Lin Fei-Xiang, Zheng Gui-Zhou, Chang Bo, Chen Rong-Chun, Zhang Qi-Hao, Xie Peng, Xie Da, Yu Guo-Yong, Hu Qin-Xiao, Liu De-Zhong, Du Shi-Xin, Li Xue-Dong
Department of Orthopedics, The Third Affiliated Hospital (The Affiliated Luohu Hospital) of Shenzhen University, Shenzhen, China.
Department of Spine Surgery, The Affiliated Ganzhou Hospital of Nanchang University (Ganzhou People's Hospital), Ganzhou, China.
Cell Physiol Biochem. 2018;47(1):161-175. doi: 10.1159/000489763. Epub 2018 May 10.
BACKGROUND/AIMS: Bone marrow stromal cells (BMSCs) are multipotent precursors that give rise to osteoblasts, and contribute directly to bone formation. Connexin 43 (Cx43) is the most ubiquitous gap junction protein expressed in bone cell types, and plays crucial roles in regulating intercellular signal transmission for bone development, differentiation and pathology. However, the precise role and mechanism of Cx43 in BMSCs are less known. Here, we investigate the function of Cx43 in osteogenic differentiation of BMSCs in vitro.
BMSCs were isolated by whole bone marrow adherent culture. Knock down of Cx43 was performed by using lentiviral transduction of Cx43 shRNA. BMSCs were induced to differentiate by culturing in a-MEM, 10% FBS, 50 µM ascorbic acid, 10 mM beta-glycerophosphate, and 100 nM dexamethasone. Alkaline phosphatase (ALP) activity and alizarin red S staining were used to evaluate osteogenic differentiation in calcium nodules. Target mRNAs and proteins were analyzed by using real-time quantitative PCR (qPCR) and western blotting.
Cx43 expression markedly increased during osteogenic differentiation. Osteogenic differentiation was suppressed following lentiviral-mediated knockdown of Cx43 expression, as judged by decreased levels of Runt-related transcription factor 2 (Runx2), bone sialoprotein (BSP), osteocalcin (Bglap), Osterix (Osx), alkaline phosphatase (ALP) activity and the number of calcium nodules in response to osteogenic differentiation stimuli. Knock down of Cx43 reduced the level of phosphorylation of GSK-3beta at Ser9 (p-GSK-3beta), resulting in decreased beta-catenin expression and activation. Furthermore, treatment of Cx43-knockdown cells with lithium chloride (LiCl), a GSK-3beta inhibitor, reduced osteogenic differentiation and decreased GSK-3beta levels, as well as partially rescued levels of both total and activated beta-catenin.
These findings indicate that Cx43 positively modulates osteogenic differentiation of BMSCs by up-regulating GSK-3beta/beta-catenin signaling pathways, suggesting a potential role for Cx43 in determining bone mass and bone mineral density by modulating osteogenesis.
背景/目的：骨髓基质细胞（BMSCs）是可分化为成骨细胞的多能前体细胞，直接参与骨形成过程。连接蛋白43（Cx43）是在骨细胞类型中表达最广泛的间隙连接蛋白，在调节骨发育、分化及病理过程中的细胞间信号传递方面发挥关键作用。然而，Cx43在BMSCs中的具体作用和机制尚不清楚。在此，我们研究Cx43在体外BMSCs成骨分化中的功能。
采用全骨髓贴壁培养法分离BMSCs。通过慢病毒转导Cx43 shRNA敲低Cx43表达。将BMSCs置于含α-MEM、10%胎牛血清、50 μM抗坏血酸、10 mM β-甘油磷酸钠和100 nM地塞米松的培养基中诱导分化。用碱性磷酸酶（ALP）活性检测及茜素红S染色评估钙结节中的成骨分化情况。采用实时定量PCR（qPCR）和蛋白质免疫印迹法分析目的mRNA和蛋白质。
在成骨分化过程中，Cx43表达显著增加。慢病毒介导的Cx43表达敲低后，成骨分化受到抑制，表现为成骨分化刺激后，与成骨相关的转录因子2（Runx2）、骨涎蛋白（BSP）、骨钙素（Bglap）、osterix（Osx）水平降低，碱性磷酸酶（ALP）活性及钙结节数量减少。敲低Cx43降低了糖原合成酶激酶-3β（GSK-3β）第9位丝氨酸的磷酸化水平（p-GSK-3β），导致β-连环蛋白表达和激活减少。此外，用GSK-3β抑制剂氯化锂（LiCl）处理Cx43敲低的细胞，可降低成骨分化，降低GSK-3β水平，并部分恢复总β-连环蛋白和激活的β-连环蛋白水平。
这些结果表明，Cx43通过上调GSK-3β/β-连环蛋白信号通路正向调节BMSCs的成骨分化，提示Cx43在通过调节成骨作用决定骨量和骨密度方面具有潜在作用。
