Runx2 is required for hypertrophic chondrocyte mediated degradation of cartilage matrix during endochondral ossification.

The RUNX2 transcription factor is a key regulator for the development of cartilage and bone. Global or resting chondrocyte-specific deletion of the gene results in failure of chondrocyte hypertrophy, endochondral ossification, and perinatal lethality. The terminally mature hypertrophic chondrocyte regulates critical steps of endochondral ossification. Importantly, expression of the gene starts in the resting chondrocyte and increases progressively, reaching the maximum level in hypertrophic chondrocytes. However, the RUNX2 role after chondrocyte hypertrophy remains unknown. To answer this question, we deleted the gene specifically in hypertrophic chondrocytes using the Col10-Cre line. Mice lacking the gene in hypertrophic chondrocytes ( ) survive but exhibit limb dwarfism. Interestingly, the length of the hypertrophic chondrocyte zone is doubled in the growth plate of mice. Expression of pro-apoptotic decreased significantly while anti-apoptotic remains unchanged leading to a four-fold increase in the / ratio in mutant mice. In line with this, a significant reduction in apoptosis of hypertrophic chondrocyte is noted. A large amount of cartilage matrix is present in the long bones that extend toward the diaphyseal region of mice. This is not due to enhanced synthesis of the cartilage matrix as the expression of both collagen type 2 and aggrecan were comparable among and WT littermates. Our qPCR analysis demonstrates the increased amount of cartilage matrix is due to impaired expression of cartilage degrading enzymes such as metalloproteinase and aggrecanase as well as tissue inhibitor of metalloproteinases. Moreover, a significant decrease of TRAP positive chondroclasts was noted along the cartilage islands in mice. Consistently, qPCR data showed an 81% reduction in the / ratio in littermates, which is inhibitory for chondroclast differentiation. Finally, we assess if increase cartilage matrix in mice serves as a template for bone and mineral deposition using micro-CT and Von Kossa. The mutant mice exhibit a significant increase in trabecular bone mass compared to littermates. In summary, our findings have uncovered a novel role of Runx2 in apoptosis of hypertrophic chondrocytes and degradation of cartilage matrix during endochondral ossification.