The transient potential receptor melastatin-2 (TRPM2) channel has emerged as an important Ca 2+ signalling mechanism in a variety of cells, contributing to cellular functions that include cytokine production, insulin release, cell motility and cell death. Its ability to respond to reactive oxygen species has made TRPM2 a potential therapeutic target for chronic inflammation, neurodegenerative diseases, and oxidative stress-related pathologies. TRPM2 is a non-selective, calcium (Ca 2+ )-permeable cation channel of the melastatin-related transient receptor potential (TRPM) ion channel subfamily. It is activated by intracellular adenosine diphosphate ribose (ADPR) through a diphosphoribose hydrolase domain in its C-terminus and regulated through a variety of factors, including synergistic facilitation by [Ca 2+ ] i , cyclic ADPR, H 2 O 2 , NAADP, and negative feedback regulation by AMP and permeating protons (pH). In addition to its role mediating Ca 2+ influx into the cells, TRPM2 can also function as a lysosomal Ca 2+ release channel, contributing to cell death. The physiological and pathophysiological context of ROS-mediated events makes TRPM2 a promising target for the development of therapeutic tools of inflammatory and degenerative diseases.

The ectoenzyme CD38 catalyzes the production of cyclic ADP-ribose (cADPR) and ADP-ribose (ADPR) from its substrate, NAD+. Both products of the CD38 enzyme reaction play important roles in signal transduction, as cADPR regulates calcium release from intracellular stores and ADPR controls cation entry through the plasma membrane channel TRPM2. We previously demonstrated that CD38 and the cADPR generated by CD38 regulate calcium signaling in leukocytes stimulated with some, but not all, chemokines and controls leukocyte migration to inflammatory sites. However, it is not known whether the other CD38 product, ADPR, also regulates leukocyte trafficking In this study we characterize 8-bromo (8Br)-ADPR, a novel compound that specifically inhibits ADPR-activated cation influx without affecting other key calcium release and entry pathways. Using 8Br-ADPR, we demonstrate that ADPR controls calcium influx and chemotaxis in mouse neutrophils and dendritic cells activated through chemokine receptors that rely on CD38 and cADPR for activity, including mouse FPR1, CXCR4, and CCR7. Furthermore, we show that the calcium and chemotactic responses of leukocytes are not dependent on poly-ADP-ribose polymerase 1 (PARP-1), another potential source of ADPR in some leukocytes. Finally, we demonstrate that NAD+ analogues specifically block calcium influx and migration of chemokine-stimulated neutrophils without affecting PARP-1-dependent calcium responses. Collectively, these data identify ADPR as a new and important second messenger of mouse neutrophil and dendritic cell migration, suggest that CD38, rather than PARP-1, may be an important source of ADPR in these cells, and indicate that inhibitors of ADPR-gated calcium entry, such as 8Br-ADPR, have the potential to be used as anti-inflammatory agents.

Chemokines induce calcium (Ca(2+)) signaling and chemotaxis in dendritic cells (DCs), but the molecular players involved in shaping intracellular Ca(2+) changes remain to be characterized. Using siRNA and knockout mice, we show that in addition to inositol 1,4,5-trisphosphate (IP(3))-mediated Ca(2+) release and store-operated Ca(2+) entry (SOCE), the transient receptor potential melastatin 2 (TRPM2) channel contributes to Ca(2+) release but not Ca(2+) influx in mouse DCs. Consistent with these findings, TRPM2 expression in DCs is restricted to endolysosomal vesicles, whereas in neutrophils, the channel localizes to the plasma membrane. TRPM2-deficient DCs show impaired maturation and severely compromised chemokine-activated directional migration as well as bacterial-induced DC trafficking to the draining lymph nodes. Defective DC chemotaxis is due to perturbed chemokine-receptor-initiated Ca(2+) signaling mechanisms, which include suppression of TRPM2-mediated Ca(2+) release and secondary modification of SOCE. DCs deficient in both TRPM2 and IP(3) receptor signaling lose their ability to perform chemotaxis entirely. These results highlight TRPM2 as a key player regulating DC chemotaxis through its function as Ca(2+) release channel and confirm ADP-ribose as a novel second messenger for intracellular Ca(2+) mobilization.

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Key pointsr The Mg 2+ and Ca 2+ conducting transient receptor potential melastatin 7 (TRPM7) channel-enzyme (chanzyme) has been implicated in immune cell function.r Mice heterozygous for a TRPM7 kinase deletion are hyperallergic, while mice with a single point mutation at amino acid 1648, silencing kinase activity, are not.r As mast cell mediators trigger allergic reactions, we here determine the function of TRPM7 in mast cell degranulation and histamine release.r Our data establish that TRPM7 kinase activity regulates mast cell degranulation and release of histamine independently of TRPM7 channel function.r Our findings suggest a regulatory role of TRPM7 kinase activity on intracellular Ca 2+ and extracellular Mg 2+ sensitivity of mast cell degranulation.Abstract Transient receptor potential melastatin 7 (TRPM7) is a divalent ion channel with a C-terminally located α-kinase. Mice heterozygous for a TRPM7 kinase deletion (TRPM7 +/ K ) are hypomagnesaemic and hyperallergic. In contrast, mice carrying a single point mutation at amino acid 1648, which silences TRPM7 kinase activity (TRPM7 KR ), are not hyperallergic and are resistant to systemic magnesium (Mg 2+ ) deprivation. Since allergic reactions are triggered by mast cell-mediated histamine release, we investigated the function of TRPM7 on mast cell degranulation and histamine release using wild-type (TRPM7 +/+ ), TRPM7 +/ K and TRPM7 KR mice. We found that degranulation and histamine release proceeded independently of TRPM7 channel function. Furthermore, extracellular Mg 2+ assured unperturbed IgE-DNP-dependent exocytosis, independently of TRPM7. However, impairment of TRPM7 kinase function suppressed IgE-DNP-dependent exocytosis, slowed the cellular degranulation rate, and diminished the sensitivity to intracellular calcium (Ca 2+ ) in G protein-induced exocytosis. In addition, G protein-coupled receptor (GPCR) stimulation revealed strong suppression of histamine release, whereas removal of extracellular Mg 2+ caused the phenotype to revert. We conclude that the TRPM7 kinase activity regulates murine mast cell degranulation by changing its sensitivity to intracellular Ca 2+ and affecting granular mobility and/or histamine contents.

Recent advances in basic cardiovascular research as well as their translation into the clinical situation were the focus at the last “New Frontiers in Cardiovascular Research meeting”. Major topics included the characterization of new targets and procedures in cardioprotection, deciphering new players and inflammatory mechanisms in ischemic heart disease as well as uncovering microRNAs and other biomarkers as versatile and possibly causal factors in cardiovascular pathogenesis. Although a number of pathological situations such as ischemia–reperfusion injury or atherosclerosis can be simulated and manipulated in diverse animal models, also to challenge new drugs for intervention, patient studies are the ultimate litmus test to obtain unequivocal information about the validity of biomedical concepts and their application in the clinics. Thus, the open and bidirectional exchange between bench and bedside is crucial to advance the field of ischemic heart disease with a particular emphasis of understanding long-lasting approaches in cardioprotection.

CD38 is a transmembrane glycoprotein that functions as an ectoenzyme and as a receptor. Based on the structural similarity between CD38 and ADP-ribosyl cyclase from Aplysia californica, it was hypothesized that CD38 is expressed as a homodimer on the surface of cells. Indeed, CD38 dimers have been reported, however, the structural requirements for their stabilization on the plasma membrane are unknown. We demonstrate that the majority of CD38 is assembled as noncovalently associated homodimers on the surface of B cells. Analysis of CD38 mutants, expressed in Ba/F3 cells, revealed that truncation of the cytoplasmic region or mutation of a single amino acid within the a1-helix of CD38 decreased the stability of the CD38 homodimers when solubilized in detergent. Cells expressing the unstable CD38 homodimers had diminished expression of CD38 on the plasma membrane and the half-lives of these CD38 mutant proteins on the plasma membrane were significantly reduced. Together, these results show that CD38 is expressed as noncovalently associated homodimers on the surface of murine B cells and suggest that appropriate assembly of CD38 homodimers may play an important role in stabilizing CD38 on the plasma membrane of B cells.Keywords: B lymphocytes; CD38; homodimer stability; NAD + glycohydrolase; protein structure.CD38 is a type II transmembrane ectoenzyme expressed by many cell types [1][2][3]. CD38 plays an important role in calcium signaling as it catalyzes the production of several calcium mobilizing metabolites including adenosine(5¢)- [4,5]. In addition to its role as an enzyme, CD38 can also serve as a receptor on the plasma membrane of leukocytes and lymphocytes. For example, incubation of B lymphocytes with agonistic antibodies to CD38 induces calcium mobilization, protein phosphorylation, proliferation, class switching, rescue from cell death and induction of apoptosis [1,[6][7][8][9][10]. In order to understand the dual receptor and enzyme properties of CD38, a number of structure/function studies have been performed. These studies have been guided by analyses of two CD38 homologues, the cytosolic Aplysia californica ADP-ribosyl cyclase [11,12] and the mammalian GPI-anchored NAD + glycohydrolase, CD157 [13,14].Crystallographic and X-ray diffraction analyses of these two proteins indicated that both proteins form noncovalently associated homodimers [15,16]. Thus, it has been proposed that CD38 is also likely to be expressed as a homodimer on the plasma membrane and, in agreement with this hypothesis, initial reports showed that high molecular mass aggregates of CD38 are formed after incubation of human erythrocytes with NAD + or 2-mercaptoethanol [17]. In addition, it was reported that CD38 formed dimers and oligomers on the membrane of CD38 transfected HeLa cells [18]. It is not clear, however, whether CD38 is always present in a dimeric form on the surface of cells as many groups have reported finding only the monomeric form of CD38 [19][20][21]. Furthermore, it remains to be determined whether CD38 dimers ar...

The precise mechanisms by which regulatory T cells operate, particularly their effect on signaling pathways leading to T cell activation, are poorly understood. In this study we have used regulatory T (Treg) cells of known Ag specificity, generated in vivo, to address their effects on early activation events occurring in naive T cells of the same Ag specificity. We found that the Treg cells need to be present at the moment of priming to suppress activation and proliferation of the naive T cell. Furthermore, the Treg cells significantly inhibit the recruitment of protein kinase Cθ (PKCθ) to the immune synapse of the naive T cell as long as both T cells are of the same Ag specificity and are contacting the same APC. Finally, naturally occurring CD4+25+ T cells seem to have the same effect on PKCθ recruitment in CD25− T cells of the same Ag specificity. These results suggest that although additional mechanisms of regulation are likely to exist, inhibition of PKCθ recruitment in the effector T cell may be a common regulatory pathway leading to the absence of NF-κB activation and contributing to the block of IL-2 secretion characteristic of immune suppression.