Targeted Deletion ofCapn4in Cells of the Chondrocyte Lineage Impairs Chondrocyte Proliferation and Differentiation

Abstract

ABSTRACTCalpains 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 geneCapn4directly 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 lineagein vitroandin vivo. To investigate a physiological role of the calpain small subunit in cells of the chondrocyte lineage, we generated chondrocyte-specificCapn4knockout mice. Mutant embryos had reduced chondrocyte proliferation and differentiation in embryonic growth plates compared with control littermates.In vitroanalysis further revealed that deletion ofCapn4in 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 p27Kip1rescued an impaired cell growth phenotype inCapn4knockdown 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.