Role of protein kinase C in B1 integrent modification on human granulocytes

Mark Rowin, Vivian W. Xue

Research output: Contribution to journalArticle

Abstract

Introduction: β1 integrins are adhesion molecules that facilitate migration of leukocytes through the extracellular matrix, β1 integrin-mediated adhesion involves altering surface number, orientation, and affinity of the integrin structure. We have shown the α isoform of protein kinase C (PKC) facilitates β1 integrin-dependent adhesion. We now examine the physiologic role of PKC in β1 integrin alteration(s) in a model of human granulocytes. Methods: DMSO-differentiated granulocytic HL-60 cells were utilized. Adhesion assays to fibronectin were performed using the PKC inhibitors staurosporin or calphostin-C, with and without stimulation by phorbol myristate acetate (PMA). Cells were analyzed by flow cytometry for total β1 integrin expression and affinity modulation (n=7). Affinity modulation was evaluated by monoclonal antibody 15/7. This recognizes a conformational change in the β1 subunit that accompanies increases in the affinity of β1 integrin for their ligand. Confocal fluorescent microscopy was performed to evaluate co-localization or "clustering" of β1 integrins. Results and Conclusions: PMA stimulated β1 integrin-dependent adhesion significantly decreased following exposure to PKC inhibitors (PMA:41± 8%, staurosporin:27± 5%, calphostin-C: 18± 4%, p<.05). Flow cytometry revealed a significant decrease in surface expression of β1 integrins on cells treated with PKC inhibitors, both at baseline (relative change-staurosporin: 0.62± .05, calphostin-C: 0.37± .08, p< .01) and following PMA stimulation (relative change-staurosporin: 0.66± .05, calphostin-C: 0.42± .09, p <.01). Affinity modulation paralleled changes in adherence. PMA stimulated an increase in 15/7 staining of 2.06, while treatment with staurosporin and calphostin showed no increase (1.01 and 1.03 respectively). Confocal fluorescent microscopy showed no difference in β1 integrin surface distribution or clustering in cells treated with PKC inhibitors when compared to controls. These data suggest a role for PKC in both modification of surface expression and affinity modulation of β1 integrins on granulocytic HL-60 cells. Clustering of β1 integrin is not effected by PKC inhibition.

Original languageEnglish (US)
JournalCritical Care Medicine
Volume27
Issue number12 SUPPL.
StatePublished - 1999
Externally publishedYes

Fingerprint

Granulocytes
Integrins
Protein Kinase C
Tetradecanoylphorbol Acetate
Protein C Inhibitor
Protein Kinase Inhibitors
Cluster Analysis
HL-60 Cells
Confocal Microscopy
Flow Cytometry
Dimethyl Sulfoxide
Fibronectins
Extracellular Matrix
Protein Isoforms
Leukocytes
Monoclonal Antibodies
Staining and Labeling
Ligands

All Science Journal Classification (ASJC) codes

  • Critical Care and Intensive Care Medicine

Cite this

Role of protein kinase C in B1 integrent modification on human granulocytes. / Rowin, Mark; Xue, Vivian W.

In: Critical Care Medicine, Vol. 27, No. 12 SUPPL., 1999.

Research output: Contribution to journalArticle

@article{fe8f67fdc6d1446dba9c92264bcdd1c3,
title = "Role of protein kinase C in B1 integrent modification on human granulocytes",
abstract = "Introduction: β1 integrins are adhesion molecules that facilitate migration of leukocytes through the extracellular matrix, β1 integrin-mediated adhesion involves altering surface number, orientation, and affinity of the integrin structure. We have shown the α isoform of protein kinase C (PKC) facilitates β1 integrin-dependent adhesion. We now examine the physiologic role of PKC in β1 integrin alteration(s) in a model of human granulocytes. Methods: DMSO-differentiated granulocytic HL-60 cells were utilized. Adhesion assays to fibronectin were performed using the PKC inhibitors staurosporin or calphostin-C, with and without stimulation by phorbol myristate acetate (PMA). Cells were analyzed by flow cytometry for total β1 integrin expression and affinity modulation (n=7). Affinity modulation was evaluated by monoclonal antibody 15/7. This recognizes a conformational change in the β1 subunit that accompanies increases in the affinity of β1 integrin for their ligand. Confocal fluorescent microscopy was performed to evaluate co-localization or {"}clustering{"} of β1 integrins. Results and Conclusions: PMA stimulated β1 integrin-dependent adhesion significantly decreased following exposure to PKC inhibitors (PMA:41± 8{\%}, staurosporin:27± 5{\%}, calphostin-C: 18± 4{\%}, p<.05). Flow cytometry revealed a significant decrease in surface expression of β1 integrins on cells treated with PKC inhibitors, both at baseline (relative change-staurosporin: 0.62± .05, calphostin-C: 0.37± .08, p< .01) and following PMA stimulation (relative change-staurosporin: 0.66± .05, calphostin-C: 0.42± .09, p <.01). Affinity modulation paralleled changes in adherence. PMA stimulated an increase in 15/7 staining of 2.06, while treatment with staurosporin and calphostin showed no increase (1.01 and 1.03 respectively). Confocal fluorescent microscopy showed no difference in β1 integrin surface distribution or clustering in cells treated with PKC inhibitors when compared to controls. These data suggest a role for PKC in both modification of surface expression and affinity modulation of β1 integrins on granulocytic HL-60 cells. Clustering of β1 integrin is not effected by PKC inhibition.",
author = "Mark Rowin and Xue, {Vivian W.}",
year = "1999",
language = "English (US)",
volume = "27",
journal = "Critical Care Medicine",
issn = "0090-3493",
publisher = "Lippincott Williams and Wilkins",
number = "12 SUPPL.",

}

TY - JOUR

T1 - Role of protein kinase C in B1 integrent modification on human granulocytes

AU - Rowin, Mark

AU - Xue, Vivian W.

PY - 1999

Y1 - 1999

N2 - Introduction: β1 integrins are adhesion molecules that facilitate migration of leukocytes through the extracellular matrix, β1 integrin-mediated adhesion involves altering surface number, orientation, and affinity of the integrin structure. We have shown the α isoform of protein kinase C (PKC) facilitates β1 integrin-dependent adhesion. We now examine the physiologic role of PKC in β1 integrin alteration(s) in a model of human granulocytes. Methods: DMSO-differentiated granulocytic HL-60 cells were utilized. Adhesion assays to fibronectin were performed using the PKC inhibitors staurosporin or calphostin-C, with and without stimulation by phorbol myristate acetate (PMA). Cells were analyzed by flow cytometry for total β1 integrin expression and affinity modulation (n=7). Affinity modulation was evaluated by monoclonal antibody 15/7. This recognizes a conformational change in the β1 subunit that accompanies increases in the affinity of β1 integrin for their ligand. Confocal fluorescent microscopy was performed to evaluate co-localization or "clustering" of β1 integrins. Results and Conclusions: PMA stimulated β1 integrin-dependent adhesion significantly decreased following exposure to PKC inhibitors (PMA:41± 8%, staurosporin:27± 5%, calphostin-C: 18± 4%, p<.05). Flow cytometry revealed a significant decrease in surface expression of β1 integrins on cells treated with PKC inhibitors, both at baseline (relative change-staurosporin: 0.62± .05, calphostin-C: 0.37± .08, p< .01) and following PMA stimulation (relative change-staurosporin: 0.66± .05, calphostin-C: 0.42± .09, p <.01). Affinity modulation paralleled changes in adherence. PMA stimulated an increase in 15/7 staining of 2.06, while treatment with staurosporin and calphostin showed no increase (1.01 and 1.03 respectively). Confocal fluorescent microscopy showed no difference in β1 integrin surface distribution or clustering in cells treated with PKC inhibitors when compared to controls. These data suggest a role for PKC in both modification of surface expression and affinity modulation of β1 integrins on granulocytic HL-60 cells. Clustering of β1 integrin is not effected by PKC inhibition.

AB - Introduction: β1 integrins are adhesion molecules that facilitate migration of leukocytes through the extracellular matrix, β1 integrin-mediated adhesion involves altering surface number, orientation, and affinity of the integrin structure. We have shown the α isoform of protein kinase C (PKC) facilitates β1 integrin-dependent adhesion. We now examine the physiologic role of PKC in β1 integrin alteration(s) in a model of human granulocytes. Methods: DMSO-differentiated granulocytic HL-60 cells were utilized. Adhesion assays to fibronectin were performed using the PKC inhibitors staurosporin or calphostin-C, with and without stimulation by phorbol myristate acetate (PMA). Cells were analyzed by flow cytometry for total β1 integrin expression and affinity modulation (n=7). Affinity modulation was evaluated by monoclonal antibody 15/7. This recognizes a conformational change in the β1 subunit that accompanies increases in the affinity of β1 integrin for their ligand. Confocal fluorescent microscopy was performed to evaluate co-localization or "clustering" of β1 integrins. Results and Conclusions: PMA stimulated β1 integrin-dependent adhesion significantly decreased following exposure to PKC inhibitors (PMA:41± 8%, staurosporin:27± 5%, calphostin-C: 18± 4%, p<.05). Flow cytometry revealed a significant decrease in surface expression of β1 integrins on cells treated with PKC inhibitors, both at baseline (relative change-staurosporin: 0.62± .05, calphostin-C: 0.37± .08, p< .01) and following PMA stimulation (relative change-staurosporin: 0.66± .05, calphostin-C: 0.42± .09, p <.01). Affinity modulation paralleled changes in adherence. PMA stimulated an increase in 15/7 staining of 2.06, while treatment with staurosporin and calphostin showed no increase (1.01 and 1.03 respectively). Confocal fluorescent microscopy showed no difference in β1 integrin surface distribution or clustering in cells treated with PKC inhibitors when compared to controls. These data suggest a role for PKC in both modification of surface expression and affinity modulation of β1 integrins on granulocytic HL-60 cells. Clustering of β1 integrin is not effected by PKC inhibition.

UR - http://www.scopus.com/inward/record.url?scp=33750680073&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33750680073&partnerID=8YFLogxK

M3 - Article

VL - 27

JO - Critical Care Medicine

JF - Critical Care Medicine

SN - 0090-3493

IS - 12 SUPPL.

ER -