Involvement of phosphoinositide 3-kinase signaling pathway in chondrocytic differentiation of ATDC5 cells: Application of a gene-trap mutagenesis

Gene-trap mutagenesis is based on the notion that the random insertion of a trapping vector may disturb the function of inserted genes. Here, we applied this method to murine mesenchymal ATDC5 cells, which differentiate into mature chondrocytes in the presence of insulin. As the trap vector we used pPT1-geo, which lacks its own promoter and enhancer, but contains a lacZ-neo fusion gene as a reporter and selection marker driven by the promoter of the trapped gene. After pPT1-geo was introduced into ATDC5 cells by electroporation, the neomycin-resistant clones were screened for beta-galactosidase activity. The selected clones were cultured in differentiation medium to evaluate the chondrogenic phenotype. The clones no. 6-30 and 6-175, which exhibited impaired and accelerated mineralization, respectively, were subjected to further analysis. In clone no. 6-30 in which the gene coding for the p85alpha subunit of phosphoinositide 3-kinase (PI3K) was trapped, the expression of marker genes of early chondrocytes including collagen type 11, aggrecan, and PTH/PTHrP receptor was delayed. The insulin-induced stimulation of growth was reduced in clone no. 6-30 compared with the parental ATDC5 cells. Moreover, treatment of parental ATDC5 cells with a specific inhibitor of PI3K, LY294002, phenocopied clone no. 6-30, suggesting the involvement of PI3K signaling in the chondrogenic differentiation of ATDC5 cells. Clone no. 6-175 with accelerated mineralization was revealed to have a gene homologous to human KIAA0312 trapped, whose function remains unclear. Taken together, the gene-trap in ATDC5 cells might be useful to identify the molecules involved in chondrogenic differentiation. (C) 2004 Wiley-Liss, Inc.