No abstract

The NADPH oxidase activity of phagocytes and its generation of reactive oxygen species (ROS) is critical for host-defense, but ROS overproduction can also lead to inflammation and tissue injury. Here we report that TRPM2, a non-selective and redox-sensitive cation channel, inhibits ROS production in phagocytic cells and prevents endotoxin-induced lung inflammation in mice. TRPM2-deficient mice challenged with endotoxin (lipopolysaccharide) showed an increased inflammatory signature and decreased survival compared to controls. TRPM2 functions by dampening NADPH oxidase-mediated ROS production through depolarization of the plasma membrane in phagocytes. Since ROS also activates TRPM2, our findings establish a negative feedback mechanism inactivating ROS production through inhibition of the membrane potential-sensitive NADPH oxidase.

Bradykinin (BK) and kallidin (Lys-BK), liberated from kininogens by kallikreins, are ligands of the BK B(2) receptor. We investigated whether kallikreins, besides releasing peptide agonist, could also activate the receptor directly. We studied the effect of porcine and human recombinant tissue kallikrein and plasma kallikrein on [Ca(2+)](i) mobilization and [(3)H]arachidonic acid release from cultured cells stably transfected to express human BK B(2) receptor (CHO/B(2), MDCK/B(2), HEK/B(2)), and endothelial cells were used as control cells. As with BK, the actions of kallikrein were blocked by the B(2) antagonist, HOE 140. Kallikrein was inactive on cells lacking B(2) receptor. Kallikrein and BK desensitized the receptor homologously but there was no cross-desensitization. Furthermore, 50 nM human cathepsin G and 50 nM trypsin also activated the receptor; this also was blocked by HOE 140. Experiments excluded a putative kinin release by proteases. [(3)H]AA release by BK was reduced by 40% by added kininase I (carboxypeptidase M); however, receptor activation by tissue kallikrein, trypsin, or cathepsin G was not affected. Prokallikrein and inhibited kallikrein were inactive, suggesting cleavage of a peptide bond in the receptor. Kallikreins were active on mutated B(2) receptor missing the 19 N-terminal amino acids, suggesting a type of activation different from that of thrombin receptor. Paradoxically, tissue kallikreins decreased the [(3)H]BK binding to the receptor with a low K(D) (3 nM) and inhibited it 78%. Thus, kallikreins and some other proteases activate human BK B(2) receptor directly, independent of BK release. The BK B(2) receptor may belong to a new group of serine protease-activated receptors.

Rationale TRPM2 (Transient Receptor Potential Melastatin-2) expressed in endothelial cells (ECs) is a cation channel mediating Ca2+ entry in response to intracellular generation of adenosine diphosphoribose (ADPR), the TRPM2 ligand. Objective Because polymorphonuclear leukocytes (PMN) interaction with endothelial cells (ECs) generates ROS, we addressed the possible role of TRPM2 expressed in ECs in the mechanism of transendothelial migration of PMNs. Methods and Results We observed defective PMN transmigration in response to LPS challenge in adult mice in which the EC expressed TRPM2 is conditionally deleted (Trpm2iΔEC). PMN interaction with ECs induced the entry of Ca2+ in ECs via the EC-expressed TRPM2. Prevention of generation of ADPR in ECs significantly reduced Ca2+ entry in response to PMN activation of TRPM2 in ECs. PMNs isolated from gp91phox−/− mice significantly reduced Ca2+ entry in ECs via TRPM2 as compared to WT PMNs and failed to induce PMN transmigration. Overexpression of the ADPR insensitive TRPM2 mutant channel (C1008→A) in ECs suppressed the Ca2+ entry response. Further the forced expression of TRPM2 C1008→A mutant channel or silencing of PARP-1 in ECs of mice prevented PMN transmigration. Conclusion Thus, endotoxin-induced transmigration of PMNs was secondary to TRPM2-activated Ca2+ signaling and VE-cadherin phosphorylation resulting in the disassembly of adherens junctions and opening of the paracellular pathways. These results suggest blocking TRPM2 activation in ECs is a potentially important means of therapeutically modifying PMN-mediated vascular inflammation.

Rationale: Oxidants generated by activated endothelial cells are known to induce apoptosis, a pathogenic feature of vascular injury and inflammation from multiple etiologies. The melastatin-family transient receptor potential 2 (TRPM2) channel is an oxidant-sensitive Ca2+ permeable channel implicated in mediating apoptosis; however, the mechanisms of gating of the supra-normal Ca2+ influx required for initiating of apoptosis are not understood. Objective: Here we addressed the role TRPM2 and its interaction with the short splice variant TRPM2-S in mediating the Ca2+ entry burst required for induction of endothelial cell apoptosis. Methods and Results: We observed that TRPM2-S was basally associated with TRPM2 in the endothelial plasmalemma and this interaction functioned to constitutively suppress TRPM2-dependent Ca2+ gating. ROS production in endothelial cells or directly applying ROS induced PKCα activation and phosphorylation of TRPM2 at Ser 39. This in turn stimulated a large entry of Ca2+ and activated the apoptosis pathway. A similar TRPM2-dependent endothelial apoptosis mechanism was seen in intact vessels. The PKCα-activated phospho-switch opened the TRPM2 channel to allow large Ca2+ influx by releasing TRPM2-S inhibition of TRPM2, which in turn activated caspase-3 and cleaved the caspase substrate poly(ADP-ribose) polymerase. Conclusions: Here we describs a fundamental mechanism by which activation of the trp super-family TRPM2 channel induces apoptosis of endothelial cells. The signaling mechanism involves ROS-induced PKCα activation resulting in phosphorylation of TRPM2-S that allows enhanced TRPM2-mediated gating of Ca2+ and activation of the apoptosis program. Strategies aimed at preventing the uncoupling of TRPM2-S from TRPM2 and subsequent Ca2+ gating during oxidative stress may mitigate endothelial apoptosis and its consequences in mediating vascular injury and inflammation.

The transient receptor potential (melastatin) 2 (TRPM2), is an oxidant-activated nonselective cation channel, that is widely expressed in mammalian tissues including the vascular endothelium. Oxidative stress, through the generation of oxygen metabolites including H 2 O 2 , stimulates intracellular ADP-ribose formation which, in turn, opens TRPM2 channels. These channels act as an endogenous redox sensor for mediating oxidative stress/ROS-induced Ca 2+ entry and the subsequent specific Ca 2+ -dependent cellular reactions such as endothelial hyper-permeability and apoptosis. This review summarizes recent findings on the mechanism by which oxidants induce TRPM2 activation, the role of these channels in the signaling vascular endothelial dysfunctions, and the modulation of oxidant-induced TRPM2 activation by PKCα and phospho-tyrosine phosphates L1.

Objective Transient Receptor Potential Melastatin-2 (TRPM2) channel is a non-selective cation channel that mediates influx of Ca2+ and Na+ with relative permeability of PCa:PNa ∼0.6 in response to cellular oxidative stress. As angiogenesis and ischemic neovascularization are both significantly dependent on oxidant signaling, here we investigated the possibile role of VEGF-induced ROS production in activating TRPM2-dependent Ca2+ signaling, and in the mechanism of angiogensis and ischemic neovascularization. Approach and Results We observed that VEGF stimulation rapidly induced the association of TRPM2 and c-Src kinase with VE-cadherin forming a signalplex at VE-cadherin junctions in endothelial cells (ECs). Using ECs isolated from TRPM2−/− mice or after siRNA depletion of TRPM2, we demonstrated that TRPM2-activated Ca2+ signaling was required for c-Src kinase-induced phosphorylation of VE-cadherin at Y658 and Y731, the crucial sites involved in VE-cadherin internalization in response to VEGF. VEGF-induced ROS generation activated TRPM2-induced Ca2+ entry whereas the ROS-insensitive TRPM2 mutant (C1008→A) showed impaired Ca2+ entry. ECs depleted of TRPM2 also displayed significantly perturbed migratory phenotype and impaired activation of c-Src in response to VEGF. TRPM2-/- mice reconstituted with wild type myeloid cells demonstrated aberrant angiogenesis and neovascularisation in the hindlimb ischemia model as compared to wild type mice. Conclusion VEGF-induced angiogeneis and post-ischemic neovascularisation in mice required ROS generation in ECs and resultant TRPM2 activation. Thus, our findings provide novel insight into the role of TRPM2 in mechanism of angiogenesis and ischemic neovascularisation.

Oxidative [Au1]stress, through the production of oxygen metabolites such as hydrogen peroxide[Au2] (H(2)O(2)), increases vascular endothelial permeability and plays a crucial role in several lung diseases. The transient receptor potential (melastatin) 2 (TRPM2) is an oxidant-sensitive, nonselective cation channel that is widely expressed in mammalian tissues, including the vascular endothelium. We have demonstrated the involvement of TRPM2 in mediating oxidant-induced calcium entry and endothelial hyperpermeability in cultured pulmonary artery endothelial cells. Here, we provide evidence that neutrophil activation-dependent increase in endothelial permeability and neutrophil extravasation requires TRPM2 in cultured endothelial cells. In addition, protein kinase Calpha (PKCalpha) that rapidly colocalizes with the short (nonconducting) TRPM2 isoform after exposure to hydrogen peroxide positively regulates calcium entry through the functional TRPM2 channel. Thus, increase in lung microvessel permeability and neutrophil sequestration depends on the activation of endothelial TRPM2 by neutrophilic oxidants and on PKCalpha regulation of TRPM2 channel activity. Manipulating TRPM2 function in the endothelium may represent a novel strategy aimed to prevent oxidative stress-related vascular dysfunction.