The cyclin-dependent kinase inhibitor p57(Kip2) mediates proliferative actions of PTHrP in chondrocytes.

Parathyroid hormone-related peptide (PTHrP) is a positive regulator of chondrocyte proliferation during bone development. In embryonic mice lacking PTHrP, chondrocytes stop proliferating prematurely, with accelerated differentiation. Because the bone phenotype of mice lacking the cyclin-dependent kinase inhibitor p57(Kip2) is the opposite of the PTHrP-null phenotype, we hypothesized that PTHrP's proliferative actions in chondrocytes might be mediated by opposing p57. We generated p57/PTHrP-null embryos, which showed partial rescue of the PTHrP-null phenotype. There was reversal of the loss of proliferative chondrocytes in most bones, with reversal of the accelerated differentiation that occurs in the PTHrP-null phenotype. p57 mRNA and protein were upregulated in proliferative chondrocytes in the absence of PTHrP. Metatarsal culture studies confirmed the action of PTHrP to decrease p57 mRNA and protein levels in a model in which parathyroid hormone (PTH), used as an analog of PTHrP, increased chondrocyte proliferation rate and the length of the proliferative domain. PTH treatment of p57-null metatarsals had no effect on proliferation rate in round proliferative chondrocytes but still stimulated proliferation in columnar chondrocytes. These studies suggest that the effects of PTHrP on both the rate and extent of chondrocyte proliferation are mediated, at least in part, through suppression of p57 expression.

Deletion of Vhlh in chondrocytes reduces cell proliferation and increases matrix deposition during growth plate development.

The von Hippel Lindau tumor suppressor protein (pVHL) is a component of a ubiquitin ligase that promotes proteolysis of the transcription factor hypoxia-inducible-factor 1alpha (HIF1alpha), the key molecule in the hypoxic response. We have used conditional inactivation of murine VHL (Vhlh) in all cartilaginous elements to investigate its role in endochondral bone development. Mice lacking Vhlh in cartilage are viable, but grow slower than control littermates and develop a severe dwarfism. Morphologically, Vhlh null growth plates display a significantly reduced chondrocyte proliferation rate, increased extracellular matrix, and presence of atypical large cells within the resting zone. Furthermore, stabilization of the transcription factor HIF1alpha leads to increased expression levels of HIF1alpha target genes in Vhlh null growth plates. Lastly, newborns lacking both Vhlh and Hif1a genes in growth plate chondrocytes display essentially the same phenotype as Hif1a null single mutant mice suggesting that the Vhlh null phenotype could result, at least in part, from increased activity of accumulated HIF1alpha. This is the first study reporting the novel and intriguing findings that pVHL has a crucial role in endochondral bone development and is necessary for normal chondrocyte proliferation in vivo.