VLA-4 Integrin Cross-linking on Human Monocytic THP-1 Cells Induces Tissue Factor Expression by a Mechanism Involving Mitogen-activated Protein Kinase

doi:10.1074/jbc.272.15.10287

VLA-4 Integrin Cross-linking on Human Monocytic THP-1 Cells Induces Tissue Factor Expression by a Mechanism Involving Mitogen-activated Protein Kinase*

From the Departments of Surgery, Toronto Hospital-General Division and the University of Toronto, Toronto, Ontario M5G 2C4, Canada

^¶ To whom correspondence should be addressed:
Toronto Hospital, 200 Elizabeth Hospital EN9-236, Toronto, Ontario M5G 2C4, Canada.
Tel.: 416-340-4988; Fax: 416-595-9486; E-mail: orotstein{at}torhosp.toronto.on.ca

Abstract

Adhesion molecules such as VLA-4 are important not only for monocyte adhesion to extracellular matrix proteins, but also for subsequent cell activation. Monocyte adherence to fibronectin or engagement of VLA-4 has been demonstrated to stimulate production of potent inflammatory mediators such as tumor necrosis factor-α, interleukin-1, and the procoagulant tissue factor protein. However, the intracellular signaling cascades leading to gene expression have not been elucidated. Using the human monocytic THP-1 cell line, VLA-4 cross-linking by monoclonal antibodies directed against its α₄ and β₁ subunits produced a time-dependent increase in tyrosine phosphorylation of a broad range of cellular proteins. Using Western blot analysis directed against the phosphorylated form of the extracellular signal-related kinase (ERK) mitogen-activated protein (MAP) kinase proteins, as well as immunoprecipitation and in vitro kinase assays, we found that VLA-4 cross-linking increased ERK1/ERK2 tyrosine phosphorylation and activity. In conjunction, integrin cross-linking also increased NF-κB nuclear translocation and 4-h expression of tissue factor. Inhibition of tyrosine kinase activity with genistein (10 μg/ml) as well as selective MAP kinase inhibition with the MEK-1 inhibitor PD98059 abolished the VLA-4-dependent ERK tyrosine phosphorylation, inhibited NF κB nuclear binding, and abrogated tissue factor expression induced by both VLA-4 cross-linking and adhesion to fibronectin in THP-1 cells and human peripheral blood monocytes. These studies point to the involvement of the MAP kinase pathway in the activation of monocytic cells during transmigration to inflammatory sites.

Footnotes

↵^‡ Recipient of a Medical Research Council/Pharmaceutical Manufacturers Association of Canada fellowship and the Bayer Surgical Infection Award.
↵^§ Supported by a Medical Research Council fellowship.
↵* This work was supported by the Medical Research Council of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

TF

tissue factor

ERK

extracellular signal-related kinase

MAP

mitogen-activated protein

PBM

peripheral blood monocytes

MEK

MAP kinase kinase

LPS

lipopolysaccharide

FCS

fetal calf serum

mAb

monoclonal antibody

Ab

antibody

PCA

procoagulant activity

PMSF

phenylmethylsulfonyl fluoride

MBP

myelin basic protein

DTT

dithiothreitol

PAGE

polyacrylamide gel electrophoresis.
- Received November 26, 1996.
- Revision received February 4, 1997.