Synthesis of cartilage by chondrocytes is an obligatory step for endochondral ossification. Global deletion of the Runx2 gene results in complete failure of the ossification process, but the underlying cellular and molecular mechanisms are not fully known. Here, we elucidated Runx2 regulatory control distinctive to chondrocyte and cartilage tissue by generating Runx2 exon 8 floxed mice. Deletion of Runx2 in chondrocytes caused failure of endochondral ossification and lethality at birth. The limbs of Runx2ΔE8/ΔE8 mice were devoid of mature chondrocytes, vasculature, and marrow. We demonstrate that the C-terminus of Runx2 drives its biological activity. Importantly, nuclear import and DNA binding functions of Runx2 are insufficient for chondrogenesis. Molecular studies revealed that despite normal level of Sox9 and PTHrP, chondrocyte differentiation and cartilage growth is disrupted in Runx2ΔE8/ΔE8 mice. Loss of Runx2 in chondrocytes also impaired OPG-RANKL signaling and chondroclast development. Dwarfism observed in Runx2 mutants was associated with the near absence of proliferative zone in the growth plates. Finally, we show Runx2 directly regulates a unique set of cell cycle genes Gpr132, Sfn, c-Myb, and Cyclin A1 to control proliferative capacity of chondrocyte. Thus, Runx2 is obligatory for both proliferation and differentiation of chondrocytes.

Coordinated activities of multiple mesenchymal cell types contribute to the development of the mammalian skeleton formed through endochondral ossification. Synthesis of a cartilage template by chondrocytes is an obligatory step for the generation of skeletal elements during endochondral ossification. Gene ablation studies have established that Runx2 is an essential transcription factor for bone formation and the differentiation of skeletal cells. However, global gene deletion has failed to discern the tissue- and cell type-specific roles of Runx2. We generated floxed mice to elucidate the Runx2 regulatory control distinctive to cartilage tissue during bone development. Exon 8 of the Runx2 gene was selectively deleted in developing chondrocytes by utilizing Col2a-Cre mice. Cell- and tissue-specific gene recombination was confirmed by β-gal activity in R26R mice. The chondrocyte-specific loss of Runx2 caused failure of endochondral ossification, impaired craniofacial development, dwarfism, and perinatal lethality. Radiographic imaging and histochemical approaches were used to characterize the skeletal phenotype. We conclude that regulatory control of Runx2 in chondrocytes is essential for endochondral ossification, and it is independent of the role of Runx2 in osteoblasts.

Glucose intolerance seen in metabolic disorders, such as type II diabetes, is commonly associated with improper execution of the insulin signaling pathway, as well as an imbalance of bone and fat tissues, such that a gain in adipose tissue occurs at the expense of bone loss. Fat-producing adipocytes and bone-forming osteoblasts stem from a common mesenchymal progenitor cell. Runx2 positively regulates the commitment of the mesenchymal cell toward osteogenesis, but its effects on energy homeostasis and the insulin signaling pathway are unknown. To investigate the connection, focused microarray profiling of genes associated with the insulin signaling pathway was performed on calvarial cells from Runx2-null embryonic mice and 3T3-L1 preadipocytes treated with control and insulin-containing media. The microarray showed that addition of insulin resulted in a robust induction of genes (>95%) in 3T3-L1 cells. Surprisingly, Runx2-null cells cultured in control media were at an elevated state of energy metabolism and addition of insulin resulted in a marked suppression of genes required for insulin signaling. Clustering analysis revealed that the suppression occurred at all stages of the insulin pathway, from the receptors and transducers to nuclear effectors and target genes. Taken together, these results demonstrate that Runx2 is central for transduction and execution of the insulin regulatory signal. In conclusion, Runx2 actively regulates the gene network required for glucose metabolism and energy homeostasis in mesenchymal cells.