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.

Rhinacanthin C is a naphthoquinone ester with anti-inflammatory activity, found in Rhinacanthus nasutus (L) Kurz (Acanthaceae). We found that rhinacanthin C inhibited osteoclast differentiation stimulated by the receptor activator of nuclear factor-κB ligand (RANKL) in mouse bone marrow macrophage cultures, although the precise molecular mechanisms underlying this phenomenon are unclear. In this study, we investigated the inhibitory mechanisms of rhinacanthin C in osteoclastogenesis. Rhinacanthin C suppressed RANKL-induced nuclear factor of activated T cells c1 (NFATc1) expression. Phosphorylation of ERK, JNK, and NF-κB, but not p38, was inhibited by rhinacanthin C, which also inhibited RANKL-stimulated TRAF6-TAK1 complex formation. Thus, the anti-osteoclastogenic effect of rhinacanthin C is mediated by a cascade of inhibition of RANKL-induced TRAF6-TAK1 association followed by activation of MAPKs/NF-κB; this leads to suppression of c-Fos and NFATc1, which regulate transcription of genes associated with osteoclast differentiation. In vivo, rhinacanthin C also reduced RANKL-induced osteoclast formation and bone resorption in mouse calvaria. Rhinacanthin C also suppressed LPS-stimulated osteoclastogenesis and bone resorption in vitro and in vivo. Rhinacanthin C may provide a novel therapy for abnormal bone lysis that occurs during inflammatory bone resorption.

Previous studies of the neuroprotective activity of polyphenols have used ununiform culture systems, making it difficult to compare their neuroprotective potency. We have established a new and simple method for preparing differentiated PC12 cells by removing the toxic coating step. Cells were induced to differentiate with the nerve growth factor (NGF) in a serum-free medium, without a medium change, but with a one-time overlay supplementation of NGF. The optimal inoculation density of the cells was 6–12 × 10³ cells/cm², and the presence of serum inhibited the differentiation. Neuroprotective activity could be quantified by the specific index (SI) value, that is, the ratio of the 50% cytotoxic concentration to the 50% effective concentration. Alkaline extract from the leaves of Sasa senanensis Rehder (SE), having had hormetic growth stimulation, showed the highest SI value, followed by epigallocatechin gallate. The SI value of curcumin and resveratrol was much lower. This simple overly method, that can prepare massive differentiated neuronal cells, may be applicable for the study of the differentiation-associated changes in intracellular metabolites, and the interaction between neuronal cells and physiological factors.

This review article summarizes the recent progress in dental applications of natural products. Catechin gel showed selective antimicrobial activity, whereas the alkaline extract of various plant species rich in lignin carbohydrate complex (LCC) showed much higher antiviral activity than lower molecular weight polyphenols. Mouthwash with the alkaline extract of a plant classified as OTC effectively reduced halitosis. Unexpectedly, many polyphenolic compounds purified from the natural kingdom showed much lower tumor-specificity against human oral squamous cell lines as compared with antitumor agents, although they showed apoptosis-inducing activity. The alkaline extract of bamboo leaf, which exerted various common biological activities with LCC, showed osteogenic activity by stimulating differentiation toward osteoblasts while inhibiting differentiation toward osteoclasts. LCC enhanced the dectin-2 mRNA expression in macrophages, whereas glucan showed anti-osteoblastic action via dectin-1. These data suggest that natural products exert their biological activity by interacting with these molecules.

Caldecrin was originally isolated from the pancreas as a factor that reduced serum calcium levels. This secreted serine protease has chymotrypsin-like activity and is also known as chymotrypsin C; it belongs to the elastase family. Although intravenous administration of caldecrin decreases the serum calcium concentration even when its protease activity is blocked, this effect does require cleavage of caldecrin’s pro-peptide by trypsin, converting it to the mature enzyme. Ectopic intramuscular expression of caldecrin prevented bone resorption in ovariectomized mice. Caldecrin inhibited parathyroid hormone-stimulated calcium release from fetal mouse long bone organ cultures. Furthermore, caldecrin suppressed the formation of osteoclasts from bone marrow cells by inhibiting the receptor activator of nuclear factor-κ B ligand (RANKL)-stimulated phospholipase Cγ-calcium oscillation-calcineurin-nuclear factor of activated T-cells, cytoplasmic 1 pathway. Caldecrin also suppressed the bone resorption activity of mature osteoclasts by preventing RANKL-stimulated Src activation, calcium entry, and actin ring formation. In vivo and in vitro studies have indicated that caldecrin is a unique multifunctional protease with anti-osteoclastogenic activities that are distinct from its protease activity. Caldecrin might be a potential therapeutic target for the treatment of osteolytic diseases such as osteoporosis and osteoarthritis. This mini-review describes caldecrin’s historical background and its mechanisms of action.

Blood calcium homeostasis is critical for biological function. Caldecrin, or chymotrypsin-like elastase, was originally identified in the pancreas as a serum calcium-decreasing factor. The serum calcium-decreasing activity of caldecrin requires the trypsin-mediated activation of the protein. Protease activity-deficient mature caldecrin can also reduce serum calcium concentration, indicating that structural processing is necessary for serum calcium-decreasing activity. Caldecrin suppresses the differentiation of bone-resorbing osteoclasts from bone marrow macrophages (BMMs) by inhibiting receptor activator of NF-κB ligand (RANKL)-induced nuclear factor of activated T-cell cytoplasmic 1 expression via the Syk–PLCγ–Ca²⁺ oscillation-calcineurin signaling pathway. It also suppresses mature osteoclastic bone resorption by RANKL-stimulated TRAF6–c-Src–Syk–calcium entry and actin ring formation. Caldecrin inhibits lipopolysaccharide (LPS)-induced osteoclast formation in RANKL-primed BMMs by inducing the NF-κB negative regulator A20. In addition, caldecrin suppresses LPS-mediated M1 macrophage polarization through the immunoreceptor triggering receptor expressed on myeloid cells (TREM) 2, suggesting that caldecrin may function as an anti-osteoclastogenic and anti-inflammatory factor via TREM2. The ectopic intramuscular expression of caldecrin cDNA prevents bone resorption in ovariectomized mice, and the administration of caldecrin protein also prevents skeletal muscle destruction in dystrophic mice. In vivo and in vitro studies have indicated that caldecrin is a unique multifunctional protease and a possible therapeutic target for skeletal and inflammatory diseases.