Protein Tyrosine Kinase Inhibitors Act Downstream of IL-1α and LPS Stimulated MAP-Kinase Phosphorylation to Inhibit Expression of E-Selectin on Human Umbilical Vein Endothelial Cells

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Abstract
HUVEC exposed to IL-1α, TNFα or LPS showed a time dependent increase in E-selectin expression which was maximal at between 4-6h after stimulation. Stimulation of HUVEC with IL-1α, TNFα or LPS for between 2 and 6h followed by removal or neutralisation of IL-la, TNFα or LPS and incubation in new media up to 6h resulted in identical levels of E-selectin expression at 6h, as cells which had been continuously stimulated for 6h with IL-1α, TNFα or LPS. These studies demonstrated that HUVEC were committed to the induction of E-selectin following a 2 hr incubation with either IL-1α, TNFα or LPS. The protein tyrosine kinase (PTK) inhibitors ST271, ST638 or genistein (0-100M) were ineffective in reducing cytokine or LPS stimulated E-selectin expression during a 2h cytokine or LPS stimulation of cells, in which inhibitors were either coincubated with cytokine/LPS or previously preincubated with the PTK inhibitors. However when PTK inhibitors were present during both agonist activation (2h) and subsequent expression of E-selectin after removal of agonist (4h) the PTK inhibitors resulted in a dose dependent reduction in both IL-1α and LPS stimulated E-selectin expression (IC50=50M). Moreover when PTK inhibitors were only incubated with cells for the 4h after cytokine or LPS activation of cells, PTK inhibitors resulted in a more effective dose dependant reduction in IL-1α or LPS stimulated E-selectin expression (IC50=10M). Determination of total and surface expressed E-selectin showed that the reduction in E-selectin expression represented a reduction in E-selectin protein and analysis of E-selectin mRNA by RT-PCR demonstrated that inhibition of E-selectin protein synthesis reflected reduced E-selectin mRNA. Other cytokine or LPS signalling pathways such as the activation of MAP-kinase (ERK-2) was unaffected by pre and coincubation with the PTK inhibitors. These studies suggest that HUVEC can become committed to the induction of E-selectin after removal of the stimulus and that protein tyrosine kinase inhibitors do not effect initial (5-30 min) cytokine or LPS signals which result in E-selectin expression but can inhibit the expression of cytokine/LPS induced E-selectin expression at a step distal to MAP-kinase activation.