The role of DNA methylation to type II collagen expression in dedifferentiation of articular chondrocytes

Kim, Yun Hee; Hahn, Soo Bong; Lee, Jin Woo

Abstract

PURPOSE
We explored the possibility of DNA methylation as a mechanism of loss of type II collagen expression in dedifferentiating chondrocytes by culturing in monolayer.
MATERIALS AND METHODS
Dedifferentiation was induced by low density subculturing primary porcine chondrocytes in vitro. The mRNA expression of Type I collagen, Type II collagen and DNA methyltransferase (DNMT) was measured by RT-PCR. Induction of redifferentiation in dedifferentiated chondrocytes was performed in 3-dimensional alginate bead culture system. As stimulating factors for reexpression of genes in dedifferentiated chondrocytes, 10 ng/ml TGF-beta1 and 5 micrometer 5-azacytidine were used.
RESULTS
Type II collagen mRNAs was expressed strongly in freshly isolated cells but had decreased in monolayer cultured cells after 3 weeks up to 40%. In contrast, type I collagen expression was increased from 21 days and kept increasing during the 86 days of study. After treatment of 5 micrometer 5-azacytidine, fibroblast like morphology was changed to round shape such as traditional chondrocyte morphology at day 4. At day 10, type II collagen expression was increased by 5-azacytidine and TGF-beta1 marginally and also integrin beta1 expression was increased in all groups. RT-PCR analysis demonstrated that DNMT3A expression increased in dedifferentiating chondrocytes when compared with control cells for 40 days.
CONCLUSION
Loss of type II collagen mRNA expression and increase of DNMT 3A expression were showed similar patterns during dedifferentiation. These results suggest that type II collagen gene expression may be influenced by DNA methylation. As stimulating factors, TGF-beta1 and 5-azacytidine have potential activity to increase the type II collagen expression in alginate culture system.