Involvement of cytoskeleton-associated proteins in the commitment of C3H10T1/2 pluripotent stem cells to adipocyte lineage induced by BMP2/4.

The developmental pathway that gives rise to mature adipocytes involves two distinct stages: commitment and terminal differentiation. Although the important proteins/factors contributing to terminal adipocyte differentiation have been well defined, the proteins/factors in the commitment of mesenchymal stem cells to the adipocyte lineage cells have not. In this study, we applied proteomics analysis profiling to characterize differences between uncommitted C3H10T1/2 pluripotent stem cells and those that have been committed to the 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. Eight proteins were found to be up-regulated by BMP2, and 27 proteins were up-regulated by BMP4, whereas five unique proteins were up-regulated at least 10-fold by both BMP2/4, including three cytoskeleton-associated proteins (i.e. lysyl oxidase (LOX), translationally controlled tumor protein 1 (TPT1), and αB-crystallin). Western blotting further confirmed the induction of the expression of these cytoskeleton-associated proteins in the committed C3H10T1/2 induced by BMP2/4. Importantly, knockdown of LOX expression totally prevented the commitment, whereas knockdown of TPT1 and αB-crystallin expression partially inhibited the commitment. Several published reports suggest that cell shape can influence the differentiation of partially committed precursors of adipocytes, osteoblasts, and chondrocytes. 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 fibers and inhibited the commitment to the adipocyte lineage. Our studies indicate that these differentially expressed cytoskeleton-associate proteins might determine the fate of mesenchymal stem cells to commit to the adipocyte lineage through cell shape regulation.