br Materials and methods br Results br Discussion Obesity

Materials and methods
Results
Discussion Obesity results when caloric intake exceeds energy expenditure, with an increase in adipocyte size or adipocyte number (Bjorntorp, 1974; Faust et al., 1978; Johnson et al., 1971, 1978; Yu et al., 1997). In the context of adipogenesis, development appears to occur in two stages, i.e. commitment of MSCs to produce preadipocytes followed by differentiation to produce adipocytes (Otto and Lane, 2005). Although it has been established that BMPs play a very important role in the processes by which MSCs undergo commitment to the adipocyte lineage (Bowers et al., 2006; Huang et al., 2009, 2011; Tang et al., 2004), little is known about the mechanism involved. The epithelial–mesenchymal transition is crucial in the differentiation of multiple tissues and organs (Dedhar et al., 2006). EMT is characterized by loss of epithelial cell polarity, loss of cell–cell contacts, and acquisition of mesenchymal markers and increased cell motility. Multiple signaling pathways such as TGF-β, Notch, Wnt and receptor tyrosine kinase-mediated signals have all been implicated as upstream initiators of the EMT process in tumor dcb and developmental cells (Heldin and Moustakas, 2007; Thiery et al., 2009; Yang and Weinberg, 2008). Herein, we clearly revealed that BMP4 induced an EMT-like phenotype during adipocyte lineage commitment on the basis of the following findings: (1) BMP4 treatment causes epithelial markers\' repression and mesenchymal markers\' induction; (2) BMP4 induces the expression of the key transcription factors that regulate the EMT process; and (3) BMP4 increases C3H10T1/2 cell motility. However, the C3H10T1/2 cells are mesenchymal stem cells and the BMP4-induced change in cell morphology is not as typical as a classical EMT, so BMP4 induced an EMT-like phenotype during adipocyte lineage commitment. We recently reported that Lox contributes to adipocyte lineage commitment (Huang et al., 2009, 2011). Lox has also been shown to be coupled to the EMT (Erler et al., 2006; Higgins et al., 2007; Schietke et al., 2010; Sion and Figg, 2006; Taylor et al., 2011). Interestingly, it has been shown that chromatin reprogramming occurs in cells undergoing EMT (McDonald et al., 2011). A mesenchymal-to-epithelial transition (MET), the reverse of EMT, is also required for the reprogramming of fibroblasts to produce induced pluripotent stem cells (iPSCs) (Chen et al., 2011, 2012; Li et al., 2010). Our current studies showed that an EMT-like feature occurs during adipocyte lineage commitment and Lox silencing induces a MET-like process in the presence of BMP4 and antagonizes adipocyte lineage commitment. The evidence presented here suggests that reprogramming could also occur during the adipocyte lineage commitment process. However, further studies will be necessary for better understanding of reprogramming during adipocyte lineage commitment. Both developmental and pathological EMTs are controlled by the same core EMT regulatory factors, which include Snai1, Snai2, Twist, and Sip1 (also known as ZEB2). Twist, a core EMT regulatory factor, can modulate the behavior and fate of cells in both development and cancer (Glackin et al., 2010; Thiery et al., 2009; Weinberg et al., 2004). We found a significant correlation between the expression of Twist and the expression of Lox during adipocyte lineage commitment, which suggested that Twist is regulated by Lox. Chromatin immunoprecipitation confirmed that Lox was specifically recruited to the Twist1 promoter, further suggesting a direct regulation of Twist by Lox. This study reveals a functional link between Lox and Twist which is consistent with a recently published paper (El-Haibi et al., 2012). In addition to silencing the expression of Cdh2 and Vim, Cdh1 and Ocln expression can also be rescued by knocking down of Twist. We therefore speculated that Twist contributes to the Lox-induced EMT-like response during adipocyte lineage commitment. It should be noted that although Lox regulates Twist and Twist is involved in the EMT-like response during adipocyte lineage commitment, Lox itself is enough to induce an EMT (Schietke et al., 2010). Snai1 and Snai2 are also induced during adipocyte lineage commitment, however, the expression pattern of Twist and Snai are quite different in Lox-silenced cells. Further studies will be needed to elucidate such differences. Overall, the present evidence indicates that Lox contributes to the EMT-like response during adipocyte lineage commitment at least in part through the activation of Twist.