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

Abstract

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. Calcium ions have numerous cellular functions, and serum calcium homeostasis is tightly regulated by the intestines, kidneys, and bones, which are governed by the key systemic hormones, parathyroid hormone, 1␣,25-dihydroxy vitamin D 3 , and calcitonin. Bone is a dynamic serum calciumregulating tissue. Bone formation by osteoblasts involves deposition of minerals, including calcium, and bone resorption by osteoclasts releases calcium from bone (1).We have purified and cloned the serum calcium-decreasing factor caldecrin from the pancreas (2-6). Caldecrin is a secretory-type serine protease that has chymotryptic activity and is also known as chymotrypsin C (EC 3.4.21.2) (8). We originally reported that the administration of caldecrin decreases mouse serum calcium concentration in a dose-dependent manner, and serum calcium decreasing activity is correlated with a decrease in serum hydroxyproline, which is included as a component of collagen and is a marker of bone resorption (2). Interestingly, this does not depend on its protease activity. Furthermore, caldecrin inhibits not only parathyroid hormone-stimulated bone destruction in mouse long bone organ culture but also bone resorption by rabbit mature osteoclasts in vitro (7). Taken together with our previous results, these observations suggest that caldecrin may suppress osteoclast differentiation and/or its activity through as yet unknown mechanisms.Osteoclasts, multinucleated giant cells that have a unique bone resorption activity, are differentiated from hematopoietic cells of the monocyte/macrophage lineage (9 -11). Osteoclast differentiation (osteoclastogenesis) from bone marrow cells (BMCs) 4 requires the cell-to-cell contact of osteoclast progenitor cells with osteoblasts or bone marrow stromal cells. The key molecule for osteoclastogenesis is a member of the tumor necrosis factor family (receptor activator of NF-B ligand (RANKL) (12), also called TRANCE (13)/ODF (14)/OPGL (15)) and is expressed in the osteoblast and bone marrow stro-

Caldecrin: A pancreas-derived hypocalcemic factor, regulates osteoclast formation and function

“…Caldecrin significantly inhibited RANKL-stimulated phosphorylation of c-Src in association with spleen tyrosine kinase, which is upstream of transient receptor potential vanilloid channel 4 and actin ring formation. On the other hand, caldecrin did not inhibit RANKL-mediated stimulation of MAPK, NF-kB, and c-Fos activation in osteoclast precursors or mature osteoclasts [34,35] . Therefore, caldecrin antagonized the RANKL-stimulated calcium signaling pathway involved in both osteoclast differentiation and activation.…”

“…RANKL induces osteoclast differentiation by activating two signaling pathways: the mitogen-activated protein kinase (MAPK), NF-kB, and c-Fos activation axis and the phospholipase C γ (PLCγ)-mediated calcium oscillation-calcineurin-nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) axis. Caldecrin did not inhibit macrophage colony-stimulating factorinduced osteoclast progenitor formation from bone marrow cells but did inhibit RANKL-induced osteoclast differentiation, even in the absence of protease activity [34] . Caldecrin inhibited the RANKL-stimulated spleen tyrosine kinase-and PLCγ-induced calcium oscillation, leading to an inhibition of calcineurin and NFATc1 activity (Figure 3).…”

Serum Calcium-decreasing Factor, Caldecrin, Inhibits Receptor Activator of NF-κB Ligand (RANKL)-mediated Ca2+ Signaling and Actin Ring Formation in Mature Osteoclasts via Suppression of Src Signaling Pathway

“…Intracellular Ca 2ϩ Measurement-Intracellular Ca 2ϩ was monitored using a Fluo-4NW calcium assay kit (Invitrogen) and Fura Red-AM according to the manufacturer's instructions and as described previously (25). RAW264.7 cell-derived mature OCs were cultured in the presence of RANKL on -dishes.…”

“…Cell Viability Assay-The same number of OCs collected from RepCell were cultured with or without RANKL or RANKL ϩ caldecrin (100 nM) for 1 day, as described above, fixed with 4% paraformaldehyde for 5 min, and stained for tartrate-resistant acid phosphatase (TRAP) as described previously (25). To evaluate the effects of caldecrin on OC survival, terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) staining was performed with an In Situ Cell Death Detection kit (Roche Applied Science).…”

“…We originally reported that the administration of caldecrin decreases mouse serum calcium concentration in a dose-dependent manner, and serum calcium-decreasing activity is correlated with a decrease in serum hydroxyproline, which is included as a component of collagen and is a marker of bone resorption (21). Recently, we reported that caldecrin inhibits osteoclast differentiation by inhibition of RANKL-mediated Ca 2ϩ oscillation, leading to suppression of NFATc1 activation (25). Interestingly, the inhibitory effect of caldecrin on OC differentiation does not depend on its protease activity.…”