Adiponectin receptor 1 regulates bone formation and osteoblast differentiation by GSK-3β/β-Catenin signaling in mice

Highlights

• AdipoR1 is a critical factor for osteoblast differentiation and bone homeostasis.

• pAdipoR1 mice had higher bone volume and trabecular number than wild-type mice.

• pAdipoR1 mice had higher expression of bone formation markers than wild-type mice.

• We demonstrated that knockdown of adipoR1 impaired osteoblast differentiation.

• AdipoR1 may be involved in bone metabolism through GSK-3β/β-Catenin signaling.

Abstract

Adiponectin and its receptors are expressed in bone marrow-derived osteoblasts. Previous studies in vivo and in vitro have produced controversial results. The purpose of this study was to use porcine adiponectin receptor 1 transgenic mice (pAdipoR1) as a model to evaluate the role of AdipoR1 on bone physiology at different ages. pAdipoR1 transgenic mice had higher bone mineral density than wild-type mice in both genders at 56 weeks of age. The bone volume and trabecular number, measured by micro-computed tomography (μCT) was significantly greater in transgenic than in wild-type female mice at both 8 and 56 weeks of age. ELISA analysis revealed that both serum osteocalcin and osteoprotegerin (OPG) were significantly increased in 8-week old pAdipoR1 transgenic mice of both genders. Furthermore, serum OPG was elevated at 32 and 56 weeks of age in female and male pAdipoR1 transgenic mice. Serum TRAP5b concentration was reduced in 8 and 56 weeks old male pAdipoR1 mice compared with wild-type male mice. Knock-down of AdipoR1 significantly decreased gene expression of osteocalcin, OPG, alkaline phosphatase and msh homeobox 2 and the mineralization in MC3T3-E1 cells and mesenchymal stem cells. In addition, pathscan analysis and real-time PCR analysis suggest AdipoR1 regulates osteoblast differentiation through GSK-3 β and β-Catenin signaling. Consequently, the lack of AdipoR1 impaired osteoblast differentiation and bone formation. We conclude that AdipoR1 is a critical factor for the osteoblast differentiation and bone homeostasis.

Introduction

The relationship between bone and fat formation within the bone marrow microenvironment is complex and remains an area of active investigation. Clinical and experimental findings suggest that acceleration of adipogenesis in the bone marrow, known as fatty marrow, is often observed in association with the progression of osteoporosis or aging [1]. Adiponectin (ApN) and its receptors play important roles in regulating glucose and lipid metabolism [2], [3], [4]. In addition, their expressions in bone marrow-derived osteoblasts and adipocytes suggest a potential role in bone metabolism [5], [6]. Several clinical studies indicate that adiponectin is negatively associated with bone formation [7], [8]. However, adiponectin increases differentiation and mineralization of bone marrow-derived mesenchymal stem cells (MSCs) [9], [10].

ApN stimulates osteoblastogenesis and suppresses osteoclastogenesis to promote bone formation [6], [9], [10]. There are three distinct ApN actions in bone formation: (1) a direct positive endocrine action through circulatory ApN; (2) an autocrine/paracrine action; and (3) indirect endocrine effects by interacting with other signaling pathways such as insulin and bone morphogenetic protein 2 (BMP2) [6], [9], [10].

Several studies confirm the positive endocrine role of ApN in bone formation. Challa et al. [11] suggest that ApN increases chondrocyte proliferation, proteoglycan synthesis and matrix mineralization by upregulating the expression of type II collagen and runt-related transcription factor 2 and increasing the activities of alkaline phosphatase. A similar effect is observed by Oshima et al. [9] who treated C57BL/6J mice with adenoviral-derived ApN and found that ApN increased trabecular bone mass and enhanced the mineralization activity of osteoblasts (Ob), accompanied with decreased number of osteoclasts and bone resorption activity. Lee and his colleagues [12] find that circulating ApN augments gene expression of several osteogenic markers and increases Ob differentiation in mesenchymal progenitor cells. Moreover, ApN activates p38 mitogen-activated protein kinase via AdipoR1, which results in c-Jun activation and up-regulation of the target gene, cyclooxygenase-2 (COX-2). ApN also stimulates BMP2 expression in a COX2-dependent manner and therefore increases Ob differentiation [12]. An alternative action of ApN by Huang et al. [13] indicates that ApN stimulates osteoblast differentiation by an increase in BMP-2 expression with involvements of AMPK, p38 and NF-κB.

ApN also regulates bone formation in an autocrine/paracrine manner. ApN and its receptors are expressed in Ob and osteoclasts, indicating ApN and its receptor participate in bone metabolism not only through an endocrine pathway, but also locally in the bone [6], [14]. Cultures of bone marrow cells from ApN-knockout (Ad^−/−) mice have significantly less osteogenesis than cultures from WT mice. Collectively, these results suggest a positive autocrine/paracrine action of ApN on bone formation.

ApN also has indirect effects on bone possibly through modulation the action of growth factor or insulin sensitivity. In the presence of insulin and BMP (but not IGF-1), ApN stimulates Ob differentiation in bone marrow cells [6]. Liu et al. [15] cultured Ob with the secretory product of adipocytes and found an inhibitory effect on Ob differentiation, which can be reversed by knockdown of AdipoR1. Other studies have produced results that do not indicate a similar effect of ApN on bone formation [16], [17].

ApN forms multi-mer complexes including trimers, hexamers and high-molecular-weight forms and various forms of ApN have distinct sensitivity for ApN receptors [3], [4]. In order to study ApN signaling specifically, we have successfully established the pAdipoR1 transgenic system and confirm the role of AdipoR1 in glucose and lipid metabolism [18]. The purpose of this study is to define the role of adiponectin receptor 1 on the differentiation of Ob using primary mesenchymal stem cells (MSCs) and preosteoblasts, and to explore mechanisms.