Abstract
Runx2 is required for chondrocyte proliferation and maturation. In the search of Runx2 target genes in chondrocytes, we found that Runx2 up-regulated the expression of hematopoietic cell kinase (Hck), which is a member of the Src tyrosine kinase family, in chondrocytes, that Hck expression was high in cartilaginous limb skeletons of wild-type mice but low in those of Runx2–/– mice, and that Runx2 bound the promoter region of Hck. To investigate the functions of Hck in chondrocytes, transgenic mice expressing a constitutively active form of Hck (HckCA) were generated using the Col2a1 promoter/enhancer. The hind limb skeletons were fused, the tibia became a large, round mass, and the growth plate was markedly disorganized. Chondrocyte maturation was delayed until E16.5 but accelerated thereafter. BrdU-labeled, but not terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive, chondrocytes were increased. Furthermore, Hck knock-down reduced the proliferation of primary chondrocytes. In microarray and real-time RT-PCR analyses using hind limb RNA from HckCA transgenic mice, the expression of Wnt (Wnt10b, Tcf7, Lef1, Dkk1) and hedgehog (Ihh, Ptch1, and Gli1) signaling pathway genes was upregulated. These findings indicated that Hck, whose expression is regulated by Runx2, is highly expressed in chondrocytes, and that HckCA activates Wnt and hedgehog signaling pathways, and promotes chondrocyte proliferation without increasing apoptosis.
Highlights
In the process of endochondral ossification, mesenchymal cells condensate and acquire the phenotype of chondrocytes to produce Col2a1 and proteoglycan
Vascular invasion occurs in the layer of terminal hypertrophic chondrocytes, the terminal hypertrophic chondrocytes transdifferentiate to osteoblastic lineage cells or die by apoptosis, osteoblast lineage cells invade from the perichondrium, and cartilage is replaced with bone [3]
We focused on the up-regulated genes because gene ontology (GO) terms related to skeletal development were enriched in the up-regulated genes, but not in the down-regulated genes, in HckCA tg embryos compared with wild-type embryos (Figure 6D and data not shown)
Summary
In the process of endochondral ossification, mesenchymal cells condensate and acquire the phenotype of chondrocytes to produce Col2a1 and proteoglycan. Sox is required for the mesenchymal cell condensation, and Sox, Sox, and Sox induce Col2a1 expression [1,2]. Hypertrophic chondrocytes terminally differentiate into terminal hypertrophic chondrocytes, which express Mmp, Ibsp, and Spp. Differentiation and proliferation are organized, and the growth plate, which is composed of resting, proliferating, prehypertrophic, hypertrophic, and terminal hypertrophic chondrocyte layers, is formed. Vascular invasion occurs in the layer of terminal hypertrophic chondrocytes, the terminal hypertrophic chondrocytes transdifferentiate to osteoblastic lineage cells or die by apoptosis, osteoblast lineage cells invade from the perichondrium, and cartilage is replaced with bone [3]
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