VLA-4 integrin cross-linking on human monocytic THP-1 cells induces tissue factor expression by a mechanism involving mitogen-activated protein kinase.

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-alpha, 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 alpha4 and beta1 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-kappaB nuclear translocation and 4-h expression of tissue factor. Inhibition of tyrosine kinase activity with genistein (10 microg/ml) as well as selective MAP kinase inhibition with the MEK-1 inhibitor PD98059 abolished the VLA-4-dependent ERK tyrosine phosphorylation, inhibited NF kappaB 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.