Background Mesenchymal stem cells (MSCs) can differentiate into cells of mesenchymal lineages, such as chondrocytes. Given the finding that MSCs exist in articular cartilage, it is conceivable that MSCs contribute to the maintenance of cartilage homeostasis and, moreover, possess an intrinsic ability to repair cartilage defects. IL-6 is known as a pleiotropic cytokine, and is constitutively and abundantly produced in MSCs. However, the detailed physiological meanings of IL-6 produced by MSCs remain unclear.
Objectives Aim of this study is to clarify the involvement of IL-6/STAT3 signaling pathway on chondrogenic differentiation of human MSCs.
Methods Human bone marrow-derived MSCs were pellet-cultured in chondrogenic induction medium containing TGF-β3. Cartilage matrix molecules, proteoglycans and type II collagen (COL2), were determined by staining with Safranin O and anti-COL2 antibody. Chondrogenic markers (COL2A1/AGGRECAN/COL10A1) were evaluated by real-time PCR. The expression and phosphorylation of IL-6 receptor (IL-6R), STAT3 and SOX9 were evaluated by western blotting. Human articular cartilage obtained from patients who had medial knee osteoarthritis (OA) and underwent total knee arthroplasty surgery was stained with anti-IL-6 and anti-CD166 Ab.
Results During pellet culture of MSCs in chondrogenic media IL-6 production was induced by day3 (10 ng/mL), which gradually reduced thereafter. Undifferentiated MSCs expressed low level of IL-6R, whereas chondrogenic culture significantly increased its expression, reaching a peak level on day7 and sustained up to day21 with a slight decline. Expression and phosphorylation of STAT3, a down-stream molecule of IL-6R, were up-regulated with similar manner of IL-6R expression. Addition of IL-6 and soluble IL-6R (sIL-6R) to the chondrogenic culture dose-dependently enhanced cartilage matrix and chondrogenic marker gene expression on day21. Although the total expression of SOX9, the master regulator of chondrogenesis, was not altered by IL-6/sIL-6R, phosphorylated SOX9 was increased at day7. Silencing of STAT3 by siRNA resulted in inhibition of cartilage matrix and chondrogenic marker gene expression. IL-6 and CD166, a surface marker of MSCs, co-localized at the superficial zone of human articular cartilage obtained from a medial knee of osteoarthritis patient.
Conclusions We have demonstrated that the activation of IL-6/STAT3 signaling pathway positively regulated chondrogenic differentiation of human MSCs through the up-regulating of Sox9 phosphorylation. Moreover, IL-6 co-localized with MSCs in the articular cartilage. These results suggest that IL-6 plays a role for maintenance of homeostasis and self-repair function by inducing chondrogenic differentiation of MSCs in human articular cartilage.
Disclosure of Interest M. Kondo Employee of: Mitsubishi Tanabe Pharma Corporation, K. Yamaoka: None declared, K. Sonomoto: None declared, K. Nakano: None declared, Y. Tanaka Grant/research support: Bristol-Myers, Mitsubishi-Tanabe, Abbvie, MSD, Chugai, Astellas, Daiichi-Sankyo, Speakers bureau: Mitsubishi-Tanabe, Eisai, Chugai, Abbott Japan, Astellas, Daiichi-Sankyo, Abbvie, Janssen, Pfizer, Takeda, Astra-Zeneca, Eli Lilly Japan, GlaxoSmithKline, Quintiles, MSD, Asahi-Kasei