lysyl oxidase ͉ preadipocyte ͉ Smad4 ͉ adipogenesis ͉ differentiation

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 cytoskeletonassociated proteins (i.e. lysyl oxidase (LOX), translationally controlled tumor protein 1 (TPT1), and ␣B-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 ␣B-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. 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 (1-5). The stromal-vascular preadipocyte arises from a multipotent stem cell population of mesodermal origin. These mesenchymal stem cells (MSCs) 1 have the capacity to commit to several distinct cell types, including adipocytes, myoblasts, osteoblasts, and chondrocytes (6 -8). The genes that are involved in the earliest stages of myoblast (MyoD) (9, 10), chondroblast (Sox9) (11,12), and osteoblast (Runx2/Cbfa1 and osterix) (13-16) lineage determination by MSCs have already been identified. However, the genes governing the earliest stages of adipocyte determination have not yet been identified.Programming the adipose lineage is a multistep process comprising an initial commitment step in which cells become restricted to the adipocyte lineage but do not yet express markers of terminal differentiation and subse...

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