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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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Inhibition of endothelial cell ERK1/2 with an ERK2 dominant negative plasmid (K52R) blocks VCAM-1-dependent leukocyte migration.Two million mHEVa endothelial cells, that were grown to 70∼80% confluence, were suspended with trypsin and transfected with GFP-ERK2 K52R or vector control using the Amaxa nucleofector method. Transfected cells were seeded onto 9 cm2 culture slides. Four hours after nucleofection, cells were examined for ERK1/2 expression or used in TEM assays. Greater than 60% of the endothelial cells were transfected as determined by flow cytometry with detection of GFP-ERK2 K52R (data not shown). The transfected endothelial cells formed confluent monolayers in 4 hours and were greater than 85% viable (data not shown). In addition, 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) The endothelial cells were washed with ice-cold phosphate-buffered saline and examined by western blot for ERK1/2 and β-actin expression. Lanes 1 and 2 are two samples of vector-treated cells. Lanes 3 and 4 are two samples of cells treated with GFP-ERK2 K52R. B) Leukocyte TEM under laminar flow at 2 dynes/cm2, C) Leukocyte association assay under laminar flow at 2 dynes/cm2. Data for each panel are from 3 experiments. *, p<0.05 compared to non-treated (NT) or vector transfected groups.
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pone-0026706-g002: Inhibition of endothelial cell ERK1/2 with an ERK2 dominant negative plasmid (K52R) blocks VCAM-1-dependent leukocyte migration.Two million mHEVa endothelial cells, that were grown to 70∼80% confluence, were suspended with trypsin and transfected with GFP-ERK2 K52R or vector control using the Amaxa nucleofector method. Transfected cells were seeded onto 9 cm2 culture slides. Four hours after nucleofection, cells were examined for ERK1/2 expression or used in TEM assays. Greater than 60% of the endothelial cells were transfected as determined by flow cytometry with detection of GFP-ERK2 K52R (data not shown). The transfected endothelial cells formed confluent monolayers in 4 hours and were greater than 85% viable (data not shown). In addition, 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) The endothelial cells were washed with ice-cold phosphate-buffered saline and examined by western blot for ERK1/2 and β-actin expression. Lanes 1 and 2 are two samples of vector-treated cells. Lanes 3 and 4 are two samples of cells treated with GFP-ERK2 K52R. B) Leukocyte TEM under laminar flow at 2 dynes/cm2, C) Leukocyte association assay under laminar flow at 2 dynes/cm2. Data for each panel are from 3 experiments. *, p<0.05 compared to non-treated (NT) or vector transfected groups.

Mentions: It was also determined whether transient transfection of mHEVa cells with the ERK2 dominant negative GFP-ERK2 K52R [24] blocks VCAM-1-dependent leukocyte TEM. The cells expressed the dominant negative ERK2 K52R as examined by western blot (Figure 2A). Under our optimized conditions for transfection as described in the methods, GFP-ERK2 K52R-transfected and vector-GFP-transfected mHEVa cells that were cultured in chamber slides for 4 hours had >70% transfection efficiency as analyzed by flow cytometry for GFP (Figure 2B). The transfection did not affect mHEVa cell expression of VCAM-1 as determined by immunolabeling and flow cytometry (data not shown). The transfected endothelial cells were plated at a density to form confluent monolayers in 4 hours. The endothelial cells were greater than 85% viable at 4 hours of culture (data not shown). In contrast, since ERK1/2 is a survival signal for endothelial cells [25], [26], [27], [28], [29], [30], at 24 hours of culture the dominant negative ERK1/2 transfected cells began to undergo cell death whereas vector transfected cells survive (data not shown). Therefore, the transient transfection studies were performed at 4 hours when confluent monolayers of viable endothelial cells are formed. At 4 hours post transfection, TEM studies were performed in the parallel plate flow chamber assay at 2 dynes/cm2 laminar flow or static conditions for 15 minutes [8]. The transfection with the dominant negative ERK2 K52R inhibited VCAM-1-dependent leukocyte TEM as compared to the vector control under laminar flow (Figure 2C) or static conditions (data not shown). Anti-VCAM-1 blocking antibodies inhibited leukocyte transendothelial migration for all the treatments compared to the vector-treated group without anti-VCAM-1 (Figure 2C). The dominant negative ERK2 K52R did not alter the number of leukocytes associated with the endothelial cells as compared to the vector-transfected cells under laminar flow conditions (Figure 2D) or static conditions (data not shown). Anti-VCAM-1 blocking antibodies inhibited leukocyte association with the endothelial cells for all the treatments compared to the corresponding groups without anti-VCAM-1 (Figure 2D). In summary, it is demonstrated for the first time that ERK2 activity is necessary for VCAM-1-dependent 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 endothelial cell ERK1/2 with an ERK2 dominant negative plasmid (K52R) blocks VCAM-1-dependent leukocyte migration.Two million mHEVa endothelial cells, that were grown to 70∼80% confluence, were suspended with trypsin and transfected with GFP-ERK2 K52R or vector control using the Amaxa nucleofector method. Transfected cells were seeded onto 9 cm2 culture slides. Four hours after nucleofection, cells were examined for ERK1/2 expression or used in TEM assays. Greater than 60% of the endothelial cells were transfected as determined by flow cytometry with detection of GFP-ERK2 K52R (data not shown). The transfected endothelial cells formed confluent monolayers in 4 hours and were greater than 85% viable (data not shown). In addition, 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) The endothelial cells were washed with ice-cold phosphate-buffered saline and examined by western blot for ERK1/2 and β-actin expression. Lanes 1 and 2 are two samples of vector-treated cells. Lanes 3 and 4 are two samples of cells treated with GFP-ERK2 K52R. B) Leukocyte TEM under laminar flow at 2 dynes/cm2, C) Leukocyte association assay under laminar flow at 2 dynes/cm2. Data for each panel are from 3 experiments. *, p<0.05 compared to non-treated (NT) or vector transfected groups.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198778&req=5

pone-0026706-g002: Inhibition of endothelial cell ERK1/2 with an ERK2 dominant negative plasmid (K52R) blocks VCAM-1-dependent leukocyte migration.Two million mHEVa endothelial cells, that were grown to 70∼80% confluence, were suspended with trypsin and transfected with GFP-ERK2 K52R or vector control using the Amaxa nucleofector method. Transfected cells were seeded onto 9 cm2 culture slides. Four hours after nucleofection, cells were examined for ERK1/2 expression or used in TEM assays. Greater than 60% of the endothelial cells were transfected as determined by flow cytometry with detection of GFP-ERK2 K52R (data not shown). The transfected endothelial cells formed confluent monolayers in 4 hours and were greater than 85% viable (data not shown). In addition, 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) The endothelial cells were washed with ice-cold phosphate-buffered saline and examined by western blot for ERK1/2 and β-actin expression. Lanes 1 and 2 are two samples of vector-treated cells. Lanes 3 and 4 are two samples of cells treated with GFP-ERK2 K52R. B) Leukocyte TEM under laminar flow at 2 dynes/cm2, C) Leukocyte association assay under laminar flow at 2 dynes/cm2. Data for each panel are from 3 experiments. *, p<0.05 compared to non-treated (NT) or vector transfected groups.
Mentions: It was also determined whether transient transfection of mHEVa cells with the ERK2 dominant negative GFP-ERK2 K52R [24] blocks VCAM-1-dependent leukocyte TEM. The cells expressed the dominant negative ERK2 K52R as examined by western blot (Figure 2A). Under our optimized conditions for transfection as described in the methods, GFP-ERK2 K52R-transfected and vector-GFP-transfected mHEVa cells that were cultured in chamber slides for 4 hours had >70% transfection efficiency as analyzed by flow cytometry for GFP (Figure 2B). The transfection did not affect mHEVa cell expression of VCAM-1 as determined by immunolabeling and flow cytometry (data not shown). The transfected endothelial cells were plated at a density to form confluent monolayers in 4 hours. The endothelial cells were greater than 85% viable at 4 hours of culture (data not shown). In contrast, since ERK1/2 is a survival signal for endothelial cells [25], [26], [27], [28], [29], [30], at 24 hours of culture the dominant negative ERK1/2 transfected cells began to undergo cell death whereas vector transfected cells survive (data not shown). Therefore, the transient transfection studies were performed at 4 hours when confluent monolayers of viable endothelial cells are formed. At 4 hours post transfection, TEM studies were performed in the parallel plate flow chamber assay at 2 dynes/cm2 laminar flow or static conditions for 15 minutes [8]. The transfection with the dominant negative ERK2 K52R inhibited VCAM-1-dependent leukocyte TEM as compared to the vector control under laminar flow (Figure 2C) or static conditions (data not shown). Anti-VCAM-1 blocking antibodies inhibited leukocyte transendothelial migration for all the treatments compared to the vector-treated group without anti-VCAM-1 (Figure 2C). The dominant negative ERK2 K52R did not alter the number of leukocytes associated with the endothelial cells as compared to the vector-transfected cells under laminar flow conditions (Figure 2D) or static conditions (data not shown). Anti-VCAM-1 blocking antibodies inhibited leukocyte association with the endothelial cells for all the treatments compared to the corresponding groups without anti-VCAM-1 (Figure 2D). In summary, it is demonstrated for the first time that ERK2 activity is necessary for VCAM-1-dependent 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