Cooperative Interaction of trp Melastatin Channel Transient Receptor Potential (TRPM2) With Its Splice Variant TRPM2 Short Variant Is Essential for Endothelial Cell ApoptosisNovelty and Significance
Rationale: Oxidants generated by activated endothelial cells are known to induce apoptosis, a pathogenic feature of vascular injury and inflammation from multiple pathogeneses. 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 supranormal Ca2+ influx required for initiating of apoptosis are not understood.
Objective: Here, we addressed the role of TRPM2 and its interaction with the short splice variant TRPM2 short 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 suppress TRPM2-dependent Ca2+ gating constitutively. Reactive oxygen species production in endothelial cells or directly applying reactive oxygen species induced protein kinase C-α 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 protein kinase C-α–activated phosphoswitch 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 describe a fundamental mechanism by which activation of the trp superfamily TRPM2 channel induces apoptosis of endothelial cells. The signaling mechanism involves reactive oxygen species–induced protein kinase C-α 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.
Melastatin-like transient receptor potential 2 (TRPM2) is an oxidant-sensitive Ca2+-permeable channel expressed in many cells, including neurons,1,2 microglia,3,4 multiple lung cell types,5,6 pancreas β cells,7–9 hematopoietic and immune cells,10,11 and vascular endothelial (VE) cells.5 However, the function of TRPM2 remains enigmatic. TRPM2 is activated by the generation of reactive oxygen species (ROS), such as H2O2 and production of adenosine diphosphate ribose (ADPR) after DNA damage and activation of the enzyme poly(ADPR) polymerase.6,12 TRPM2 has been implicated in mediating of oxidant-induced apoptosis secondary to Ca2+ influx that may initiate apoptosis program via the caspase pathway.1,13,14 Although apoptosis is important in normal biological processes and development, apoptosis of endothelial cells, which have low turnover in vessels,15 is a fundamental pathogenic feature of inflammatory and vascular diseases, such as acute lung injury16 and sepsis.17 Our studies have demonstrated a key role of TRPM2 in mediating oxidative injury of the endothelium,5 resulting in disruption of endothelial barrier and tissue edema.18–20 A component of endothelial disruption seen in these studies may well have been because of TRPM2-induced apoptosis.
TRPM2 channel opening after exposure to H2O2 and other ROS is induced by the binding of ADPR to the Nudix box sequence motif (nucleoside diphosphate type motif 9 protein) in the carboxyl-terminal domain of TRPM2.5,6,10,12,21–23 H2O2 produced in the cell5 also activated the production of ADPR,6,10,23,24 which functioned by binding to the TRPM2 Nudix motif.6,10,12,24,25 In addition, other mechanisms of TRPM2 activation, such as direct oxidative modification of the channel, have been proposed.26
Besides TRPM2,5,27 several splice variants of TRPM2 associated with TRPM2 in the plasma membrane have also been identified.28 Their role in regulating TRPM2 function and mediating oxidant-induced apoptosis remains obscure. Of particular interest is the short splice variant (TRPM2-S) that functions as a dominant-negative to inhibit TRPM2 channel activity14,28 but which itself lacks both the carboxyl terminus present in the long isoform TRPM2 and the Ca2+-permeable pore present in TRPM2.28 In cells in which both isoforms are expressed, TRPM2-S interact with TRPM2 to inhibit formation of functional homotetrameric channels.14 Here, we investigated the interaction of TRPM2-S with TRPM2 and how the component cooperated to signal oxidant-induced apoptosis in endothelial cells. The study presents a new mechanism of endothelial apoptosis involving ROS-induced and protein kinase C (PKC)-α phosphorylation-dependent disruption of the interaction of TRPM2 with TRPM2-S and opening of the channel to allow sufficient Ca2+ entry required for activation of the apoptosis program.
An expanded Materials and Methods is available in the online Data Supplement.
Endothelial Cell Culture and Transfection
Isolation of Mouse Endothelial Cells
Endothelial cells were isolated from lungs of wild-type (WT), PKCα–/– (obtained from Dr Jeffrey D. Molkentin, University of Cincinnati, Cincinnati, OH) and TRPM2–/– mice (GlaxoSmithKline). The cells were used between passages 2 and 5.
Human pulmonary artery endothelial cells (HPAEC; Clonetics, La Jolla, CA) were cultured in gelatin-coated flasks and used between passages 3 and 6. Human TRPM2-S splice variant, tagged with poly-His (His6-TRPM2-S), was inserted into a pcDNA3 expression vector (Invitrogen). Phosphorylation-defective TRPM2-S was generated by alanine substitution (S39A), and phosphorylation-mimetic TRPM2-S was generated by aspartic substitution (S39D). Transfection of TRPM2-S constructs using fuGENE HD was verified by Western blotting. Control cells received vector alone.
HPAECs were transiently transfected with TRPM2 or PKCα siRNAs (100 nmol/L; Santa Cruz Biotechnology, Santa Cruz, CA) using TransIT-TKO transfection reagent (Mirus, Madison, WI); nonspecific siRNA served as control (Ambion, Austin, TX). Transfection efficiency was >75%.
Immunoprecipitation and Phosphorylation Studies
Untransfected, His6-(S39A)TRPM2-S and His6-(S39D)TRPM2-S–transfected HPAEC cultures were treated with 300 µmol/L H2O2 for indicated times (37°C). In some experiments, cells were pretreated with 3,4-dihydro-5[4-(1-piperindinyl)butoxy]-1(2H)-isoquinoline or PKC inhibitors 30 minutes before the assay. In other experiments, cells first received siRNA to suppress TRPM2 or PKCα expression. TRPM2 or PKCα immune complexes were precipitated with protein A-Sepharose beads (Sigma) for 2 h at 4°C as described.5
Generation of H2O2 Using Glucose Oxidase/Glucose
H2O2 production in vitro was induced by glucose (1 mmol/L) and glucose oxidase (GO; 1–2.5 mU/mL) and was measured spectrophotometrically from the generation of resorufin (absorbance, 565 nm; extinction coefficient, 58 000 mol/L per cm). GO produced H2O2 at a constant rate (320 nmol/L H2O2/min).
Analysis for Apoptosis
Apoptosis was identified by double-fluorescent staining with phycoerythrin annexin V-fluorescein isothiocyanate and 7-aminoactinomycin D, which detected apoptotic and dead cells, respectively. Confluent endothelial monolayers, without or with PKCα inhibition or silencing, were incubated in 300 µmol/L H2O2 for 6 or 24 hours (37°C). Cells were washed twice with PBS and trypsinized; samples of 1×106 cells were incubated with 5 μL of phycoerythrin-labeled annexin V and 5 μL of 7-aminoactinomycin D (BD Bioscience, Rockville, MD) for 20 minutes at 24°C in the dark and analyzed with a Beckman Coulter CyAn II cytometer (Beckman Coulter, Miami, FL). We also assessed apoptosis in intact lung vascular endothelium by immunofluorescence and terminal deoxynucleotidyl transferase dUTP nick-end labeling assay. Lungs of TRPM2–/– and WT mice were perfused (2 mL/min; 37°C) for 3 hours with recirculation of Roswell Park Memorial Institute medium 1640 (5 mL) containing H2O2 (300 μmol/L) or GO. Lungs were removed and frozen by the optimal cutting temperature method. Frozen lungs, sectioned (5 μm) and fixed in 3.7% formaldehyde, were permeabilized with 0.2% triton X-100. Tissue sections were blocked with 10% fetal bovine serum and incubated with goat anti– VE-cadherin and rabbit anti–poly(ADP-ribose) polymerase (PARP; ie, the cleaved 89-kDa fragment; 1:200 dilution) overnight (4°C). The sections were incubated with secondary antibodies conjugated to Alexa Fluor 488 and 594 (Invitrogen). As an alternative to cleaved PARP antibody, terminal deoxynucleotidyl transferase dUTP nick-end labeling staining was performed according to the manufacturer’s protocol (Roche Diagnostics Corp, Indianapolis, IN). Nuclei were visualized by 4,6-diamidino-2-phenylindole (Sigma-Aldrich, Saint Louis, MO). Slides were analyzed under a Zeiss fluorescence microscope using AxioVision software. Apoptotic cells were identified by double staining (PARP+VE-cadherin or terminal deoxynucleotidyl transferase dUTP nick-end labeling+VE-cadherin).
Statistical comparisons were made with the 2-tailed Student t test. The significance level was P<0.05.
Institutional Study Approval
All studies were conducted after review by the Institutional Animal Care and Use Committee at the University of Illinois at Chicago where the work was performed and in accordance with the Policy on the Care, Welfare, and Treatment of Laboratory Animals.
Methods in Supplement
Methods for [Ca2+]i measurements, TRPM2 protein purification, in vitro phosphorylation assay, bone marrow transplantation, murine model of endotoxin-mediated mortality, and Western blotting are given in the Online Data Supplement.
TRPM2 Is Required for H2O2-Induced Endothelial Cell Apoptosis
Apoptosis was determined by staining endothelial cells with annexin V-phycoerythrin and 7-aminoactinomycin D followed by fluorescence-activated cell sorting assessment (Figure 1A and 1B). H2O2 in a concentration-dependent manner induced apoptosis within 24 hours with an EC50 value of 136±6 µmol/L (Figure 1A). Inhibition of TRPM2 by an anti-TRPM2 blocking antibody or TRPM2 siRNA silencing prevented the apoptosis (Figure 1B). The flow cytometry dot plot data demonstrating apoptosis are shown in Online Figure I. The sustained generation of H2O2 (320 nmol/L per minutes for 90 minutes) by GO with glucose substrate also induced endothelial apoptosis, which was blocked by TRPM2 silencing or inhibition of channel activity (Figure 1B). To address in vivo relevance, we also examined apoptosis in endothelial cells of lung vessels in WT and TRPM2 knockout mice perfused with a solution containing H2O2 or GO/glucose (this generated 320 nmol/L per minute H2O2).
Because Ca2+ signaling activates the apoptotic pathway through activation of caspase-3 followed by cleavage of caspase substrates, such as 113-kDa PARP,29,30 we also determined expression of the 17- and 20-kDa caspase-3 fragments and 89- or 24-kDa caspase cleavage fragments of PARP.29,30 Data from lungs showed that cleaved PARP characteristically colocalized with the endothelial cell marker VE-cadherin, whereas deletion of TRPM2 (TRPM2–/– mice) markedly reduced endothelial apoptosis (Figure 1C). Western blotting confirmed the activation of caspase-3 and cleaved PARP in lungs of WT and not TRPM2–/– mice (Figure 1D). Using another apoptosis assay, terminal deoxynucleotidyl transferase dUTP nick-end labeling staining, we observed fewer endothelial cells undergoing apoptosis after H2O2 or GO/glucose infusion in TRPM2–/– mice when compared with WT (Online Figure II).
H2O2 Induces PKCα Phosphorylation of TRPM2-S
We next determined the role of TRPM2 and its binding partner TRPM2-S in the mechanism of apoptosis. Western blotting showed that both TRPM2-S and TRPM2 (90 and 171 kDa, respectively) were basally expressed in HPAECs (Figure 2A) and in multiple other endothelial cells examined (unpublished observations). TRPM2 and TRPM2-S expressions were not modified by H2O2 exposure per se (Figure 2A). Using the motif scanning graphic software (Merck Genome Research Institute), we identified a putative high-affinity binding site for PKCα near the N terminus of TRPM2-S, at Ser 39. This site was predicted as a possible domain that could be phosphorylated by PKCα. We observed that PKCα was basally expressed in these cells, and its expression was not modified by H2O2 (Figure 2A). Depleting PKCα using siRNA did not modify the expression of either TRPM2 or TRPM2-S (Figure 2A), whereas TRPM2 depletion as expected suppressed the expression of its splice variants (Figure 2A).
To address whether H2O2 was involved in the phosphorylation at Ser39 on TRPM2-S, we used HPAEC monolayers treated with PKCα inhibitors (Gö6976 or PKCα blocking peptide [PKCα inhibitor peptide]) or transfected with siRNA to knockdown PKCα expression. PKCα was immunoprecipitated from lysates of cells exposed to H2O2, and coimmunoprecipitated TRPM2 and TRPM2-S were detected using an antibody recognizing each form (Figure 2B). H2O2 rapidly induced the association of PKCα with TRPM2-S, but not with TRPM2, and the response persisted up to 5 minutes (Figure 2B). Immunoprecipitation was reduced when PKCα activation was inhibited (Figure 2B). Treatment with PKCβII inhibitory peptide, used as control for nonspecific effects of Gö6976 in blocking activation of both PKCα and PKCβII,31 did not modify the H2O2-induced TRPM2-S association with PKCα (Figure 2B). PKCα-TRPM2-S immunoprecipitation was also suppressed predictably by TRPM2 silencing (Figure 2B). Control experiments showed that transfection of endothelial cells with TRPM2 siRNA significantly reduced the expression of both TRPM2 and TRPM2-S (Figure 2A). Control experiments confirmed that siRNA effectively suppressed PKCα expression (Figure 2B). Treatment of cells with PKCα inhibitors (Gö6976 or PKCα inhibitor peptide) and PKCβII blocking peptide did not modify PKCα expression (Figure 2B).
Because the above studies dealt with the role of H2O2 in activating the phosphorylation of TRPM2-S, we next examined whether key alterations could be replicated using a physiological stimulus to generated oxidants. Here, we used tumor necrosis factor-α (TNF-α), a generator of intracellular oxidants and potent inducer of endothelial cell apoptosis.32 We observed that TNF-α induced the association of PKCα with TRPM2-S, whereas suppressing PKCα activity prevented the association (Online Figure IIIA).
To determine whether PKCα was responsible for phosphorylating TRPM2-S after H2O2 challenge, in other studies TRPM2 proteins in cell lysates were precipitated using antibodies recognizing TRPM2 and TRPM2-S, and phosphorylated proteins were visualized using antiphospho-Ser antibody. Western blotting demonstrated that only TRPM2-S was phosphorylated, which occurred within 1 minute of H2O2 exposure with maximum response seen at 2 minutes, whereas there was no phosphorylation of TRPM2 (Figure 2C). Western blotting also showed that TRPM2-S but not TRPM2 was phosphorylated within the same time frame after TNFα exposure (Online Figure IIIB). Phosphorylation of PKCα (82 kDa) was detected as a comigrating band on the gel (seen in top blot of Figure 2C; Online Figure IIIB), an indication that the kinase was in the active state.
We next determined phosphorylation of PKCα using an antibody recognizing phosphorylated on Ser 657, the crucial PKCα catalytic domain.33 H2O2 rapidly induced phosphorylation of PKCα at this site and the phosphorylated PKCα comigrated with TRPM2 (middle blot; Figure 2C). Treatment with Gö6976 (but not with control PKCβII inhibitor peptide) inhibited not only PKCα phosphorylation but also H2O2-induced phosphorylation of TRPM2-S (Figure 2C). These results thus show time-dependent and reversible association between TRPM2-S and PKCα induced by PKCα activation (Figure 2D).
S39 Phosphorylation of TRPM2-S Activates TRPM2 and Supranormal Ca2+ Influx
We used the Fura-2 dye to study the Ca2+ entry response activated by TRPM2 interaction with TRPM2-S, In addition, we used the Ca2+ add-back protocol to rule out any indirect effects of H2O2 on Ca2+ entry secondary to Ca2+-store depletion.5 In the absence of extracellular Ca2+, H2O2 did not produce a Ca2+ transient (Figure 3A and 3B), indicating that H2O2 did not deplete intracellular Ca2+ stores. By contrast, extracellular Ca2+ repletion in the continued presence of H2O2 elicited a sharp and marked increase in intracellular Ca2+ concentration secondary to Ca2+ entry (Figure 3A and 3B). TRPM2 knockdown markedly suppressed the Ca2+ transient (Figure 3A and 3B), showing that H2O2-induced Ca2+ entry required TRPM2. Gö6976 significantly decreased the amplitude of Ca2+repletion-dependent transients by 66±9%, and PKCα inhibitor peptide reduced Ca2+ transient by 46±10% (Figure 3A and 3B). PKCα silencing also reduced Ca2+ entry by 43±6% (Figure 3A and 3B). Treatment of cells with control PKCβII peptide inhibitor, however, did not modify H2O2 -activated Ca2+ entry via TRPM2 channels (Figure 3A and 3B). Along the same lines, TNFα-induced Ca2+ entry in endothelial cells (Online Figure IIID) was also decreased by inhibiting PKCα.
We next addressed the role of the S39 phosphoswitch on TRPM2-S in mediating TRPM2 channel activity. Here, we determined whether mutation of TRPM2-S at Ser 39 (S39A), the PKCα phosphorylation site disrupted Ca2+ signaling. The mutant was tagged on its C terminus with a poly-His fusion protein. Transfected HPAECs showed protein expression of the (S39A)-TRPM2-S mutant (Figure 4A). Western blotting showed that S39A mutation of TRPM2-S abrogated the migration of TRPM2-S with PKCα on the gel (Figure 4B) and phosphorylation of TRPM2-S by PKCα after H2O2 challenge (Figure 4C). PKCα also did not migrate with (S39A)-TRPM2-S mutant (Figure 4C).
We next determined the functional significance of the failure of PKCα to bind to and phosphorylate TRPM2-S on the TRPM2-mediated Ca2+ entry. Intracellular Ca2+ transient elicited by H2O2 was markedly reduced in cells transduced with the TRPM2-S phosphodefective mutant (Figure 4D). To validate the finding that PKCα was indeed responsible for phosphorylation of TRPM2-S at S39, we treated an extract of native protein with recombinant active PKCα. We observed that active PKCα induced phosphorylation of WT TRPM2-S but not of S39A mutant (Online Figure IV).
PKCα Phosphorylation of TRPM2-S Induces TRPM2-S Dissociation From TRPM2
We next determined whether PKCα phosphorylation of TRPM2-S in some manner interfered with TRPM2-S association with TRPM2, thus permitting TRPM2 to gate Ca2+ at sufficient level to activate the apoptosis program. Because TRPM2 variants generated by alternative splicing differed only in their C terminal,28 we immunoprecipitated TRPM2 from cell lysates using an anti-TRPM2 antibody, recognizing the region present solely in TRPM2 form. TRPM2-S, which in the plasma membrane basally associated with TRPM2, dissociated within minutes from TRPM2 after H2O2 addition (Figure 5A). Inhibition of PKCα activation suppressed this TRPM2-S dissociation from TRPM2 (Figure 5A). S39A mutation of TRPM2-S also suppressed the dissociation of TRPM2 from TRPM2-S (Figure 5A). PKCα-dependent phosphorylation of TRPM2-S at Ser 39 blocked the interaction of TRPM2-S with TRPM2 (summarized in Figure 5B). These results show that phosphorylation of TRPM2-S at Ser 39 was responsible for releasing the TRPM2-S inhibition of TRPM2 and thus mediated the increased Ca2+ entry needed for apoptosis.
To reinforce the crucial role of TRPM2-S S39 phosphorylation in mediating TRPM2 channel activity, we mutated TRPM2-S S39 to aspartate (S39D) to mimic the effects of phosphorylation. This poly-His tagged phosphomimetic mutant was expressed in HPAECs (Online Figure VA), and we examined its ability to associate with TRPM2, and influence Ca2+ entry. Western blotting showed that phosphomimetic of TRPM2-S promoted TRPM2-S interaction with PKCα (Online Figure VB), but impaired its association with TRPM2 (Online Figure VC) under basal condition in the absence of H2O2 and after H2O2 challenge. Moreover, S39D mutation of TRPM2-S enhanced TRPM2-mediated Ca2+ entry after H2O2 challenge (Online Figure VD) consistent with the key role of PKCα phosphorylation of TRPM2-S in activating TRPM2 channel activity.
To elucidate the mechanism of PKCα regulation of TRPM2 channel activity further, we next coexpressed the TRPM2-S phosphorylation mutants with either PKCα siRNA or control siRNA in endothelial cells (Online Figure VI). Expression of (S39A)-TRPM2-S phosphodefective mutant in control siRNA-transduced cells as expected inhibited H2O2-elicited Ca2+ entry when compared with control cells, whereas expression of phosphomimetic mutant enhanced this response. The decreased H2O2-activated Ca2+ entry caused by depletion of PKCα was restored by expression of the (S39D)-TRPM2-S but not the (S39A)-TRPM2-S mutant, consistent with the essential role of PKCα phosphorylation of TRPM2-S in activating TRPM2 channel activity.
PKCα Mediates H2O2-Induced Apoptosis Through Activation of TRPM2
Fluorescence-activated cell sorting analysis showed that inhibition of PKCα activation or its silencing protected the cells from H2O2-induced apoptosis (Figure 6A and 6B). The role of PKCα in regulating TRPM2-mediated apoptosis was also seen in endothelial cells transduced with the (S39A)-TRPM2-S mutant (Figure 6A). Using lung endothelial cells cultured from TRPM2 or PKCα knockout mice to validate the above studies in human endothelial cells, we observed normal expression of TRPM2 in endothelial cells from PKCα knockout mice and normal expression of PKCα in endothelial cells from TRPM2 knockout mice (Figure 6A and 6B). The H2O2-induced Ca2+ entry was virtually abolished in TRPM2-null cells and was reduced by 40% in PKCα-null cells (Figure 6C). As in the human cells, H2O2-mediated apoptosis in WT mouse endothelial cells was concentration dependent (Figure 6D). Deletion of TRPM2 caused 2.4-fold rightward shift in the concentration–response curve for H2O2-induced apoptosis (EC50 shift, 213–553 µmol/L), indicating the crucial role of TRPM2 in mediating H2O2-induced apoptosis (Figure 6D). Deletion of the PKCα gene similarly inhibited apoptosis (EC50 shift, 213–512 µmol/L; Figure 6D).
Deletion of PKCα Gene in Mice Reduces TRPM2-Induced Endothelial Cell Apoptosis Improves Survival in Endotoxemia
To address the pathophysiological significance of PKCα phosphorylation of TRPM2 channel activity in mediating apoptosis in vivo, we examined the apoptosis response in mouse lung endothelial cells and survival of mice after intraperitoneal challenge with lipopolysaccharide (30 mg/kg), the Gram-negative bacterial endotoxin, which produces ROS in endothelial cells.34,35 Because TRPM2 and PKCα expressed in myeloid cells may also play a role in ROS production and apoptosis,36 we generated chimeric mice in which the PKCα- and TRPM2-deficient mice were transplanted with bone marrow cells from WT mice. These mice showed comparable TRPM2 and PKCα protein expression as WT (Figure 7A). We observed that either TRPM2 or PKCα deletion markedly reduced endothelial cell apoptosis in lungs 4 hours after lipopolysaccharide treatment when compared with WT mice (Figure 7B). In a positive control experiment, administration of the oxidant scavenger Tempol in mice 30 minutes before lipopolysaccharide also reduced oxidant-mediated lipopolysaccharide-induced apoptosis (Figure 7B). In addition, deletion of either TRPM2 or PKCα significantly improved survival rate of lipopolysaccharide-challenged mice (Figure 7C).
In the present study, we addressed the role of the ROS-activated TRPM2 channel in mediating endothelial cell apoptosis. We identified that the interaction of the 171-kDa TRPM210,12,27,37 with its 90-kDa splice variant TRPM2-S28 in the endothelial cell plasmalemma. This interaction functioned constitutively to restrain TRPM2 Ca2+ entry. However, ROS-induced activation of PKCα and resulting phosphorylation of TRPM2-S at Ser39 released the TRPM2-S inhibition of TRPM2 to induce the large Ca2+ influx required for activation of the caspase apoptosis program.
PKCα phosphorylation of TRPM2-S and the dissociation of TRPM2-S from TRPM2 increased the Ca2+ concentration in endothelial cells to 4-fold the baseline levels within the range of the Ca2+ burst required to signal apoptosis, which has the intracellular Ca2+ concentration threshold of 200 to 500 nmol/L.38 Inhibition of PKCα by preventing the phosphorylation of TRPM2-S reduced Ca2+ entry through TRPM2 by half this level, well below the Ca2+ threshold required for activation of caspase-mediated apoptosis.
A stop codon (TAG) on the TRPM2 gene is located at the splice junction between exons 16 and 17; hence, alternative splicing resulted in deletion of the 4 C-terminal transmembrane domains in TRPM2-S, the putative Ca2+-permeable pore region.28 The observation that TRPM2-S served as a dominant-negative for TRPM2, and its uncoupling from TRPM2 required for the full gating of Ca2+ identifies TRPM2-S as an important intrinsic negative regulator of endothelial cell apoptosis. H2O2 exposure or oxidants generated by mediators, such as TNF-α, induced the interaction between PKCα and TRPM2-S permitting the channel to open for Ca2+ entry. Thus, TRPM2-S only functioned to induce apoptosis when PKCα was activated and induced TRPM2-S phosphorylation. That TRPM2-S mediated Ca2+ gating through heterodimerization with TRPM2 is reminiscent of the finding in melanocytes that another splice variant member of the trp gene family TRPM1 interacted with full-length long form of TRPM1 to suppressed its activity.39
PKCα activation was shown to be crucial for the mechanism of H2O2-induced apoptosis through its binding to and phosphorylation of TRPM2-S. Mutation of the sole PKCα phosphorylation site on Ser39 of TRPM2-S N terminus to Ala resulted in failure of PKCα to phosphorylate TRPM2-S. This mutation in turn prevented TRPM2-S dissociation from TRPM2 and hence the apoptosis-inducing Ca2+ entry signal. Both K562 myeloid leukemia cell line that do not express the TRPM2-S14 and Jurkat t-lymphocyte cell line that expresses the short isoform at very low levels14,40 also did not undergo apoptosis secondary to TRPM2 activation,14,40 consistent with the critical role of TRPM2-S as the apoptosis-suppressing partner of TRPM2. Although PKCα activation contributed to TRPM2-induced endothelial apoptosis in the present through phosphorylation of TRPM2-S, it is important to note that PKCα signaling in other signaling pathways can induce endothelial injury. We have shown that PKCα phosphorylation of p120-catenin mediates disassociation of p120-catenin from VE-cadherin that resulted in disassembly of adherens junctions and disruption of VE barrier function.41 These studies showed the role of PKCα in mediating phosphorylation of p120-catenin in response to endotoxin and resultant increased lung vascular permeability.41 Thus, PKCα can function in a complex matter at multiple levels to induce endothelial dysfunction either via injury or through activating the apoptosis program.
The signaling pathway downstream of Ca2+ entry leading to cell death involves the activation of intrinsic executioner caspases (caspase-9) and extrinsic caspases (caspase-8) that activate the effector caspase-3 and caspase-7.14,42,43 These cleave cellular substrates, disrupting survival pathways and inducing membrane blebbing, cell shrinkage, and apoptotic body formation. PARP is part of a protective mechanism involved in repair of DNA damage44 and DNA stability.29 Inactivation of PARP by cleavage of the enzymatic domain after oxidant activation of TRPM2 also caused apoptosis14 similar to that seen with PKCα-induced uncoupling of TRPM2-S from TRPM2 in the present study.
We have uncovered in these studies a novel mechanism of TRPM2 activation resulting in Ca2+ entry secondary to PKCα-induced phosphorylation of TRPM2-S. A question arises about the relationship of this mechanism with TRPM2 activation induced by the generation of ADPR after the activation of poly(ADPR) polymerase.6,12 It is possible that both mechanisms function to activate TRPM2 secondary ROS stimulation (see Model Figure 8). ADPR generation after activation of poly(ADPR) polymerase may help to amplify the Ca2+ entry response. However, in the event that both TRPM2-S and TRPM2 are coexpressed as they are in endothelial cells, it is likely as the present results show that TRPM2-S functions by restraining the activity of TRPM2 (and hence suppresses apoptosis). However, when TRPM2-S is not expressed or poorly expressed, ADPR binding to Nudix box sequence would by default be the primary mechanism of TRPM2 activation, but it is not clear whether Ca2+ entry by this mechanism is sufficient to activate the proapoptotic caspases.
In summary, we identified a fundamental relationship between oxidant-activated TRPM2 channel and its associated short splice variant TRPM2-S in the gating of large Ca2+ influx and the critical role of loss of this interaction in mediating oxidant-induced apoptosis of endothelial cells. We demonstrated that apoptosis induced by this mechanism contributed to the mortality seen in endotoxin-challenged mice. PKCα functioned to induce phosphorylation of TRPM2-S, which prevented its association with TRPM2, and thereby activated Ca2+ gating and caspases. Thus, disabling TRPM2-S and TRPM2 interaction such as by inhibiting PKCα activation represents a novel strategy for abrogating apoptosis and resultant vascular injury and inflammation associated with apoptosis in diseases, such as acute lung injury and vascular inflammation.
We thank M. Ushio-Fukai for her insights. We also thank B.A. Miller (Pennsylvania State University School of Medicine) for kindly supplying the GFP-TRPM2-S construct, and GlaxoSmithKline for providing the Trpm2−/− C57BL/6 mice used in these experiments. We are also grateful to Dr Jeffrey Molkentin for PKCα–/– mice.
Sources of Funding
This work was supported by National Institutes of Health grant P01 HL077806-07 to A.B. Malik and by grant 10SDG2610057 from American Heart Association Midwest affiliate to C.M. Hecquet.
In November 2013, the average time from submission to first decision for all original research papers submitted to Circulation Research was 14.6 days.
The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.114.302414/-/DC1.
- Nonstandard Abbreviations and Acronyms
- adenosine diphosphate ribose
- fluorescence-activated cell sorting
- human pulmonary artery endothelial cells
- poly(ADPR) polymerase
- protein kinase C-α
- reactive oxygen species
- serine 39 mutated to alanine
- tumor necrosis factor-α
- melastatin-like transient receptor potential 2
- TRPM2 short variant
- vascular endothelial
- Received August 13, 2013.
- Revision received December 10, 2013.
- Accepted December 11, 2013.
- © 2013 American Heart Association, Inc.
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- Langhorst MF,
- Meinke S,
- Hein D,
- Krüger S,
- Weber K,
- Heiner I,
- Oppenheimer N,
- Schwarz JR,
- Guse AH
- Vandenbroucke St Amant E,
- Tauseef M,
- Vogel SM,
- Gao X-P,
- Mehta D,
- Komarova YA,
- Malik AB
- Wiseman H,
- Halliwell B
Novelty and Significance
What Is Known?
Oxidants induce injury to the vascular endothelium resulting in endothelial denudation, permeability alterations, edema formation, and inflammation.
Endothelial cell loss via apoptosis is a crucial feature of vascular injury and is implicated in the mechanism of lung injury induced by sepsis and other vascular diseases.
Activation of the cation (primarily Ca2+) permeable melastatin transient receptor potential 2 (TRPM2) channel during oxidative stress is linked to cell death.
What New Information Does This Article Contribute?
Oxidant activation of TRPM2 mediates lung endothelial cell apoptosis and is critically regulated by protein kinase C-α (PKCα).
TRPM2 in endothelial cells is normally impermeable to Ca2+ because of its binding to the short splice variant TRPM2 short variant (TRPM2-S).
Oxidants induce PKCα phosphorylation of TPPM2-S at Ser 39, which functions by releasing TRPM2-S inhibition of TRPM2 channel and thereby induces Ca2+ entry and sequestration to activate the apoptotic program.
Endothelial cell apoptosis is a crucial feature of vascular injury that leads to disruption of the endothelial barrier and to inflammation. Understanding the molecular mechanisms regulating apoptosis is vital for identifying novel targets for treating vascular injury and inflammatory disorders. We have uncovered a role of PKCα phosphorylation of the short splice variant of TRPM2, TRPM2-S, which acts as a phosphoswitch to regulate channel activity and results in calcium overload. Phosphorylation of TRPM2-S at Ser 39 caused release of TRPM2-S inhibition of TRPM2 and thereby activated Ca2+ gating. This unique mechanism of TRPM2 channel activation was crucial for induction of oxidant-mediated apoptosis of endothelial cells. Thus, oxidant-induced gating of Ca2+ via TRPM2 and apoptosis are critically dependent on PKCα phosphorylation of TRPM2-S at a specific site identifying a novel potential anti-inflammatory target.