Abstract
The RUNX2 transcription factor is a key regulator for the development of cartilage and bone. Global or resting chondrocyte-specific deletion of the Runx2 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 Runx2 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 Runx2 gene specifically in hypertrophic chondrocytes using the Col10-Cre line. Mice lacking the Runx2 gene in hypertrophic chondrocytes (Runx2HC/HC ) survive but exhibit limb dwarfism. Interestingly, the length of the hypertrophic chondrocyte zone is doubled in the growth plate of Runx2HC/HC mice. Expression of pro-apoptotic Bax decreased significantly while anti-apoptotic Bcl2 remains unchanged leading to a four-fold increase in the Bcl2/Bax ratio in mutant mice. In line with this, a significant reduction in apoptosis of Runx2HC/HC hypertrophic chondrocyte is noted. A large amount of cartilage matrix is present in the long bones that extend toward the diaphyseal region of Runx2HC/HC 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 Runx2HC/HC 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 Runx2HC/HC mice. Consistently, qPCR data showed an 81% reduction in the Rankl/Opg ratio in Runx2HC/HC littermates, which is inhibitory for chondroclast differentiation. Finally, we assess if increase cartilage matrix in Runx2HC/HC 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.