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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

Inhibition of MEK, which is known to activate ERK1/2, blocks VCAM-1-dependent spleen leukocyte TEM.Confluent monolayers of mHEVa cells were nontreated (NT) or treated for 30 minutes with the MEK inhibitors PD98059 (20 µM) or U0126 (20–40 µM). To block leukocyte binding to VCAM-1, the endothelial cells were treated with a blocking anti-VCAM-1 antibody without a secondary crosslinking antibody. A–D) Splenic leukocytes were added to the endothelial monolayer, allowed to briefly settle to mediate cell contact and then exposed to 2 dynes/cm2 laminar flow for 15 minutes to examine migration (A and B) or 2 minutes to examine leukocyte-endothelial cell association (C and D). Then, cells were washed and fixed in 3% paraformaldehyde for 1 hour and examined by phase contrast microscopy [7], [8], [65]. Non-migrated leukocytes are phase-light and migrated leukocytes appear as phase-dark [7], [8], [65]. We previously reported that the leukocytes that migrated are >88% lymphocytes as determined by flow cytometry [6]. E) Relative cytotoxicity was determined by the G6PDH assay; PD98059 (20 µM) and U0126 (40 µM) were not cytotoxic as compared to the nontreated control cells. Data in each panel are from 3 experiments. *, p<0.05 compared to A,C) NT groups, B,D) 0 minutes groups, or compared to DMSO-treated or last washes (data not shown). Inhibitors had no effect on cell viability, as determined by Trypan blue exclusion (data not shown).
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pone-0026706-g001: Inhibition of MEK, which is known to activate ERK1/2, blocks VCAM-1-dependent spleen leukocyte TEM.Confluent monolayers of mHEVa cells were nontreated (NT) or treated for 30 minutes with the MEK inhibitors PD98059 (20 µM) or U0126 (20–40 µM). To block leukocyte binding to VCAM-1, the endothelial cells were treated with a blocking anti-VCAM-1 antibody without a secondary crosslinking antibody. A–D) Splenic leukocytes were added to the endothelial monolayer, allowed to briefly settle to mediate cell contact and then exposed to 2 dynes/cm2 laminar flow for 15 minutes to examine migration (A and B) or 2 minutes to examine leukocyte-endothelial cell association (C and D). Then, cells were washed and fixed in 3% paraformaldehyde for 1 hour and examined by phase contrast microscopy [7], [8], [65]. Non-migrated leukocytes are phase-light and migrated leukocytes appear as phase-dark [7], [8], [65]. We previously reported that the leukocytes that migrated are >88% lymphocytes as determined by flow cytometry [6]. E) Relative cytotoxicity was determined by the G6PDH assay; PD98059 (20 µM) and U0126 (40 µM) were not cytotoxic as compared to the nontreated control cells. Data in each panel are from 3 experiments. *, p<0.05 compared to A,C) NT groups, B,D) 0 minutes groups, or compared to DMSO-treated or last washes (data not shown). Inhibitors had no effect on cell viability, as determined by Trypan blue exclusion (data not shown).

Mentions: To examine endothelial MEK1/2 function in VCAM-1-dependent TEM, confluent monolayers of mHEVa cells were nontreated or pretreated for 30 minutes with two inhibitors of MEK1/2, PD98059 or U0126. PD98059 was chosen since reports show that it inhibits activation of inactive MEK1 and MEK 2 with IC50 values of 4 µM and 50 µM, respectively [17], [18], [19]. U0126 irreversibly inhibits both active and inactive MEK1/2 [19]. U0126 has been found to be a selective and highly potent selective inhibitor of MAPK cascade with IC50 values of 70 and 60 nM for purified MEK1 and MEK2, respectively [19], [20]. The endothelial cells were pretreated with MEK inhibitors PD98059 (20 µM) or U0126 (10–40 µM) at doses typically used (50 µM) for inhibition of this enzyme in endothelial cells [21], [22]. After treatment with these MEK1/2 inhibitors, the endothelial cells were washed five times. The last wash was added to a set of untreated endothelial cells to demonstrate that effective concentrations of free inhibitor were removed since there was no effect of the last wash on TEM (data not shown). Spleen leukocytes which were >90% lymphocytes were added. After 15 minutes at 2 dynes/cm2 laminar flow, the monolayers were washed and fixed; leukocyte transendothelial migration was quantified by phase contrast microscopy [23]. Anti-VCAM-1 blocking antibodies inhibited leukocyte transendothelial migration and leukocyte adhesion (Figure 1A–D) but migration was not blocked by isotype control antibodies as we previously reported [5]. The MEK inhibitors did not affect cell viability as determined by trypan blue exclusion (data not shown), did not increase relative cytotoxicity as compared to the nontreated control as determined by the G6PDH release assay (Figure 1E) and did not affect the expression of VCAM-1 as determined by flow cytometry (data not shown). PD98059 (20 µM) and U0126 (30–40 µM) significantly inhibited leukocyte transmigration (Figure 1A,B). Furthermore, neither PD98059 nor U0126 inhibited leukocyte adhesion (Figure 1C,D), indicating that endothelial cell MEK1/2 is not required for leukocyte adhesion but is required for the leukocyte TEM.


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)

Inhibition of MEK, which is known to activate ERK1/2, blocks VCAM-1-dependent spleen leukocyte TEM.Confluent monolayers of mHEVa cells were nontreated (NT) or treated for 30 minutes with the MEK inhibitors PD98059 (20 µM) or U0126 (20–40 µM). To block leukocyte binding to VCAM-1, the endothelial cells were treated with a blocking anti-VCAM-1 antibody without a secondary crosslinking antibody. A–D) Splenic leukocytes were added to the endothelial monolayer, allowed to briefly settle to mediate cell contact and then exposed to 2 dynes/cm2 laminar flow for 15 minutes to examine migration (A and B) or 2 minutes to examine leukocyte-endothelial cell association (C and D). Then, cells were washed and fixed in 3% paraformaldehyde for 1 hour and examined by phase contrast microscopy [7], [8], [65]. Non-migrated leukocytes are phase-light and migrated leukocytes appear as phase-dark [7], [8], [65]. We previously reported that the leukocytes that migrated are >88% lymphocytes as determined by flow cytometry [6]. E) Relative cytotoxicity was determined by the G6PDH assay; PD98059 (20 µM) and U0126 (40 µM) were not cytotoxic as compared to the nontreated control cells. Data in each panel are from 3 experiments. *, p<0.05 compared to A,C) NT groups, B,D) 0 minutes groups, or compared to DMSO-treated or last washes (data not shown). Inhibitors had no effect on cell viability, as determined by Trypan blue exclusion (data not shown).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198778&req=5

pone-0026706-g001: Inhibition of MEK, which is known to activate ERK1/2, blocks VCAM-1-dependent spleen leukocyte TEM.Confluent monolayers of mHEVa cells were nontreated (NT) or treated for 30 minutes with the MEK inhibitors PD98059 (20 µM) or U0126 (20–40 µM). To block leukocyte binding to VCAM-1, the endothelial cells were treated with a blocking anti-VCAM-1 antibody without a secondary crosslinking antibody. A–D) Splenic leukocytes were added to the endothelial monolayer, allowed to briefly settle to mediate cell contact and then exposed to 2 dynes/cm2 laminar flow for 15 minutes to examine migration (A and B) or 2 minutes to examine leukocyte-endothelial cell association (C and D). Then, cells were washed and fixed in 3% paraformaldehyde for 1 hour and examined by phase contrast microscopy [7], [8], [65]. Non-migrated leukocytes are phase-light and migrated leukocytes appear as phase-dark [7], [8], [65]. We previously reported that the leukocytes that migrated are >88% lymphocytes as determined by flow cytometry [6]. E) Relative cytotoxicity was determined by the G6PDH assay; PD98059 (20 µM) and U0126 (40 µM) were not cytotoxic as compared to the nontreated control cells. Data in each panel are from 3 experiments. *, p<0.05 compared to A,C) NT groups, B,D) 0 minutes groups, or compared to DMSO-treated or last washes (data not shown). Inhibitors had no effect on cell viability, as determined by Trypan blue exclusion (data not shown).
Mentions: To examine endothelial MEK1/2 function in VCAM-1-dependent TEM, confluent monolayers of mHEVa cells were nontreated or pretreated for 30 minutes with two inhibitors of MEK1/2, PD98059 or U0126. PD98059 was chosen since reports show that it inhibits activation of inactive MEK1 and MEK 2 with IC50 values of 4 µM and 50 µM, respectively [17], [18], [19]. U0126 irreversibly inhibits both active and inactive MEK1/2 [19]. U0126 has been found to be a selective and highly potent selective inhibitor of MAPK cascade with IC50 values of 70 and 60 nM for purified MEK1 and MEK2, respectively [19], [20]. The endothelial cells were pretreated with MEK inhibitors PD98059 (20 µM) or U0126 (10–40 µM) at doses typically used (50 µM) for inhibition of this enzyme in endothelial cells [21], [22]. After treatment with these MEK1/2 inhibitors, the endothelial cells were washed five times. The last wash was added to a set of untreated endothelial cells to demonstrate that effective concentrations of free inhibitor were removed since there was no effect of the last wash on TEM (data not shown). Spleen leukocytes which were >90% lymphocytes were added. After 15 minutes at 2 dynes/cm2 laminar flow, the monolayers were washed and fixed; leukocyte transendothelial migration was quantified by phase contrast microscopy [23]. Anti-VCAM-1 blocking antibodies inhibited leukocyte transendothelial migration and leukocyte adhesion (Figure 1A–D) but migration was not blocked by isotype control antibodies as we previously reported [5]. The MEK inhibitors did not affect cell viability as determined by trypan blue exclusion (data not shown), did not increase relative cytotoxicity as compared to the nontreated control as determined by the G6PDH release assay (Figure 1E) and did not affect the expression of VCAM-1 as determined by flow cytometry (data not shown). PD98059 (20 µM) and U0126 (30–40 µM) significantly inhibited leukocyte transmigration (Figure 1A,B). Furthermore, neither PD98059 nor U0126 inhibited leukocyte adhesion (Figure 1C,D), indicating that endothelial cell MEK1/2 is not required for leukocyte adhesion but is required for the leukocyte TEM.

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