CCAR 1 regulates adipogenesis through glucocorticoid receptor 1

Glucocorticoids contribute to adipocyte differentiation by cooperating with transcription factors, such as CCAAT/enhancer-binding protein  (C/EBP), to stimulate transcription of the gene encoding peroxisome proliferatoractivated receptor (PPAR), a master regulator of adipogenesis. However, the mechanism of PPAR gene regulation by glucocorticoids, the glucocorticoid receptor (GR) and its coregulators is poorly understood. Here we show that two GR binding regions (GBRs) in the mouse PPAR gene were responsive to glucocorticoid, and treatment of 3T3-L1 preadipocytes with glucocorticoid alone induced GR occupancy and chromatin remodeling at PPAR GBRs, which also contain binding sites for C/EBP and PPAR proteins. GR recruited cell cycle and apoptosis regulator 1 (CCAR1), a transcription coregulator, to the PPAR gene GBRs. Notably, CCAR1 was required for GR occupancy and chromatin remodeling at one of the PPAR gene GBRs. Moreover, depletion of CCAR1 markedly suppressed differentiation of mouse mesenchymal stem cells and 3T3-L1 preadipocytes to mature adipocytes, and decreased induction of PPAR, C/EBP and C/EBP. While CCAR1 was required for stimulation of several GRregulated adipogenic genes in 3T3-L1 preadipocytes by glucocorticoid, it was not required for GR-activated transcription of certain anti-inflammatory genes in human A549 lung epithelial cells. Overall, our results highlighted the novel and specific roles of GR and CCAR1 in adipogenesis. http://www.jbc.org/cgi/doi/10.1074/jbc.M114.548081 The latest version is at JBC Papers in Press. Published on May 8, 2014 as Manuscript M114.548081 Copyright 2014 by The American Society for Biochemistry and Molecular Biology, Inc. at SU N Y A T SE N U N IV E R IT Y on Jauary 1, 2015 hp://w w w .jb.org/ D ow nladed from CCAR1 regulates adipogenesis through glucocorticoid receptor 2 INTRODUCTION Obesity is a major risk factor for many diseases, such as type-2 diabetes, hypertension, and cardiovascular disease (1). Hence, it is recognized as a prevalent health hazard worldwide. This excess of white adipose tissue caused by increases in the size and number of white adipocytes is the major cause of obesity. The number of white adipocytes present in an organism is determined largely by adipocyte differentiation (1). Therefore, elucidation of mechanisms of adipocyte differentiation is essential for understanding the pathogenesis of obesity and obesity-related diseases and thereby providing important information for developing new strategies to prevent and treat obesity. Adipocytes, the major fat-containing components of adipose tissue, are developed from mesenchymal stem cells (MSC) in adipose tissue. This process involves an initial commitment phase where MSC are committed to the preadipocyte lineage, followed by a differentiation phase (adipogenesis) where preadipocytes differentiate into mature fat-laden adipocytes. The major discoveries of key adipogenic signaling pathways have relied heavily on investigations in preadipocyte cell lines, such as mouse 3T3-L1 cells, which undergo a highly conserved and efficient program of adipogenesis in culture and upon transplantation in vivo (2). Since subsequent experiments in mice have convincingly validated the physiological significance of these major signaling pathways, the in vitro differentiation model represents a powerful and valid tool for deciphering the complex regulatory network of adipocyte differentiation. The adipogenic conversion of most mouse and human preadipocyte cell lines requires stimulation with a hormonal cocktail that consists of the synthetic glucocorticoid analogue dexamethasone (dex), insulin and the cAMP phosphodiesterase inhibitor isobutylmethylxanthine (IBMX). Temporary exposure of preadipocytes to the adipogenic stimuli induces a complex network of proadipogenic transcription factors that act at different stages of differentiation and cooperatively promote adipogenesis. Significant players in this transcriptional cascade include CCAAT/enhancer binding protein (C/EBP) family members (i.e. C/EBP, C/EBPβ, and C/EBP) and the nuclear receptor peroxisome proliferator-activated receptor  (PPAR), which is considered as the master positive regulator of preadipocyte differentiation to adipocytes and is both indispensable and under some conditions sufficient for adipogenesis (2). The expression levels of C/EBPβ and C/EBP are low in preadipocytes, but are greatly enhanced after treatment of the cells with adipogenic cocktail. During the first hours of adipogenesis, both of these C/EBPs associate with regulatory elements of a variety of adipogenic genes, including the two major late-acting transcription factors, C/EBP and PPAR, and subsequently activate gene expression. Raised levels of C/EBPα and PPAR then induce each other’s expression in a positive feedback loop promoting and maintaining the differentiated state of the mature adipocyte (3). Dex is a key component of the adipogenic cocktail and is involved in transcriptional regulation of adipogenesis via binding to and activating the glucocorticoid receptor (GR), a hormone-dependent transcription factor (4). Several lines of evidence have demonstrated the important role of GR in adipogenesis. Firstly, cells with decreased GR levels were unable to efficiently store lipids and express adipocyteselective proteins C/EBP and PPAR in response to adipogenic stimuli. Secondly, GR is involved in induction of key pro-adipogenic transcription factor C/EBP (4) as well as several other adipogenic genes such as RGS2 (5) and EPAS1 (6). Finally, during the early stages of adipogenesis, transient enrichment of GR, C/EBPβ, mediator subunit 1 (Med1), p300 and histone acetylation at regulatory sites near adipogenic genes has been previously reported (7); target genes include the gene encoding the master regulator of adipogenesis, PPAR. Thus, GR cooperates with C/EBPβ to initiate transcriptional activation of key adipogenic genes that are required to specify adipogenic cell fate. However, both the precise molecular contribution by GR and the identities of the transcriptional coregulators that support and modulate GR activity during the adipocyte differentiation process remain largely at SU N Y A T SE N U N IV E R IT Y on Jauary 1, 2015 hp://w w w .jb.org/ D ow nladed from CCAR1 regulates adipogenesis through glucocorticoid receptor 3 unexplored and are the subject of the study presented here. In addition to the role of glucocorticoids and GR in regulating adipogenesis, glucocorticoids are well-known for their anti-inflammatory and immunosuppressive properties and are therefore widely and successfully used in the treatment of numerous inflammatory conditions, including auto-immune diseases, inflammatory bowel disease, and lymphoma. Nevertheless, glucocorticoids are also infamous for adverse metabolic side effects associated with the chronic administration required to treat these diseases effectively. For approximately two decades, great efforts have been made to identify and develop for clinical use selective GR ligands that dissociate beneficial anti-inflammatory effects from adverse metabolic effects of glucocorticoids (8-11), but unfortunately with little success. Another possible approach to this problem may be offered by coregulators, which have been shown to function in a gene-specific manner with GR and other transcription factors, and therefore may represent potential targets for combination therapy to accompany the use of classical anti-inflammatory steroids such as dex. For example, inhibition of a coregulator required for regulation of genes involved in the metabolic actions of glucocorticoids but not required for regulation of genes involved in the antiinflammatory response to glucocorticoids could limit some side effects associated with glucocorticoid treatment of inflammatory diseases. Toward that end we performed a limited screen of coregulators by assessing the effect of depleting each coregulator from 3T3-L1 cells on the induction of several adipogenic genes by dex. In parallel, we tested the effect of coregulator depletion on dex-induced expression of several anti-inflammatory genes in A549 human lung adenocarcinoma cells. From this screen cell cycle and apoptosis regulator 1 (CCAR1), previously identified as a coactivator for GR (12), was preferentially required for dex-induced expression of adipogeneic genes versus antiinflammatory genes. Subsequently, we characterized the role of CCAR1 in the differentiation of 3T3-L1 cells to mature adipocytes and the mechanisms by which GR and CCAR1 contribute to the activation of the key adipogenic transcription factor, PPAR. We found that CCAR1 determines adipogenic competency, acting, in part, through positive regulation of GR transcriptional activity that drives early adipogenesis. Thus, targeting glucocorticoid coregulators, such as CCAR1, could be an effective approach to separate beneficial anti-inflammatory effects of glucocorticoids from certain unfavorable effects of glucocorticoids, such as obesity involving aberrantly activated adipogenesis. EXPERIMENTAL PROCEDURES Materials Antibodies against the following proteins were purchased and used for this study: GR from Santa Cruz Biotechnology; β-actin and αtubulin from Sigma; CCAR1 and IgG from Bethyl Laboratory. Secondary antibodies (horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG) were purchased from Promega. Insulin, dex, and IBMX were purchased from Sigma Aldrich. Transfection reagent Dharmafect Duo was purchased from Thermo Fisher Scientific-Dharmacon, and Oligofectamine was purchased from Invitrogen. Lentivirus-mediated coregulator depletion For lentivirus production, the packaging vector pCMV-ΔR8.91, the envelop plasmid pMD.G1 and the transfer vector pHRCMVpuroSin8 targeting non-specific sequence as control were described previously (13). For depletion of mouse CCAR1, two pLKO.1 vectors expressing shRNA against different regions