Chloride channels regulate chondrogenesis in chicken mandibular mesenchymal cells

Voltage gated chloride channels (ClCs) play an important role in the regulation of intracellular pH and cell volume homeostasis. Mutations of these genes result in genetic diseases with abnormal bone deformation and body size, indicating that ClCs may have a role in chondrogenesis. In the present study, we isolated chicken mandibular mesenchymal cells (CMMC) from Hamburg–Hamilton (HH) stage 26 chick embryos and induced chondrocyte maturation by using ascorbic acid and β-glycerophosphate (AA-BGP). We also determined the effect of the chloride channel inhibitor NPPB 5-nitro-2-(3-phenylpropylamino) benzoic acid] on regulation of growth, differentiation, and gene expression in these cells using MTT and real-time PCR assays. We found that CLCN1 and CLCN3–7 mRNA were expressed in CMMC and NPPB reduced expression of CLCN3, CLCN5, and CLCN7 mRNA in these cells. At the same time, NPPB inhibited the growth of the CMMC, but had no effect on the mRNA level of cyclin D1 and cyclin E (P > 0.05) with/without AA-BGP treatment. AA-BGP increased markers for early chondrocyte differentiation including type II collagen, aggrecan (P < 0.01) and Sox9 (P < 0.05), whilst had no effect on the late chondrocyte differentiation marker type X collagen. NPPB antagonized AA-BGP-induced expression of type II collagen and aggrecan (P < 0.05). Furthermore, NPPB downregulated type X collagen (P < 0.05) with/without AA-BGP treatment. We conclude that abundant chloride channel genes in CMMC play important roles in regulating chondrocyte proliferation and differentiation. Type X collagen might function as a target of chloride channel inhibitors during the differentiation process.