Method: An immortalized BMSC line (imBMSC) from a primary culture of FVB/NJ mouse cells were used. Sfrp2 siRNA was used to knockdown expression, and cells were cultured with 1 mM β-glycerol phosphate, supplemented with either dexamethasone or rhBMP2. qRT-PCR assessed the relative expression of osteogenic transcription factors, runx2 and osterix. Western blot analysis detected the phosphorylation of Lrp6. Expression of wnt target genes, axin2, c-myc, and cyclin D1 was determined by qRT-PCR. In in-vivo transplantation assays, primary BMSCs isolated from sfrp2 +/- and sfrp2 -/- mice were transplanted with collagen sponges into immunocompromised mice. Ossicles were harvested and stained with hematoxylin and eosin, and examined histologically.
Result: We found that knockdown of sfrp2 decreased expression of osteogenic transcription factors, runx2 and osterix, and resulted in decreased ability to support the formation of a mineralized matrix. Sfrp2 knockdown decreased the phosphorylation of the Wnt co-receptor, Lrp6, as well as of the expression of Wnt target genes, axin2, cyclin D1, and c-myc. The addition of recombinant sfrp2 increased the phosphorylation of Lrp6, and partially rescued the expression of axin2 and c-myc.
Conclusion: Our data indicate that sfrp2 expression plays an important role in mediating the osteogenic differentiation of murine BMSCs, and suggests that it may exert its influence by enhancing canonical wnt signaling.
Keywords: Bone, Fibroblasts, Mineralization and Stem Cells