Abstract
Wdr5 is developmentally expressed in osteoblasts and accelerates osteoblast differentiation in vitro and in vivo. To address whether Wdr5 is essential for osteoblast differentiation, plasmid-based small interfering RNAs were used to stably suppress endogenous Wdr5 protein levels in MC3T3-E1 cells. Reduction of endogenous Wdr5 levels markedly inhibited osteoblast differentiation, evidenced by a significant decrease in alkaline phosphatase activity, Runx-2 and osteocalcin mRNAs, and absence of mineralized matrix formation. Wdr5 suppression also resulted in a reduction of histone H3 lysine 4 trimethylation, confirming its critical role in this modification. Because Wdr5 overexpression enhances canonical Wnt signaling in osteoblasts in vivo, the effects of Wdr5 silencing on this pathway were examined. The expression of the canonical Wnt target gene, c-myc, was decreased, whereas that of sfrp2, which is repressed by Wnt signaling, was increased with Wdr5 knockdown. Although only a minimal increase in apoptosis was observed, the antiapoptotic effect of Wnt signaling was also impaired with Wdr5 silencing. The expression of canonical Wnts was significantly decreased with Wdr5 knockdown, resulting in a decrease in nuclear beta-catenin protein levels. Activation of the canonical Wnt signaling pathway did not overcome the effects of Wdr5 knockdown on the expression of Wnt target genes. Chromatin immunoprecipitation demonstrated that Wdr5 is present on the Wnt1 promoter and on canonical Wnt response elements of the c-myc and Runx-2 promoters. These studies demonstrate that Wdr5 suppression interferes with the canonical Wnt signaling pathway at multiple stages and that optimal Wdr5 levels are required for induction of the osteoblast phenotype.
Highlights
MC3T3-E1 cells have been used by other investigators to study the canonical Wnt signaling pathway (29 –31)
The expression of Runx-2 and c-myc, which are induced in response to activation of the canonical Wnt signaling pathway [26, 27], was significantly decreased at both 4 and 7 days in culture (Figs. 2B and 3), whereas mRNA levels of secreted Frizzled-related protein 2 (Sfrp2), the expression of which is repressed by canonical Wnt signaling, was significantly increased in the siRNA-2 and siRNA-4 cells relative to the siRNA-S cells (Fig. 3)
Since BMP-2 has been shown to activate canonical Wnt sig- ablated do not uniformly identify an essential role for these naling, we addressed whether our observations could reflect factors in osteoblast differentiation
Summary
The expression of Runx-2 and c-myc, which are induced in response to activation of the canonical Wnt signaling pathway [26, 27], was significantly decreased at both 4 and 7 days in culture 2B and 3), whereas mRNA levels of secreted Frizzled-related protein 2 (Sfrp2), the expression of which is repressed by canonical Wnt signaling, was significantly increased in the siRNA-2 and siRNA-4 cells relative to the siRNA-S cells (Fig. 3).
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