Effects of protein tyrosine kinase inhibitors on cytokine‐induced adhesion molecule expression by human umbilical vein endothelial cells

Abstract

. Endothelial cells can be stimulated by the pro‐inflammatory cytokines interleukin (IL)‐1α and tumour necrosis factor (TNF)α to express the leukocyte adhesion molecules E‐selectin, vascular cell adhesion molecule (VCAM)‐1 and intercellular adhesion molecule (ICAM)‐1 but the intracellular signalling mechanisms leading to this expression are incompletely understood. We have investigated the role of protein tyrosine kinases (PTK) in adhesion molecule expression by cytokine‐activated human umbilical vein endothelial cells (HUVEC) using the PTK inhibitors genistein and herbimycin A, and the protein tyrosine phosphatase (PTP) inhibitor sodium orthovanadate. . Maximal E‐selectin expression induced by incubation of HUVEC for 4 h with IL‐1α (100 u ml−1) and TNFα (100 u ml−1) was dose‐dependently inhibited by genistein and herbimycin A. Although similar effects were seen on phorbol 12‐myristate, 13‐acetate (PMA)‐induced expression, this was not due to inhibition of protein kinase C (PKC) activity as the selective inhibitors of PKC, bisindolylmaleimide (BIM), Ro31‐7549 or Ro31‐8220 did not affect IL‐1α‐ or TNFα‐induced E‐selectin expression at concentrations which maximally inhibited PMA‐induced expression. . Genistein inhibited VCAM‐1 expression induced by incubation of HUVEC for 24 h with TNFα or IL‐1α whereas it did not affect ICAM‐1 expression induced by 24 h incubation with either of these cytokines. Herbimycin A inhibited both VCAM‐1 and ICAM‐1 expression induced by TNFα. . Basal expression of E‐selectin, VCAM‐1 and ICAM‐1 was dose‐dependently enhanced by sodium orthovanadate. In contrast, vanadate differentially affected TNFα‐induced expression of these molecules with maximal E‐selectin and ICAM‐1 expression being slightly enhanced and VCAM‐1 expression dose‐dependently reduced. . We also studied the effects of PTK and PTP inhibitors on adhesion of the human pre‐myeloid cell line U937 to TNFα‐stimulated HUVEC. Adhesion of U937 cells to HUVEC pretreated for 4 or 24 h with TNFα was dose‐dependently inhibited by genistein and herbimycin A but unaffected by daidzein. Adhesion of U937 cells after 4 h was partially inhibited by blocking antibodies against both E‐selectin and VCAM‐1 but after 24 h was only inhibited by anti‐VCAM‐1. . Sodium orthovanadate had no effect on TNFα‐induced U937 adhesion but dose‐dependently enhanced adhesion to unstimulated HUVEC. Vanadate‐induced adhesion was inhibited by an antibody against VCAM‐1. . These results demonstrate that PTK‐mediated phosphorylation events are important for the regulation of adhesion molecule expression by human endothelial cells, and additionally show that PTK inhibitors differentially affect upregulation of different adhesion molecules, implicating divergent regulatory pathways for cytokine‐induced adhesion molecule expression.

Inhibition of MAP kinase kinase (MEK) blocks endothelial PGI2 release but has no effect on von Willebrand factor secretion or E‐selectin expression

“…Thus, we have shown that thrombin, histamine or cytokines promote changes in the phosphotyrosine content of endogenous HUVEC proteins and that inhibitors of tyrosine kinases can attenuate distinct agonist-induced tyrosine phosphorylation events in parallel with PGIz release [2] or adhesion molecule expression [3]. One of the major tyrosine phosphorylated proteins observed in GPCA- [2] or cytokine-stimulated HUVEC [4] corresponds to the 42 kDa form of MAP kinase (p42 mapk) which is activated by phosphorylation on tyrosine and threonine residues catalysed by an upstream, dual-specificity MAP kinase kinase (MEK) [5]. In addition, there is a strong correlation between agonist-induced p42 mapk activation and some of the functional responses of HUVEC and we have hypothesised that activation of the MAP kinase cascade may be nec-*Corresponding author.…”

“…Agonist-induced expression of E-selectin was then assessed by enzyme-linked immunosorbent assay [3].…”

“…Our recent studies in human umbilical vein endothelial cells (HUVEC) have implicated tyrosine kinases as regulators of (i) the rapid synthesis and release of prostacyclin (PGI2) in response to G protein-coupled agonists (GPCA) [2], and (ii) the expression of leucocyte adhesion molecules following exposure to inflammatory cytokines [3]. Thus, we have shown that thrombin, histamine or cytokines promote changes in the phosphotyrosine content of endogenous HUVEC proteins and that inhibitors of tyrosine kinases can attenuate distinct agonist-induced tyrosine phosphorylation events in parallel with PGIz release [2] or adhesion molecule expression [3]. One of the major tyrosine phosphorylated proteins observed in GPCA- [2] or cytokine-stimulated HUVEC [4] corresponds to the 42 kDa form of MAP kinase (p42 mapk) which is activated by phosphorylation on tyrosine and threonine residues catalysed by an upstream, dual-specificity MAP kinase kinase (MEK) [5].…”

“…HUVEC, human umbilical vein endothelial cell; MAP kinase, mitogen activated protein kinase; MEK, MAP kinase kinase; p42 mapk, 42 kDa MAP kinase; p44 mapk, 44 kDa MAP kinase; PGI2, prostacyclin; vWF, von Willebrand Factor; GPCA, G protein-coupled agonist 0014-5793/96/$12.00 © 1996 Federation of European Biochemical Societies. PH SO0 1 4-5793(96)00547-9 essary both for GPCA-driven PGI2 synthesis [2], and for cytokine-induced adhesion molecule expression by human endothelium [4].…”

Activation of extracellular signal regulated kinase 1/2 in human dermal microvascular endothelial cells stimulated by anti-endothelial cell antibodies in sera of patients with Behçet's disease

“…JNK and p38 cascade participate in the control of IL-3 mRNA [21], and p38 cascade also participates in the induction of VCAM-1 expression by TNFa at human umbilical vein endothelial cells [11] and in the ICAM-1 expression of human pulmonary microvascular endothelial cells [10]. Also, there was a report that protein tyrosine kinase participates in the adhesion molecule expression of human umbilical vein endothelial cells by TNFa and IL-1a [22], but there have not been any reports on the signal transduction pathway of HDMEC ICAM-1 expression on HDMEC which is induced by AECA.…”