Effects of Secreted Frizzled-Related Protein 3 on Osteoblasts In Vitro

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Abstract
To examine if sFRP3s act as decoy receptors for Wnt, we examined the effects of recombinant sFRP3 on mouse osteoblast proliferation and differentiation. We found that sFRP3 unexpectedly increased osteoblast differentiation, suggesting it may act through other mechanisms besides acting as a decoy receptor for Wnt's. Introduction: Secreted frizzled-related proteins (sFRPs) are a truncated form of frizzled receptor, missing both the transmembrane and cytosolic domains. Because previous studies have shown that sFRPs bind and act as decoy receptors for Wnt proteins that promote osteoblast differentiation, we postulated that sFRP3 acts as an inhibitor of osteoblast differentiation. Materials and Methods: We examined the effects of mouse recombinant sFRP3 and/or Wnt-3A on cell proliferation and differentiation using MC3T3-E1 mouse osteoblasts and primary cultures of mouse bone marrow stromal cells. We evaluated the effects of sFRP3 on β-catenin levels using Western immunoblot analyses. Results: We found that sFRP3 suppressed osteoblast cell number in a dose-dependent manner that was the result of a decrease in proliferation and not because of an increase in apoptosis. Surprisingly, sFRP3 increased osteoblast differentiation, which could not be explained based on sFRP3 acting as a decoy receptor for stimulatory Wnt's. Furthermore, sFRP3 did not inhibit Wnt3A-induced increase in alkaline phosphatase (ALP) activity. Wnt3A, but not sFRP3 treatment, increased cellular β-catenin levels, and sFRP3 failed to block Wnt3A-induced increase in cellular β-catenin levels. Treatment with endostatin, an agent known to degrade β-catenin, did not inhibit sFRP3-induced increase in ALP activity. sFRP1, like sFRP3, inhibited proliferation and stimulated ALP activity in MC3T3-E1 mouse osteoblasts. Conclusions: Based on our findings, we conclude that sFRP3 decreased osteoblast proliferation and unexpectedly increased parameters of osteoblast differentiation. Based on our findings, we propose that sFRP3 may stimulate differentiation through a β-catenin-independent pathway in addition to its previously known function as a decoy receptor for Wnt's.