Define The Role Of Cytoskeleton-associated Proteins During The Commitment Of C3h10t1/2 Pluripotent Stem Cells To The Adipocyte Lineage Induced By Bmp2/4

Adipose tissue plays an important role in energy storage. Obesity results when caloric intake exceeds energy expenditure, leading to adipocyte hypertrophy and hyperplasia, including the recruitment of stem cells and subsequent differentiation of stromal-vascular preadipocytes. The stromal-vascular preadipocyte arises from a multi-potent stem cell population of mesodermal origin. These mesenchymal stem cells (MSCs) have the capacity to commit into several distinct cell types, including adipocytes, myoblasts, osteoblasts, and chondrocytes.The developmental pathway that gives rise to mature adipocytes involves three distinct stages:1) commitment; 2) mitotic clonal expansion; 3) terminal differentiation. Although the important proteins/factors contributing to terminal adipocyte differentiation have been well defined, the proteins/factors in commitment of mesenchymal stem cells (MSCs) to the adipocyte lineage cells have not.Our previous findings indicate that BMP2/4 treatment of C3H10T1/2 cells induces nearly complete commitment to the adipocyte lineage. BMP/Smad and BMP/p38MAPK signaling pathway is required for BMP-induced adipocyte lineage commitment of C3H10T1/2 stem cells. In our research, we applied proteomic analysis profiling to characterize differences between uncommitted C3H10T1/2 pluripotent stem cells and those which ha ve been committed to adipocyte lineage by BMP4 or BMP2 with the goal to identify such proteins/factors and to understand the molecular mechanisms that govern the earliest stages of adipocyte lineage commitment.In research,8 proteins were found to be up-regulated by BMP2 and 28 proteins were up-regulated by BMP4, while a total of 5 unique proteins were up-regulated at least 10-fold by both BMP2/4, including 3 cytoskeleton-associated proteins (i.e. lysyl oxidase (lox), translationally-controlled tumor protein 1 (tpt1) and crystallin alpha B). Western blotting further confirmed the induction of the expression of these cytoskeleton-associated proteins in the committed C3H10T1/2 induced by BMP2/4.To investigate whether the differentially expressed proteins identified above are downstream target genes of BMP signaling pathways, the Smad4/p38MAPK RNAi knockdown experiments were repeated and the effects on the expression of the identified differentially expressed proteins were examined. We found that Smad4 RNAi inhibited lox and crystallin aB expression, while knockdown of p38MAPK had a slight inhibitory effect on lox and crystallin aB expression, suggesting a more vital role for BMP/Smad in the induction of these two proteins. For tptl, both Smad4 and p38MAPK RNAi can down regulate its expression equal efficiently. While Smad4 RNAi combined with p38MAPK RNAi can almost totally block the expression of tptl, lox and crystallin aB. Our results indicated that lox, tptl, and crystallin aB are target proteins of the BMP signaling pathway.To further ascertain whether lox, tptl and crystallin aB are required for the adipocyte lineage commitment process, the expression of these proteins was knocked down in proliferating C3H10T1/2 cells with RNAi with BMP treatment. It was found that knockdown of lox expression totally prevented the commitment, while knockdown of tptl and crystallin aB expression partially inhibit the commitment. Our results confirmed that these three cytoskeleton-associated proteins are involved in the commitment of C3H10T1/2 stem cells to preadipocytes, while lox plays a more important role for the adipocyte commitment. We constructed a plasmid of constitutively expression lox and then transferred it into C3H10T1/2 using a retroviral system. The results showed that overexpression of lox cann't induce the commitment of C3H10T1/2 stem cells to adipocyte lineage. Above data show that lox is required but not sufficient for the commitment of C3H10T1/2 stem cells to preadipocytes.Several published reports suggest that cell shape is involved in the adipose commitment and development, and the cytoskeleton plays important roles in determining/maintaining cell shape. We observed a dramatic change of cell shape during the commitment process, and we showed that knockdown of these cytoskeleton-associated proteins prevented the cell shape change and restored F-actin organization into stress fiber and inhibited the commitment to adipocyte lineage. Our studies indicate that these differentially expressed cytoskeleton-associate proteins might determine the fate of MSCs to commit to adipocyte lineage through cell shape regulation.