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1.  Ca2+ is a Regulator of the WNK/OSR1/NKCC Pathway in a Human Salivary Gland Cell Line 
Wnk kinase maintains cell volume, regulating various transporters such as sodium-chloride cotransporter, potassium-chloride cotransporter, and sodium-potassium-chloride cotransporter 1 (NKCC1) through the phosphorylation of oxidative stress responsive kinase 1 (OSR1) and STE20/SPS1-related proline/alanine-rich kinase (SPAK). However, the activating mechanism of Wnk kinase in specific tissues and specific conditions is broadly unclear. In the present study, we used a human salivary gland (HSG) cell line as a model and showed that Ca2+ may have a role in regulating Wnk kinase in the HSG cell line. Through this study, we found that the HSG cell line expressed molecules participating in the WNK-OSR1-NKCC pathway, such as Wnk1, Wnk4, OSR1, SPAK, and NKCC1. The HSG cell line showed an intracellular Ca2+ concentration ([Ca2+]i) increase in response to hypotonic stimulation, and the response was synchronized with the phosphorylation of OSR1. Interestingly, when we inhibited the hypotonically induced [Ca2+]i increase with nonspecific Ca2+ channel blockers such as 2-aminoethoxydiphenyl borate, gadolinium, and lanthanum, the phosphorylated OSR1 level was also diminished. Moreover, a cyclopiazonic acid-induced passive [Ca2+]i elevation was evoked by the phosphorylation of OSR1, and the amount of phosphorylated OSR1 decreased when the cells were treated with BAPTA, a Ca2+ chelator. Finally, through that process, NKCC1 activity also decreased to maintain the cell volume in the HSG cell line. These results indicate that Ca2+ may regulate the WNK-OSR1 pathway and NKCC1 activity in the HSG cell line. This is the first demonstration that indicates upstream Ca2+ regulation of the WNK-OSR1 pathway in intact cells.
PMCID: PMC4422965  PMID: 25954130
Ca2+ signaling; NKCC; OSR1; Salivary gland; WNK
2.  Bacterial PAMPs and Allergens Trigger Increase in [Ca2+]i-induced Cytokine Expression in Human PDL Fibroblasts 
An oral environment is constantly exposed to environmental factors and microorganisms. The periodontal ligament (PDL) fibroblasts within this environment are subject to bacterial infection and allergic reaction. However, how these condition affect PDL fibroblasts has yet to be elucidated. PDL fibroblasts were isolated from healthy donors. We examined using reverse transcription-polymerase chain reaction and measuring the intracellular Ca2+ concentration ([Ca2+]i). This study investigated the receptors activated by exogenous bacterial pathogens (Lipopolysaccharide and peptidoglycan) and allergens (German cockroach extract and house dust mite) as well as these pathogenic mediators-induced effects on the intracellular Ca2+ signaling in human PDL fibroblasts. Moreover, we evaluated the expression of pro-inflammatory cytokines (interleukin (IL)-1β, IL-6, and IL-8) and bone remodeling mediators (receptor activator of NF-κB ligand and osteoprotegerin) and intracellular Ca2+-involved effect. Bacterial pathogens and allergic mediators induced increased expression of pro-inflammatory cytokines, and these results are dependent on intracellular Ca2+. However, bacterial pathogens and allergic mediators did not lead to increased expression of bone remodeling mediators, except lipopolysaccharide-induced effect on receptor activator of NF-κB ligand expression. These experiments provide evidence that a pathogens and allergens-induced increase in [Ca2+]i affects the inflammatory response in human PDL fibroblasts.
PMCID: PMC4422971  PMID: 25954136
Calcium signaling; Cytokines; Inflammation; Interleukins; Periodontal ligament
3.  Peptidoglycan Induces the Production of Interleukin-8 via Calcium Signaling in Human Gingival Epithelium 
The etiology of periodontal disease is multifactorial. Exogenous stimuli such as bacterial pathogens can interact with toll-like receptors to activate intracellular calcium signaling in gingival epithelium and other tissues. The triggering of calcium signaling induces the secretion of pro-inflammatory cytokines such as interleukin-8 as part of the inflammatory response; however, the exact mechanism of calcium signaling induced by bacterial toxins when gingival epithelial cells are exposed to pathogens is unclear. Here, we investigate calcium signaling induced by bacteria and expression of inflammatory cytokines in human gingival epithelial cells. We found that peptidoglycan, a constituent of gram-positive bacteria and an agonist of toll-like receptor 2, increases intracellular calcium in a concentration-dependent manner. Peptidoglycan-induced calcium signaling was abolished by treatment with blockers of phospholipase C (U73122), inositol 1,4,5-trisphosphate receptors, indicating the release of calcium from intracellular calcium stores. Peptidoglycan-mediated interleukin-8 expression was blocked by U73122 and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl ester). Moreover, interleukin-8 expression was induced by thapsigargin, a selective inhibitor of the sarco/endoplasmic reticulum calcium ATPase, when thapsigargin was treated alone or co-treated with peptidoglycan. These results suggest that the gram-positive bacterial toxin peptidoglycan induces calcium signaling via the phospholipase C/inositol 1,4,5-trisphosphate pathway, and that increased interleukin-8 expression is mediated by intracellular calcium levels in human gingival epithelial cells.
PMCID: PMC4297762  PMID: 25605997
Ca2+ signaling; Cytokine; Human oral epithelial cells; Inflammation; Toll like receptors
4.  DA-6034 Induces [Ca2+]i Increase in Epithelial Cells 
DA-6034, a eupatilin derivative of flavonoid, has shown potent effects on the protection of gastric mucosa and induced the increases in fluid and glycoprotein secretion in human and rat corneal and conjunctival cells, suggesting that it might be considered as a drug for the treatment of dry eye. However, whether DA-6034 induces Ca2+ signaling and its underlying mechanism in epithelial cells are not known. In the present study, we investigated the mechanism for actions of DA-6034 in Ca2+ signaling pathways of the epithelial cells (conjunctival and corneal cells) from human donor eyes and mouse salivary gland epithelial cells. DA-6034 activated Ca2+-activated Cl- channels (CaCCs) and increased intracellular calcium concentrations ([Ca2+]i) in primary cultured human conjunctival cells. DA-6034 also increased [Ca2+]i in mouse salivary gland cells and human corneal epithelial cells. [Ca2+]i increase of DA-6034 was dependent on the Ca2+ entry from extracellular and Ca2+ release from internal Ca2+ stores. Interestingly, these effects of DA-6034 were related to ryanodine receptors (RyRs) but not phospholipase C/inositol 1,4,5-triphosphate (IP3) pathway and lysosomal Ca2+ stores. These results suggest that DA-6034 induces Ca2+ signaling via extracellular Ca2+ entry and RyRs-sensitive Ca2+ release from internal Ca2+ stores in epithelial cells.
PMCID: PMC3994308  PMID: 24757369
Calcium signaling; DA-6034; Epithelial cells; Eupatilin
5.  Activation of G Proteins by Aluminum Fluoride Enhances RANKL-Mediated Osteoclastogenesis 
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.
PMCID: PMC3823956  PMID: 24227944
AlF4-; Ca2+ signaling; G protein; MAPK activation; Osteoclastogenesis
6.  TRPM7 Is Essential for RANKL-Induced Osteoclastogenesis 
The transient receptor potential melastatin type 7 (TRPM7) channel is a widely expressed non-selective cation channel with fusion to the C-terminal alpha kinase domain and regarded as a key regulator of whole body Mg2+ homeostasis in mammals. However, the roles of TRPM7 during osteoclastogenesis in RAW264.7 cells and bone marrow-derived monocyte/macrophage precursor cells (BMMs) are not clear. In the present study, we investigate the roles of TRPM7 in osteoclastogenesis using methods of small interfering RNA (siRNA), RT-PCR, patch-clamp, and calcium imaging. RANKL (receptor activator of NF-κB ligand) stimulation did not affect the TRPM7 expression and TRPM7-mediated current was activated in HEK293, RAW264.7, and BMM cells by the regulation of Mg2+. Knock-down of TRPM7 by siTRPM7 reduced intracellular Ca2+ concentration ([Ca2+]i) increases by 0 mM [Mg2+]e in HEK293 cells and inhibited the generation of RANKL-induced Ca2+ oscillations in RAW264.7 cells. Finally, knock-down of TRPM7 suppressed RANKL-mediated osteoclastogenesis such as activation and translocation of NFATc1, formation of multinucleated cells, and the bone resorptive activity, sequentially. These results suggest that TRPM7 plays an essential role in the RANKL-induced [Ca2+]i oscillations that triggers the late stages of osteoclastogenesis.
PMCID: PMC3579107  PMID: 23440520
Calcium signaling; Osteoclastogenesis; RANKL; TRPM7
7.  Effects of Inositol 1,4,5-triphosphate on Osteoclast Differentiation in RANKL-induced Osteoclastogenesis 
The receptor activator of NF-κB ligand (RANKL) signal is an activator of tumor necrosis factor receptor-associated factor 6 (TRAF6), which leads to the activation of NF-κB and other signal transduction pathways essential for osteoclastogenesis, such as Ca2+ signaling. However, the intracellular levels of inositol 1,4,5-trisphosphate (IP3) and IP3-mediated cellular function of RANKL during osteoclastogenesis are not known. In the present study, we determined the levels of IP3 and evaluated IP3-mediated osteoclast differentiation and osteoclast activity by RANKL treatment of mouse leukemic macrophage cells (RAW 264.7) and mouse bone marrow-derived monocyte/macrophage precursor cells (BMMs). During osteoclastogenesis, the expression levels of Ca2+ signaling proteins such as IP3 receptors (IP3Rs), plasma membrane Ca2+ ATPase, and sarco/endoplasmic reticulum Ca2+ ATPase type2 did not change by RANKL treatment for up to 6 days in both cell types. At 24 h after RANKL treatment, a higher steady-state level of IP3 was observed in RAW264.7 cells transfected with green fluorescent protein (GFP)-tagged pleckstrin homology (PH) domains of phospholipase C (PLC) δ, a probe specifically detecting intracellular IP3 levels. In BMMs, the inhibition of PLC with U73122 [a specific inhibitor of phospholipase C (PLC)] and of IP3Rs with 2-aminoethoxydiphenyl borate (2APB; a non-specific inhibitor of IP3Rs) inhibited the generation of RANKL-induced multinucleated cells and decreased the bone-resorption rate in dentin slice, respectively. These results suggest that intracellular IP3 levels and the IP3-mediated signaling pathway play an important role in RANKL-induced osteoclastogenesis.
PMCID: PMC3298823  PMID: 22416217
Inositol 1,4,5-trisphosphate; RANKL; Osteoclastogenesis; Ca2+ signaling
8.  Role of Regulators of G-Protein Signaling 4 in Ca2+ Signaling in Mouse Pancreatic Acinar Cells 
Regulators of G-protein signaling (RGS) proteins are regulators of Ca2+ signaling that accelerate the GTPase activity of the G-protein α-subunit. RGS1, RGS2, RGS4, and RGS16 are expressed in the pancreas, and RGS2 regulates G-protein coupled receptor (GPCR)-induced Ca2+ oscillations. However, the role of RGS4 in Ca2+ signaling in pancreatic acinar cells is unknown. In this study, we investigated the mechanism of GPCR-induced Ca2+ signaling in pancreatic acinar cells derived from RGS4-/- mice. RGS4-/- acinar cells showed an enhanced stimulus intensity response to a muscarinic receptor agonist in pancreatic acinar cells. Moreover, deletion of RGS4 increased the frequency of Ca2+ oscillations. RGS4-/- cells also showed increased expression of sarco/endoplasmic reticulum Ca2+ ATPase type 2. However, there were no significant alterations, such as Ca2+ signaling in treated high dose of agonist and its related amylase secretion activity, in acinar cells from RGS4-/- mice. These results indicate that RGS4 protein regulates Ca2+ signaling in mouse pancreatic acinar cells.
PMCID: PMC3282226  PMID: 22359476
RGS4; Ca2+ signaling; Pancreatic acinar cells
9.  Deletion of TRPC3 in mice reduces Store-Operated Ca2+ influx and the severity of acute pancreatitis 
Gastroenterology  2009;137(4):1509-1517.
Background and Aims
Receptor–stimulated Ca2+ influx is a critical component of the Ca2+ signal and mediates all cellular functions regulated by Ca2+. However, excessive Ca2+ influx is highly toxic resulting in cell death, which is the nodal point in all forms of pancreatitis. Ca2+ influx is mediated by store-operated channels (SOCs). The identity and function of the native SOCs in most cells is unknown.
Here, we determine the role of deletion of Trpc3 in mice on Ca2+ signaling, exocytosis, intracellular trypsin activation and pancreatitis.
Deletion of TRPC3 reduced the receptor-stimulated and SOCs-mediated Ca2+ influx by about 50%, indicating that TRPC3 functions as SOC in vivo. The reduced Ca2+ influx in TRPC3−/− acini resulted in reduced frequency of the physiological Ca2+ oscillations and of the pathological sustained [Ca2+]i increase caused by supramaximal stimulation and by the toxins bile acids and palmitoleic acid ethyl ester. Consequently, deletion of TRPC3 shifted the dose response for receptor-stimulated exocytosis, and prevented the pathological inhibition of digestive enzyme secretion at supramaximal agonist concentrations. Accordingly, deletion of TRPC3 markedly reduced intracellular trypsin activation and excessive actin depolymerization in vitro and the severity of pancreatitis in vivo.
These findings establish the native TRPC3 as a SOC in vivo and a role for TRPC3-mediated Ca2+ influx in the pathogenesis of acute pancreatitis and suggest that TRPC3 should be considered a target for prevention of the pancreatic damage in acute pancreatitis.
PMCID: PMC2757493  PMID: 19622358
10.  Loss of miR-200c up-regulates CYP1B1 and confers docetaxel resistance in renal cell carcinoma 
Oncotarget  2015;6(10):7774-7787.
Despite high protein expression and enzymatic activity of cytochrome P450 1B1 (CYP1B1) in renal cell cancer (RCC), its functional significance has not been elucidated. Here we explored the functional role and regulatory mechanism of CYP1B1 in RCC. Reduction of CYP1B1 levels fail to prevent in vitro tumorigenicity such as proliferation, apoptosis, and cell cycle progression of RCC cells. Moreover, the expression levels are not associated with tumor type, stage, Fuhrman grade and 5-year survival probability after surgery. Instead, alteration of CYP1B1 expression regulates the chemosensitivity of RCC cells to docetaxel suggesting its critical contribution to the chemoresistance. Additionally, miR-200c, which is significantly down-regulated in RCC regulates CYP1B1 expression and activity. An inverse association was also observed between the expression levels of miR-200c and CYP1B1 protein in RCC tissues. Finally, alteration of miR-200c levels affects the chemosensitivity of RCC cells. Restoration of docetaxel resistance by exogenous expression of CYP1B1 in miR-200c-over-expressing cells indicates that CYP1B1 is a functional target of miR-200c. These results suggest that CYP1B1 up-regulation mediated by low miR-200c is one of the mechanisms underlying resistance of RCC cells to docetaxel. Therefore, expression of CYP1B1 and miR-200c in RCC may be useful as a prediction for docetaxel response.
PMCID: PMC4480715  PMID: 25860934
Renal cell carcinoma; CYP1B1; miR-200c; chemotherapy; docetaxel
11.  Alteration of Expression of Ca2+ Signaling Proteins and Adaptation of Ca2+ Signaling in SERCA2+/- Mouse Parotid Acini 
Yonsei Medical Journal  2008;49(2):311-321.
The sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), encoded by ATP2A2, is an essential component for G-protein coupled receptor (GPCR)-dependent Ca2+ signaling. However, whether the changes in Ca2+ signaling and Ca2+ signaling proteins in parotid acinar cells are affected by a partial loss of SERCA2 are not known.
Materials and Methods
In SERCA2+/- mouse parotid gland acinar cells, Ca2+ signaling, expression levels of Ca2+ signaling proteins, and amylase secretion were investigated.
SERCA2+/- mice showed decreased SERCA2 expression and an upregulation of the plasma membrane Ca2+ ATPase. A partial loss of SERCA2 changed the expression level of 1, 4, 5-tris-inositolphosphate receptors (IP3Rs), but the localization and activities of IP3Rs were not altered. In SERCA2+/- mice, muscarinic stimulation resulted in greater amylase release, and the expression of synaptotagmin was increased compared to wild type mice.
These results suggest that a partial loss of SERCA2 affects the expression and activity of Ca2+ signaling proteins in the parotid gland acini, however, overall Ca2+ signaling is unchanged.
PMCID: PMC2615323  PMID: 18452270
sarco/endoplasmic reticulum Ca2+-ATPase; Ca2+ signaling proteins; parotid gland acinar cells
12.  Initiation Site of Ca2+ Entry Evoked by Endoplasmic Reticulum Ca2+ Depletion in Mouse Parotid and Pancreatic Acinar Cells 
Yonsei Medical Journal  2007;48(3):526-530.
In non-excitable cells, which include parotid and pancreatic acinar cells, Ca2+ entry is triggered via a mechanism known as capacitative Ca2+ entry, or store-operated Ca2+ entry. This process is initiated by the perception of the filling state of endoplasmic reticulum (ER) and the depletion of internal Ca2+ stores, which acts as an important factor triggering Ca2+ entry. However, both the mechanism of store-mediated Ca2+ entry and the molecular identity of store-operated Ca2+ channel (SOCC) remain uncertain.
Materials and Methods
In the present study we investigated the Ca2+ entry initiation site evoked by depletion of ER to identify the localization of SOCC in mouse parotid and pancreatic acinar cells with microfluorometeric imaging system.
Treatment with thapsigargin (Tg), an inhibitor of sarco/ endoplasmic reticulum Ca2+-ATPase, in an extracellular Ca2+ free state, and subsequent exposure to a high external calcium state evoked Ca2+ entry, while treatment with lanthanum, a non-specific blocker of plasma Ca2+ channel, completely blocked Tg-induced Ca2+ entry. Microfluorometric imaging showed that Tg-induced Ca2+ entry started at a basal membrane, not a apical membrane.
These results suggest that Ca2+ entry by depletion of the ER initiates at the basal pole in polarized exocrine cells and may help to characterize the nature of SOCC.
PMCID: PMC2628100  PMID: 17594163
Parotid; Ca2+ signaling; store-operated calcium channel
13.  Small heterodimer partner interacts with NLRP3 and negatively regulates activation of the NLRP3 inflammasome 
Nature Communications  2015;6:6115.
Excessive activation of the NLRP3 inflammasome results in damaging inflammation, yet the regulators of this process remain poorly defined. Herein, we show that the orphan nuclear receptor small heterodimer partner (SHP) is a negative regulator of NLRP3 inflammasome activation. NLRP3 inflammasome activation leads to an interaction between SHP and NLRP3, proteins that are both recruited to mitochondria. Overexpression of SHP competitively inhibits binding of NLRP3 to apoptosis-associated speck-like protein containing a CARD (ASC). SHP deficiency results in increased secretion of proinflammatory cytokines IL-1β and IL-18, and excessive pathologic responses typically observed in mouse models of kidney tubular necrosis and peritoneal gout. Notably, the loss of SHP results in accumulation of damaged mitochondria and a sustained interaction between NLRP3 and ASC in the endoplasmic reticulum. These data are suggestive of a role for SHP in controlling NLRP3 inflammasome activation through a mechanism involving interaction with NLRP3 and maintenance of mitochondrial homeostasis.
Excessive NLRP3 inflammasome activation underlies inflammatory diseases such as gout. Here the authors show that orphan nuclear receptor small heterodimer partner protein (SHP) negatively regulates NLRP3, and its loss leads to accumulation of damaged mitochondria and gout-like immunopathology.
PMCID: PMC4347017  PMID: 25655831
14.  Expression of Ca2+-dependent Synaptotagmin Isoforms in Mouse and Rat Parotid Acinar Cells 
Yonsei Medical Journal  2006;47(1):70-77.
Synaptotagmin is a Ca2+ sensing protein, which triggers a fusion of synaptic vesicles in neuronal transmission. Little is known regarding the expression of Ca2+-dependent synaptotagmin isoforms and their contribution to the release of secretory vesicles in mouse and rat parotid acinar cells. We investigated a type of Ca2+-dependent synaptotagmin and Ca2+ signaling in both rat and mouse parotid acinar cells using RT-PCR, microfluorometry, and amylase assay. Mouse parotid acinar cells exhibited much more sensitive amylase release in response to muscarinic stimulation than did rat parotid acinar cells. However, transient [Ca2+]i increases and Ca2+ influx in response to muscarinic stimulation in both cells were identical, suggesting that the expression or activity of the Ca2+ sensing proteins is different. Seven Ca2+-dependent synaptotagmins, from 1 to 7, were expressed in the mouse parotid acinar cells. However, in the rat parotid acinar cells, only synaptotagmins 1, 3, 4 and 7 were expressed. These results indicate that the expression of Ca2+-dependent synaptotagmins may contribute to the release of secretory vesicles in parotid acinar cells.
PMCID: PMC2687583  PMID: 16502487
Synaptotagmin; calcium signaling; exocytosis; parotid acinar cells
15.  Zinc inhibits osteoclast differentiation by suppression of Ca2+-Calcineurin-NFATc1 signaling pathway 
Zinc, 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.
In 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.
Taken 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.
PMCID: PMC3851046  PMID: 24088289
Zinc; Bone loss; Osteoclast; NFATc1; Calcineurin; Ca2+ oscillation
16.  Vitamin D Is Required for IFN-γ–Mediated Antimicrobial Activity of Human Macrophages 
Science Translational Medicine  2011;3(104):104ra102.
Control of tuberculosis worldwide depends on our understanding of human immune mechanisms, which combat the infection. Acquired T cell responses are critical for host defense against microbial pathogens, yet the mechanisms by which they act in humans remain unclear. We report that T cells, by the release of interferon-γ (IFN-γ), induce autophagy, phagosomal maturation, the production of antimicrobial peptides such as cathelicidin, and antimicrobial activity against Mycobacterium tuberculosis in human macrophages via a vitamin D–dependent pathway. IFN-γ induced the antimicrobial pathway in human macrophages cultured in vitamin D–sufficient sera, but not in sera from African-Americans that have lower amounts of vitamin D and who are more susceptible to tuberculosis. In vitro supplementation of vitamin D–deficient serum with 25-hydroxyvitamin D3 restored IFN-γ–induced antimicrobial peptide expression, autophagy, phagosome-lysosome fusion, and antimicrobial activity. These results suggest a mechanism in which vitamin D is required for acquired immunity to overcome the ability of intracellular pathogens to evade macrophage-mediated antimicrobial responses. The present findings underscore the importance of adequate amounts of vitamin D in all human populations for sustaining both innate and acquired immunity against infection.
PMCID: PMC3269210  PMID: 21998409
17.  Protease-Activated Receptor 2 Mediates Mucus Secretion in the Airway Submucosal Gland 
PLoS ONE  2012;7(8):e43188.
Protease-activated receptor 2 (PAR2), a G protein-coupled receptor expressed in airway epithelia and smooth muscle, plays an important role in airway inflammation. In this study, we demonstrated that activation of PAR2 induces mucus secretion from the human airway gland and examined the underlying mechanism using the porcine and murine airway glands. The mucosa with underlying submucosal glands were dissected from the cartilage of tissues, pinned with the mucosal side up at the gas/bath solution interface of a physiological chamber, and covered with oil so that secretions from individual glands could be visualized as spherical bubbles in the oil. Secretion rates were determined by optical monitoring of the bubble diameter. The Ca2+-sensitive dye Fura2-AM was used to determine intracellular Ca2+ concentration ([Ca2+]i) by means of spectrofluorometry. Stimulation of human tracheal mucosa with PAR2-activating peptide (PAR2-AP) elevated intracellular Ca2+ and induced glandular secretion equal to approximately 30% of the carbachol response in the human airway. Porcine gland tissue was more sensitive to PAR2-AP, and this response was dependent on Ca2+ and anion secretion. When the mouse trachea were exposed to PAR2-AP, large amounts of secretion were observed in both wild type and ΔF508 cystic fibrosis transmembrane conductance regulator mutant mice but there is no secretion from PAR-2 knock out mice. In conclusion, PAR2-AP is an agonist for mucus secretion from the airway gland that is Ca2+-dependent and cystic fibrosis transmembrane conductance regulator-independent.
PMCID: PMC3419645  PMID: 22916223
18.  Genetic and Pharmacological Inhibition of the Ca2+ Influx Channel TRPC3 Protects Secretory Epithelia from Ca2+-Dependent Toxicity 
Gastroenterology  2011;140(7):2107-2115.e4.
Background & Aims
Excessive Ca2+ influx mediates many cytotoxic processes, including those associated with autoimmune inflammatory diseases such as acute pancreatitis and Sjögren's syndrome. TRPC3 is a major Ca2+ influx channel in pancreatic and salivary gland cells. We investigated whether genetic or pharmacological inhibition of TRPC3 protects pancreas and salivary glands from Ca2+-dependent damage.
We developed a Ca2+-dependent model of cell damage for salivary gland acini. Acute pancreatitis was induced by injection of cerulein into wild-type and Trpc3−/− mice. Mice were also given the Trpc3-selective inhibitor pyrazole 3 (Pyr3).
Salivary glands and pancreas of Trpc3−/− mice were protected from Ca2+-mediated cell toxicity. Analysis of Ca2+ signaling in wild-type and Trpc3−/− acini showed that Pyr3 is highly specific inhibitor of Tprc3; it protected salivary glands and pancreas cells from Ca2+-mediated toxicity by inhibiting the Trpc3-mediated component of Ca2+ influx.
TRPC3-mediated Ca2+ influx mediates damage to pancreas and salivary glands. Pharmacological inhibition of TRPC3 with the highly selective TRPC3 inhibitor Pyr3 might be developed for treatment of patients with acute pancreatitis and Sjögren's syndrome.
PMCID: PMC3109139  PMID: 21354153
Ca2+ influx; inflammation; cell death; therapeutics
19.  The Role of NLR-related Protein 3 Inflammasome in Host Defense and Inflammatory Diseases 
Among a number of innate receptors, the nucleotide-binding domain leucine-rich repeat containing (NLR) nucleotide oligomerization domain (NOD)-like receptor families are involved in the recognition of cytosolic pathogen- or danger-associated molecules. Activation of these specific sets of receptors leads to the assembly of a multiprotein complex, the inflammasome, leading to the activation of caspase-1 and maturation of the cytokines interleukin (IL)-1β, IL-18, and IL-33. Among NLRs, NLR-related protein 3 (NLRP3) is one of the best-characterized receptors that activates the inflammasome. There is no doubt that NLRP3 inflammasome activation is important for host defense and effective pathogen clearance against fungal, bacterial, and viral infection. In addition, mounting evidence indicates that the NLRP3 inflammasome plays a role in a variety of inflammatory diseases, including gout, atherosclerosis, and type II diabetes, as well as under conditions of cellular stress or injury. Here, we review recent advances in our understanding of the role of the NLRP3 inflammasome in host defense and various inflammatory diseases.
PMCID: PMC3321399  PMID: 22500248
Inflammasomes; Defense mechanisms; Inflammation
20.  Polarized but differential localization and recruitment of STIM1/Orai1 and STIM1/TRPC channels in secretory cells 
Traffic (Copenhagen, Denmark)  2010;12(2):232-245.
Polarized Ca2+ signals in secretory epithelial cells are determined by compartmentalized localization of Ca2+ signaling proteins at the apical pole. Recently the ER Ca2+ sensor STIM1 and the Orai channels were shown to play a critical role in store-dependent Ca2+ influx. STIM1 also gates the TRPC channels. Here, we asked how cell stimulation affects the localization, recruitment and function of the native proteins in polarized cells. Inhibition of Orai1, STIM1, or deletion of TRPC1 reduces Ca2+ influx and frequency of Ca2+ oscillations. Orai1 localization is restricted to the apical pole of the lateral membrane. Surprisingly, cell stimulation does not lead to robust clustering of native Orai1, as is observed with expressed Orai1. Unexpectedly, cell stimulation causes polarized recruitment of native STIM1 to both the apical and lateral regions, thus to regions with and without Orai1. Accordingly, STIM1 and Orai1 show only 40% co-localization. Consequently, STIM1 shows higher co-localization with the basolateral membrane marker E-cadherin than does Orai1, while Orai1 showed higher co-localization with the tight junction protein ZO1. TRPC1 is expressed in both apical and basolateral regions of the plasma membrane. Co-IP of STIM1/Orai1/IP3Rs/TRPCs is enhanced by cell stimulation and disrupted by 2APB. The polarized localization and recruitment of these proteins results in preferred Ca2+ entry that is initiated at the apical pole. These findings reveal that in addition to Orai1, STIM1 likely regulates other Ca2+ permeable channels, such as the TRPCs. Both channels contribute to the frequency of [Ca2+] oscillations and thus impact critical cellular functions.
PMCID: PMC3021582  PMID: 21054717
STIM1; Orai1; TRPC1; polarized; recruitment; epithelial cells
21.  Mycobacterial signaling through toll-like receptors 
Studies over the past decade have helped to decipher molecular networks dependent on Toll-like receptor (TLR) signaling, in mycobacteria-infected macrophages. Stimulation of TLRs by mycobacteria and their antigenic components rapidly induces intracellular signaling cascades involved in the activation of nuclear factor-κB and mitogen-activated protein kinase pathways, which play important roles in orchestrating proinflammatory responses and innate defense through generation of a variety of antimicrobial effector molecules. Recent studies have provided evidence that mycobacterial TLR-signaling cross talks with other intracellular antimicrobial innate pathways, the autophagy process and functional vitamin D receptor (VDR) signaling. In this article we describe recent advances in the recognition, responses, and regulation of mycobacterial signaling through TLRs.
PMCID: PMC3504976  PMID: 23189273
mycobacteria; vitamin D; autophagy; antimicrobial peptides; innate immunity
22.  Antimicrobial Peptides in Innate Immunity against Mycobacteria 
Immune Network  2011;11(5):245-252.
Antimicrobial peptides/proteins are ancient and naturallyoccurring antibiotics in innate immune responses in a variety of organisms. Additionally, these peptides have been recognized as important signaling molecules in regulation of both innate and adaptive immunity. During mycobacterial infection, antimicrobial peptides including cathelicidin, defensin, and hepcidin have antimicrobial activities against mycobacteria, making them promising candidates for future drug development. Additionally, antimicrobial peptides act as immunomodulators in infectious and inflammatory conditions. Multiple crucial functions of cathelicidins in antimycobacterial immune defense have been characterized not only in terms of direct killing of mycobacteria but also as innate immune regulators, i.e., in secretion of cytokines and chemokines, and mediating autophagy activation. Defensin families are also important during mycobacterial infection and contribute to antimycobacterial defense and inhibition of mycobacterial growth both in vitro and in vivo. Hepcidin, although its role in mycobacterial infection has not yet been characterized, exerts antimycobacterial effects in activated macrophages. The present review focuses on recent efforts to elucidate the roles of host defense peptides in innate immunity to mycobacteria.
PMCID: PMC3242998  PMID: 22194707
Antimicrobial peptides; Innate Immunity; Mycobacteria
23.  A Comparison of 2-Octyl Cyanoacrylate Adhesives versus Conventional Suture Materials for Eyelid Wound Closure in Rabbits 
To evaluate the clinical efficacy and histopathological tolerance of 2-octyl cyanoacrylate versus conventional suture materials for eyelid wound closure in rabbits.
We performed an experimental study on 16 eyes of eight New Zealand albino rabbits. Eyelid incisions of 15 mm were done 4mm from the upper eyelid margin in both eyes. The eyes of the rabbits were divided into two groups: eyelid incisions of the right eye were closed by a 2-octyl cyanoacrylate adhesive (group A) and eyelid incisions of the left eye were closed by 7-0 nylon sutures (group B). At 1, 2, 4, and 8 weeks after surgery, the rabbits were macroscopically examined and then sacrificed. The specimens of their eyelid tissues were stained by a hematoxylin and eosin stain and Masson-trichrome stain, and were observed under microscope.
Both eyelid surgical closure methods were found to be equally efficacious in fixing the eyelids of groups A and B, and their clinical efficacy was similar. Histopathological findings of the hematoxylin and eosin stain of group A showed less inflammatory infiltration than group B at 2 weeks. There were no significant histopathological differences between the two groups at 1, 4, and 8 weeks. The degree of fibrosis of the Masson-trichrome stain was similar between the two groups at 8 weeks.
The 2-octyl cyanoacrylate adhesive proved to be an effective eyelid closure method and was very well tolerated by the skin surface. 2-Octyl cyanoacrylate could be used as an alternative tissue adhesive for eyelid wound closure along with conventional suture materials.
PMCID: PMC3060389  PMID: 21461225
Eyelid wound closure; Octyl 2-cyanoacrylate
24.  Mycobacterium tuberculosis Eis Regulates Autophagy, Inflammation, and Cell Death through Redox-dependent Signaling 
PLoS Pathogens  2010;6(12):e1001230.
The “enhanced intracellular survival” (eis) gene of Mycobacterium tuberculosis (Mtb) is involved in the intracellular survival of M. smegmatis. However, its exact effects on host cell function remain elusive. We herein report that Mtb Eis plays essential roles in modulating macrophage autophagy, inflammatory responses, and cell death via a reactive oxygen species (ROS)-dependent pathway. Macrophages infected with an Mtb eis-deletion mutant H37Rv (Mtb-Δeis) displayed markedly increased accumulation of massive autophagic vacuoles and formation of autophagosomes in vitro and in vivo. Infection of macrophages with Mtb-Δeis increased the production of tumor necrosis factor-α and interleukin-6 over the levels produced by infection with wild-type or complemented strains. Elevated ROS generation in macrophages infected with Mtb-Δeis (for which NADPH oxidase and mitochondria were largely responsible) rendered the cells highly sensitive to autophagy activation and cytokine production. Despite considerable activation of autophagy and proinflammatory responses, macrophages infected with Mtb-Δeis underwent caspase-independent cell death. This cell death was significantly inhibited by blockade of autophagy and c-Jun N-terminal kinase-ROS signaling, suggesting that excessive autophagy and oxidative stress are detrimental to cell survival. Finally, artificial over-expression of Eis or pretreatment with recombinant Eis abrogated production of both ROS and proinflammatory cytokines, which depends on the N-acetyltransferase domain of the Eis protein. Collectively, these data indicate that Mtb Eis suppresses host innate immune defenses by modulating autophagy, inflammation, and cell death in a redox-dependent manner.
Author Summary
Tuberculosis is a global health problem: at least one-third of the world's population is infected with Mycobacterium tuberculosis (Mtb). Mtb is a successful pathogen that enhances its own intracellular survival by arresting phagolysosomal fusion. Recently, autophagy has emerged as a host defense strategy against Mtb infection, through stimulation of the fusion of phagosomes and lysosomes. However, excessive and uncontrolled autophagic activity can be detrimental to host cells and can result in their death. The Mtb “enhanced intracellular survival” (eis) gene has been implicated in the intracellular survival of M. smegmatis. However, its exact role and how it regulates host innate immune responses have not been fully explained. Here, we provide evidence that Eis suppresses macrophage autophagy, inflammation, and cell death through the inhibition of reactive oxygen species (ROS) generation. Although it has previously been demonstrated that autophagy is a key host defense response to mycobacterial infections, our data indicate that excessive autophagy, and the resulting cell death, do not significantly affect host defense responses to mycobacteria. Additionally, our data reveal that Eis's ability to regulate ROS generation and proinflammatory responses depends on its N-acetyltransferase domain. These results underscore a previously unrecognized role of Eis in modulating host inflammatory responses, oxidative stress, and cell survival/death during mycobacterial infection.
PMCID: PMC3002989  PMID: 21187903
25.  Mycobacterial lipoprotein activates autophagy via TLR2/1/CD14 and a functional vitamin D receptor signalling 
Cellular microbiology  2010;12(11):1648-1665.
In human monocytes, Toll-like receptor (TLR) 2/1 activation leads to vitamin D3-dependent antimycobacterial activities, but the molecular mechanisms by which TLR2/1 stimulation induces antimicrobial activities against mycobacteria remain unclear. Here we show that TLR2/1/CD14 stimulation by mycobacterial lipoprotein LpqH can robustly activate antibacterial autophagy through vitamin D receptor signalling activation and cathelicidin induction. We found that CCAAT/enhancer-binding protein (C/EBP)-β-dependent induction of 25-hydroxycholecalciferol-1α-hydroxylase (Cyp27b1) hydroxylase was critical for LpqH-induced cathelicidin expression and autophagy. In addition, increases in intracellular calcium following AMP-activated protein kinase (AMPK) activation played a crucial role in LpqH-induced autophagy. Moreover, AMPK-dependent p38 mitogen-activated protein kinase (MAPK) activation was required for LpqH-induced Cyp27b1 expression and autophagy activation. Collectively, these data suggest that TLR2/1/CD14-Ca2+-AMPK-p38 MAPK pathways contribute to C/EBP-β-dependent expression of Cyp27b1 and cathelicidin, which played an essential role in LpqH-induced autophagy. Furthermore, these results establish a previously uncharacterized signalling pathway of antimycobacterial host defence through a functional link of TLR2/1/CD14-dependent sensing to the induction of autophagy.
PMCID: PMC2970753  PMID: 20560977

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