Differential expression of CCN4/Wisp1 mRNA splicing variants in chondrocytes

Objectives: CCN4/ Wnt-induced secreted protein 1 (WISP1) is one of the members of the CCN (CTGF/Cyr61/Nov) family proteins. They have typical structures composed of four conserved cysteine-rich modular domains, but variants of CCN members lacking one or more modules that were generated by alternative splicing or gene mutations, have been described in various pathological conditions. WISP1 gene was first described as a downstream target of the Wnt signaling pathway, which is frequently altered in human hepatocellular carcinoma (HCC). Several reports described a WISP1 variant (WISP1v) lacking VWC in a few malignancies, but no information concerning the production of WISP1 variants in normal tissue is currently available. In this study, expression of WISP1 mRNA and its variants was analyzed in chondrocytic cells. Methods: WISP1 mRNA expression was analyzed by RT-PCR in a human chondrosarcoma-derived chondrocytic cell line (HCS-2/8) and rabbit primary growth plate chondrocytes RGC, as well as other cell lines of different origins. To monitor the expression of WISP1 mRNA along chondrocyte differentiation, rabbit growth cartilage cells were cultured and sampled following a long time course until they were mineralized. Quantitative RT-PCR was carried out with a real-time PCR system. Result: Our results showed that WISP1, WISP1v, and a novel variant of WISP (WISP1vx) were expressed in HCS-2/8 cell line. This new variant lacking the exons for VWC and TSP domains and a part of IGFBP domain. Moreover, we identified a Wisp1v orthologue splicing variant in normal rabbit chondrocytes. Interestingly, WISP1v mRNA level was increased along with the terminal differentiation of growth plate chondrocytes. Conclusion: The differentiation-dependent expression of WISP1v suggests a critical role of WISP1v in normal endochondral ossification, while WISP1vx may be associated with malignant phenotypes. We are currently investigating the function of WISP1 and WISP1v in normal chondrocyte cells.