Protein tyrosine kinase inhibitors act downstream of IL-1 alpha and LPS stimulated MAP-kinase phosphorylation to inhibit expression of E-selectin on human umbilical vein endothelial cells.
HUVEC exposed to IL-1 alpha, TNF alpha 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-1 alpha, TNF alpha or LPS for between 2 and 6h followed by removal or neutralisation of IL-1 alpha, TNF alpha 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-1 alpha, TNF alpha or LPS. These studies demonstrated that HUVEC were committed to the induction of E-selectin following a 2 hr incubation with either IL-1 alpha, TNF alpha 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-1 alpha 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 dependent reduction in IL-1 alpha 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.
Adamson
P
P
Vascular Biology Research Centre, Kings College London.
Tighe
M
M
Pearson
J D
JD
D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000032:analysis / Q000378:metabolism*; D002478:Cells, Cultured; D019040:E-Selectin / Q000235:genetics*; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology*; D007529:Isoflavones / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology*; D019950:Mitogen-Activated Protein Kinase 1; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000502:physiology*; D012333:RNA, Messenger / Q000032:analysis; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D014471:Umbilical Veins
D019040:E-Selectin; D004791:Enzyme Inhibitors; D007375:Interleukin-1; D007529:Isoflavones; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; C004742:daidzein; D011505:Protein-Tyrosine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1