Dimethyl sulfoxide (DMSO) is the most effective solvent that mixes readily with most water-insoluble and water-soluble substances. Similar to retinoic acid, DMSO demonstrates diverse differentiation-inducing effects on many cell types, including leukemia cells, myoblasts and embryonic stem cells. Recently, it is demonstrated that the DMSO-induced differentiation of leukemia cells is mediated via the upregulation of the tumor suppressor PTEN, which leads to the decrease of Akt phosphorylation. Although DMSO serves as solvent for most ligand-receptor studies of bone formation, the effect of DMSO on bone formation remains obscure. In this study, the effect of DMSO on osteoblast differentiation and phenotype-specific gene expression was examined using murine preosteoblast MC3T3E1 clone 4 (MC) cells as model systems. At concentrations of 0.5--1%, DMSO enhanced the osteoblast differentiation of MC cells as demonstrated by Von Kossa staining. Using real-time RT-PCR, DMSO was shown to upregulate the expression of alkaline phosphatase and collagen type la. To examine if DMSO acted on specific stage, osteogenic medium was supplemented with 1% DMSO transiently during osteoblast differentiation of MC cells. It was observed that maximum bone nodule formation was resulted when DMSO was supplemented at day 0--5 when compared with control. Expression of osteoblast-specific transcription factors, Runx2 and osterix, was upregulated during the differentiation process. DMSO treatment at day 5--10 or 10--15 resulted in no significant increase in bone nodule formation, suggesting that DMSO affects mainly the differentiation process but not the maturation, nor matrix mineralization process of osteoblast differentiation. While the expression of activated p42/p44 MAP kinases (MAPK) was downregulated, blockage of activated MAPK further enhanced the DMSO-mediated osteoblast differentiation. These data demonstrate that DMSO enhances osteoblast differentiation of MC cells by affecting the differentiation process via the inhibition of MAPK pathway.

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