Serum Calcium-decreasing Factor, Caldecrin, Inhibits Osteoclast Differentiation by Suppression of NFATc1 Activity

Caldecrin/chymotrypsin C is a novel secretory-type serine protease that was originally isolated as a serum calcium-decreasing factor from the pancreas. Previously, we reported that caldecrin suppressed the bone-resorbing activity of rabbit mature osteoclasts (Tomomura, A., Yamada, H., Fujimoto, K., Inaba, A., and Katoh, S. (2001) FEBS Lett. 508, 454–458). Here, we investigated the effects of caldecrin on mouse osteoclast differentiation induced by macrophage-colony stimulating factor and the receptor activator of NF-κB ligand (RANKL) from the monocyte/macrophage cell lineage of bone marrow cells. Wild-type and protease-deficient mutant caldecrin dose-dependently inhibited RANKL-stimulated tartrate-resistant acid phosphatase-positive osteoclast formation from bone marrow cells. Caldecrin did not affect macrophage colony formation from monocyte/macrophage lineage cells or osteoclast progenitor generation in cultures of bone marrow cells. Caldecrin inhibited accumulation of the RANKL-stimulated nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) mRNA in bone marrow cells, which is a key transcription factor for the differentiation of osteoclasts. Caldecrin also suppressed RANKL-induced differentiation of the RAW264.7 monocyte/macrophage cell line into osteoclasts. Caldecrin reduced the transcriptional activity of NFATc1 in RAW264.7 cells, whereas those of NF-κB and c-Fos, which are also transcription factors involved in osteoclast differentiation, were unaffected. Caldecrin inhibited RANKL-stimulated nuclear translocation of NFATc1 and the activity of the calcium/calmodulin-dependent phosphatase, calcineurin. Caldecrin inhibited phospholipase Cγ1-mediated Ca2+ oscillation evoked by RANKL stimulation. RANKL-stimulated phosphorylation of spleen tyrosine kinase (Syk) was also attenuated by caldecrin. Taken together, these results indicate that caldecrin inhibits osteoclastogenesis, without its protease activity, by preventing a phospholipase Cγ1-mediated Ca2+oscillation-calcineurin-NFATc1 pathway.