Huang Jian, Hsu Yi-Hsiang, Mo Chenglin, Abreu Eduardo, Kiel Douglas P, Bonewald Lynda F, Brotto Maxrco, Karasik David
Muscle Biology Research Group, Schools of Nursing & Health Studies, University of Missouri Kansas City, 2464 Charlotte Street, Kansas City, MO.
Institute for Aging Research, Hebrew SeniorLife, Department of Medicine, Beth Israel Deaconess Medical Center, Boston, MA, USA.
J Bone Miner Res. 2014 Jul;29(7):1531-1540. doi: 10.1002/jbmr.2200.
Sarcopenia and osteoporosis are important public health problems that occur concurrently. A bivariate genome-wide association study (GWAS) identified METTL21c as a suggestive pleiotropic gene for both bone and muscle. The METTL21 family of proteins methylates chaperones involved in the etiology of both myopathy and inclusion body myositis with Paget's disease. To validate these GWAS results, Mettl21c mRNA expression was reduced with siRNA in a mouse myogenic C2C12 cell line and the mouse osteocyte-like cell line MLO-Y4. At day 3, as C2C12 myoblasts start to differentiate into myotubes, a significant reduction in the number of myocytes aligning/organizing for fusion was observed in the siRNA-treated cells. At day 5, both fewer and smaller myotubes were observed in the siRNA-treated cells as confirmed by histomorphometric analyses and immunostaining with myosin heavy chain (MHC) antibody, which only stains myocytes/myotubes but not myoblasts. Intracellular calcium (Ca(2+)) measurements of the siRNA-treated myotubes showed a decrease in maximal amplitude peak response to caffeine, suggesting that less Ca(2+) is available for release due to the partial silencing of Mettl21c, correlating with impaired myogenesis. In siRNA-treated MLO-Y4 cells, 48 hours after treatment with dexamethasone there was a significant increase in cell death, suggesting a role of Mettl21c in osteocyte survival. To investigate the molecular signaling machinery induced by the partial silencing of Mettl21c, we used a real-time PCR gene array to monitor the activity of 10 signaling pathways. We discovered that Mettl21c knockdown modulated only the NF-κB signaling pathway (ie, Birc3, Ccl5, and Tnf). These results suggest that Mettl21c might exert its bone-muscle pleiotropic function via the regulation of the NF-κB signaling pathway, which is critical for bone and muscle homeostasis. These studies also provide rationale for cellular and molecular validation of GWAS, and warrant additional in vitro and in vivo studies to advance our understanding of role of METTL21C in musculoskeletal biology.
肌肉减少症和骨质疏松症是同时出现的重要公共卫生问题。一项双变量全基因组关联研究（GWAS）将METTL21c鉴定为骨骼和肌肉的一个提示性多效基因。METTL21蛋白家族可使参与肌病和伴有佩吉特病的包涵体肌炎病因的伴侣蛋白甲基化。为了验证这些GWAS结果，在小鼠成肌C2C12细胞系和小鼠骨细胞样细胞系MLO-Y4中，用小干扰RNA（siRNA）降低了Mettl21c mRNA的表达。在第3天，当C2C12成肌细胞开始分化为肌管时，在经siRNA处理的细胞中观察到排列/组织融合的肌细胞数量显著减少。在第5天，经组织形态计量分析和用肌球蛋白重链（MHC）抗体免疫染色证实，经siRNA处理的细胞中肌管数量更少且更小，MHC抗体仅对肌细胞/肌管染色而不对成肌细胞染色。对经siRNA处理的肌管进行细胞内钙（Ca(2+)）测量显示，对咖啡因的最大振幅峰值反应降低，这表明由于Mettl21c的部分沉默，可用于释放的Ca(2+)减少，这与肌生成受损相关。在经siRNA处理的MLO-Y4细胞中，用地塞米松处理48小时后细胞死亡显著增加，这表明Mettl21c在骨细胞存活中起作用。为了研究Mettl21c部分沉默诱导的分子信号机制，我们使用实时PCR基因芯片监测10条信号通路的活性。我们发现敲低Mettl21c仅调节NF-κB信号通路（即Birc3、Ccl5和Tnf）。这些结果表明，Mettl21c可能通过调节NF-κB信号通路发挥其骨骼-肌肉多效功能，而NF-κB信号通路对骨骼和肌肉的稳态至关重要。这些研究也为GWAS的细胞和分子验证提供了理论依据，并需要更多的体外和体内研究来加深我们对METTL21C在肌肉骨骼生物学中作用的理解。
