Abstract
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.
Highlights
From the ‡The Key Laboratory of Molecular Medicine, the Ministry of Education, Department of Biochemistry and Molecular Biology, Fudan University Shanghai Medical College, Shanghai 200032, China, §Institute of Stem Cell Research and Regenerative Medicine, Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, China, and ʈDivision of Endocrinology, Department of Internal Medicine, Huashan Hospital, Fudan University, Shanghai 200032, China
Investigators have suggested differences in the effects of BMP2 and BMP4 on a variety of cell types (27–32), the similar effect of these two factors on the commitment of stem cells to preadipocytes allowed us to identify collectively expressed proteins induced by BMP2/4 that may contribute to adipocyte lineage commitment
The C3H10T1/2 pluripotent stem cell line behaves to mesenchymal stem cells, which have the potential to develop into osteoblasts, chondrocytes, and adipocytes (38 – 41)
Summary
Cell Culture and Induction of Commitment/Differentiation—To induce adipocyte lineage commitment, C3H10T1/2 stem cells were plated at low density and cultured in DMEM containing 10% calf serum without or with purified recombinant BMP2 (50 ng/ml) or BMP4 (10 ng/ml). On day 8, the cells were washed three times with phosphate-buffered saline (PBS) and fixed for 10 min with 3.7% formaldehyde. Immunofluorescence—C3H10T1/2 cells were plated on the coverslips and treated as described above; at postconfluency, cells were washed three times with PBS and fixed in 4% (w/v) formaldehyde for 10 min at room temperature. Gel pieces were destained with a solution of 15 mM potassium ferricyanide and 50 mM sodium thiosulfate (1:1) for 20 min at room temperature. They were washed twice with deionized water and shrunk by dehydration in ACN. PMF Acceptance Criteria—If the ion score was equal to or greater than the Mascot significance level calculated for the search, the peptide identification was considered to be statistically non-random at the 95% confidence interval
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