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Mechanisms for vascular cell adhesion molecule-1 activation of ERK1/2 during leukocyte transendothelial migration.

Abdala-Valencia H, Berdnikovs S, Cook-Mills JM - PLoS ONE (2011)

Bottom Line: In this study, we identified a mechanism for VCAM-1 activation of ERK1/2 in human and mouse endothelial cells.VCAM-1 signaling, which occurs through endothelial cell NADPH oxidase, protein kinase Cα (PKCα), and protein tyrosine phosphatase 1B (PTP1B), activates endothelial cell ERK1/2.Inhibition of these signals blocked VCAM-1 activation of ERK1/2, indicating that ERK1/2 is activated downstream of PTP1B during VCAM-1 signaling.

View Article: PubMed Central - PubMed

Affiliation: Allergy-Immunology Division, Northwestern University Feinberg School of Medicine, Chicago, Illinois, United States of America.

ABSTRACT

Background: During inflammation, adhesion molecules regulate recruitment of leukocytes to inflamed tissues. It is reported that vascular cell adhesion molecule-1 (VCAM-1) activates extracellular regulated kinases 1 and 2 (ERK1/2), but the mechanism for this activation is not known. Pharmacological inhibitors of ERK1/2 partially inhibit leukocyte transendothelial migration in a multi-receptor system but it is not known whether VCAM-1 activation of ERK1/2 is required for leukocyte transendothelial migration (TEM) on VCAM-1.

Methodology/principal findings: In this study, we identified a mechanism for VCAM-1 activation of ERK1/2 in human and mouse endothelial cells. VCAM-1 signaling, which occurs through endothelial cell NADPH oxidase, protein kinase Cα (PKCα), and protein tyrosine phosphatase 1B (PTP1B), activates endothelial cell ERK1/2. Inhibition of these signals blocked VCAM-1 activation of ERK1/2, indicating that ERK1/2 is activated downstream of PTP1B during VCAM-1 signaling. Furthermore, VCAM-1-specific leukocyte migration under physiological laminar flow of 2 dynes/cm(2) was blocked by pretreatment of endothelial cells with dominant-negative ERK2 K52R or the MEK/ERK inhibitors, PD98059 and U0126, indicating for the first time that ERK regulates VCAM-1-dependent leukocyte transendothelial migration.

Conclusions/significance: VCAM-1 activation of endothelial cell NADPH oxidase/PKCα/PTP1B induces transient ERK1/2 activation that is necessary for VCAM-1-dependent leukocyte TEM.

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Mechanisms for VCAM-1 activation of ERK1/2 in endothelial cell lines.Monolayers of mHEVa cells were nontreated (NT) or incubated for 30 minutes with apocynin (4 mM), PD98059 (30uM), U0126 (40 µM), Gö-6976 (2.3 nM), CinnGEL 2-methylester (10 µM), or catalase (5000 U/ml) where indicated. These are the optimal doses for these inhibitors as we have previously described [5], [7], [8]. At these concentrations, none of the inhibitors had any significant effects on the basal level of ERK1/2 in the absence of anti-VCAM-1 stimulation (data not shown). After treatement with the inhibitor, the endothelial cells were stimulated with anti-VCAM-1 antibody plus a secondary antibody to crosslink VCAM-1 for 30 min under static conditions. We examined phosphorylation of A,B,F) ERK1/2 Thr202/Tyr204 (P-ERK1/2), C) PKCα Thr638 (P-PKCα), or D) MEK1/2 Ser217/221 (P-MEK1/2) by western blot. E) mHEVa cells were treated with exogenous 1 µM H2O2 for 10–60 minutes and ERK1/2 Ser217/221 phosphorylation was determined by western blot. The phosphorylation status of ERK1/2 Thr202/Tyr204, PKCα Thr638, or MEK1/2 Ser217/221 is presented as the fold increase in the ratio of the relative intensity of the phosphorylated enzyme to total ERK1/2, total PKCα or total MEK1/2 expression. Representative western blots are shown and data are presented as the mean ± standard deviation from 3 experiments. (A, B) *, p<0.05 less than anti-VCAM-1-treated group. (C–F) *, p<0.05 greater than NT.
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pone-0026706-g004: Mechanisms for VCAM-1 activation of ERK1/2 in endothelial cell lines.Monolayers of mHEVa cells were nontreated (NT) or incubated for 30 minutes with apocynin (4 mM), PD98059 (30uM), U0126 (40 µM), Gö-6976 (2.3 nM), CinnGEL 2-methylester (10 µM), or catalase (5000 U/ml) where indicated. These are the optimal doses for these inhibitors as we have previously described [5], [7], [8]. At these concentrations, none of the inhibitors had any significant effects on the basal level of ERK1/2 in the absence of anti-VCAM-1 stimulation (data not shown). After treatement with the inhibitor, the endothelial cells were stimulated with anti-VCAM-1 antibody plus a secondary antibody to crosslink VCAM-1 for 30 min under static conditions. We examined phosphorylation of A,B,F) ERK1/2 Thr202/Tyr204 (P-ERK1/2), C) PKCα Thr638 (P-PKCα), or D) MEK1/2 Ser217/221 (P-MEK1/2) by western blot. E) mHEVa cells were treated with exogenous 1 µM H2O2 for 10–60 minutes and ERK1/2 Ser217/221 phosphorylation was determined by western blot. The phosphorylation status of ERK1/2 Thr202/Tyr204, PKCα Thr638, or MEK1/2 Ser217/221 is presented as the fold increase in the ratio of the relative intensity of the phosphorylated enzyme to total ERK1/2, total PKCα or total MEK1/2 expression. Representative western blots are shown and data are presented as the mean ± standard deviation from 3 experiments. (A, B) *, p<0.05 less than anti-VCAM-1-treated group. (C–F) *, p<0.05 greater than NT.

Mentions: Then, we determined whether ERK1/2 is activated by VCAM-1′s signaling cascade (NADPH oxidase, 1 µM H2O2, PKCα and PTP1B). Anti-VCAM-1 activation of ERK1/2 Thr202/Tyr204 phosphorylation in mHEVa cells under static conditions was blocked by the NADPH oxidase inhibitor apocynin or scavenging extracellular ROS with catalase (Figure 4A, C), suggesting that ERK1/2 functions downstream of NADPH oxidase. In the absence of VCAM-1 stimulation, apocynin and catalase did not alter background ERK1/2 phosphorylation (data not shown). Since we previously reported that binding to VCAM-1 stimulates endothelial cell NADPH oxidase, resulting in the generation of 1 µM H2O2 [5] and that exogenous addition of 1 µM H2O2 is sufficient for the activation of PKCα and PTP1B in endothelial cells at 10 minutes [7], [8], it was determined whether exogenous 1 µM H2O2 activated endothelial cell ERK1/2. At 10 minutes, 1 µM H2O2 significantly increased ERK1/2 Thr202/Tyr204 phosphorylation in mHEVa cells (Figure 4D). Therefore, exogenous ROS, at concentrations that are generated by VCAM-1-outside-in signals [6], stimulate a significant increase in ERK1/2 phosphorylation. Anti-VCAM-1 activation of ERK1/2 Thr202/Tyr204 phosphorylation in mHEVa cells was also blocked by the PKCα inhibitor Gö-6976, and the PTP1B inhibitor CinnGEL 2-methyl ester (Figure 4B). At these concentrations, none of the inhibitors had any significant effects on the basal level of ERK1/2 in the absence of anti-VCAM-1 stimulation (data not shown). The inhibitors used at these optimal doses did not affect cell viability as we previously reported [7], [8].


Mechanisms for vascular cell adhesion molecule-1 activation of ERK1/2 during leukocyte transendothelial migration.

Abdala-Valencia H, Berdnikovs S, Cook-Mills JM - PLoS ONE (2011)

Mechanisms for VCAM-1 activation of ERK1/2 in endothelial cell lines.Monolayers of mHEVa cells were nontreated (NT) or incubated for 30 minutes with apocynin (4 mM), PD98059 (30uM), U0126 (40 µM), Gö-6976 (2.3 nM), CinnGEL 2-methylester (10 µM), or catalase (5000 U/ml) where indicated. These are the optimal doses for these inhibitors as we have previously described [5], [7], [8]. At these concentrations, none of the inhibitors had any significant effects on the basal level of ERK1/2 in the absence of anti-VCAM-1 stimulation (data not shown). After treatement with the inhibitor, the endothelial cells were stimulated with anti-VCAM-1 antibody plus a secondary antibody to crosslink VCAM-1 for 30 min under static conditions. We examined phosphorylation of A,B,F) ERK1/2 Thr202/Tyr204 (P-ERK1/2), C) PKCα Thr638 (P-PKCα), or D) MEK1/2 Ser217/221 (P-MEK1/2) by western blot. E) mHEVa cells were treated with exogenous 1 µM H2O2 for 10–60 minutes and ERK1/2 Ser217/221 phosphorylation was determined by western blot. The phosphorylation status of ERK1/2 Thr202/Tyr204, PKCα Thr638, or MEK1/2 Ser217/221 is presented as the fold increase in the ratio of the relative intensity of the phosphorylated enzyme to total ERK1/2, total PKCα or total MEK1/2 expression. Representative western blots are shown and data are presented as the mean ± standard deviation from 3 experiments. (A, B) *, p<0.05 less than anti-VCAM-1-treated group. (C–F) *, p<0.05 greater than NT.
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Related In: Results  -  Collection

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pone-0026706-g004: Mechanisms for VCAM-1 activation of ERK1/2 in endothelial cell lines.Monolayers of mHEVa cells were nontreated (NT) or incubated for 30 minutes with apocynin (4 mM), PD98059 (30uM), U0126 (40 µM), Gö-6976 (2.3 nM), CinnGEL 2-methylester (10 µM), or catalase (5000 U/ml) where indicated. These are the optimal doses for these inhibitors as we have previously described [5], [7], [8]. At these concentrations, none of the inhibitors had any significant effects on the basal level of ERK1/2 in the absence of anti-VCAM-1 stimulation (data not shown). After treatement with the inhibitor, the endothelial cells were stimulated with anti-VCAM-1 antibody plus a secondary antibody to crosslink VCAM-1 for 30 min under static conditions. We examined phosphorylation of A,B,F) ERK1/2 Thr202/Tyr204 (P-ERK1/2), C) PKCα Thr638 (P-PKCα), or D) MEK1/2 Ser217/221 (P-MEK1/2) by western blot. E) mHEVa cells were treated with exogenous 1 µM H2O2 for 10–60 minutes and ERK1/2 Ser217/221 phosphorylation was determined by western blot. The phosphorylation status of ERK1/2 Thr202/Tyr204, PKCα Thr638, or MEK1/2 Ser217/221 is presented as the fold increase in the ratio of the relative intensity of the phosphorylated enzyme to total ERK1/2, total PKCα or total MEK1/2 expression. Representative western blots are shown and data are presented as the mean ± standard deviation from 3 experiments. (A, B) *, p<0.05 less than anti-VCAM-1-treated group. (C–F) *, p<0.05 greater than NT.
Mentions: Then, we determined whether ERK1/2 is activated by VCAM-1′s signaling cascade (NADPH oxidase, 1 µM H2O2, PKCα and PTP1B). Anti-VCAM-1 activation of ERK1/2 Thr202/Tyr204 phosphorylation in mHEVa cells under static conditions was blocked by the NADPH oxidase inhibitor apocynin or scavenging extracellular ROS with catalase (Figure 4A, C), suggesting that ERK1/2 functions downstream of NADPH oxidase. In the absence of VCAM-1 stimulation, apocynin and catalase did not alter background ERK1/2 phosphorylation (data not shown). Since we previously reported that binding to VCAM-1 stimulates endothelial cell NADPH oxidase, resulting in the generation of 1 µM H2O2 [5] and that exogenous addition of 1 µM H2O2 is sufficient for the activation of PKCα and PTP1B in endothelial cells at 10 minutes [7], [8], it was determined whether exogenous 1 µM H2O2 activated endothelial cell ERK1/2. At 10 minutes, 1 µM H2O2 significantly increased ERK1/2 Thr202/Tyr204 phosphorylation in mHEVa cells (Figure 4D). Therefore, exogenous ROS, at concentrations that are generated by VCAM-1-outside-in signals [6], stimulate a significant increase in ERK1/2 phosphorylation. Anti-VCAM-1 activation of ERK1/2 Thr202/Tyr204 phosphorylation in mHEVa cells was also blocked by the PKCα inhibitor Gö-6976, and the PTP1B inhibitor CinnGEL 2-methyl ester (Figure 4B). At these concentrations, none of the inhibitors had any significant effects on the basal level of ERK1/2 in the absence of anti-VCAM-1 stimulation (data not shown). The inhibitors used at these optimal doses did not affect cell viability as we previously reported [7], [8].

Bottom Line: In this study, we identified a mechanism for VCAM-1 activation of ERK1/2 in human and mouse endothelial cells.VCAM-1 signaling, which occurs through endothelial cell NADPH oxidase, protein kinase Cα (PKCα), and protein tyrosine phosphatase 1B (PTP1B), activates endothelial cell ERK1/2.Inhibition of these signals blocked VCAM-1 activation of ERK1/2, indicating that ERK1/2 is activated downstream of PTP1B during VCAM-1 signaling.

View Article: PubMed Central - PubMed

Affiliation: Allergy-Immunology Division, Northwestern University Feinberg School of Medicine, Chicago, Illinois, United States of America.

ABSTRACT

Background: During inflammation, adhesion molecules regulate recruitment of leukocytes to inflamed tissues. It is reported that vascular cell adhesion molecule-1 (VCAM-1) activates extracellular regulated kinases 1 and 2 (ERK1/2), but the mechanism for this activation is not known. Pharmacological inhibitors of ERK1/2 partially inhibit leukocyte transendothelial migration in a multi-receptor system but it is not known whether VCAM-1 activation of ERK1/2 is required for leukocyte transendothelial migration (TEM) on VCAM-1.

Methodology/principal findings: In this study, we identified a mechanism for VCAM-1 activation of ERK1/2 in human and mouse endothelial cells. VCAM-1 signaling, which occurs through endothelial cell NADPH oxidase, protein kinase Cα (PKCα), and protein tyrosine phosphatase 1B (PTP1B), activates endothelial cell ERK1/2. Inhibition of these signals blocked VCAM-1 activation of ERK1/2, indicating that ERK1/2 is activated downstream of PTP1B during VCAM-1 signaling. Furthermore, VCAM-1-specific leukocyte migration under physiological laminar flow of 2 dynes/cm(2) was blocked by pretreatment of endothelial cells with dominant-negative ERK2 K52R or the MEK/ERK inhibitors, PD98059 and U0126, indicating for the first time that ERK regulates VCAM-1-dependent leukocyte transendothelial migration.

Conclusions/significance: VCAM-1 activation of endothelial cell NADPH oxidase/PKCα/PTP1B induces transient ERK1/2 activation that is necessary for VCAM-1-dependent leukocyte TEM.

Show MeSH
Related in: MedlinePlus