BackgroundZinc, an essential trace element, inhibits osteoclast differentiation in vitro and in vivo. The molecular mechanism for the inhibitory effect of zinc, however, is poorly understood. The purpose of this study was to investigate the effect of zinc and determine its molecular mechanism on receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis in mouse bone marrow-derived monocyte cells (BMMs) and RAW264.7 cells.ResultsIn BMMs, zinc treatment during osteoclast differentiation decreased RANKL-induced osteoclast formation in a dose-dependent manner. We show that zinc suppressed the mRNA levels of nuclear factor of activated T-cells, cytoplasmic 1 (Nfatc1). Zinc also accumulated phospho-Nfatc1 (p-Nfatc1) in the cytosol in a dose-dependent manner and inhibited the translocation of Nfatc1 to the nucleus in RAW264.7 cells. Zinc suppressed the activities of Nfatc1 in the nucleus without changing the activities of NF-κB in RAW264.7 cells. In contrast, calcineurin activity decreased in response to zinc but its protein level was unchanged. RANKL-induced Ca2+ oscillations were inhibited by zinc treatment, but phospho-phospholipase Cγ1 (p-PLCγ1), the upstream signaling molecule of Ca2+ oscillations, was unaffected. Moreover, a constitutively active form of Nfatc1 obviously rescued suppression of osteoclastogenesis by zinc.ConclusionsTaken together, these results demonstrate for the first time that the inhibitory effect of zinc during osteoclastogesis is caused by suppressing the Ca2+-Calcineurin-NFATc1 signaling pathway. Thus, zinc may be a useful therapeutic candidate for the prevention of bone loss caused by NFATc1 activation in osteoclasts.

In the lifecycle of microorganisms, prolonged starvation is prevalent and sustaining life during starvation periods is a vital task. In the literature, it is commonly assumed that survival kinetics of starving microbes follows exponential decay. This assumption, however, has not been rigorously tested. Currently, it is not clear under what circumstances this assumption is true. Also, it is not known when such survival kinetics deviates from exponential decay and if it deviates, what underlying mechanisms for the deviation are. Here, to address these issues, we quantitatively characterized dynamics of survival and death of starving E. coli cells. The results show that the assumption – starving cells die exponentially – is true only at high cell density. At low density, starving cells persevere for extended periods of time, before dying rapidly exponentially. Detailed analyses show intriguing quantitative characteristics of the density-dependent and biphasic survival kinetics, including that the period of the perseverance is inversely proportional to cell density. These characteristics further lead us to identification of key underlying processes relevant for the perseverance of starving cells. Then, using mathematical modeling, we show how these processes contribute to the density-dependent and biphasic survival kinetics observed. Importantly, our model reveals a thrifty strategy employed by bacteria, by which upon sensing impending depletion of a substrate, the limiting substrate is conserved and utilized later during starvation to delay cell death. These findings advance quantitative understanding of survival of microbes in oligotrophic environments and facilitate quantitative analysis and prediction of microbial dynamics in nature. Furthermore, they prompt revision of previous models used to analyze and predict population dynamics of microbes.

Mammalian chitinase released by airway epithelia is thought to be an important mediator of disease manifestation in an experimental model of asthma. However, the intracellular signaling mechanisms engaged by exogenous chitinase in human airway epithelial cells are unknown. Here, we investigated the direct effects of exogenous chitinase from Streptomyces griseus on Ca(2+) signaling in human airway epithelial cells. Spectrofluorometry was used to measure intracellular Ca(2+) concentration ([Ca(2+)](i)) in fura-2-AM-loaded cells. S. griseus chitinase induced dose-dependent [Ca(2+)](i) increases in normal human bronchial epithelial cells and promoted [Ca(2+)](i) oscillations in H292 cells. Chitinase-induced [Ca(2+)](i) oscillations were independent of extracellular Ca(2+), suggesting that the observed [Ca(2+)](i) increases were due to Ca(2+) release from intracellular stores. Accordingly, after depleting endoplasmic reticulum (ER) Ca(2+) with the ER Ca(2+) ATPase inhibitor, thapsigargin, chitinase-mediated [Ca(2+)](i) increases were abolished. Treatment with the phospholipase C (PLC) inhibitor U73122 or the 1, 4, 5-trisinositolphosphate (IP(3)) receptor inhibitor 2-APB attenuated chitinase-induced [Ca(2+)](i) increases. Desensitization of protease-activated receptor-2 (PAR-2) by repetitive agonist stimulation or siRNA-mediated PAR-2 knock-down revealed that chitinase-mediated [Ca(2+)](i) increases were exclusively mediated by PAR-2 activation. Finally, chitinase was found to cleave a model peptide representing the cleavage site of PAR-2 and enhanced IL-8 production. These results indicate that exogenous chitinase is a potent proteolytic activator of PAR-2 that can directly induce PLC/IP(3)-dependent Ca(2+) signaling in human airway epithelial cells.

Mucus hypersecretion is a prominent feature of respiratory diseases, and MUC5B is a major airway mucin. Mucin gene expression can be affected by inflammatory mediators, including prostaglandin (PG) D 2, an inflammatory mediator synthesized by hematopoietic PGD synthase (H-PGDS). PGD 2 binds to either D-prostanoid receptor (DP1) or chemoattractant receptor homologous molecule expressed on T-helper type 2 cells (CRTH2). We investigated the mechanisms by which PGD 2 induces MUC5B gene expression in airway epithelial cells. Western blot analysis showed that H-PGDS was highly expressed in nasal polyps. Similar results were obtained for PGD 2 expression. In addition, we could clearly detect the expressions of both H-PGDS and DP1 in nasal epithelial cells but not CRTH2. We demonstrated that PGD 2 increased MUC5B gene expression in normal human nasal epithelial cells as well as in NCI-H292 cells in vitro. S5751, a DP1 antagonist, inhibited PGD 2 -induced MUC5B expression, whereas a CRTH2 antagonist (OC0459) did not. These data suggest that PGD 2 induced MUC5B expression via DP1. Pretreatment with extracellular signal-regulated kinase (ERK) inhibitor (PD98059) blocked both PGD 2 -induced ERK mitogen-activated protein kinase (MAPK) activation and MUC5B expression. Proximity ligation assays showed direct interaction between RSK1 and cAMP response element-binding protein (CREB). Stimulation with PGD 2 caused an increase in intracellular cAMP levels, whereas intracellular Ca 2؉ did not have such an effect. PGD 2 -induced MUC5B mRNA levels were regulated by CREB via direct interaction with two cAMP-response element sites (؊921/؊914 and ؊900/؊893). Finally, we demonstrated that PGD 2 can induce MUC5B overproduction via ERK MAPK/ RSK1/CREB signaling and that DP1 receptor may have suppressive effects in controlling MUC5B overproduction in the airway.Mucus secretion in the airway, including the nasal and paranasal sinus cavities, is drained by the mucociliary transport system (1). Normal mucus secretion is essential for survival (2), but upregulation of mucin gene expression is a major manifestation of chronic airway diseases such as allergic rhinitis, asthma, and cystic fibrosis (3, 4). Patients suffering from these diseases have pathological abnormalities in both the submucosal glands and surface epithelium, characterized by inflammation, increased mucus cell number, and increased airway mucus. Several classes of inflammatory mediators have been implicated in the process of mucus hypersecretion based on their ability to stimulate secretion from cultured cells and tissue explants (5). These inflammatory mediators are lipopolysaccharides (6), reactive oxygen species (7), and arachidonic acid metabolites (8, 9).A total of 20 different mucin genes have been identified and subdivided into two groups, membrane-bound and secreted mucins, according to Human Genome Mapping conventions. The secreted mucins are MUC5AC, MUC5B, MUC6, and MUC19 (10 -13). Mucins are primarily synthesized by two different cell types in the airway tract, namely...

Receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis is accompanied by intracellular Ca2+ mobilization in a form of oscillations, which plays essential roles by activating sequentially Ca2+/calmodulin-dependent protein kinase, calcineurin and NFATc1, necessary in the osteoclast differentiation. However, it is not known whether Ca2+ mobilization which is evoked in RANKL-independent way induces to differentiate into osteoclasts. In present study, we investigated Ca2+ mobilization induced by aluminum fluoride (AlF4-), a G-protein activator, with or without RANKL and the effects of AlF4- on the osteoclastogenesis in primary cultured mouse bone marrow-derived macrophages (BMMs). We show here that AlF4- induces intracellular Ca2+ concentration ([Ca2+]i) oscillations, which is dependent on extracellular Ca2+ influx. Notably, co-stimulation of AlF4- with RANKL resulted in enhanced NFATc1 expression and formation of tartrate-resistant acid phosphatase (TRAP) positive multinucleated cells. Additionally, we confirmed that mitogen-activated protein kinase (MAPK) is also activated by AlF4-. Taken together, these results demonstrate that G-protein would be a novel modulator responsible for [Ca2+]i oscillations and MAPK activation which lead to enhancement of RANKL-mediated osteoclastogenesis.