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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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Related in: MedlinePlus

Mechanisms for VCAM-1 activation of ERK1/2 in HMVEC-L.Treatment of HMVEC-L cells overnight with 10ng/ml TNF-α induced VCAM-1 expression (data not shown). A) Confluent monolayers of TNF-α-treated HMVEC-Ls were nontreated (NT) or treated with 27 µg/ml anti-VCAM-1 plus 15 µg/ml of a secondary antibody to crosslink and stimulate VCAM-1. Phosphorylation of ERK1/2 Thr202/Tyr204 (P-ERK1/2) and total expression of ERK1/2 was examined by western blot using rabbit anti-phospho ERK1/2 Thr202/Tyr204 (1/1000) followed by HRP-conjugated anti-rabbit (1/2000) and ECL detection. B) Confluent monolayers of TNF-α stimulated HMVEC-L cells in 12 well plates were nontreated or incubated for 30 minutes with the solvent control DMSO, apocynin (4 mM), Gö-6976 (2.3 nM) or CinnGEL 2-methylester (10 µM). These endothelial cells were then stimulated with anti-VCAM-1 antibody plus a secondary antibody for 15 minutes. The apocynin, DMSO, Gö-6976 or CinnGEL 2-methylester had no effect on endothelial cell viability as determined by trypan blue exclusion and had no effect on VCAM-1 expression as determined by flow cytometry (data not shown). Representative western blots are shown. Data presented are the mean ± standard deviation from 3 experiments. The phosphorylation status of ERK1/2 is presented as the fold increase in the ratio of the relative intensity of P-ERK1/2 divided by the relative intensity of the loading control (total ERK1/2). *, p<0.05 compared to A) NT cells or B) anti-VCAM-1 stimulated cells.
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pone-0026706-g005: Mechanisms for VCAM-1 activation of ERK1/2 in HMVEC-L.Treatment of HMVEC-L cells overnight with 10ng/ml TNF-α induced VCAM-1 expression (data not shown). A) Confluent monolayers of TNF-α-treated HMVEC-Ls were nontreated (NT) or treated with 27 µg/ml anti-VCAM-1 plus 15 µg/ml of a secondary antibody to crosslink and stimulate VCAM-1. Phosphorylation of ERK1/2 Thr202/Tyr204 (P-ERK1/2) and total expression of ERK1/2 was examined by western blot using rabbit anti-phospho ERK1/2 Thr202/Tyr204 (1/1000) followed by HRP-conjugated anti-rabbit (1/2000) and ECL detection. B) Confluent monolayers of TNF-α stimulated HMVEC-L cells in 12 well plates were nontreated or incubated for 30 minutes with the solvent control DMSO, apocynin (4 mM), Gö-6976 (2.3 nM) or CinnGEL 2-methylester (10 µM). These endothelial cells were then stimulated with anti-VCAM-1 antibody plus a secondary antibody for 15 minutes. The apocynin, DMSO, Gö-6976 or CinnGEL 2-methylester had no effect on endothelial cell viability as determined by trypan blue exclusion and had no effect on VCAM-1 expression as determined by flow cytometry (data not shown). Representative western blots are shown. Data presented are the mean ± standard deviation from 3 experiments. The phosphorylation status of ERK1/2 is presented as the fold increase in the ratio of the relative intensity of P-ERK1/2 divided by the relative intensity of the loading control (total ERK1/2). *, p<0.05 compared to A) NT cells or B) anti-VCAM-1 stimulated cells.

Mentions: We have reported that, in primary cultures of human endothelial cells, VCAM-1 signals through NADPH oxidase, PKCα and PTP1B [7], [8]. Therefore, we determined whether VCAM-1 activates ERK1/2 downstream of NADPH oxidase, PKCα, and PTP1B in primary cultures of human microvascular endothelial cells from the lung (HMVEC-L). For these studies, HMVEC-Ls were treated overnight with 10 ng/ml TNFα to induce expression of several adhesion molecules, including VCAM-1 and ICAM-1 [7], [8], [35], [36]. TNFα induced expression of VCAM-1 by HMVEC-L cells as determined by immunolabeling and fluorescence microscopy (data not shown). Therefore, since TNFα induces expression of several adhesion molecules, VCAM-1 on TNFα-treated HMVEC-Ls was specifically activated by crosslinking with anti-VCAM-1 plus a secondary antibody for 10 to 30 min and then analyzed by Western Blot for Thr202/Tyr204 phosphorylation of ERK1/2. Stimulation of VCAM-1 at 15 minutes induced Thr202/Tyr204 phosphorylation of ERK1/2 in TNFα -pretreated cultures of HMVEC-L cells without altering total ERK1/2 (Figure 5A, B). This time point for human VCAM-1 activation of ERK1/2 in human endothelial cells is generally consistent with the time course for mouse VCAM-1 activation of ERK1/2 in the murine endothelial cell lines, even though the anti-human VCAM-1 and anti-mouse VCAM-1 antibodies differ. In addition, the time course for VCAM-1 activation of ERK1/2 in HMVEC-Ls is consistent with the time course for VCAM-1 signaling in HMVEC-Ls including maximal stimulation of PKCα at 10 minutes and PTP1B at 15 minutes [7], [8]. To determine whether ERK1/2 functions downstream of NADPH oxidase, PKCα and PTP1B during VCAM-1 signaling, TNF-α stimulated HMEVC-L cells were treated with the vehicle control 0.1% DMSO, the NADPH oxidase inhibitor apocynin (4 mM), the PKCα inhibitor Gö-6976 (2.3 nM) or the PTP1B inhibitor CinnGEL 2-methylester (10 µM) for 30 min and then stimulated with anti-VCAM-1 plus secondary antibody for 15 min. The inhibitors were used at the optimal doses and they did not affect endothelial cell viability as we previously reported [5], [7], [8]. None of the inhibitors had any significant effects on the basal level of ERK1/2 protein expression in the absence of anti-VCAM-1 as determined by western blot (data not shown). Anti-VCAM-1-activated Thr202/Tyr204 phosphorylation of ERK1/2 was blocked by inhibitors of NADPH oxidase, PTP1B and PKCα (Figure 5B). Together, these data indicate that VCAM-1 activates ERK1/2 downstream of NADPH oxidase, PKCα and PTP1B in mouse endothelial cell lines (mHEVa cells) and primary cultures of endothelial cells (HMVEC-L cells). This is the first report for a mechanism for VCAM-1 activation of ERK1/2.


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 HMVEC-L.Treatment of HMVEC-L cells overnight with 10ng/ml TNF-α induced VCAM-1 expression (data not shown). A) Confluent monolayers of TNF-α-treated HMVEC-Ls were nontreated (NT) or treated with 27 µg/ml anti-VCAM-1 plus 15 µg/ml of a secondary antibody to crosslink and stimulate VCAM-1. Phosphorylation of ERK1/2 Thr202/Tyr204 (P-ERK1/2) and total expression of ERK1/2 was examined by western blot using rabbit anti-phospho ERK1/2 Thr202/Tyr204 (1/1000) followed by HRP-conjugated anti-rabbit (1/2000) and ECL detection. B) Confluent monolayers of TNF-α stimulated HMVEC-L cells in 12 well plates were nontreated or incubated for 30 minutes with the solvent control DMSO, apocynin (4 mM), Gö-6976 (2.3 nM) or CinnGEL 2-methylester (10 µM). These endothelial cells were then stimulated with anti-VCAM-1 antibody plus a secondary antibody for 15 minutes. The apocynin, DMSO, Gö-6976 or CinnGEL 2-methylester had no effect on endothelial cell viability as determined by trypan blue exclusion and had no effect on VCAM-1 expression as determined by flow cytometry (data not shown). Representative western blots are shown. Data presented are the mean ± standard deviation from 3 experiments. The phosphorylation status of ERK1/2 is presented as the fold increase in the ratio of the relative intensity of P-ERK1/2 divided by the relative intensity of the loading control (total ERK1/2). *, p<0.05 compared to A) NT cells or B) anti-VCAM-1 stimulated cells.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198778&req=5

pone-0026706-g005: Mechanisms for VCAM-1 activation of ERK1/2 in HMVEC-L.Treatment of HMVEC-L cells overnight with 10ng/ml TNF-α induced VCAM-1 expression (data not shown). A) Confluent monolayers of TNF-α-treated HMVEC-Ls were nontreated (NT) or treated with 27 µg/ml anti-VCAM-1 plus 15 µg/ml of a secondary antibody to crosslink and stimulate VCAM-1. Phosphorylation of ERK1/2 Thr202/Tyr204 (P-ERK1/2) and total expression of ERK1/2 was examined by western blot using rabbit anti-phospho ERK1/2 Thr202/Tyr204 (1/1000) followed by HRP-conjugated anti-rabbit (1/2000) and ECL detection. B) Confluent monolayers of TNF-α stimulated HMVEC-L cells in 12 well plates were nontreated or incubated for 30 minutes with the solvent control DMSO, apocynin (4 mM), Gö-6976 (2.3 nM) or CinnGEL 2-methylester (10 µM). These endothelial cells were then stimulated with anti-VCAM-1 antibody plus a secondary antibody for 15 minutes. The apocynin, DMSO, Gö-6976 or CinnGEL 2-methylester had no effect on endothelial cell viability as determined by trypan blue exclusion and had no effect on VCAM-1 expression as determined by flow cytometry (data not shown). Representative western blots are shown. Data presented are the mean ± standard deviation from 3 experiments. The phosphorylation status of ERK1/2 is presented as the fold increase in the ratio of the relative intensity of P-ERK1/2 divided by the relative intensity of the loading control (total ERK1/2). *, p<0.05 compared to A) NT cells or B) anti-VCAM-1 stimulated cells.
Mentions: We have reported that, in primary cultures of human endothelial cells, VCAM-1 signals through NADPH oxidase, PKCα and PTP1B [7], [8]. Therefore, we determined whether VCAM-1 activates ERK1/2 downstream of NADPH oxidase, PKCα, and PTP1B in primary cultures of human microvascular endothelial cells from the lung (HMVEC-L). For these studies, HMVEC-Ls were treated overnight with 10 ng/ml TNFα to induce expression of several adhesion molecules, including VCAM-1 and ICAM-1 [7], [8], [35], [36]. TNFα induced expression of VCAM-1 by HMVEC-L cells as determined by immunolabeling and fluorescence microscopy (data not shown). Therefore, since TNFα induces expression of several adhesion molecules, VCAM-1 on TNFα-treated HMVEC-Ls was specifically activated by crosslinking with anti-VCAM-1 plus a secondary antibody for 10 to 30 min and then analyzed by Western Blot for Thr202/Tyr204 phosphorylation of ERK1/2. Stimulation of VCAM-1 at 15 minutes induced Thr202/Tyr204 phosphorylation of ERK1/2 in TNFα -pretreated cultures of HMVEC-L cells without altering total ERK1/2 (Figure 5A, B). This time point for human VCAM-1 activation of ERK1/2 in human endothelial cells is generally consistent with the time course for mouse VCAM-1 activation of ERK1/2 in the murine endothelial cell lines, even though the anti-human VCAM-1 and anti-mouse VCAM-1 antibodies differ. In addition, the time course for VCAM-1 activation of ERK1/2 in HMVEC-Ls is consistent with the time course for VCAM-1 signaling in HMVEC-Ls including maximal stimulation of PKCα at 10 minutes and PTP1B at 15 minutes [7], [8]. To determine whether ERK1/2 functions downstream of NADPH oxidase, PKCα and PTP1B during VCAM-1 signaling, TNF-α stimulated HMEVC-L cells were treated with the vehicle control 0.1% DMSO, the NADPH oxidase inhibitor apocynin (4 mM), the PKCα inhibitor Gö-6976 (2.3 nM) or the PTP1B inhibitor CinnGEL 2-methylester (10 µM) for 30 min and then stimulated with anti-VCAM-1 plus secondary antibody for 15 min. The inhibitors were used at the optimal doses and they did not affect endothelial cell viability as we previously reported [5], [7], [8]. None of the inhibitors had any significant effects on the basal level of ERK1/2 protein expression in the absence of anti-VCAM-1 as determined by western blot (data not shown). Anti-VCAM-1-activated Thr202/Tyr204 phosphorylation of ERK1/2 was blocked by inhibitors of NADPH oxidase, PTP1B and PKCα (Figure 5B). Together, these data indicate that VCAM-1 activates ERK1/2 downstream of NADPH oxidase, PKCα and PTP1B in mouse endothelial cell lines (mHEVa cells) and primary cultures of endothelial cells (HMVEC-L cells). This is the first report for a mechanism for VCAM-1 activation of ERK1/2.

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