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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.

SUBMITTER: Hasegawa H

PROVIDER: S-EPMC2919108 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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Serum calcium-decreasing factor, caldecrin, inhibits osteoclast differentiation by suppression of NFATc1 activity.

Hasegawa Hiroya H Kido Seisui S Tomomura Mineko M Fujimoto Kengo K Ohi Michi M Kiyomura Masaru M Kanegae Haruhide H Inaba Akemi A Sakagami Hiroshi H Tomomura Akito A

The Journal of biological chemistry 20100614 33

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 rec ...[more]

PMID: 20547767

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Project description:Bone diseases such as postmenopausal osteoporosis are primarily caused by excessive formation and activity of osteoclasts (OCLs). Receptor activator of nuclear factor-?B ligand (RANKL) is a key initiating cytokine for OCL differentiation and function. RANKL induces calcium (Ca(2+)) oscillations, resulting in selective and robust induction of nuclear factor of activated T cells c1 (NFATc1), a Ca(2+)-responsive transcription factor that drives osteoclastogenesis. Store-operated Ca(2+) entry (SOCE) is a major Ca(2+) influx pathway in most nonexcitable cell types and is activated by any stimulus that depletes Ca(2+) stores in the endoplasmic reticulum. Although the role of Orai1, a SOCE channel in the plasma membrane, in maintaining Ca(2+) oscillations and transactivation of NFAT in other cell types is well known, its contribution to osteoclastogenesis remains unclear. We show here that silencing of the Orai1 gene with viral delivery of shRNA reduces SOCE and inhibits RANKL-induced osteoclastogenesis of RAW264.7 cells, a murine monocyte/macrophage cell line, by suppressing the induction of NFATc1. This was accompanied by defective induction of OCL-specific genes, such as tartrate-resistant acid phosphatase and immunoreceptor OCL-associated receptor, which are known to be direct transcriptional targets of NFATc1 during osteoclastogenesis. In addition, maturation of OCLs was abrogated by defective cell fusion of pre-OCLs depleted of Orai1, consistent with defective RANKL-mediated induction of d2 isoform of vacuolar ATPase V(o) domain that is involved in cell fusion of pre-OCLs. We found that the functional bone resorbing capacity was severely impaired in OCLs depleted of Orai1, potentially related to the observed decrease in the induction of cathepsin K, a major bone matrix degrading protease. Our results indicate that Orai1 plays a critical role in the differentiation and function of OCLs, suggesting that Orai1 might be a potential therapeutic target for the treatment or prevention of bone loss caused by OCLs.

Project description:We recently reported that oral administration of a (+)-vitisin A-enriched product prepared from Vitis thunbergii obviously ameliorated bone loss in ovariectomized mice and (+)-vitisin A was able to inhibit receptor activator of NF-?B ligand (RANKL)-induced osteoclast differentiation in RAW264.7 cells. Here we further clarified the mechanism(s) by which (+)-vitisin A targets osteoclastic differentiation and activity. Osteoclast-characteristic enzyme activity was determined using gel zymography or spectroflurometric-based assay. Expression of signal molecules was analyzed via Western blot or immunoprecipitation. Results showed that (+)-vitisin A suppressed RANKL-induced multinuclear cells (MNCs) formation and bone resorption which was accompanied with reduction in ?3 integrin, osteoclast stimulatory transmembrane protein (OC-STAMP), matrix metalloproteinase-9 (MMP-9) and cathepsin K proteins expression. (+)-Vitisin A also down-regulated the proteolytic activities of MMP-9 and cathepsin K via targeting at the late stage function. (+)-Vitisin A prominently abrogated RANKL-triggered nuclear translocations of NF-?B, AP-1 (c-Fos/c-Jun dimer) and associated induction and nuclear accumulation of nuclear factor of activated T cells c1 (NFATc1). The upstream I?B degradation as well as ERK and JNK phosphorylation were also substantially repressed. Transfection with siRNA targeting tumor necrosis factor receptor associated factor 6 (TRAF6) clearly restrained RANKL-induced MNCs formation and NFATc1 induction. Interesting, RANKL triggered poly-ubiquitination of TRAF6 and associated TRAF6-TAK1 (transforming growth factor ?-activated kinase 1) complex formation was prominently attenuated by (+)-vitisin A. Furthermore, the interaction between c-src tyrosine kinase (c-Src) and ?3 was markedly induced by RANKL stimulation. (+)-Vitisin A significantly attenuated this interaction when concomitant treated with RANKL in RAW264.7 cells, but failed to affect c-Src/?3 complex formation when post-cultured with MNCs. Taken together, (+)-vitisin A suppressed bone resorption possibly via interruption of RANKL-induced TRAF6 ubiquitination and associated downstream signaling pathways. Furthermore, action through negative regulation of the proteolytic activity of MMP-9 and cathepsin K might also contribute to the anti-resorption effect of (+)-vitisin A.

Dataset Information

Serum calcium-decreasing factor, caldecrin, inhibits osteoclast differentiation by suppression of NFATc1 activity.

Publications

Serum calcium-decreasing factor, caldecrin, inhibits osteoclast differentiation by suppression of NFATc1 activity.

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