ABSTRACT. Previous studies revealed that bone morphogenetic protein (BMP) induces commitment to the adipocyte lineage in pluripotent stem cells. The present study explored the role of endogenous BMP activity in 3T3-L1 preadipocytes. The expression of phosphoSmad1/5/8 was monitored because BMP transmits its signal through Smad1/5/8 phosphorylation. Phosphorylated Smad1/5/8 was higher in proliferating preadipocytes, and lower in differentiating adipocytes after removing differentiation inducers. Reporter assays revealed that dorsomorphin predominantly inhibits the BMP pathway but not the structurally related TGF-/activin pathway. The addition of dorsomorphin to the culture medium prior to treatment with differentiation inducers impaired lipid accumulation in 3T3-L1 cells. The present study indicated that activation of BMP signaling in preadipocytes is required for these cells to initiate the adipogenic program. The amount of adipose tissue in the body is an important determinant of energy metabolism in animals, and is related to various (patho-)physiological conditions [18]. Adipocytes also integrate a wide array of homeostatic processes by secreting various cytokines [22]. The size of adipocytes varies markedly and largely reflects the amount of stored triglyceride, whereas the number of adipocytes increases as a result of the proliferation of preadipocytes and their subsequent differentiation into adipocytes. Various growth factors/hormones affect adipocyte differentiation by regulating the expression and activity of adipogenic transcription factors [21].Members of the transforming growth factor- (TGF-) family, which is composed of three subgroups, TGF-, activin and bone morphogenetic protein (BMP), have diverse physiological roles, including the control of cell division, early embryonic patterning, differentiation, and cell determination [3]. They are also involved in the regulation of adipocyte differentiation; TGF- blocked adipogenic differentiation both in vitro [4,11] and in vivo [5]. Treatment with activin A, an activin isoform, inhibited the differentiation of 3T3-L1 preadipocytes [9]. By contrast, the roles of BMP are controversial. Treatment with BMP-2 decreased lipid accumulation in 3T3-F442A preadipocytes, suggesting the activity of BMP-2 as an inhibitor of adipocyte differentiation [19]. BMP-4 addition to the culture medium, however, conversely stimulated adipocyte differentiation of multipotential C3H10T1/2 cells [1,20], suggesting that BMP induces to commit to the adipocyte lineage. Although endogenous BMP-4 expression is sufficient for the commitment in C3H10T1/2 cells [2], endogenous BMP signaling in preadipocytes and its role for adipocyte differentiation are unknown.The present study examined the effects of endogenous BMP activity on adipocyte differentiation. We used 3T3-L1 preadipocytes as a cell model for adipocyte differentiation; growth-arrested confluent 3T3-L1 preadipocytes are treated with appropriate hormonal agents, which induce synchronous reentrance into the cell cycle and unde...

We previously revealed that endogenous bone morphogenetic protein (Bmp) activity is required for lipid accumulation in 3T3-L1 adipocytes. The present study characterized the role of endogenous Bmp activity in preadipocytes. Endogenous Bmp activity was monitored by analyzing the level of phosphorylation of Smad1/5/8, downstream molecules in the Bmp pathway. Higher levels of phosphorylated Smad1/5/8 were detected in adipogenic cells but not in non-adipogenic cells prior to differentiation induction. The inhibition of the Bmp pathway during this period decreased the expression of Pparγ2 and C/ebpα, which are transcription factors responsible for adipocyte differentiation. The expression of these transcription factors were also down-regulated by Bmp4 knockdown. In addition, endogenous Bmp4 was required for the repression of Intrleukin-11 expression. Endogenous Bmp4 in preadipocytes is indispensable for the onset of the adipogenic program, and may help to maintain the preadipocytic state during adipocyte differentiation.

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