Vitamin E isoforms differentially regulate intercellular adhesion molecule-1 activation of PKCα in human microvascular endothelial cells

Abstract

Aims: ICAM-1-dependent leukocyte recruitment in vivo is inhibited by the vitamin E isoform d-α-tocopherol and elevated by d-γ-tocopherol. ICAM-1 is reported to activate endothelial cell signals including protein kinase C (PKC), but the PKC isoform and the mechanism for ICAM-1 activation of PKC are not known. It is also not known whether ICAM-1 signaling in endothelial cells is regulated by tocopherol isoforms. We hypothesized that d-α-tocopherol and d-γ-tocopherol differentially regulate ICAM-1 activation of endothelial cell PKC.

Results: ICAM-1 crosslinking activated the PKC isoform PKCα but not PKCβ in TNFα-pretreated human microvascular endothelial cells. ICAM-1 activation of PKCα was blocked by the PLC inhibitor U73122, ERK1/2 inhibitor PD98059, and xanthine oxidase inhibitor allopurinol. ERK1/2 activation was blocked by inhibition of XO and PLC but not by inhibition of PKCα, indicating that ERK1/2 is downstream of XO and upstream of PKCα during ICAM-1 signaling. During ICAM-1 activation of PKCα, the XO-generated ROS did not oxidize PKCα. Interestingly, d-α-tocopherol inhibited ICAM-1 activation of PKCα but not the upstream signal ERK1/2. The d-α-tocopherol inhibition of PKCα was ablated by the addition of d-γ-tocopherol.

Conclusions: Crosslinking ICAM-1 stimulated XO/ROS which activated ERK1/2 that then activated PKCα. ICAM-1 activation of PKCα was inhibited by d-α-tocopherol and this inhibition was ablated by the addition of d-γ-tocopherol. These tocopherols regulated ICAM-1 activation of PKCα without altering the upstream signal ERK1/2. Thus, we identified a mechanism for ICAM-1 activation of PKC and determined that d-α-tocopherol and d-γ-tocopherol have opposing regulatory functions for ICAM-1-activated PKCα in endothelial cells.

Figure 1. ICAM-1 activates PKCα but not PKCβII in HMVECLs. A

) At 70% confluence, HMVECLs cells pretreated with 10 ng/ml TNFα to induce ICAM-1 expression. At 24 hrs, the cells were suspended and immunolabeled with anti-ICAM-1 antibodies and examined by flow cytometry for ICAM-1 expression. B–D) HMVECLs were pretreated with TNFα as in panel A. At 24 hrs, the endothelial cells were nonstimulated (NS) or stimulated with a confluent monolayer of anti-ICAM-1-coated beads or the control anti-PECAM-1-coated beads for 20 minutes in B and D or for the times indicated in C. The cells were lysed and the activation of PKCβII and PKCα was examined by western blot with B) anti-phospho-PKCβII^Thr641 or anti-PKCβII or C–D) anti-phospho- PKCα^Thr638 or anti-PKCα. Shown are representative blots. Shown are the mean ± SEM of 3 experiments. NT, nontreated. *, p<0.05 as compared to the nontreated (NT) groups.

Figure 2. Anti-ICAM-1 stimulation induces an increase in PKCα^{Thr 638} phosphorylation through XO/PLC/ERK1/2 activities but not by oxidation. A,B,D

) 70% confluent monolayers of HMVECLs were pretreated with TNFα to induce ICAM-1 expression. Then, the endothelial cells were were treated for 1 hr with the pharmacological inhibitors allopurinol (0.3 mg/ml), PD98056 (20 µM), U73122 (10 µM), apocynin (4 mM), Ly294002 (100 nM), Go6976 (2.3 nM), PP2 (10 µM), apocynin (4 mM) or the solvent control DMSO (0.01%). The cells were stimulated with anti-ICAM-1-coated beads and examined for (A,B) PKCα^Thr638 phosphorylation and total PKCα by western blot or examined for (D) cytotoxicity by the Vybrant cytotoxicity assay. In panel D, 200 µM H₂O₂ was used as a positive control for cytotoxicity. C) To label non-oxidized cysteines, BIAM was added to the cell lysates. PKCα was immunoprecipitated and BIAM labeling was detected by western blot with HRP-conjugated streptavidin. The blots were reprobed for total PKCα. The positive control for oxidation includes lysates oxidized with 200 µM H₂O₂ for 20 min before addition of BIAM. Loss of BIAM labeling in the western blot indicates PKCα oxidation. Shown are the means ± SEM from 3 experiments. *, p<0.05 compared to nonstimulated (NS) controls.

Figure 3. ICAM-1 activation of PKCα phosphorylation in HMVECLs is mediated by xanthine oxidase stimulation of ERK1/2.

70% confluent monolayers of HMVECLs were pretreated with TNFα to induce ICAM-1 expression. A) Time course for anti-ICAM-1-coated bead stimulation of ERK1/2 Thr²⁰²/Tyr²⁰⁴ phosphorylation (P-ERK1/2). *, p<0.05 as compared to the nontreated control group. B) TNFα-pretreated HMVECLs were incubated for 1 hr with the pharmacological inhibitors allopurinol (0.3 mg/ml), U73122 (10 µM), and Go6976 (2.3 nM), apocynin (4 mM), or the solvent control DMSO (0.01%). The cells were nonstimulated (NS) or stimulated with anti-ICAM-1-coated beads and examined by western blot for ERK1/2 Thr²⁰²/Tyr²⁰⁴ phosphorylation and total ERK1/2. Shown are the means ± SEM from 3 experiments. Panel A: *, p<0.05 as compared to 0 minutes. Panel B: *, p<0.05 as compared to the anti-ICAM-1-stimulated group.

Figure 4. Tocopherol treatment of TNFα-stimulated HMVECLs was not cytotoxic and did not alter ICAM-1 expression.

70% confluent monolayers of HMVECLs were pretreated for 6 hrs with TNFα (10 ng/ml) to induce ICAM-1 expression. Then, the endothelial cells were treated for 16 hrs with α-tocopherol (α-toc) (40, 60 or 80 µM), γ-tocopherol (γ-toc) (1, 2 or 4 µM) or the vehicle control DMSO (0.01%). The cells were examined for cytotoxicity with the A) Vybrant Cytotoxicity Assay or examined by B,C) immunolabeling with FITC-labeled anti-ICAM-1 and flow cytometry. Shown are the means ± SEM from 3 experiments. MFI, mean fluorescence intensity. *, p<0.05 compared with non-treated (NT) cells.

Figure 5. D-α-tocopherol inhibits ICAM-1-activated PKCα but not ERK1/2 in HMVECLs and the inhibition by d-α-tocopherol is abrogated by d-γ-tocopherol.

At 70% confluence, HMVECLs cells were pretreated with TNFα for 6hrs and then treated with tocopherols or the solvent control 0.01% DMSO for 16 hrs. A) d-α-tocopherol (α-toc) regulation of ICAM-1-stimulated PKCα Thr⁶³⁸ phosphorylation (PKCα P-Thr⁶³⁸). B) d-γ-tocopherol (γ-toc) regulation of ICAM-1-stimulated PKCα P-Thr⁶³⁸. C) d-α-tocopherol + d-γ-tocopherol regulation of ICAM-1-stimulated PKCα P-Thr⁶³⁸. D) d-α-tocopherol does not alter background PKCα P-Thr⁶³⁸ (no anti-ICAM-1). E) d-α-tocopherol does not regulate ERK1/2 Thr²⁰²/Tyr²⁰⁴ phosphorylation (P-ERK1/2). F) d-γ-tocopherol does not regulate ERK1/2 Thr²⁰²/Tyr²⁰⁴ phosphorylation. Shown are the means ± SEM from 3 experiments. *, p<0.05 as compared with the anti-ICAM-1-stimulated groups.