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Objective Synovial fibroblasts (SFs) in rheumatoid arthritis (RA) and osteoarthritis (OA) play biphasic roles in joint destruction and regeneration of bone/cartilage as mesenchymal stem cells (MSCs). Although MSCs contribute to joint homeostasis, such function is impaired in arthritic joints. We have identified functionally distinct three SF subsets characterized by the expression of CD34 and THY1 as follows: CD34+THY1+, CD34−THY1−, and CD34−THY1+. The objective of this study was to clarify the differentiation potentials as MSCs in each SF subset since both molecules would be associated with the MSC function. Methods SF subsets were isolated from synovial tissues of 70 patients (RA: 18, OA: 52). Expressions of surface markers associated with MSCs (THY1, CD34, CD73, CD271, CD54, CD44, and CD29) were evaluated in fleshly isolated SF subsets by flow cytometry. The differentiation potentials of osteogenesis, chondrogenesis, and adipogenesis were evaluated with histological staining and a quantitative polymerase chain reaction of differentiation marker genes. Small interfering RNA was examined to deplete THY1 in SFs. Results The expression levels of THY1+, CD73+, and CD271+ were highest and those of CD54+ and CD29+ were lowest in CD34+THY1+ among three subsets. Comparing three subsets, the calcified area, alkaline phosphatase (ALP)-stained area, and cartilage matrix subset were the largest in the CD34+THY1+ subset. Consistently, the expressions of differentiation markers of the osteoblasts (RUNX2, ALPL, and OCN) or chondrocytes (ACAN) were the highest in the CD34+THY1+ subset, indicating that the CD34+THY1+ subset possessed the highest osteogenic and chondrogenic potential among three subsets, while the differentiation potentials to adipocytes were comparable among the subsets regarding lipid droplet formations and the expression of LPL and PPARγ. The knockdown of THY1 in bulk SFs resulted in impaired osteoblast differentiation indicating some functional aspects in this stem-cell marker. Conclusion The CD34+THY1+ SF subset has high osteogenic and chondrogenic potentials. The preferential enhancement of MSC functions in the CD34+THY1+ subset may provide a new treatment strategy for regenerating damaged bone/cartilage in arthritic joints.

Introduction Increased activity of osteoclasts is responsible for bone loss and joint destruction in rheumatoid arthritis. For osteoclast development and bone resorption activity, cytoskeletal organization must be properly regulated. MicroRNAs (miRNAs) are endogenous small noncoding RNAs that suppress expression of their target genes. This study was conducted to identify crucial miRNAs to control osteoclasts. Methods miRNA expression in the bone marrow-derived macrophages (BMM) with or without receptor activator of nuclear factor κB ligand (RANKL) stimulation was analyzed by miRNA array. To examine the role of specific miRNAs in osteoclast formation, bone resorption activity and actin ring formation, the BMM were retrovirally transduced with miRNA antagomirs. To confirm whether the suppressive effects on osteoclastogenesis by miR-31 inhibition were mediated by targeting RhoA, osteoclast formation was analyzed in the presence of the RhoA inhibitor, exoenzyme C3. Results miR-31 was identified as one of the highly upregulated miRNAs during osteoclast development under RANKL stimulation. Inhibition of miR-31 by specific antagomirs suppressed the RANKL-induced formation of osteoclasts and bone resorption. Phalloidin staining of osteoclasts revealed that actin ring formation at the cell periphery was severely impaired by miR-31 inhibition, and clusters of small ringed podosomes were observed instead. In these osteoclasts, expression of RhoA, one of the miR-31 target genes, was upregulated by miR-31 inhibition in spite of the impaired osteoclastogenesis. Treatment with the RhoA inhibitor, exoenzyme C3, rescued the osteoclastogenesis impaired by miR-31 inhibition. Conclusions miR-31 controls cytoskeleton organization in osteoclasts for optimal bone resorption activity by regulating the expression of RhoA.