Investigation of the mechanism of proliferation and differentiation of cartilage tissue using chondrocytes derived from p53-/-mouse.

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1) Investigation of PGE2 signalImmunohistochemical and RT-PCR analysis showed that the EP2 is the major receptor for PGE2 in articular chondrocytes. EP2 specific agonist induced a dose-dependent increase of cAMP in MMA2, which is a chondrocyte cell line derived from articular cartilage of p53-/-mice. Gene-expression profile of MMA2 before and after the treatment with EP2 agonist was compared, and 22 genes up-regulated genes by EP2 agonist were identified including several growth-promoting and apoptotis-inhibiting genes. On treatment with the EP2 agonist, human articular chondrocytes showed an increase in the incorporation of BrdU, and the organ culture of rat femur showed an increase in PCNA staining in articular chondrocytes. These results suggested that PGE2 signal through EP2 enhances the growth of articular chondrocytes, and the EP2 agonist is a candidate for a new therapeutic compound for the treatment of cartilage degenerative disease.2) Isolation of factors involved in the calcification in growth plate.Gene expression profiles of MMR14 and MMR17, of which the former but not the later produced the calcified nodules in monolayer culture, was compared to isolate the factors involved in the process of calcification in the growth plate. Several cell-adhesion molecule, extracellular matrix, signal molecule and transcriptional factor genes were isolated, as differentially expressed, genes including the OB-cadherin. Expression of OB-cadherin both at mRNA and protein levels increased with times in MMR14 in the monolayer culture, but no expression was observed in MMR17. In the growth plate of rib, OB-cadherin was detected in calcifying cartilages, suggesting its involvement of the step of final differentiation of growth plate chondrocytes.