Objective: Human synovium–derived mesenchymal stem cells (MSCs) can efficiently differentiate into mature chondrocytes. It has been suggested that DNA methylation is one mechanism that regulates human chondrogenesis; however, the methylation status of genes related to chondrogenic differentiation is not known. The purpose of this study was to investigate the CpG methylation status in human synovium–derived MSCs during experimental chondrogenesis, with a view toward potential therapeutic use in osteoarthritis.Methods: Human synovium–derived MSCs were subjected to chondrogenic pellet culture for 3 weeks. The methylation status of 12 regions in the promoters of 10 candidate genes (SOX9, RUNX2, CHM1, FGFR3, CHAD, MATN4, SOX4, GREM1, GPR39, and SDF1) was analyzed by bisulfite sequencing before and after differentiation. The expression levels of these genes were analyzed by real‐time reverse transcription–polymerase chain reaction. Methylation status was also examined in human articular cartilage.Results: Bisulfite sequencing analysis indicated that 10 of the 11 CpG‐rich regions analyzed were hypomethylated in human progenitor cells before and after 3 weeks of pellet culture, regardless of the expression levels of the genes. The methylation status was consistently low in SOX9, RUNX2, CHM1, CHAD, and FGFR3 following an increase in expression upon differentiation and was low in GREM1 and GPR39 following a decrease in expression upon chondrogenesis. One exceptional instance of a differentially methylated CpG‐rich region was in a 1‐kb upstream sequence of SDF1, the expression of which decreased upon differentiation. Paradoxically, the hypermethylation status of this region was reduced after 3 weeks of pellet culture.Conclusion: The DNA methylation levels of CpG‐rich promoters of genes related to chondrocyte phenotypes are largely kept low during chondrogenesis in human synovium–derived MSCs.

Significance: Osteoporosis is a highly prevalent disease affecting nearly 20 million people in the United States and is life-threatening in elderly patients. However, underlying pathophysiology regarding the posttranscriptional control of bone resorption is incompletely understood. CNOT3 is a molecule involved in mRNA stability in yeast to mammalian cells, but its role in bone regulation is not known. We discovered that Cnot3 deficiency specifically enhances receptor activator of NF-κB (RANK) mRNA stability and leads to osteopenia in healthy young adult animals. Moreover, Cnot3 levels are reduced in ageing-induced osteoporosis, and Cnot3 deficiency further exacerbates such osteoporosis significantly. As a mechanism, Cnot3 binds to RANK mRNA and its 3′-UTR renders Cnot3 -dependent instability to the reporter gene. Our results reveal Cnot3 regulation in aging-induced osteoporosis.

Significance: Osteoporosis is a major health problem worldwide, as the aging population is soaring. However, molecules involved in the regulation of bone remodeling are still incompletely understood. Nck (noncatalytic region of tyrosine kinase) is an adaptor molecule linking cytoskeleton and cell motility. We now identify that Nck regulates cell migration in vitro as well as in vivo and regulates bone mass via bone formation activity. Nck positively regulates repair of bone injury. This discovery provides a unique insight into the understanding of bone remodeling.