Cooperative interaction of trp melastatin channel transient receptor potential (TRPM2) with its splice variant TRPM2 short variant is essential for endothelial cell apoptosis

Abstract

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.

Keywords: apoptosis; capillary permeability; endothelium; inflammation.

Figure 1. TRPM2 is required for H₂O₂-mediated apoptosis of endothelial cells

(A-B) Confluent endothelial cell monolayers (HPAECs) challenged with H₂O₂ or glucose oxidase/glucose were labeled with PE Annexin V-FITC and 7-AAD and analyzed by flow cytometry. (A) Concentration-response curve for H₂O₂-induced apoptosis after 24-h period of H₂O₂ exposure. Mean apoptotic values (± SEM) obtained by flow cytometry (top panel) are plotted as % apoptotic cells vs. [H₂O₂] (EC₅₀ = 136 μmol/L ; bottom panel) (n=3). (B) Left, representative flow cytometry histograms 6 or 24 h after exposure to 300 μmol/L H₂O₂ or glucose oxidase/glucose (90 min), with or without prior TRPM2 silencing or inhibition with blocking TRPM2 antibody. Right panel, mean percent apoptotic cells at 0, 6, or 24 h after H₂O₂ treatment (± SEM, n=3). Baseline values were not significantly altered by TRPM2 silencing or TRPM2 blocking Ab. (C-D) Apoptosis in lungs of TRPM2^−/− and wild-type (WT) mice measured 3 h after perfusion with solution containing H₂O₂ (300 μmol/L) or glucose oxidase/glucose (75 min). (C) Right, immunofluorescent staining of frozen lung sections using VE-cadherin (red) and cleaved-PARP (green) antibodies and DAPI (blue)(n=3). Scale bar: 50 μm. Left, quantification of apoptotic endothelial cells (±SEM; n=6), * p ≤ 0.007 vs. WT lungs (t-test). (D) Western immunoblots (Right) and quantification of inactive or cleaved caspase-3 and active or cleaved PARP in lung homogenates (Left). GAPDH was used as loading control. (±SEM; n=3), * p ≤ 0.002, ** p ≤ 0.009 vs. WT perfused-lungs.

Figure 2. PKCα binding and phosphorylation of TRPM2-S

HPAECs were transduced with siRNA to suppress the expression of TRPM2 or PKCα or pretreated with PKC inhibitors (100 nmol/L Gö6976, 1 μmol/L PKCαi or 1 μmol/L PKCβIIi). Cells were challenged with 300 μmol/L H₂O₂ for the indicated times at 37°C. (A) Western blots from HPAEC lysates identifying expression of TRPM2 isoforms and PKCα; GAPDH was used as loading control. Endothelial cell expression of TRPM2-S (90 kDa) and two TRPM2 splice variants (140 and 171 kDa) were suppressed by TRPM2 silencing and PKCα protein expression (82 kDa) was blocked by PKCα silencing. (B) H₂O₂ induced association of PKCα and TRPM2-S. PKCα was immunoprecipitated from cell lysates. Following SDS-PAGE and electrophoretic transfer, co-immunoprecipitated TRPM2 was detected using an antibody recognizing both TRPM2 and TRPM2-S. TRPM2-S associated with PKCα immediately after H₂O₂ exposure whereas inhibition of PKCα activation prevented the association. (C) H₂O₂ induced PKCα-dependent phosphorylation of 90kDa TRPM2-S splice variant. TRPM2 was immunoprecipitated from same cell lysates using an Ab that recognized either TRPM2 isoform. Top panel: Blots showing phosphorylation of TRPM2 using monoclonal anti-phospho-Ser antibody. Membrane was reblotted with anti-phospho-PKCα (Ser 657) Ab to check the amount of phosphorylated PKCα in the complex (middle panel). Immunoprecipitation of TRPM2 was verified using an anti-TRPM2 Ab (lower panel). (D) Mean densitometric values (± SEM; n=3-4) obtained in B-C showing that PKCα inhibition prevented H₂O₂-induced association of PKCα with TRPM2-S and phosphorylation of TRPM2-S

Figure 3. PKCα and TRPM2 cooperation mediates Ca²⁺ entry in endothelial cells required for signaling apoptosis

(A) Ca²⁺ repletion transients generated by “Ca²⁺-add-back” in the presence of H₂O₂. Cultured HPAECs were loaded with Fura-2 Ca²⁺ dye, washed, and transferred to Ca²⁺-free medium. In control cells, H₂O₂ (100 μM) elicited a marked Ca²⁺ transient on Ca repletion (red trace). Ca²⁺ transients were blocked by TRPM2 silencing (grey trace) and reduced by Gö6976 (100 nmol/L; green trace), PKCαi (1 μmol/L; cyan trace), or after PKCα silencing (yellow trace); PKCβIIi (1 μmol/L, navy trace) and control siRNA (purple trace) had no effect. Ordinate gives [Ca²⁺]_i as 340:380 nm ratio. (B) Summary of mean ratiometric data (± SEM) for the peak intracellular [Ca²⁺]_i obtained in (E) (n = 3 to 5). *P ≤ 0.0002 vs. control (t-test).

Figure 4. PKCα binds TRPM2-S at Ser 39 and activates apoptosis-inducing Ca²⁺ entry signal in endothelial cells

The sole predicted PKCα phosphorylation site near the TRPM2-S N-terminus at Ser 39 was mutated by Ala substitution (resulting in phosphodefective mutant). HPAEC monolayers transduced with mutant TRPM2-S (tagged on its carboxy-terminal end with poly-His residues) were grown to confluence for Western blot analysis (A through C) or intracellular Ca²⁺ measurements using fura-2 (D). (A-C) Cells were exposed to 300 μM H₂O₂ for the indicated times. (A) Western blots for TRPM2, PKCα, and GAPDH expression in cells transduced with phosphodefective construct. Transfected protein was detected with an anti-His Ab confirming the expression of mutant TRPM2-S construct. (B) PKCα was immunoprecipitated from cell lysates with an antibody and co-immunoprecipitated TRPM2 protein was detected using an Ab recognizing both forms of TRPM2. Graph in B shows mean densitometric values (± SEM; n=3-4). Mutation of Ser 39 with Ala in TRPM2-S prevented TRPM2-S association with PKCα. (C) TRPM2 was immunoprecipitated from the same lysates and phosphorylated TRPM2 was detected using anti-phospho-Ser Ab. Graph in C shows mean densitometric values (± SEM; n=3-4). Ala substitution at Ser 39 abrogated H₂O₂-induced phosphorylation of TRPM2-S confirming the importance of the PKCα phosphorylation site on TRPM2-S at Ser 39. (D) Ca²⁺ mobilization assay was carried out using the “Ca²⁺ add-back” protocol. Transduction of phosphodefective TRPM2-S mutant suppressed H₂O₂-induced Ca²⁺ entry. * p = 0.0001 compared with control (t-test). (n = 3 per bar); error bars, ± SEM.

Figure 5. PKCα phosphorylation of TRPM2-S mediates TRPM2-S dissociation from TRPM2 resulting apoptosis-inducing Ca²⁺ entry signal

HPAEC monolayers were pretreated with PKCα and PKCβII (control) inhibitors 45 min prior to experiments. (A) TRPM2 was immunoprecipitated from cell lysates with an anti-TRPM2 antibody recognizing the region present only on the long isoform following H₂O₂ (300 μM) exposure for indicated times. Co-immunoprecipitated short isoform was then detected using an Ab that recognizes both TRPM2 and TRPM2-S. At 0 time, TRPM2 in plasma membrane was associated with its short isoform, and H₂O₂ application induced rapid dissociation of TRPM2-S from TRPM2. H₂O₂-mediated dissociation was suppressed by PKCα inhibition and did not occur when Ser 39 of TRPM2-S was substituted by Ala. (B) Western blots were quantified by densitomitry. Co-immunoprecipitated TRPM2-S was quantified as ratio to TRPM2 and plotted relative to zero time value (mean ± SEM; n = 3)..

Figure 6. H₂O₂-induced endothelial cell apoptosis resulting from TRPM2-mediated Ca²⁺ influx

HPAECs (A) and mouse lung endothelial cells (B-D) challenged with H₂O₂ were labeled with PE-Annexin/7-AAD. (A) Flow-cytometry histograms (Left) and summary plots of apoptosis (Right) 0, 6, and 24 h after challenge with H₂O₂ (300 μmol/L) or glucose oxidase/glucose to generate 320 nmol/L H₂O₂/min for 90 min, as a function of PKCα inhibition or silencing (± SEM, n=5). (B-D) Mouse endothelial cells were isolated from lungs of TRPM2^−/−, PKCα^−/−, and WT mice. (B) Western blot verifying absence of PKCα expression in PKCα^−/− cells and of TRPM2 expression in TRPM2^−/− cells. (C) Left, Ca²⁺ mobilization assay using “Ca²⁺ add-back” protocol with the Fluor-3 Ca²⁺ indicator. Right, Mean ratiometric values (± SEM) for steady-state [Ca²⁺]i (n=6). *p ≤ 0.0001 vs. WT cells (t-test). (D) Dose-response curve for H₂O₂-induced apoptosis in mouse endothelial cells detected by flow cytometry. Deletion of PKCα or TRPM2 caused ~2.5-fold rightward shift in the dose-response curve.

Figure 7. PKCα interaction with TRPM2 in mice is required lung endothelial cell apoptosis in response to LPS and contributes to mortality

WT, TRPM2^−/− and PKCα^−/− mice were transplanted with bone marrow cells isolated from WT mice 8 weeks prior to Western blotting (A), apoptosis (B-C) and survival (D) studies. (A) Representative Western blots verifying expression of TRPM2, PKCα, and β-actin in bone marrow of transplanted mice (molecular masses of 171, 82, and 45 kDa, respectively). Left, protein was quantified by densitometry. TRPM2 and PKCα densities were normalized to β-actin and plotted as percentage of untreated control (mean ± SEM for n=3). (B-C) Endothelial apoptosis (B) and oxidant production (C) were determined in lungs of mice 4h after intraperitoneal injection of lipopolysaccharide (LPS, 40 mg/kg) and in lungs of mice treated with the oxidant scavenger Tempol (100 mg/kg, IP) 30 min before injection of LPS. (B) Right, immunofluorescent staining of frozen lung sections using VE-cadherin (red) antibody, TUNEL (green), and DAPI (blue)(n=3). Scale bar: 50 μm. Left, quantification of apoptotic endothelial cells (±SEM; n=3), * p ≤ 0.001 and ^# p ≤ 0.005 vs. LPS-treated WT lungs. TRPM2 and PKCα deletion significantly reduced LPS-induced endothelial apoptosis. (C) Lungs were homogenized and assayed for H₂O₂ accumulation using the horseradish peroxide-linked Amplex Red assay. H₂O₂ was determined spectrophotometrically from its absorbance at 570 nM and corrected for total protein (± SEM, n=3). * p ≤ 0.005 vs. LPS-treated control lungs (D) Deletion of either TRPM2 or PKCα reduced LPS-induced lethality in mice. LPS (30 mg/kg) was injected intraperitoneally and survival was assessed every 12 h during the experiment (WT, n=16; TRPM2^−/−, n= 16; and PKCα^−/−, n=12). Statistical analysis was performed using the log-rank test. *p ≤ 0.03 and ^#p ≤ 0.05 vs. WT cells.

Figure 8. PKCα-induced phosphorylation of TRPM2-S mediates oxidant-induced TRPM2 channel opening and supra-normal Ca²⁺ entry required for activation of the apoptosis program

In resting cells, TRPM2-S associates with TRPM2 to restrict Ca²⁺ entry (the channel is in a closed conformation state). Oxidative stress induces PKCα activation resulting in binding and phosphorylation of TRPM2-S on Ser 39. TRPM2-S uncoupling from TRPM2 in turn opens the channel and Ca²⁺ entry resulting in activation of executioner caspases and endothelial apoptosis. In this model the generation of the endogenous TRPM2 agonist, ADPR may also contribute to opening TRPM2 channel.