A. Kashiwagi, M. Fein, E. Schipani, P. Greer, M. Shimada

Calpains are Ca-dependent intracellular cysteine proteases, which include widely 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 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 in vivo, we generated chondrocyte-specific Capn4 knockout mice by mating transgenic mice expressing Cre-recombinase under the control of the collagen IIα1 promoter (Col2-Cre) with Capn4^flox/flox mice. Efficiency of Cre activity assessed by real-time qPCR of RNA isolated from E15.5 mutant metatarsals was about 80%. Mutant (Col.2-Cre^+/-Capn4^flox/flox) embryos had slightly smaller skeletons. The mean total length of the mutant proliferating chondrocyte zone was shorter than that of controls (Col.2-Cre^+/-Capn4^flox/+) due to the significantly reduced length of the columnar proliferating chondrocyte layer (Control, 5.5±0.1 vs. Mutant, 4.8±0.1μm, *p<0.005), while the lengths of the periarticular and hypertrophic chondrocyte layers were indistinguishable between two groups. The shortened columnar proliferating chondrocyte layer was also observed at birth. The reduced length of the proliferating chondrocyte layer in mutant embryos could be a consequence of either decreased proliferation or increased apoptosis. Thus, proliferation of cells of the chondrocyte lineage was examined by BrdU incorporation in E15.5 and E18.5 embryos. Differently from the periarticular region, the number of BrdU-positive cells detected in the columnar proliferating region of the proximal tibial growth plate was significantly reduced in mutants compared with controls. No significant difference in the number of apoptotic cells determined by TUNEL staining was detected in E18.5 mutants vs. controls. In vitro analysis further revealed that deletion of Capn4 in cells of the chondrocyte lineage showed impaired cell cycle progression at G1/S transition, reduced Cyclin D1 transcription and accumulation of cell cycle proteins including cyclin D and E, and p27^Kip1. Collectively, our findings provide the in vivo evidence that the calpain small subunit is essential for proper chondrocyte proliferation in embryonic growth plate.

Disclosures: None