Dental Science Research Institute, Medical Research Center for Biomineralization Disorders, School of Dentistry, Chonnam National University, Gwangju, Korea.
J Bone Miner Res. 2014 Jul;29(7):1586-96. doi: 10.1002/jbmr.2197.
Relaxin (Rln), a polypeptide hormone of the insulin superfamily, is an ovarian peptide hormone that is involved in a diverse range of physiological and pathological reactions. In this study, we investigated the effect of Rln on bone morphogenetic protein 2 (BMP-2)-induced osteoblast differentiation and bone formation. Expression of Rln receptors was examined in the primary mouse bone marrow stem cells (BMSCs) and mouse embryonic fibroblast cell line C3H/10T1/2 cells by RT-PCR and Western blot during BMP-2-induced osteoblast differentiation. The effect of Rln on osteoblast differentiation and mineralization was evaluated by measuring the alkaline phosphatase activity, osteocalcin production, and Alizarin red S staining. For the in vivo evaluation, BMP-2 and/or Rln were administered with type I collagen into the back of mice, and after 3 weeks, bone formation was analyzed by micro-computed tomography (µCT). Western blot was performed to determine the effect of Rln on osteoblast differentiation-related signaling pathway. Expression of Rxfp 1 in BMSCs and C3H/10T1/2 cells was significantly increased by BMP-2. In vitro, Rln augmented BMP-2-induced alkaline phosphatase expression, osteocalcin production, and matrix mineralization in BMSCs and C3H/10T1/2 cells. In addition, in vivo administration of Rln enhanced BMP-2-induced bone formation in a dose-dependent manner. Interestingly, Rln synergistically increased and sustained BMP-2-induced Smad, p38, and transforming growth factor-β activated kinase (TAK) 1 phosphorylation. BMP-2-induced Runx 2 expression and activity were also significantly augmented by Rln. These results show that Rln enhanced synergistically BMP-2-induced osteoblast differentiation and bone formation through its receptor, Rxfp 1, by augmenting and sustaining BMP-2-induced Smad and p38 phosphorylation, which upregulate Runx 2 expression and activity. These results suggest that Rln might be useful for therapeutic application in destructive bone diseases.
松弛素（Rln）是胰岛素超家族的一种多肽激素，是一种参与多种生理和病理反应的卵巢肽激素。在这项研究中，我们研究了 Rln 对骨形态发生蛋白 2（BMP-2）诱导的成骨细胞分化和骨形成的影响。通过 RT-PCR 和 Western blot 检测 Rln 受体在 BMP-2 诱导的成骨细胞分化过程中在原代小鼠骨髓基质细胞（BMSCs）和小鼠胚胎成纤维细胞系 C3H/10T1/2 细胞中的表达。通过测量碱性磷酸酶活性、骨钙素产生和茜素红 S 染色来评估 Rln 对成骨细胞分化和矿化的影响。为了进行体内评价，将 BMP-2 和/或 Rln 与 I 型胶原一起注入小鼠背部，3 周后，通过微计算机断层扫描（µCT）分析骨形成。进行 Western blot 以确定 Rln 对成骨细胞分化相关信号通路的影响。BMP-2 显著增加了 BMSCs 和 C3H/10T1/2 细胞中 Rxfp1 的表达。在体外，Rln 增强了 BMP-2 诱导的 BMSCs 和 C3H/10T1/2 细胞中碱性磷酸酶表达、骨钙素产生和基质矿化。此外，体内给予 Rln 以剂量依赖的方式增强了 BMP-2 诱导的骨形成。有趣的是，Rln 协同增强和维持了 BMP-2 诱导的 Smad、p38 和转化生长因子-β激活激酶（TAK）1 磷酸化。Rln 还显著增强了 BMP-2 诱导的 Runx2 表达和活性。这些结果表明，Rln 通过其受体 Rxfp1 协同增强 BMP-2 诱导的成骨细胞分化和骨形成，通过增强和维持 BMP-2 诱导的 Smad 和 p38 磷酸化，上调 Runx2 表达和活性。这些结果表明，Rln 可能对破坏性骨疾病的治疗应用有用。
