The Role And Mechanism Of Gdf6 And Egr2 In Oriental Adipocyte Orientation Of Pluripotent Stem Cells

Obesity results from caloric intake persistently exceeding energy expenditure, leading to adipocyte hypertrophy and hyperplasia due to the recruitment of stem cells and subsequent differentiation of stromal-vascular preadipocytes. Mesenchymal stem cells (MSCs) are multipotent stem cells that can differentiate into many cell types, including osteoblasts, chondrocytes, myocytes and adipocytes. The developmental pathway that gives rise to mature adipocytes involves2distinct stages, commitment and terminal differentiation. Factors are key in regulating MSC commitment to adipocyte lineage. The C3H10T1/2mesenchymal cell line derived from C3H mouse embryos has characteristics that make it suitable for studying MSC commitment and differentiation.Growth differentiation factor6(Gdf6), also known as BMP13, is a member of the transforming growth factor β superfamily. In this study, we report that Gdf6can induce the commitment of C3H10T1/2cells to preadipocytes that, when subjected to an adipocyte differentiation protocol (MDI), develop into cells of the adipocyte phenotype.To define which signal pathway downstream of Gdf6is responsible for the commitment process, we blocked Smad and p38MAPK pathways by specific RNA interference. We found that knockdown of Smad or p38MAPK inhibited the commitment of C3H10T1/2stem cells to preadipocyte, and the inhibitory effect could be enhanced by co-transfection with Smad4and p38RNAi remarkably. So, we conclude that both Smad and p38pathways are responsible for the commitment of C3H10T1/2stem cells to the adipocyte lineage induced by Gdf6.BMPs exhibit their biological effects through the sequential activation of two types of transmembrane receptors, namely BMPR-Ⅰ and BMPR-Ⅱ, which possess intrinsic serine/threonine kinase activity. In this article, we examined the expression Type Ⅰ receptor Bmprla and type Ⅱ receptors Bmpr2and Acvr2a in C3H10T1/2cells by RT-PCR.Bmpr2expression was higher than Acvr2a expression, whereas Bmprlb and Acvr2b were not detected. RNAi silencing experiments suggested that Gdf6signaled through type Ⅰ receptor Bmprla and type Ⅱ receptors Bmpr2and Acvr2a to activate its signaling pathways and induce the commitment of C3H10T1/2cells to adipocyte lineage.Runxltl, also known as ETO or MTG8, was first described due to its involvement in a chromosomal translocation that causes the t (8;21) form of acute myeloid leukemia. Runxltl was expressed in a number of normal human tissues, including brain, heart, skeletal muscle, and adipose tissue. Runx1t1is also an inhibitor of terminal differentiation in3T3-L1preadipocytes, but its role in the commitment process from pluripotent mesenchymal cells to preadipocytes is unclear. We found that the expression of Runx1t1was down-regulated in Gdf6-expressed C3H10T1/2cells and BMP4-treated C3H10T1/2cells. To further confirm Runx1t1function during adipocyte lineage commitment, we overexpressed Runxltl in Gdf6-expressed C3H10T1/2and found the commitment induced by Gdf6was strongly inhibited.Taken together, we report that Gdf6signals through type Ⅰ receptor Bmprla and type Ⅱ receptors Bmpr2and Acvr2a, which actives Smad and p38MAPK pathways in pluripotent mesenchymal C3H10T1/2cells and induces its commitment to the adipocyte lineage. Downregulation of Runxltl is required for adipocyte lineage commitment process. Obesity is characterized by an expansion of fat mass that results from adipocyte hypertrophy and hyperplasia. The increase in adipocyte number is mainly due to the recruitment of new preadipocytes from mesenchymal stem cells (MSCs) and by mitotic clonal expansion of the preadipocytes. These mesenchymal stem cells (MSCs) have the potential to undergo commitment into adipocyte, myocyte, osteocyte, or chondrocyte lineages. The developmental pathway that gives rise to mature adipocytes involves two distinct stages:commitment and terminal differentiation. The core of the transcriptional cascade contributes to terminal differentiation has been well established, while the transcription factors involved in the commitment process has not. The C3H10T1/2stem cell line derived from C3H mouse embryos has characteristics suitable for studies of the stem cell commitment program. Our previous findings indicate that bone morphogenetic protein4(BMP4) treatment of C3H10T1/2cells induces its nearly complete commitment to the adipocyte lineage.Meanwhile, transforming growth factor β1(Tgfβ1), a member of Tgfβ superfamily, has been shown to promote differentiation of several kinds of precursor cells or differentiated cells, such as C3H10T1/2stem cells, neural crest Monc-1cells (pluripotent neural crest stem cells) and macrophages, into smooth muscle cell (SMC)-like cells which have polarized and elongated SMC morphology and express one or more SMC specific genes. For example, C3H10T1/2cells express SMC specific genes, including α-SMA and Sm22α when treated with Tgfβ1.Early growth response2(Egr2, also known as Krox20) is a member of a family of zinc-finger transcription factors (Egr1-4) shared a highly conserved DNA-binding domain composed of three C2H2zinc fingers, which binds the consensus nucleotide sequence GCGGGGGCG to regulate expression of target genes. Egr2is induced in response to multiple extracellular signals, such as growth factors, cytokines, hypoxia and mechanical forces associated with injury and cellular stress. Egr2is an important regulator of cell differentiation, proliferation and apoptosis. Egr2is essential for hindbrain development and peripheral nerve myelination, also it is involved in vertebrate tendon differentiation by regulating type I collagen production. Egr2functions as an intracellular mediator of collagen gene stimulation and related fibrotic responses elicited by Tgfβ1, and as a positive regulator of adipocyte differentiation via both C/EBPβ-dependent and-independent mechanisms. However, its role in commitment from pluripotent stem cells to the adipocyte lineage has not been investigated.The aim of this study was to investigate whether Egr2played a role in the process of adipocyte lineage commitment and to further provide insights into its mechanism. Here we showed that Egr2was down-regulated during BMP4-induced commitment of C3H10T1/2stem cells to adipocyte lineage, and down-regulation of Egr2was mediated by BMP-Smad/p38MAPK signaling pathways. Over-expression of Egr2completely prevented BMP4-induced adipocyte lineage commitment of C3H10T1/2stem cells, while simultaneously stimulated differentiation to smooth muscle cell (SMC)-like cells. We further demonstrated that Egr2-induced SMC-like cells differentiation is Tgfβ1-independent, whereas by binding and activating the promoters of SMC specific genes Sm22a and Cnnl. Taken together, our results indicate a novel role for Egr2in repressing adipocyte lineage commitment and promoting a switch to smooth muscle cell lineage.