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.

No abstract

Azole derivatives exert direct anti-inflammatory activity in fungal infections and other cutaneous inflammatory conditions. Inhibition of PMN chemotaxis and leukotriene biosynthesis has been suggested to explain this phenomenon, yet the molecular mechanism of action for these drugs remains unknown. Recently we showed that NAD+ derived metabolites, cADPR and ADPR; regulate PMN chemotaxis by inducing extracellular Ca2+ influx via the ion channel TRPM2. Here we investigated whether azole derivatives inhibit PMN chemotaxis in vitro by altering the production of NAD+-derived metabolites or by blocking plasma membrane channel-conducted Ca2+ influx. First, we demonstrated that econazole and clotrimazole significantly inhibited PMN directionality and speed in an EZ-Taxiscan chemotaxis assay. Interestingly, econazole and clotrimazole, as well as FA, another compound proposed to block the TRPM2 channel, blocked fMLP-induced Ca2+ influx, rendering defective Ca2+ responses comparable to those responses observed in fMLP-stimulated TRPM2 KO PMN. Moreover, we found that pre-treatment of neutrophils with econazole or clotrimazole did not alter NAD+ metabolism, but its effect was by blocking a membrane channel with physiological properties similar to TRPM2. Collectively, these data suggest that compounds, which antagonize TRPM2-mediated Ca2+ signaling pathway, may function as effective inhibitors of PMN recruitment, and will likely prove useful in the treatment of inflammatory diseases.