Targeted Deletion of Capn4 in Cells of the Chondrocyte Lineage Impairs Chondrocyte Proliferation and Differentiation

Abstract

Calpains are calcium-dependent intracellular cysteine proteases, which include ubiquitously expressed μ- and m-calpains. Both calpains are heterodimers consisting of a large catalytic subunit and a small regulatory subunit. The calpain small subunit encoded by the gene Capn4 directly binds to the intracellular C-terminal tail of the receptor for the parathyroid hormone (PTH) and PTH-related peptide and modulates cellular functions in cells of the osteoblast lineage in vitro and in vivo. To investigate a physiological role of the calpain small subunit in cells of the chondrocyte lineage, we generated chondrocyte-specific Capn4 knockout mice. Mutant embryos had reduced chondrocyte proliferation and differentiation in embryonic growth plates compared with control littermates. In vitro analysis further revealed that deletion of Capn4 in cells of the chondrocyte lineage correlated with impaired cell cycle progression at the G₁/S transition, reduced cyclin D gene transcription, and accumulated cell cycle proteins known as calpain substrates. Moreover, silencing of p27^Kip1 rescued an impaired cell growth phenotype in Capn4 knockdown cells, and reintroducing the calpain small subunit partially normalized cell growth and accumulated cyclin D protein levels in a dose-dependent manner. Collectively, our findings suggest that the calpain small subunit is essential for proper chondrocyte functions in embryonic growth plates.

We thank Henry M. Kronenberg for critical review of the manuscript and helpful discussion and Andrew P. McMahon (Department of Molecular and Cellular Biology, Harvard University) for a generous gift of Osx-Cre^+/− transgenic mice. We also thank Kimberly Atkin and Kerline Cepoudy at the Endocrine Unit and Laura B. Prickett-Rice at the Center for Regenerative Medicine in MGH for technical support.

This work was supported by grants from the National Institutes of Health Grant R01 DK072102 (M.S.) and the Canadian Institute of Health Research MOP-81189 (P.A.G.).