Adipose tissue mass is modulated by SLUG (SNAI2)

Pedro Antonio Pe rez-Mancera 2 Camino Bermejo-Rodrguez 2 Ine s Gonza lez-Herrero 2 Michel Herranz 2 Teresa Flores 1 Rafael Jime nez 0 Isidro Sa nchez-Garca 2 0 Departamento de Fisiologa y Farmacologa, Universidad de Salamanca , Campus M. Unamuno, 37007 Salamanca, Spain 1 Servicio de Anatoma Patolo gica 2 Experimental Therapeutics and Translational Oncology Program, Instituto de Biologa Molecular y Celular del Ca ncer (IBMCC) , CSIC The zinc-finger transcription factor SLUG (SNAI2) triggers epithelial - mesenchymal transitions (EMTs) and plays an important role in the developmental processes. Here, we show that SLUG is expressed in white adipose tissue (WAT) in humans and its expression is tightly controlled during adipocyte differentiation. Slugdeficient mice exhibit a marked deficiency in WAT size, and Slug-overexpressing mice (Combi-Slug) exhibit an increase in the WAT size. Consistent with in vivo data, Slug-deficient mouse embryonic fibroblasts (MEFs) showed a dramatically reduced capacity for adipogenesis in vitro and there was extensive lipid accumulation in Combi-Slug MEFs. The analysis of adipogenic gene expression both in vivo and in vitro showed that peroxisome proliferator-activated factor g2 (PPARg2) expression was altered. Complementation studies rescued this phenotype, indicating that WAT alterations induced by Slug are reversible. Our results further show a differential histone deacetylase recruitment to the PPARg2 promoter in a tissue- and Slug-dependent manner. Our results connect, for the first time, adipogenesis with the requirement of a critical level of an EMT regulator in mammals. This work may lead to the development of targeted drugs for the treatment of patients with obesity and/or lipodystrophy. - INTRODUCTION Being overweight or obese increases the risk of many diseases and health conditions. Therefore, a better knowledge of the molecular mechanisms that control adipose tissue development and function is an important goal for understanding the causes, prevention and treatment of obesity. Previous studies have identified a number of transcription factors involved in adipocyte differentiation. These include peroxisome proliferatoractivated factor g (PPARg) and members of the C/EBP family of transcription factors (1,2). Although many of the components of the gene regulatory network that controls the differentiation of adipocytes have been elucidated in studies of cultured 3T3-L1 pre-adipocytes and primary mouse embryonic fibroblasts (MEFs), recent evidence has suggested that additional factors are likely to be necessary in vivo (3,4). The zinc-finger transcription factor SLUG (SNAI2) is a member of the Snail family of zinc-finger transcription factors that share an evolutionary conserved role in mesoderm formation in invertebrates and vertebrates. SLUG is an important regulator of normal and tumour development (5,6). Slug controls key aspects of stem cell function, suggesting that similar mechanisms control normal development and cancer stem cell properties (7 10). The post-natal expression of SLUG (SNAI2) and the effects of SLUG deletion and overexpression are similar in mouse and human (8,10 15). Homozygous-null Slug mice have a white forehead blaze, patchy depigmentation of the ventral body, tail and feet and macrocytic anaemia and infertility, inferring an essential role for Slug in melanocytes, haematopoietic stem cells and germ cells (8). Heterozygous deletion of the SNAI2 gene results in human piebaldism (14), whereas a homozygous deletion has been detected in two individuals with Waardenburg disease type 2 (12). Recent studies showed that Slug is tightly controlled temporally and spatially in a number of sites including the neural crest and haematopoietic system (7,8). Regarding the major adult tissues, transcripts of the Slug gene are present in white adipose tissue (WAT) in mice (10), suggesting a potential role for Slug in adipogenesis. However, the functions of SLUG in adipocyte development in vivo and in vitro remain unknown. In this study, we found that SLUG is expressed in human WAT tissue. Slug expression is tightly controlled during adipocyte differentiation in both 3T3-L1 and primary murine embryonic fibroblast (MEFs), suggesting that Slug is also required for adipogenesis. Slug-deficient mice carried much less WAT mass than wild-type mice, showing Slug also plays a role in WAT development in vivo. In agreement with these results, mice carrying a tetracycline-repressible Slug transgene (Combi-Slug) exhibit an increase in the WAT mass, and this increase in the WAT tissue was restored by suppression of the Slug transgene. Thus, it seems likely that failure to regulate Slug expression explains why Combi-Slug mice develop obesity. Consistent with in vivo data, Slugdeficient MEFs showed a dramatically reduced capacity for adipogenesis in vitro when compared with wild-type MEFs. However, there was extensive lipid accumulation in CombiSlug MEFs. We therefore analysed the molecular mechanism by which Slug controls WAT development and found that PPARg2 expression is altered both in vivo in WAT of Slugdeficient and Combi-Slug mice and in vitro in Slug-deficient and Combi-Slug MEFs during the course of adipocytic differentiation. Complementation studies in Slug-deficient MEFs confirmed this regulation. Histone acetylation status is related to Slug expression in adipose tissue, and chromatin immunoprecipitation (ChIP) assays show differential histone deacetylase (HDAC) recruitment to the PPARg2 promoter in a tissue- and Slug-dependent manner. These results provide evidence that Slug is a key regulator of the adipocyte differentiation both in vivo and in vitro and indicate that loss of tight control of Slug expression can induce obesity and/or lipodystrophy in mice. Because Slug is also expressed in human white fat, this work may lead to the development of targeted drugs for the treatment of these pathologies in humans. SLUG is expressed in white fat in humans SLUG (SNAI2) expression and the effects of its deletion and overexpression are similar in mouse and human (8,10 15). Our previous observations indicated that Slug was also present in mouse adipose tissue (10 and Fig. 1A E). Thus, now we first studied whether human adipose tissue expressed SLUG. Expression of human SLUG was analysed by reverse transcriptase polymerase chain reaction (RT PCR). The PCR products were transferred to a nylon membrane and analysed by hybridization with a specific probe. SLUG expression was identified in human subcutaneous adipose tissues (Fig. 1B and C). These and previous observations (10) indicate that expression of SLUG is a common finding in both human and mouse WAT, suggesting a role for SLUG in WAT development. Slug expression is tightly controlled during adipocyte differentiation To determine the function of Slug in WAT development, we first examined expression of Slug during adipocyte differentiation. 3T3-L1 pre-adipocytes are a well-chara (...truncated)