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.
OBJECTIVE
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.


METHODS
We investigated the effects of tumour necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), 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-kappaB) signalling pathway was assessed. Methods included semi-quantitative and quantitative RT-PCR, [(3)H]inositol phosphate (IP) accumulation and Ca(2+)-dependent fluorescence.


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 TNFalpha 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. TNFalpha -induced enhancement of PAR2 stimulated [(3)H]-inositol phosphate accumulation (IP) and Ca(2+) signalling was abolished following SB203580 pre-treatment. Infection with adenovirus encoding dominant-negative IKKbeta (Ad.IKKbeta(+/-)) and to a lesser extent dominant-negative IKKalpha (Ad.IKKalpha(+/-)), substantially reduced both control and IL-1beta- induced expression of both PAR2 and PAR4 mRNA and enhancement of PAR2-stimulated IP accumulation and Ca(2+) mobilisation.


CONCLUSIONS
These data reveal for the first time the signalling events involved in the upregulation of both PAR2 and PAR4 during pro-inflammatory challenge.
Ritchie
E
E
The Department of Physiology and Pharmacology, The University of Strathclyde, Strathclyde Institute for Biomedical Sciences, Glasgow, UK.
Saka
M
M
Mackenzie
C
C
Drummond
R
R
Wheeler-Jones
C
C
Kanke
T
T
Plevin
R
R
D000256:Adenoviridae / Q000235:genetics; D020013:Calcium Signaling / Q000187:drug effects; D002478:Cells, Cultured; D016207:Cytokines / Q000502:physiology*; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D005799:Genes, Dominant; D006801:Humans; D051550:I-kappa B Kinase / Q000235:genetics / Q000378:metabolism*; D007093:Imidazoles / Q000494:pharmacology; D007295:Inositol Phosphates / Q000378:metabolism; D053583:Interleukin-1beta / Q000502:physiology; D020935:MAP Kinase Signaling System / Q000187:drug effects; D009842:Oligopeptides / Q000494:pharmacology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis; D044464:Receptor, PAR-2 / Q000096:biosynthesis* / Q000235:genetics; D018179:Receptors, Thrombin / Q000096:biosynthesis* / Q000235:genetics; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology; D015854:Up-Regulation / Q000187:drug effects; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism*
C471410:2-furoyl-leucyl-isoleucyl-glycyl-lysyl-valine; D016207:Cytokines; D007093:Imidazoles; D007295:Inositol Phosphates; D053583:Interleukin-1beta; D009842:Oligopeptides; D047428:Protein Kinase Inhibitors; D011725:Pyridines; D012333:RNA, Messenger; D044464:Receptor, PAR-2; D018179:Receptors, Thrombin; D014409:Tumor Necrosis Factor-alpha; D051550:I-kappa B Kinase; D048051:p38 Mitogen-Activated Protein Kinases; D014357:Trypsin; C112908:protease-activated receptor 4; C093642:SB 203580