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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

VCAM-1 induces an increase in phosphorylation of ERK1/2 in endothelial cell lines under static and laminar flow conditions.Confluent monolayers of mHEVa cells were stimulated with 27 µg/ml anti-VCAM-1 (or the binding control anti-CD98) plus 15 µg/ml of a secondary antibody. ERK1/2 Thr202/Tyr204 phosphorylation (P-ERK1/2) and total ERK1/2 was determined by western blot. A) Time course for anti-VCAM-1 activation of ERK1/2 Thr202/Tyr204 phosphorylation under static conditions. B) Stimulation of VCAM-1 for 30 minutes and time course for antibody crosslinking of the control CD98 under static conditions. C) Confluent monolayers of HEV were non-treated (NT) or stimulated with anti-VCAM-1 or the control anti-CD98 under 2 dynes/cm2 laminar flow or nontreated under static conditions for 15 minutes. *, p<0.05 compared to A) 0 minutes, B) NT and C) the NT,flow group. In panel C) **, p<0.5 compared to the anti-VCAM-1, flow group.
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pone-0026706-g003: VCAM-1 induces an increase in phosphorylation of ERK1/2 in endothelial cell lines under static and laminar flow conditions.Confluent monolayers of mHEVa cells were stimulated with 27 µg/ml anti-VCAM-1 (or the binding control anti-CD98) plus 15 µg/ml of a secondary antibody. ERK1/2 Thr202/Tyr204 phosphorylation (P-ERK1/2) and total ERK1/2 was determined by western blot. A) Time course for anti-VCAM-1 activation of ERK1/2 Thr202/Tyr204 phosphorylation under static conditions. B) Stimulation of VCAM-1 for 30 minutes and time course for antibody crosslinking of the control CD98 under static conditions. C) Confluent monolayers of HEV were non-treated (NT) or stimulated with anti-VCAM-1 or the control anti-CD98 under 2 dynes/cm2 laminar flow or nontreated under static conditions for 15 minutes. *, p<0.05 compared to A) 0 minutes, B) NT and C) the NT,flow group. In panel C) **, p<0.5 compared to the anti-VCAM-1, flow group.

Mentions: To examine the time course for anti-VCAM-1 activation of ERK1/2 under static and laminar flow conditions, confluent monolayers of mHEVa cells were stimulated with a confluent monolayer of anti-VCAM-1 antibody-coated beads for 10 to 60 min. Antibody crosslinking of VCAM-1 mimics physical leukocyte interaction with endothelial cells for the activation of VCAM-1-dependent signals [5], [6], [7], [8], [16]. The negative control included treatment with a confluent monolayer of anti-CD98 antibody-coated beads for 10 to 60 min, since CD98 is expressed by mHEVa cells, but does not signal through ERK1/2 [31]. After stimulation with antibody-coated beads, the cells were examined by Western blot for phosphorylation of ERK1/2 Thr202/Tyr204, the active form of ERK1/2. Antibody crosslinking of VCAM-1 under static conditions induced an increase in ERK1/2 phosphorylation at 15–30 minutes in mHEVa cells (Figure 3A). VCAM-1 stimulation did not increase total ERK1/2 expression (Figure 3A–B). There was no increase in ERK1/2 phosphorylation with the negative control anti-CD98 coated beads (Figure 3B). To study the effect of laminar flow on ERK1/2 activation, confluent monolayers of endothelial cells were assembled in parallel plate flow chambers, anti-VCAM-1-coated beads or anti-CD98-coated beads were added to the flow chamber and then laminar flow at 2 dynes/cm2 was applied for 15 minutes. Equivalent numbers of the anti-VCAM-1-coated beads or anti-CD98-coated beads were loaded onto the endothelial cells and were bound to the endothelial cells (data not shown). To avoid stimulation of the endothelial cells by serum growth factors in fresh culture medium, we perfused the laminar flow chambers with media taken from cultured mHEVa cells. ERK1/2 Thr202/Tyr204 phosphorylation was significantly increased when mHEVa cells were stimulated with anti-VCAM-1 beads, compared to nontreated or the anti-CD98 binding controls under flow conditions (Figure 3C). When we compared nontreated endothelial cells under static conditions with nontreated endothelial cells under flow conditions, there was a significant but modest increase in ERK1/2 Thr202/Tyr204 phosphorylation in nontreated cells under laminar flow (Figure 3C), consistent with previous reports that laminar flow induces some ERK1/2 activation [32], [33]. Anti-VCAM-1 under flow induced a greater increase in ERK1/2 phosphorylation compared to stimulation under static conditions (5 fold increase under flow versus 3 ½ fold increase under static conditions) (Figure 3). Together, these data suggest that VCAM-1 stimulates an increase in endothelial ERK1/2 activity under both static and laminar flow at 2 dynes/cm2.


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)

VCAM-1 induces an increase in phosphorylation of ERK1/2 in endothelial cell lines under static and laminar flow conditions.Confluent monolayers of mHEVa cells were stimulated with 27 µg/ml anti-VCAM-1 (or the binding control anti-CD98) plus 15 µg/ml of a secondary antibody. ERK1/2 Thr202/Tyr204 phosphorylation (P-ERK1/2) and total ERK1/2 was determined by western blot. A) Time course for anti-VCAM-1 activation of ERK1/2 Thr202/Tyr204 phosphorylation under static conditions. B) Stimulation of VCAM-1 for 30 minutes and time course for antibody crosslinking of the control CD98 under static conditions. C) Confluent monolayers of HEV were non-treated (NT) or stimulated with anti-VCAM-1 or the control anti-CD98 under 2 dynes/cm2 laminar flow or nontreated under static conditions for 15 minutes. *, p<0.05 compared to A) 0 minutes, B) NT and C) the NT,flow group. In panel C) **, p<0.5 compared to the anti-VCAM-1, flow group.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198778&req=5

pone-0026706-g003: VCAM-1 induces an increase in phosphorylation of ERK1/2 in endothelial cell lines under static and laminar flow conditions.Confluent monolayers of mHEVa cells were stimulated with 27 µg/ml anti-VCAM-1 (or the binding control anti-CD98) plus 15 µg/ml of a secondary antibody. ERK1/2 Thr202/Tyr204 phosphorylation (P-ERK1/2) and total ERK1/2 was determined by western blot. A) Time course for anti-VCAM-1 activation of ERK1/2 Thr202/Tyr204 phosphorylation under static conditions. B) Stimulation of VCAM-1 for 30 minutes and time course for antibody crosslinking of the control CD98 under static conditions. C) Confluent monolayers of HEV were non-treated (NT) or stimulated with anti-VCAM-1 or the control anti-CD98 under 2 dynes/cm2 laminar flow or nontreated under static conditions for 15 minutes. *, p<0.05 compared to A) 0 minutes, B) NT and C) the NT,flow group. In panel C) **, p<0.5 compared to the anti-VCAM-1, flow group.
Mentions: To examine the time course for anti-VCAM-1 activation of ERK1/2 under static and laminar flow conditions, confluent monolayers of mHEVa cells were stimulated with a confluent monolayer of anti-VCAM-1 antibody-coated beads for 10 to 60 min. Antibody crosslinking of VCAM-1 mimics physical leukocyte interaction with endothelial cells for the activation of VCAM-1-dependent signals [5], [6], [7], [8], [16]. The negative control included treatment with a confluent monolayer of anti-CD98 antibody-coated beads for 10 to 60 min, since CD98 is expressed by mHEVa cells, but does not signal through ERK1/2 [31]. After stimulation with antibody-coated beads, the cells were examined by Western blot for phosphorylation of ERK1/2 Thr202/Tyr204, the active form of ERK1/2. Antibody crosslinking of VCAM-1 under static conditions induced an increase in ERK1/2 phosphorylation at 15–30 minutes in mHEVa cells (Figure 3A). VCAM-1 stimulation did not increase total ERK1/2 expression (Figure 3A–B). There was no increase in ERK1/2 phosphorylation with the negative control anti-CD98 coated beads (Figure 3B). To study the effect of laminar flow on ERK1/2 activation, confluent monolayers of endothelial cells were assembled in parallel plate flow chambers, anti-VCAM-1-coated beads or anti-CD98-coated beads were added to the flow chamber and then laminar flow at 2 dynes/cm2 was applied for 15 minutes. Equivalent numbers of the anti-VCAM-1-coated beads or anti-CD98-coated beads were loaded onto the endothelial cells and were bound to the endothelial cells (data not shown). To avoid stimulation of the endothelial cells by serum growth factors in fresh culture medium, we perfused the laminar flow chambers with media taken from cultured mHEVa cells. ERK1/2 Thr202/Tyr204 phosphorylation was significantly increased when mHEVa cells were stimulated with anti-VCAM-1 beads, compared to nontreated or the anti-CD98 binding controls under flow conditions (Figure 3C). When we compared nontreated endothelial cells under static conditions with nontreated endothelial cells under flow conditions, there was a significant but modest increase in ERK1/2 Thr202/Tyr204 phosphorylation in nontreated cells under laminar flow (Figure 3C), consistent with previous reports that laminar flow induces some ERK1/2 activation [32], [33]. Anti-VCAM-1 under flow induced a greater increase in ERK1/2 phosphorylation compared to stimulation under static conditions (5 fold increase under flow versus 3 ½ fold increase under static conditions) (Figure 3). Together, these data suggest that VCAM-1 stimulates an increase in endothelial ERK1/2 activity under both static and laminar flow at 2 dynes/cm2.

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