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

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-alpha ( TNF-alpha), interleukin-1 beta ( IL-1 beta), 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-kappa B) signalling pathway was assessed. Methods included semi-quantitative and quantitative RT-PCR, [ H-3] 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 TNF alpha 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. TNF alpha-induced enhancement of PAR2 stimulated [ H-3]-inositol phosphate accumulation ( IP) and Ca2+ signalling was abolished following SB203580 pre-treatment. Infection with adenovirus encoding dominant-negative IKK beta ( Ad.IKK beta(+/-)) and to a lesser extent dominant-negative IKK alpha ( Ad.IKK alpha(+/-)), substantially reduced both control and IL-1 beta-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.