CD38/ADP-ribose/TRPM2-mediated nuclear Ca2+ signaling is essential for hepatic gluconeogenesis in fasting and diabetes.

Hepatic glucose production by glucagon is crucial for glucose homeostasis during fasting, yet the underlying mechanisms remain incompletely delineated. Although CD38 has been detected in the nucleus, its function in this compartment is unknown. Here, we demonstrate that nuclear CD38 (nCD38) controls glucagon-induced gluconeogenesis in primary hepatocytes and liver in a manner distinct from CD38 occurring in the cytoplasm and lysosomal compartments. We found that the localization of CD38 in the nucleus is required for glucose production by glucagon and that nCD38 activation requires NAD+ supplied by PKCδ-phosphorylated connexin 43. In fasting and diabetes, nCD38 promotes sustained Ca2+ signals via transient receptor potential melastatin 2 (TRPM2) activation by ADP-ribose, which enhances the transcription of glucose-6 phosphatase and phosphoenolpyruvate carboxykinase 1. These findings shed light on the role of nCD38 in glucagon-induced gluconeogenesis and provide insight into nuclear Ca2+ signals that mediate the transcription of key genes in gluconeogenesis under physiological conditions.

NAD+-boosting molecules suppress mast cell degranulation and anaphylactic responses in mice.

Nicotinamide adenine dinucleotide (NAD+) acts as a cofactor for multiple biological processes. While previous research has revealed that the NAD+ declines associated with aging contributes to an impairment of immune cells, its role in mast cell function, especially in response to an anaphylactic condition, has remained unexplored. We tested whether the restoration of cellular NAD+ concentration by the supplementation of NAD+ boosting molecules prevented mast cell degranulation and anaphylactic responses. Methods: Bone marrow derived mast cells (BMMCs) and human cord blood derived mast cells were treated with NAD+ precursors nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR), and FcεRI downstream signaling was assessed. Animal models of passive systemic anaphylaxis (PSA) and passive cutaneous anaphylaxis (PCA) were used to investigate the effects of NAD+ precursors in the anaphylactic responses of mice. Results: Treatment of murine BMMCs and human cord blood derived mast cells with NAD+ precursors repressed intracellular signaling downstream of FcεRI, as well as the release of inflammatory cytokines and lipid mediators. The intraperitoneal administration of NMN or NR also markedly attenuated IgE-mediated anaphylactic responses in mouse models of PSA and PCA. These beneficial effects of NAD+ precursors, however, were attenuated in mast cell-specific Sirt6 knockout mice, indicating a Sirt6 dependency for their action. Conclusion: NAD+ precursors may serve as an effective therapeutic strategy that limits mast cell-mediated anaphylactic responses.

PERK activation by SB202190 ameliorates amyloidogenesis via the TFEB-induced autophagy-lysosomal pathway.

The protein kinase R (PKR)-like endoplasmic reticulum (ER) kinase (PERK), a key ER stress sensor of the unfolded protein response (UPR), can confer beneficial effects by facilitating the removal of cytosolic aggregates through the autophagy-lysosome pathway (ALP). In neurodegenerative diseases, the ALP ameliorates the accumulation of intracellular protein aggregates in the brain. Transcription factor-EB (TFEB), a master regulator of the ALP, positively regulates key genes involved in the cellular degradative pathway. However, in neurons, the role of PERK activation in mitigating amyloidogenesis by ALP remains unclear. In this study, we found that SB202190 selectively activates PERK independently of its inhibition of p38 mitogen-activated protein kinase, but not inositol-requiring transmembrane kinase/endoribonuclease-1α (IRE1α) or activating transcription factor 6 (ATF6), in human neuroblastoma cells. PERK activation by SB202190 was dependent on mitochondrial ROS production and promoted Ca2+-calcineurin activation. The activation of the PERK-Ca2+-calcineurin axis by SB202190 positively affects TFEB activity to increase ALP in neuroblastoma cells. Collectively, our study reveals a novel physiological mechanism underlying ALP activation, dependent on PERK activation, for ameliorating amyloidogenesis in neurodegenerative diseases.

Sirtuin 6 is a negative regulator of FcεRI signaling and anaphylactic responses.

BACKGROUND: Binding IgE to a cognate allergen causes aggregation of Fcε receptor I (FcεRI) in mast cells, resulting in activation of receptor-associated Src family tyrosine kinases, including Lyn and Syk. Protein tyrosine phosphatase, receptor type C (PTPRC), also known as CD45, has emerged as a positive regulator of FcεRI signaling by dephosphorylation of the inhibitory tyrosine of Lyn. OBJECTIVE: Sirtuin 6 (Sirt6), a NAD+-dependent deacetylase, exhibits an anti-inflammatory property. It remains to be determined, however, whether Sirt6 attenuates mast cell-associated diseases, including anaphylaxis. METHODS: FcεRI signaling and mast cell degranulation were measured after IgE cross-linking in murine bone marrow-derived mast cells (BMMCs) and human cord blood-derived mast cells. To investigate the function of Sirt6 in mast cell activation in vivo, we used mast cell-dependent animal models of passive systemic anaphylaxis (PSA) and passive cutaneous anaphylaxis (PCA). RESULTS: Sirt6-deficient BMMCs augmented IgE-FcεRI-mediated signaling and degranulation compared to wild-type BMMCs. Reconstitution of mast cell-deficient KitW-sh/W-sh mice with BMMCs received from Sirt6 knockout mice developed more severe PSA and PCA compared to mice engrafted with wild-type BMMCs. Similarly, genetic overexpression or pharmacologic activation of Sirt6 suppressed mast cell degranulation and blunted responses to PCA. Mechanistically, Sirt6 deficiency increased PTPRC transcription via acetylating histone H3, leading to enhanced aggregation of FcεRI in BMMCs. Finally, we recapitulated the Sirt6 regulation of PTPRC and FcεRI signaling in human mast cells. CONCLUSIONS: Sirt6 acts as a negative regulator of FcεRI signaling cascade in mast cells by suppressing PTPRC transcription. Activation of Sirt6 may therefore represent a promising and novel therapeutic strategy for anaphylaxis.

Reactive Oxygen Species-Mediated Autophagy by Ursolic Acid Inhibits Growth and Metastasis of Esophageal Cancer Cells.

Ursolic acid (UA) possesses various pharmacological activities, such as antitumorigenic and anti-inflammatory effects. In the present study, we investigated the mechanisms underlying the effects of UA against esophageal squamous cell carcinoma (ESCC) (TE-8 cells and TE-12 cells). The cell viability assay showed that UA decreased the viability of ESCC in a dose-dependent manner. In the soft agar colony formation assay, the colony numbers and size were reduced in a dose-dependent manner after UA treatment. UA caused the accumulation of vacuoles and LC3 puncta, a marker of autophagosome, in a dose-dependent manner. Autophagy induction was confirmed by measuring the expression levels of LC3 and p62 protein in ESCC cells. UA increased LC3-II protein levels and decreased p62 levels in ESCC cells. When autophagy was hampered using 3-methyladenine (3-MA), the effect of UA on cell viability was reversed. UA also significantly inhibited protein kinase B (Akt) activation and increased p-Akt expression in a dose-dependent manner in ESCC cells. Accumulated LC3 puncta by UA was reversed after wortmannin treatment. LC3-II protein levels were also decreased after treatment with Akt inhibitor and wortmannin. Moreover, UA treatment increased cellular reactive oxygen species (ROS) levels in ESCC in a time- and dose-dependent manner. Diphenyleneiodonium (an ROS production inhibitor) blocked the ROS and UA induced accumulation of LC3-II levels in ESCC cells, suggesting that UA-induced cell death and autophagy are mediated by ROS. Therefore, our data indicate that UA inhibits the growth of ESCC cells by inducing ROS-dependent autophagy.

Interleukin-8 drives CD38 to form NAADP from NADP+ and NAAD in the endolysosomes to mobilize Ca2+ and effect cell migration.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent Ca2+ mobilizing second messenger whose formation has remained elusive. In vitro, CD38-mediated NAADP synthesis requires an acidic pH and a nonphysiological concentration of nicotinic acid (NA). We discovered that CD38 catalyzes synthesis of NAADP by exchanging the nicotinamide moiety of nicotinamide adenine dinucleotide phosphate (NADP+ ) for the NA group of nicotinic acid adenine dinucleotide (NAAD) inside endolysosomes of interleukin 8 (IL8)-treated lymphokine-activated killer (LAK) cells. Upon IL8 stimulation, cytosolic NADP+ is transported to acidified endolysosomes via connexin 43 (Cx43) and gated by cAMP-EPAC-RAP1-PP2A signaling. CD38 then performs a base-exchange reaction with the donor NA group deriving from NAAD, produced by newly described endolysosomal activities of NA phosphoribosyltransferase (NAPRT) and NMN adenyltransferase (NMNAT) 3. Thus, the membrane organization of endolysosomal CD38, a signal-mediated transport system for NADP+ and luminal NAD+ biosynthetic enzymes integrate signals from a chemokine and cAMP to specify the spatiotemporal mobilization of Ca2+ to drive cell migration.

Cross-talk between CD38 and TTP Is Essential for Resolution of Inflammation during Microbial Sepsis.

The resolution phase of acute inflammation is essential for tissue homeostasis, yet the underlying mechanisms remain unclear. We demonstrate that resolution of inflammation involves interactions between CD38 and tristetraprolin (TTP). During the onset of acute inflammation, CD38 levels are increased, leading to the production of Ca2+-signaling messengers, nicotinic acid adenine dinucleotide phosphate (NAADP), ADP ribose (ADPR), and cyclic ADPR (cADPR) from NAD(P)+. To initiate the onset of resolution, TTP expression is increased by the second messengers, NAADP and cADPR, which downregulate CD38 expression. The activation of TTP by Sirt1-dependent deacetylation, in response to increased NAD+ levels, suppresses the acute inflammatory response and decreases Rheb expression, inhibits mTORC1, and induces autophagolysosomes for bacterial clearance. TTP may represent a mechanistic target of anti-inflammatory agents, such as carbon monoxide. TTP mediates crosstalk between acute inflammation and autophagic clearance of bacteria from damaged tissue in the resolution of inflammation during sepsis.

Anti-inflammatory effects of Artemisia scoparia and its active constituent, 3,5-dicaffeoyl-epi-quinic acid against activated mast cells.

OBJECTIVES: Artemisia scoparia Waldst. et Kit. (AS) has been used to treat inflammation, urticaria and hepatitis. However, the scientific studies of AS and its active compound for inflammatory reactions in activated human mast cell line, HMC-1 cells have not yet been elucidated. MATERIALS AND METHODS: Here, we isolated 3,5-dicaffeoyl-epi-quinic acid (DEQA) from AS butanol fraction. The anti-inflammatory effect of AS and its new active compound, DEQA was examined in HMC-1 cells by studying the following markers: phorbol 12-myristate 13-acetate and calcium ionophore A23187 (PMACI)-induced thymic stromal lymphopoietin (TSLP), tumor necrosis factor (TNF)-α, interleukin (IL)-1ß and IL-6 secretion and mRNA expression by ELISA and RT-PCR, respectively. Furthermore, mechanism related to anti-inflammatory was examined by Western blotting. RESULTS: We reported that AS and its new active compound, DEQA significantly reduced TSLP, TNF-α, IL-1ß and IL-6 production levels through the reduction of caspase-1 activity. The mRNA expression of these inflammatory cytokine was also reduced via blocking nuclear factor-κB nuclear translocation by AS and DEQA. In addition, AS significantly reduced phosphorylated-c-Jun N-terminal kinase level and DEQA significantly reduced both phosphorylated-c-Jun N-terminal kinase and -p38 mitogen-activated protein kinase levels. CONCLUSIONS: Therefore, these results indicated that AS and its active compound, DEQA may improve mast cell-mediated inflammatory diseases.

Anti-inflammatory effects of a traditional Korean medicine: Ojayeonjonghwan.

OBJECTIVE: To study the anti-inflammatory properties of OJ. CONTEXT: Ojayeonjonghwan (OJ) is a traditional Korean prescription, which has been widely used for the treatment of prostatitis. However, no scientific study has been performed of the anti-inflammatory effects of OJ. MATERIALS AND METHODS: Peritoneal macrophages were isolated 3-4 days after injecting a C57BL/6J mouse with thioglycollate. They were then treated with OJ water extract (0.01, 0.1, and 1 mg/mL) for 1 h and stimulated with lipopolysaccharide (LPS) for different times. Nitric oxide (NO), inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2, and proinflammatory cytokine levels were determined by NO assay, Western blotting, RT-PCR and ELISA. RESULTS: NO generation and iNOS induction were increased in the LPS-activated mouse peritoneal macrophages. However, NO generation and iNOS induction by LPS were suppressed by treatment with OJ for the first time. The IC50 value of OJ with respect to NO production was 0.09 mg/mL. OJ did not influence LPS-stimulated COX-2 induction, but did significantly decrease LPS-stimulated secretions and mRNA expressions of tumour necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1ß. Inhibition rates of TNF-α, IL-6, and IL-1ß at an OJ concentration of 1 mg/mL were 77%, 88%, and 50%, respectively. OJ also suppressed the LPS-induced nuclear translocation of NF-κB. High-performance liquid chromatography showed schizandrin and gomisin A are major components of OJ. CONCLUSIONS: OJ reduces inflammatory response, and this probably explains its positive impact on the prostatitis associated inflammation.

NAADP-mediated Ca2+ signaling promotes autophagy and protects against LPS-induced liver injury.

LPS has been shown to induce hepatocyte autophagy, but little is known about how LPS is able to do this during acute toxic liver injury. Our aim was to determine the existence of any selective Ca2+ signaling coupling to hepatocyte autophagy in response to LPS. LPS increased the autophagic process in hepatocytes, and CD38 knockdown prevented this response. Ned19, a specific inhibitor for nicotinic acid adenine dinucleotide phosphate (NAADP), prevented LPS-mediated Ca2+ signaling and autophagosome formation in hepatocytes. CD38 overexpression protected the liver from LPS/d-galactosamine (GalN)-induced injury, and NAADP administration promoted autophagosome formation and protected hepatocytes from injury induced by LPS/GalN. Autophagy was promoted by the up-regulation of autophagy-related gene expression via NAADP-mediated Ca2+ signaling in response to LPS. However, CD38-knockout mice displayed down-regulation in hepatocyte gene expression. Ned19 also inhibited the NAADP-stimulated induction of gene expression by inhibiting the LPS-induced nuclear translocation of transcription factor EB (TFEB). Hepatocyte autophagy protects against LPS-induced liver injury via the CD38/NAADP/Ca2+/TFEB pathway. The role of NAADP-mediated Ca2+ signaling in the autophagic process will help elucidate the complexities of autophagy regulation, which is essential toward the discovery of new therapeutic tools against acute liver injury.-Rah, S.-Y., Lee, Y.-H., Kim, U.-H. NAADP-mediated Ca2+ signaling promotes autophagy and protects against LPS-induced liver injury.

RhBMP-2 Activates Hippo Signaling through RASSF1 in Esophageal Cancer Cells.

Despite that recombinant human bone morphogenetic protein-2 (rhBMP-2) has been reported as a stimulatory effecter of cancer cell growth because of its characteristic like morphogen, the biological functions of rhBMP-2 in human esophageal cancer cells are unknown. The purpose of this study was to investigate whether rhBMP-2 has an inhibitory effect on the growth of human esophageal squamous carcinoma cells (ESCC). RhBMP-2 significantly inhibited proliferation of ESCC cells in a dose-dependent manner in the MTT assay. Cell cycle arrest at the G1 phase was induced 24 h after rhBMP2 treatment. RhBMP-2 also reduced cyclin D1, cyclin-dependent kinase (CDK) 4 and CDK 6 activities, and stimulated p-Smad1/5/8, p53, and p21 levels at 12 h. In contrast, rhBMP-2 diminished poly (ADP-ribose) polymerase (PARP) protein expression levels and activated cleaved PARP, cleaved caspase-7, and cleaved-caspase 9 levels in ESCC cells. In addition, rhBMP-2 increased MST1, MOB1, and p-YAP protein levels and the RASSF1 binds Mst1 more upon treatment with rhBMP2. The induced p-YAP expression in TE-8 and TE-12 cells by rhBMP-2 was reversed by the RASSF1 knockdown. In vivo study, rhBMP-2 decreased tumor volume following subcutaneous implantation and showed higher radiologic score (less bony destruction) after femoral implantation compared to those in a control group. These results suggest that rhBMP-2 inhibits rather than activates proliferation of human esophageal cancer cells which is mediated through activating the hippo signaling pathway.

Impaired learning and memory in CD38 null mutant mice.

CD38 is an enzyme that catalyzes the formation of cyclic ADP ribose and nicotinic acid adenine dinucleotide phosphate, both of which are involved in the mobilization of Ca(2+) from intracellular stores. Recently, CD38 has been shown to regulate oxytocin release from hypothalamic neurons. Importantly, CD38 mutations are associated with autism spectrum disorders (ASD) and CD38 knockout (CD38(-/-)) mice display ASD-like behavioral phenotypes including deficient parental behavior and poor social recognition memory. Although ASD and learning deficits commonly co-occur, the role of CD38 in learning and memory has not been investigated. We report that CD38(-/-) mice show deficits in various learning and memory tasks such as the Morris water maze, contextual fear conditioning, and the object recognition test. However, either long-term potentiation or long-term depression is not impaired in the hippocampus of CD38(-/-) mice. Our results provide convincing evidence that CD38(-/-) mice show deficits in various learning and memory tasks including spatial and non-spatial memory tasks. Our data demonstrate that CD38 is critical for regulating hippocampus-dependent learning and memory without modulating synaptic plasticity.

CD38-mediated Ca(2+) signaling contributes to glucagon-induced hepatic gluconeogenesis.

CD38 is a multifunctional enzyme for the synthesis of Ca(2+) second messengers. Glucagon promotes hepatic glucose production through Ca(2+) signaling in the fasting condition. In this study, we investigated the role of CD38 in the glucagon signaling of hepatocytes. Here, we show that glucagon induces cyclic ADP-ribose (cADPR) production and sustained Ca(2+) increases via CD38 in hepatocytes. 8-Br-cADPR, an antagonistic cADPR analog, completely blocked glucagon-induced Ca(2+) increases and phosphorylation of cAMP response element-binding protein (CREB). Moreover, glucagon-induced sustained Ca(2+) signals and translocation of CREB-regulated transcription coactivator 2 to the nucleus were absent and glucagon-induced glucose production and expression of glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (Pck1) are remarkably reduced in hepatocytes from CD38(-/-) mice. Furthermore, in the fasting condition, CD38(-/-) mice have decreased blood glucose and hepatic expression of G6Pase and Pck1 compared to wild type mice. Our data suggest that CD38/cADPR-mediated Ca(2+) signals play a key role in glucagon-induced gluconeogenesis in hepatocytes, and that the signal pathway has significant clinical implications in metabolic diseases, including type 2 diabetes.

ADP-ribose/TRPM2-mediated Ca2+ signaling is essential for cytolytic degranulation and antitumor activity of natural killer cells.

Natural killer (NK) cells are essential for immunosurveillance against transformed cells. Transient receptor potential melastatin 2 (TRPM2) is a Ca(2+)-permeable cation channel gated by ADP-ribose (ADPR). However, the role of TRPM2-mediated Ca(2+) signaling in the antitumor response of NK cells has not been explored. Here, we show that ADPR-mediated Ca(2+) signaling is important for cytolytic granule polarization and degranulation but not involved in target cell recognition by NK cells. The key steps of this pathway are: 1) the activation of intracellular CD38 by protein kinase A following the interaction of the NK cell with a tumor cell results in the production of ADPR, 2) ADPR targets TRPM2 channels on cytolytic granules, and 3) TRPM2-mediated Ca(2+) signaling induces cytolytic granule polarization and degranulation, resulting in antitumor activity. NK cells treated with 8-Br-ADPR, an ADPR antagonist, as well as NK cells from Cd38(-/-) mice showed reduced tumor-induced granule polarization, degranulation, granzyme B secretion, and cytotoxicity of NK cells. Furthermore, TRPM2-deficient NK cells showed an intrinsic defect in tumoricidal activity. These results highlight CD38, ADPR, and TRPM2 as key players in the specialized Ca(2+) signaling system involved in the antitumor activity of NK cells.

Seminal CD38 is a pivotal regulator for fetomaternal tolerance.

A successful pregnancy depends on a complex process that establishes fetomaternal tolerance. Seminal plasma is known to induce maternal immune tolerance to paternal alloantigens, but the seminal factors that regulate maternal immunity have yet to be characterized. Here, we show that a soluble form of CD38 (sCD38) released from seminal vesicles to the seminal plasma plays a crucial role in inducing tolerogenic dendritic cells and CD4(+) forkhead box P3(+) (Foxp3(+)) regulatory T cells (Tregs), thereby enhancing maternal immune tolerance and protecting the semiallogeneic fetus from resorption. The abortion rate in BALB/c females mated with C57BL/6 Cd38(-/-) males was high compared with that in females mated with Cd38(+/+) males, and this was associated with a reduced proportion of Tregs within the CD4(+) T-cell pool. Direct intravaginal injection of sCD38 to CBA/J pregnant mice at preimplantation increased Tregs and pregnancy rates in mice under abortive sonic stress from 48 h after mating until euthanasia. Thus, sCD38 released from seminal vesicles to the seminal plasma acts as an immunoregulatory factor to protect semiallogeneic fetuses from maternal immune responses.

The therapeutic efficacy of α-pinene in an experimental mouse model of allergic rhinitis.

In the present study, the therapeutic effect and underlying mechanism of α-pinene (α-PN) in the ovalbumin (OVA)-sensitized allergic rhinitis (AR) model were investigated. Our results showed that pretreatment with α-PN caused a decrease in clinical symptoms, including a decrease in the number of nasal, eye, and ear rubs, and spleen weight in the OVA-sensitized mice. The level of interleukin (IL)-4 was decreased on the spleen tissue of α-PN treated mice. Pretreatment with α-PN significantly decreased levels of nasal immunoglobulin E. Protein levels of tumor necrosis factor-α, intercellular adhesion molecule-1, and macrophage inflammatory protein-2 were decreased by the administration of α-PN in the nasal mucosa of the OVA-sensitized mice. The increased numbers of eosinophils and mast cells infiltrating the nasal mucosal tissue of mice with AR were decreased following oral administration of α-PN. Post-treatment with α-PN 1h after OVA challenge also resulted in a significant reduction of clinical symptoms and IgE levels. In addition, the expression and phosphorylation of receptor-interacting protein 2 (RIP2) and IκB kinase (IKK)-ß and activation of nuclear factor-κB (NF-κB), and caspase-1 were all increased in the activated human mast cell line, HMC-1 cells, however, increased activations of RIP2, IKK-ß, NF-κB, and caspase-1 were inhibited by treatment with α-PN. Taken together, we suggest that α-PN is a promising anti-allergic agent and may be useful in the clinical management of AR.

Connexin-43 hemichannels mediate cyclic ADP-ribose generation and its Ca2+-mobilizing activity by NAD+/cyclic ADP-ribose transport.

The ADP-ribosyl cyclase CD38 whose catalytic domain resides in outside of the cell surface produces the second messenger cyclic ADP-ribose (cADPR) from NAD(+). cADPR increases intracellular Ca(2+) through the intracellular ryanodine receptor/Ca(2+) release channel (RyR). It has been known that intracellular NAD(+) approaches ecto-CD38 via its export by connexin (Cx43) hemichannels, a component of gap junctions. However, it is unclear how cADPR extracellularly generated by ecto-CD38 approaches intracellular RyR although CD38 itself or nucleoside transporter has been proposed to import cADPR. Moreover, it has been unknown what physiological stimulation can trigger Cx43-mediated export of NAD(+). Here we demonstrate that Cx43 hemichannels, but not CD38, import cADPR to increase intracellular calcium through RyR. We also demonstrate that physiological stimulation such as Fcγ receptor (FcγR) ligation induces calcium mobilization through three sequential steps, Cx43-mediated NAD(+) export, CD38-mediated generation of cADPR and Cx43-mediated cADPR import in J774 cells. Protein kinase A (PKA) activation also induced calcium mobilization in the same way as FcγR stimulation. FcγR stimulation-induced calcium mobilization was blocked by PKA inhibition, indicating that PKA is a linker between FcγR stimulation and NAD(+)/cADPR transport. Cx43 knockdown blocked extracellular cADPR import and extracellular cADPR-induced calcium mobilization in J774 cells. Cx43 overexpression in Cx43-negative cells conferred extracellular cADPR-induced calcium mobilization by the mediation of cADPR import. Our data suggest that Cx43 has a dual function exporting NAD(+) and importing cADPR into the cell to activate intracellular calcium mobilization.

Generation of cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate by CD38 for Ca2+ signaling in interleukin-8-treated lymphokine-activated killer cells.

We have previously demonstrated that cyclic ADP-ribose (cADPR) is a calcium signaling messenger in interleukin 8 (IL-8)-induced lymphokine-activated killer (LAK) cells. In this study we examined the possibility that IL-8 activates CD38 to produce another messenger, nicotinic acid adenine dinucleotide phosphate (NAADP), in LAK cells, and we showed that IL-8 induced NAADP formation after cADPR production. These calcium signaling messengers were not produced when LAK cells prepared from CD38 knock-out mice were treated with IL-8, indicating that the synthesis of both NAADP and cADPR is catalyzed by CD38 in LAK cells. Application of cADPR to LAK cells induced NAADP production, whereas NAADP failed to increase intracellular cADPR levels, confirming that the production of cADPR precedes that of NAADP in IL-8-treated LAK cells. Moreover, NAADP increased intracellular Ca(2+) signaling as well as cell migration, which was completely blocked by bafilomycin A1, suggesting that NAADP is generated in lysosome-related organelles after cADPR production. IL-8 or exogenous cADPR, but not NAADP, increased intracellular cAMP levels. cGMP analog, 8-(4-chlorophenylthio)-guanosine 3',5'-cyclic monophosphate, increased both cADPR and NAADP production, whereas the cAMP analog, 8-(4-chlorophenylthio)-cAMP, increased only NAADP production, suggesting that cAMP is essential for IL-8-induced NAADP formation. Furthermore, activation of Rap1, a downstream molecule of Epac, was required for IL-8-induced NAADP formation in LAK cells. Taken together, our data suggest that IL-8-induced NAADP production is mediated by CD38 activation through the actions of cAMP/Epac/protein kinase A/Rap1 in LAK cells and that NAADP plays a key role in Ca(2+) signaling of IL-8-induced LAK cell migration.

Molecular mechanism of ADP-ribosyl cyclase activation in angiotensin II signaling in murine mesangial cells.

ADP-ribosyl cyclase (ADPR-cyclase) produces a Ca(2+)-mobilizing second messenger cyclic ADP-ribose (cADPR) from NAD(+). In this study, we investigated the molecular basis of ADPR-cyclase activation and the following cellular events in angiotensin II (ANG II) signaling in mouse mesangial cells (MMCs). Treatment of MMCs with ANG II induced an increase in intracellular Ca(2+) concentrations through a transient Ca(2+) release via an inositol 1,4,5-trisphosphate receptor and a sustained Ca(2+) influx via L-type Ca(2+) channels. The sustained Ca(2+) signal, but not the transient Ca(2+) signal, was blocked by a cADPR antagonistic analog, 8-bromo-cADPR (8-Br-cADPR), and an ADPR-cyclase inhibitor, 4,4'-dihydroxyazobenzene (DHAB). In support of the results, ANG II stimulated cADPR production in a time-dependent manner, and DHAB inhibited ANG II-induced cADPR production. Application of pharmacological inhibitors revealed that activation of ADPR-cyclase by ANG II involved ANG II type 1 receptor, phosphoinositide 3-kinase, protein tyrosine kinase, and phospolipase C-gamma1. Moreover, DHAB as well as 8-Br-cADPR abrogated ANG II-mediated Akt phosphorylation, nuclear translocation of nuclear factor of activated T cell, and uptake of [(3)H]thymidine and [(3)H]leucine in MMCs. These results demonstrate that ADPR-cyclase in MMCs plays a pivotal role in ANG II signaling for cell proliferation and protein synthesis.

Extracellular NAD is a regulator for FcgammaR-mediated phagocytosis in murine macrophages.

NAD is available in the extracellular environment and elicits immune modulation such as T cell apoptosis by being used as the substrate of cell surface ADP-ribosyl transferase. However, it is unclear whether extracellular NAD affects function of macrophages expressing cell surface ADP-ribosyl transferase. Here we show that extracellular NAD enhances Fcgamma receptor (FcgammaR)-mediated phagocytosis in J774A.1 macrophages via the conversion into cyclic ADP-ribose (cADPR), a potent calcium mobilizer, by CD38, an ADP-ribosyl cyclase. Extracellular NAD increased the phagocytosis of IgG-coated sheep red blood cells (IgG-SRBC) in J774A.1 macrophages, which was completely abolished by pretreatment of 8-bromo-cADPR, an antagonist of cADPR, or CD38 knockdown. Extracellular NAD increased basal intracellular Ca(2+) concentration, which also was abolished by pretreatment of 8-bromo-cADPR or CD38 knockdown. Moreover, the chelation of intracellular calcium abolished NAD-induced enhancement of phagocytosis of IgG-SRBC. Our results suggest that extracellular NAD act as a regulator for FcgammaR-mediated phagocytosis in macrophages.