Repression of Cellular Retinoic Acid-binding Protein II during Adipocyte Differentiation

Hooman Soltanian,Noa Noy,Daniel C. Berry

doi:10.1074/jbc.m110.110635

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.

Highlights

The individual components of the mixture that trigger differentiation facilitate different aspects of adipogenesis
It has been demonstrated in regard to this that RA is delivered to RA receptor (RAR) by the intracellular lipidbinding proteins (iLBP) cellular retinoic acid-binding protein II (CRABP-II) and that it is transported to PPAR␤/␦ by the iLBP termed fatty acid-binding protein 5 (FABP5) (18, 20 –24)
Available information indicates that the ability of RA to inhibit adipogenesis is exerted by activities of the hormone that are mediated by CRABP-II and its cognate receptor RAR in preadipocytes and suggests that downregulation of CRABP-II is a critical component of the differentiation process

Summary

EXPERIMENTAL PROCEDURES

Reagents—Antibodies against CREM, phospho-CREB, and GR were from Santa Cruz Biotechnology. C/EBP␣ and CREB antibodies were from Cell Signaling and Millipore, respectively. The triglyceride assay kit was obtained from Zen-Bio. Cells—3T3-L1s were obtained from ATCC and cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% calf serum. Cells were grown 2 days postconfluence and induced to differentiate with Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 10 ␮g/ml insulin, 0.5 mM IBMX, and 0.25 mM dexamethasone. Chromatin Immunoprecipitation (ChIP) Assays—Preadipocytes were treated with IBMX (0.5 mM, 4 h) or dexamethasone (250 nM, 2 h) prior to cross-linking with formaldehyde. Q-PCR was carried out using TaqMan chemistry and Assays-on-Demand probes (Applied Biosystems) for CRABP-II Mm00801691_ m1, CREM Mm00516346_m1, and CREB Mm00501607_m1. Cells were centrifuged (500 ϫ g, 10 min, 4 °C), resuspended in Dulbecco’s modified Eagle’s/F-12 media containing 10% calf serum, and cultured (37 °C, 5% CO2).

RESULTS

RA regulates gene transcription by activating the nuclear receptor

The observations that suppression