We previously showed that type X collagen, a marker of late stage chondrocyte hypertrophy (associated with endochondral ossification), is constitutively expressed by mesenchymal stem cells (MSCs) from osteoarthritis patients and this may be related to Naproxen (Npx), a nonsteroidal anti-inflammatory drug used for therapy. Hedgehog (HH) signaling plays an important role during the development of bone. We tested the hypothesis that Npx affected osteogenic differentiation of human MSCs through the expression of Indian hedgehog (IHH), Patched-1 (PTC1) and GLI family members GLI1, GLI2, GLI3 in vitro.
MSCs were cultured in osteogenic differentiation medium without (control) or with 0.5 μM Npx. The expression of collagen type X, alpha 1 (COL10A1), alkaline phosphatase (ALP), osteopontin (OPN), osteocalcin (OC), collagen type I, alpha 1 (COL1A1) was analyzed with real-time reverse transcription (RT) PCR, and the ALP activity was measured. The osteogenesis of MSCs was monitored by mineral staining and quantification with alizarin red S. To examine whether Npx affects osteogenic differentiation through HH signaling, the effect of Npx on the expression of IHH, GLI1, GLI2, GLI3 and PTC1 was analyzed with real-time RT PCR. The effect of cyclopamine (Cpn), a HH signaling inhibitor, on the expression of COL10A1, ALP, OC and COL1A1 was also determined.
When MSCs were cultured in osteogenic differentiation medium, Npx supplementation led to a significant decrease in ALP gene expression as well as its activity, and had a tendency to decrease mineral deposition. It also decreased the expression of COL1A1 significantly. In contrast, the gene expression of COL10A1 and OPN were upregulated significantly by Npx. No significant effect was found on OC expression. The expression of IHH, PTC1, GLI1, and GLI2 was increased by Npx, while no significant difference was observed on GLI3 expression. Cpn reversed the effect of Npx on the expression of COL10A1, ALP, OPN and COL1A1.
These results indicate that Npx can affect gene expression during osteogenic differentiation of MSCs, and downregulate mineral deposition in the extracellular matrix through IHH signaling. Therefore, Npx could affect MSC-mediated repair of subchondral bone in OA patients.