RUNX2

Runx2 Control Chondrocyte Proliferation through Direct Regulation of Cell Cycle Genes

"we generated mice with a conditional ablation of Runx2 in chondrocyte. Selective deletion of Runx2 in chondrocyte by Col2a-Cre was confirmed with Rosa26-tdTomato mice. Homozygeous mice exhibited developmentally failed endochondral ossification and dies shortly after birth. Chondroprogenitors in the hyaline cartilage of wild type mice showed uni-directional differentiation to hypertrophic chondrocytes. Runx2 deficiency resulted in disruption of chondrocyte differentiation as determined by absence of pre/hypertrophy and Collagen type X expression. To our surprise, chondrocyte proliferation which is an integral component of endochondral ossification was severely impaired. We observed a dramatic decrease (50%) in the total cell number in both proliferative and pre-hypertrophic zones of homozygous mutant compared to wild type. Similar reduction in chondrocyte cell number was noted in hyaline cartilage of trachea. Furthermore, in vivo BrdU labeling confirmed this reduction results from a decreased mitotic activity of chondrocyte. This unique role of Runx2 to enhance chondrocyte proliferation is consistent with the growth failure and primordial dwarfism in mutant mice. To mechanistically understand alteration in chondrocyte proliferation rate, we determined genes associated with cell cycle cascade in the Runx2 null growth plate. Our gene microarray identified 5 genes with reproducible differences in expression levels ranging from 3-50 folds. In Runx2 null chondrocytes Sfn mRNA was upregulated, while expression of Gpr132, c-Myb, cyclin A1, A2 were strongly inhibited. Interestingly, the differentially expressed gene set encompass functional grouping across cell cycle from G1/S phase to cell cycle check points and arrest. To assess if Runx2 directly regulate their transcription, we cloned 2kb promoter of all five genes. Multiple high affinity Runx recognition motifs were noted in these promoters. Chromatin immunoprecipitation revealed that in chondrocytes Runx2 is bound to these sites. Consistent with in vivo promoter occupancy, promoter activity of these genes was enhanced by Runx2. Our results establish for the first time that Runx2 is obligatory for chondrocyte proliferation and regulate cell cycle genes. "