Abstract
BackgroundReceptor tyrosine kinase-like orphan receptor 2 (Ror2) plays a key role in bone formation, but its signaling pathway is not completely understood. Signal transducer and activator of transcription 3 (Stat3) takes part in maintaining bone homeostasis. The aim of this study is to reveal the role and mechanism of Ror2 in the osteogenic differentiation from mouse bone marrow mesenchymal stem cells (mBMSCs) and to explore the effect of Stat3 on Ror2-mediated osteogenesis.MethodsRor2 CKO mice were generated via the Cre-loxp recombination system using Prrx1-Cre transgenic mice. Quantitative real-time PCR and western blot were performed to assess the expression of Stat3 and osteogenic markers in Ror2-knockdown mBMSCs (mBMSC-sh-Ror2). After being incubated in osteogenic induction medium for 3 weeks, Alizarin Red staining and western blot were used to examine the calcium deposit and osteogenic markers in Stat3 overexpression in mBMSC-sh-Ror2.ResultsLoss of Ror2 in mesenchymal or osteoblast progenitor cells led to a dwarfism phenotype in vivo. The mRNA expression of osteogenic markers (osteocalcin, osteopontin (OPN), and collagen I) in the ulna proximal epiphysis of Ror2 CKO mice was significantly decreased (P < 0.05). The mRNA and protein expression of Stat3 and osteogenic markers (Runx2, osterix, and OPN) decreased in mBMSC-sh-Ror2 cells (P < 0.05). The overexpression of Stat3 in mBMSC-sh-Ror2 cells rescued the calcium deposit and expression of Runx2, osterix, and OPN to a level comparable to normal mBMSCs.ConclusionsRor2 was essential for skeleton development by regulating mBMSCs’ osteogenesis and osteoblast differentiation. Loss of Ror2 may impair the osteogenesis of mBMSCs by inhibiting Stat3.
Highlights
Receptor tyrosine kinase-like orphan receptor 2 (Ror2) plays a key role in bone formation, but its signaling pathway is not completely understood
Whole-mount skeletal staining by Alizarin Red and Alcian Blue showed that the length of forelimb and hindlimb was distinctly reduced in Ror2 Conditional knockout (CKO) mice (Fig. 1c, e) when compared with littermate controls (Fig. 1d, f)
IHF staining showed that the osteogenic differentiation markers osterix (OSX), osteopontin (OPN), collagen I (COL1A1), and Sox9 were dramatically diminished in Ror2 CKO mice (Fig. 3a)
Summary
Receptor tyrosine kinase-like orphan receptor 2 (Ror2) plays a key role in bone formation, but its signaling pathway is not completely understood. The aim of this study is to reveal the role and mechanism of Ror in the osteogenic differentiation from mouse bone marrow mesenchymal stem cells (mBMSCs) and to explore the effect of Stat on Ror2-mediated osteogenesis. The process involves in multiple regulatory signaling pathways and transcription factors [1, 2] Both canonical and non-canonical Wnt signaling pathways play essential roles in mesenchymal stem cells differentiation into osteoblasts along with their proliferation and mineralization [1, 3, 4]. When Prrx1-Cre transgenic mice are crossed with a strain containing a loxp site-flanked sequence of interest, Cre-mediated recombination results in deletion of the floxed sequence in all mesenchyme-derived cells in the limbs and craniofacial tissue [19,20,21,22]. In order to elucidate the role of Ror in osteogenesis of osteoblastic cell lineages in vivo, Ror2f/f transgenic mice which possess loxp sites flanking exons 3–4 of Ror gene [23] and the Prrx1-Cre transgenic mice were used to generate mice with Ror gene-specific deletion in mesenchymal progenitors
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