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Fig. 5. Chondrocyte proliferation and differentiation are decreased and apoptosis is increased in Col2a1-ICAT transgenic mice. (A,B) Ki-67 and DAPI double staining was performed using anti-Ki-67 antibody in tissue sections collected from 2-week-old Col2a1-ICAT transgenic mice and their WT littermates. The proliferation rate was determined by counting the numbers of Ki-67-positive cells at the proliferating zone divided by the DAPI-positive cell number. The proliferation of growth plate chondrocytes is decreased by 24% in 2-week-old Col2a1-ICAT transgenic mice. *P<0.05, unpaired t-test, n=3. (C) The expression of cyclin D1, cyclin D2, cyclin A, PCNA, total and phosphorylated β-catenin and phosphorylated Rb was examined by western blotting. The expression of cyclin D1, cyclin D2 and PCNA was significantly decreased in primary chondrocytes derived from Col2a1-ICAT transgenic mice compared with those derived from WT mice. By contrast, the protein levels of cyclin A, total and phosphorylated β-catenin and phosphorylated Rb was not significantly changed. (D) Total RNA was extracted from primary sternal chondrocytes isolated from Col2a1-ICAT transgenic mice and WT littermates. The expression of chondrocyte differentiation marker genes, such as collagen type X (colX), Vegf, Mmp13 and Alp was determined by real-time reverse transcriptase (RT)-PCR and was normalized to β-actin levels. The expression of chondrocyte marker genes was significantly reduced in Col2a1-ICAT transgenic mice. *P<0.05, unpaired t-test, n=3. (E) The apoptosis of growth-plate chondrocytes was determined by TUNEL staining using 2-week-old WT and Col2a1-ICAT transgenic mice. Increased cell apoptosis was observed in the hypertrophic region of the growth plate in Col2a1-ICAT transgenic mice.
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