METTL21C is a potential pleiotropic gene for osteoporosis and sarcopenia acting through the modulation of the NFκB signaling pathway Jian Huang #1 , Yi-Hsiang Hsu #2,3 , Chenglin Mo 1 , Eduardo Abreu 1 , Douglas P. Kiel 2,3 , Lynda F. Bonewald 4 , Maxrco Brotto #1,† , and David Karasik #2,† 1 Muscle Biology Research Group, Schools of Nursing & Health Studies, University of Missouri Kansas City, 2464 Charlotte Street, Kansas City, MO 2 Institute for Aging Research, Hebrew SeniorLife, Department of Medicine, Beth Israel Deaconess Medical Center, Boston, MA, USA 3 Harvard Medical School, Boston, MA, USA 4 Department of Oral and Craniofacial Sciences, School of Dentistry, University of Missouri, Kansas City, MO, USA # These authors contributed equally to this work. Abstract 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. METTL21 family of proteins methylates chaperones involved in the etiology of both 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 monitored with a real-time PCR gene array the activity of 10 signaling pathways. We discovered that Mettl21c knockdown modulated only the NFκB signaling pathway (i.e., Birc3, Ccl5 and Tnf). † corresponding authors Muscle Biology Research Group, Schools of Nursing & Health Studies, University of Missouri Kansas City, 2464 Charlotte Street, Kansas City, MO 64108 brottom@umkc.edu Institute for Aging Research, Hebrew SeniorLife, 1200 Center Street, Boston, MA 02131, USA. karasik@hrca.harvard.edu. Disclosures: All authors state that they have no conflicts of interest. NIH Public Access Author Manuscript J Bone Miner Res. Author manuscript; available in PMC 2015 July 01. Published in final edited form as: J Bone Miner Res. 2014 July ; 29(7): 1531–1540. doi:10.1002/jbmr.2200. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript