Mesenchymal stem cells (MSC) can differentiate into osteoblasts upon activation of Wnt signaling. Identifying targets of Wnt signaling in MSC may help promote MSC osteoblast differentiation for bone regeneration. In this study, using microarray analysis we found that Wnt3a upregulates neuregulin 1 (NRG-1) during Wnt3a-induced osteoblast differentiation in primary human MSC and murine C3H10T1/2 mesenchymal cells. Western blot and qPCR analyses confirmed that NRG-1 is upregulated by Wnt3a, and that this effect was counterbalanced by decreased expression of the NRG-1 receptor ErbB3. Consistently, exogenous NRG-1 had no effect on alkaline phosphatase (ALP) activity, an early marker of osteoblast differentiation. In contrast, small interfering RNA-mediated silencing of endogenous NRG-1 increased basal and Wnt3a-induced ALP activity in MSC. We showed that short hairpin (sh) ErbB3 and Wnt3a additively increased β-catenin transcriptional activity and ALP activity in MSC. These effects were abrogated by DKK1, indicating that cross-talk between Wnt3a and ErbB3 control MSC osteoblast differentiation via Wnt/β-catenin signaling. Furthermore, ErbB3 silencing decreased Src expression. Pharmacological inhibition of Src signaling promoted ErbB3- and Wnt-induced ALP activity, suggestive of a role of Src signaling in the modulation of osteoblast differentiation by ErbB3 and Wnt3a. The results indicate that downregulation of ErbB3 induced by Wnt3a contributes to Wnt3a-induced early osteoblast differentiation of MSCs through increased canonical Wnt/β-catenin signaling and decreased Src signaling.
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