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. (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-kappaB 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-kappaB 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 Cgamma1-mediated Ca(2+) 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 Cgamma1-mediated Ca(2+)oscillation-calcineurin-NFATc1 pathway.

FIGURE 1.

Caldecrin inhibits RANKL-stimulated OC formation from BMCs and RAW264.7 cells without protease activity. A, BMCs were cultured for 3 days with M-CSF (M, 10 ng/ml) and further cultured for 3–4 days with M-CSF alone or M-CSF plus RANKL (R, 10 ng/ml) (M/R) with or without WT or Sm caldecrin (C, 3 μg/ml). The cells were stained for TRAP activity. The scale bar shows 200 μm. B, macrophage colony formation assay (left) and frequency analysis (right) were performed as described under “Experimental Procedures.” C, BMCs were cultured as shown in A except various concentrations of WT or Sm caldecrin were added, and TRAP activities of the culture media were compared (mean ± S.D. of three experiments; **, p < 0.01 versus RANKL-treated group). D, BMCs were cultured as shown in A and stained for TRAP activity. TRAP-positive mononucleated osteoclasts (MonoNOC) and multinucleated osteoclasts (MultiNOC) were counted (mean ± S.D. of three experiments; **, p < 0.01 versus the RANKL-treated mononucleated or multinucleated osteoclast control group, respectively). E, RAW264.7 cells were cultured for 3–4 days with RANKL (10 ng/ml) or RANKL plus various concentrations of WT or Sm caldecrin, and TRAP activities were measured. F, RAW264.7 cells were cultured as shown in E and stained for TRAP activity. TRAP-positive mononucleated and multinucleated osteoclasts were counted and compared with respective controls (mean ± S.D. of three experiments; **, p < 0.01 versus the RANKL-treated mononucleated or multinucleated osteoclast control group, respectively).

FIGURE 2.

Caldecrin inhibits RANKL-stimulated NFATc1 activity. A, BMCs were cultured as shown in Fig. 1A. At 8, 24, and 48 h after treatment with RANKL with or without WT caldecrin (3 μg/ml), total RNAs were isolated, and NFATc1 mRNA was evaluated by TaqMan real-time PCR (mean ± S.D. of three experiments; *, p < 0.05 versus RANKL-treated group with the same incubation time). B, RAW264.7 cells were incubated with RANKL alone (R) or in combination with WT caldecrin (9 μg/ml, shaded) for 30 min (R+C). Nuclear extracts were prepared, and the transcriptional activities of NFATc1, NF-κΒ, and c-Fos were measured (mean ± S.D. of three experiments: *, p < 0.05 versus RANKL-treated group). C, RAW264.7 cells were stimulated for 40 min with RANKL or RANKL plus WT caldecrin (5 μg/ml). Cytosolic and nuclear fractions were prepared from untreated (−), RANKL-stimulated (R), or RANKL plus WT caldecrin-treated (R+C) cells and analyzed by Western blotting with anti-NFATc1 antibody. The purity of the subcellular fractions and equal sample loading were controlled by analyzing β-actin and histone H1. Phosphorylation of NFATc1 in the whole-cell lysates from the same cells were evaluated by Western blotting with anti-phospho-NFATc1. D, RAW264.7 cells were incubated and stimulated for 40 min with RANKL or RANKL plus WT caldecrin (5 μg/ml). Cells were fixed and stained for anti-NFATc1 (green) and nucleus (red). The scale bar shows 10 μm.

FIGURE 3.

Caldecrin inhibits RANKL-stimulated calcineurin activity in (RANKL) RAW 264.7 cells. A, RAW264.7 cells were cultured and stimulated for 30 min by RANKL with (R+Cal) or without (RANKL) WT caldecrin (9 μg/ml). Cell homogenates were prepared, and calcineurin activities were compared (mean ± S.D. of three experiments; *, p < 0.05 versus RANKL-treated group). B, RAW264.7 cells were cultured as shown in A, and calcineurin contents in the cells prepared from unstimulated (−), RANKL-stimulated (R), or RANKL plus WT caldecrin (R+C) were compared by Western blotting with anti-calcineurin antibody.

FIGURE 4.

Caldecrin inhibits PLCγ1 mediated Ca²⁺ oscillation evoked by RANKL in BMCs and RAW 264.7 cells. A, BMCs were cultured for 1 day with M-CSF (10 ng/ml) plus RANKL (10 ng/ml). Intracellular [Ca²⁺]_i in single cells was measured as described under “Experimental Procedures.” After a 10- min observation of Ca²⁺ oscillation with the addition of RANKL, WT caldecrin (10 nm) was further added. Each color indicates an individual cell in the same field. At the end of the experiment, ionomycin was added. B, [Ca²⁺]_i in RAW264.7 cells was monitored as shown in A. Effects of BAPTA-AM (100 nm) and U73122 (100 nm) were evaluated (middle panels). The cells showed Ca²⁺ oscillation without RANKL (−), RANKL-triggered Ca²⁺ oscillation-acquired cells (R), and caldecrin-responsive cells in which RANKL-triggered Ca²⁺ oscillation was inhibited by WT caldecrin (R+C) were scored in the same field (mean ± S.D. of three experiments: *, p < 0.01 versus RANKL-treated group). C, RAW264.7 cells were incubated for 30 min with or without RANKL (10 ng/ml, R) or RANKL plus WT caldecrin (5 μg/ml, R+C). Cell lysates were subjected to Western blotting with anti-PLCγ1 or anti-phospho-PLCγ1 antibody.

FIGURE 5.

Caldecrin suppresses RANKL-stimulated Syk phosphorylation in RAW264.7 cells. A, RANKL-evoked Ca²⁺ oscillation was monitored as shown in Fig. 4B. The Syk inhibitor ER-27319 (100 nm) was added at 10 min after RANKL treatment. B, RAW264.7 cells were incubated for 6 min with RANKL (R, 10 ng/ml) or RANKL plus WT caldecrin (R+C, 5 μg/ml). Total cell lysates were subjected to immunoprecipitation (IP) with anti-Syk antibody. The immunoprecipitates were separated by SDS-PAGE and immunoblotted with anti-phosphotyrosine antibody.