Alternative Splicing of Type II Procollagen Gene in the Dedifferentiation of Rat Epiphyseal Chondrocytes Serially Cultured in Monolayer

Abstract

Type II procollagen is synthesized in two forms (IIA and IIB), generated by the alternative splicing of its precursor mRNA. The purpose of this study was to determine whether alternative splicing of type II procollagen pre-mRNA occurs during the dedifferentiation of chondrocytes in monolayer culture. We serially cultured rat epiphyseal chondrocytes for eight weeks in monolayer, and investigated the expression patterns of the two forms of type II procollagen transcripts according to the duration of the culture period. Reverse transcription-polymerase chain reaction and northern analysis revealed that both transcripts peaked on the day of cell isolation, but the expression level of IIB form was much higher than that of IIA form. The expression of IIA form decreased gradually, whereas the expression of IIB form decreased so rapidly that the ratio of type IIB and type IIA transcripts was reversed in the second week. In situ hybridization showed that all cells expressed type IIA mRNA throughout the culture period. The signal intensity for type IIA transcript decreased gradually in accordance with the results of RT-PCR and northern analysis. In situ hybridization of type IIB mRNA showed different results from in situ hybridization of type IIA mRNA. Like type IIA transcript, the freshly isolated chondrocytes and the cells in the first week showed positive signals for type IIB transcript. However, in the second week, positive signal was detected neither in polygonal cells nor large fibroblastic cells. Only a few cells of small size were weakly positive. From the third to the eighth week, no positive signal was detected. These findings indicate that alternative splicing of type II procollagen gene does occur and is regulated during the dedifferentiation of chondrocytes in monolayer culture.