The CCN2-inducer Harmine promotes chondrogenesis and protection against TNF-induced inflammation

Purpose: Previous studies demonstrated the capability of CCN2 to regenerate surgical defects in articular cartilage of rats, and that transgenic mice over-expressing cartilage-specific CCN2 were more resistant to aging-related cartilage degradation. This study aimed to comprehensively search for a compound that can increase cartilage-derived CCN2 in order to prevent or even repair cartilage degeneration. Methods: In the screening process, human chondrosarcoma-derived cells (HCS-2/8) were cultured in 96-well plates and stimulated with 84 orphan-ligands (Enzo Life Sciences) for 6 hours. CCN2 protein levels in the culture media were quantified by ELISA. The toxicity of the selected molecule to HCS-2/8 cells was evaluated by MTS (Promega) and LDH-cytotoxicity (Roche) assays. Expression levels of CCN2, SOX-9, aggrecan and collagen-II were analyzed by either real-time RT-PCR or western blot. To evaluate the effect of Harmine under inflammatory condition, HCS-2/8 cells and human osteoarthritic chondrocytes (hOACs) were stimulated with TNFα. The ability to promote chondrogenesis was assessed by Alcian blue staining of 10-day micromass cultures of ATDC5 cells. Results: The screening process selected harmine as the single molecule that increased CCN2 levels, and 5μM concentration yielded the highest gene expression of cartilage markers. Harmine clearly induced chondrogenesis in ATDC5 micromass cultures. Interestingly, harmine could rescue TNFα-induced decrease in expression of cartilage markers in both HCS-2/8 cells and hOACs. Conclusion: Harmine, identified as an inducer of CCN2, presented notable chondrogenic and chondroprotective effects which indicate its utility as a potential drug for prevention and/or repair of cartilage degradation due to aging or osteoarthritis.