Cytokine upregulation of proteinase‐activated‐receptors 2 and 4 expression mediated by p38 MAP kinase and inhibitory kappa B kinaseβin human endothelial cells

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Abstract
Background and purpose: Up‐regulation of proteinase‐activated receptor‐2 (PAR2) is a factor in a number of disease states and we have therefore examined the signalling pathways involved in the expression of the receptor.Experimental approach: We investigated the effects of tumour necrosis factor‐α (TNF‐α), interleukin‐1β (IL‐1β), trypsin and the PAR2 activating peptide, 2‐furoyl(2f)‐LIGKV‐OH on both mRNA and functional expression of PAR2 in human umbilical vein endothelial cells (HUVECs). The effect of specific chemical inhibitors and dominant negative adenovirus constructs of the mitogen‐activated protein kinase (MAPK) cascade and the nuclear factor kappa B (NF‐κB) signalling pathway was assessed. Methods included semi‐quantitative and quantitative RT‐PCR, [3H]inositol phosphate (IP) accumulation and Ca2+‐dependent fluorescence.Key results: The above agonists induced both mRNA and functional expression of PAR2; PAR4 mRNA, but not that for PAR1 or PAR‐3, also increased following TNFα treatment. Inhibition of p38 MAP kinase reduced PAR2 and PAR4 expression, whilst inhibition of MEK1/ERK/JNK was without effect. A similar dependency upon p38 MAP kinase was observed for the expression of PAR4. TNFα ‐induced enhancement of PAR2 stimulated [3H]‐inositol phosphate accumulation (IP) and Ca2+signalling was abolished following SB203580 pre‐treatment. Infection with adenovirus encoding dominant‐negative IKKβ (Ad.IKKβ+/−) and to a lesser extent dominant‐negative IKKα (Ad.IKKα+/−), substantially reduced both control and IL‐1β‐ induced expression of both PAR2 and PAR4 mRNA and enhancement of PAR2‐stimulated IP accumulation and Ca2+mobilisation.Conclusions and implications: These data reveal for the first time the signalling events involved in the upregulation of both PAR2 and PAR4 during pro‐inflammatory challenge.British Journal of Pharmacology(2007)150, 1044–1054. doi:10.1038/sj.bjp.0707150

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