitation.

FIGURE 7.

C/EBPα represses CRABP-II in mature adipocytes. a, five C/EBPα sites are located in the CRABP-II promoter. b, top, ChIP assays indicate recruitment of C/EBPα to the promoter region containing the putative C/EBPα sites in mature adipocytes. Bottom, ChIP assays indicating that in mature adipocytes CREM does not occupy the Cre in the CRABP-II promoter (compare with Fig. 5j). c and d, expression of C/EBPα in mature adipocytes was decreased using a lentivirus harboring C/EBPα shRNA. CRABP-II expression was analyzed by Q-PCR (b) or immunoblot (c). IP, immunoprecipitation. Error bars indicate S.D.

FIGURE 8.

Model for the regulation of CRABP-II expression during adipogenesis and in mature adipocytes. The classical adipocyte differentiation inducers, insulin, IBMX, and dexamethasone (dex), all repress the expression of CRABP-II. IBMX increases cellular levels of cAMP leading to activation of PKA. Activated PKA phosphorylates CREB, which positively regulates the expression of its direct target gene CREM. In turn, CREM associates with a CRE in the first intron of CRABP-II and recruits HDAC1 to repress CRABP-II transcription. Dexamethasone activates GR. In turn, GR binds to a GRE in the CRABP-II promoter and negatively regulates CRABP-II expression. The details of the mechanism through which insulin represses CRABP-II expression remain to be explored. In mature adipocytes, low levels of expression of CRABP-II are maintained by direct repression by C/EBPα.