| The increase of adipose tissue mass associated with obesity is due in part to an increase in the number of adipocytes (hyperplasia). This hyperplasia results from the recruitment of pluripotent stem cells present in the vascular stroma of adipose tissue. These stem cells have the capacity to undergo commitment to adipose, muscle, bone or cartilage lineages.The development of mesenchymal stem cells into terminally differentiated adipocytes can be divided into four stages: lineage commitment, preadipocyte proliferation, growth arrest and terminal differentiation. Many of the key players in the latter phases of the adipocyte development program, i.e., mitotic clonal expansion and terminal differentiation, have been well characterized. However, little is known about the factors/genes that trigger the commitment process. The goal of our research is to define the role of BMP signaling pathway during the commitment of C3H10T1/2 pluripotent stem cells to the adipocyte lineage.In this article, we firstly report that bone morphogenetic protein 2 (BMP-2), as well as BMP-4, can induce the commitment of C3H10T1/2 cells to preadipocytes that, when subjected to an adipocyte differentiation protocol (MDI), develop into cells of the adipocyte phenotype.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. BMPR-I is composed of many subtypes, of which BMPR-IA and BMPR-IB are downstream of BMP-2 and BMP-4. In this article, we examined the expression of BMP receptors in C3H10T1/2 stem cells by RT-PCR. Data show that C3H10T1/2 cells express BMPR-IA and BMPR-II but not BMPR-IB.A variety of studies indicate that BMPR-IA and BMPR-IB may transmit different signals during the specification and differentiation of mesenchymal lineages. To investigate the role of BMPR-IA and BMPR-IB in the BMP-2 or BMP-4 signaling pathways in C3H10T1/2, we constructed constitutively active BMPR-IA (Ca-BMPR-IA), constitutively active BMPR-IB (Ca-BMPR-IB), dominant-negative BMPR-IA (DN-BMPR-IA) plasmids and then transferred them into C3H10T1/2 by using a retroviral system. The results showed that C3H10T1/2 cells that overexpressed Ca-BMPR-IA and Ca-BMPR-IB underwent differentiation into adipocyes after MDI induction, while overexpression of DN-BMPR-IA decreased the proportion of adipocyte induced by BMP-2 or BMP-4 and subsequent MDI. Since BMPR-IB is absent in this cell line, above data show that BMP-2 or BMP-4 induces the commitment of C3H10T1/2 stem cells to the adipocyte lineage through BMPR-IA.To define which signal pathway in the downstream of BMP-2 or BMP-4 is responsible for the commitment process, we blocked Smad, p38 MAPK and ERK pathway by specific chemical inhibtors and/or RNA interference. We found SB203580, a chemical inhibitor of p38 MAPK, inhibited the commitment of C3H10T1/2 stem cells to the adipocyte lineage induced by BMP-2 or BMP-4, and p38 Stealth ? RNAi also decreased the proportion of adipocyte induced by BMP and MDI. PD98059, a chemical inhibitor of MEK1, kinase of ERK, did not have influence on the commitment process. We also found that Smad4 Stealth ? RNAi reduced the proportion of adipocyte induced by BMP and MDI. And the inhibitory effect could be enhanced by co-transfection with Smad4 and p38 RNAi remarkably. So, we conclude that both Smad and p38 MAPK pathways are required for the commitment of C3H10T1/2 stem cells to the adipocyte lineage induced by BMP-2 or BMP-4, while ERK is not involved in the process.Taken together, our study identified BMP-2 and BMP-4 induced the commitment of C3H10T1/2 stem cells to the adipocyte lineage through BMPR-IA and its downstream signal molecules—Smad, p38 were responsible for this process. |
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