Wnt5a signaling is a substantial constituent in bone morphogenetic protein-2-mediated osteoblastogenesis

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Abstract

Wnts are secreted glycoproteins that mediate developmental and post-developmental physiology by regulating cellular processes including proliferation, differentiation, and apoptosis through β-catenin-dependent canonical and β-catenin-independent noncanonical pathway. It has been reported that Wnt5a activates noncanonical Wnt signaling through receptor tyrosine kinase-like orphan receptor 2 (Ror2). Although it appears that Wnt5a/Ror2 signaling supports normal bone physiology, the biological significance of noncanonical Wnts in osteogenesis is essentially unknown. In this study, we identified expression of Wnt5a in osteoblasts in the ossification zone of the tibial growth plate as well as bone marrow of the rat tibia as assessed by immunohistochemistry. In addition, we show that osteoblastic differentiation mediated by BMP-2 is associated with increased expression of Wnt5a and Ror2 using cultured pre-osteoblasts, MC3T3-E1 cells. Silencing gene expression of Wnt5a and Ror2 in MC3T3-E1 cells results in suppression of BMP-2-mediated osteoblastic differentiation, suggesting that Wnt5a and Ror2 signaling are of substantial importance for BMP-2-mediated osteoblastic differentiation. BMP-2 stimulation induced phosphorylation of Smad1/5/8 in a similar fashion in both siWnt5a-treated cells and control cells, suggesting that Wnt5a was dispensable for the phosphorylation of Smads by BMP-2. Taken together, our results suggest that Wnt5a/Ror2 signaling appears to be involved in BMP-2-mediated osteoblast differentiation in a Smad independent pathway.

Highlights

► Wnt5a is identified in osteoblasts in tibial growth plate and bone marrow. ► Osteoblastic differentiation is associated with increased expression of Wnt5a/Ror2. ► Wnt5a/Ror2 signaling is important for BMP-2-mediated osteoblastic differentiation. ► Wnt5a/Ror2 operates independently of BMP-Smad pathway.

Introduction

Wnts are a family of 19 secreted glycoproteins that mediate developmental and post-developmental physiology by regulating cellular processes including proliferation, differentiation, and apoptosis [1]. Wnts are classified into two sub-families based on downstream mediated signaling. The Wnt1 sub-family (e.g. Wnt1, Wnt3a, and Wnt8) activates the canonical Wnt/β-catenin pathway, whereas the Wnt5a sub-family (e.g. Wnt5a and Wnt11) activates noncanonical pathways [1].

The canonical Wnt/β-catenin signaling pathway has been well-characterized and implicated in promotion of bone formation [1], [2]. Loss-of-function mutations of low-density lipoprotein receptor related protein (LRP)-5, a co-receptor for Wnt/β-catenin signaling, leads to low bone mass accompanied by fractures causing osteoporosis pseudoglioma syndrome [3], [4]. LRP-5 gain-of-function mutations in humans result in high bone mass syndrome [5], [6]. On the other hand, noncanonical Wnts activate the β-catenin-independent signaling pathway, including the Wnt/Ca2+ pathway and Wnt/ planar cell polarity (PCP) pathway, and are supposed to have crucial functions in the regulation of cell migration and polarity during embryogenesis [7]. It has been reported that receptor tyrosine kinase-like orphan receptor 2 (Ror2), a member of the Ror-family of receptor tyrosine-protein kinases [8], acts as a receptor or co-receptor for Wnt5a [9], [10]. Ror2 by itself or in combination with Frizzled protein through its Frizzled-like cysteine-rich domain [9] mediates diverse Wnt5a signaling by activating the Wnt-c-Jun N-terminal kinase PCP pathway [9] and inhibiting the β-catenin-T cell factor/lymphoid enhancer factor pathway [10]. Loss-of-function mutations in Wnt5a and Ror2 cause autosomal recessive Robinow syndrome, characterized by midfacial hypoplasia, limb bone shortening, and genital abnormalities [11], [12], suggesting that the Wnt5a/Ror2 signaling pathway is important in human craniofacial and skeletal development. Overexpression of Ror2 increases osteoblast differentiation of human mesenchymal cells and osteoblastic MC3T3-E1 cells [13]. Wnt5a induces osteoblastogenesis through attenuation of peroxisome proliferator-activated receptor-γ-induced adipogenesis in mesenchymal stem cells of bone marrow [14]. Thus, it is likely that noncanonical Wnt signaling supports normal bone physiology. However, a specific role of Wnt5a in osteogenesis has not been determined.

Bone morphogenetic proteins (BMPs) are structurally related to the transforming growth factor-β superfamily and were originally identified by their capacity to induce ectopic bone formation in rodents [15]. Among the BMP family members, BMP-2 has been extensively studied for its various biological functions, particularly during osteogenic differentiation [16].

In this study, we demonstrated that osteoblastic differentiation mediated by BMP-2 is associated with increased expression of Wnt5a and Ror2 in vivo and in vitro, and that silencing gene expression of Wnt5a and Ror2 results in the suppression of BMP-2-induced expression of alkaline phosphatase (ALP) and osteocalcin (OCN), suggesting that Wnt5a and Ror2 signaling is a substantial constituent for BMP-2-mediated osteoblastic differentiation.

Section snippets

Reagents

Recombinant human BMP-2 was purchased from R&D systems (Minneapolis, MN, USA). Ascorbic acid, Triton X-100, phenylmethylsulfonyl fluoride (PMSF), and p-nitrophenyl phosphate (pNPP) were purchased from Sigma Chemical Co. (St. Louis, MO, USA).

Tissue preparation and immunohistochemistry (IHC)

Serial sections (5-μm) of the tibia from male Wistar rats (Kumagai-shigeyasu Co., Sendai, Japan) in their second postnatal week were used for IHC, as previously described [17]. Fixed specimens, with 4% paraformaldehyde in phosphate-buffer saline (PBS) at 4 °C

Expression of Wnt5a on osteoblasts and its induction upon stimulation with BMP-2

We investigated the expression of Wnt5a on osteoblasts in the tibia from two-week-old Wistar rats by IHC. Expression of Wnt5a was detected in osteoblasts at the ossification zone (OZ) of the tibial growth plate (Fig. 1A). Osteoblasts in bone marrow (BM) of the rat tibia also showed strong immunoreactivity, while no expression was detected in osteocytes (arrowhead) located in bone lacunae. (Fig. 1B) Negative control sections showed no immunoreactivities as shown in Fig. 1C. Because Wnt5a was

Discussion

We show that Wnt5a was expressed by osteoblasts in the ossification zone of the growth plate as well as bone marrow in the rat tibia, but was not expressed by osteocytes in bone lacunae. Furthermore, we demonstrate that expression levels of Wnt5a and Ror2 on cultured osteoblasts were upregulated by stimulation with BMP-2. Alterations in expression levels during osteoblast differentiation shown in this study are consistent with previous in vitro studies using conventional osteogenic medium

Acknowledgments

This work was supported by a Grant-in-Aid for Scientific Research (21390552 and 23390475) and Grant-in-Aid for Exploratory Research (21659437) from the Japan Society for the Promotion of Science.

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