Regulation of proteinese-activated-receptors 2 and 4 expression by p38 map kinase and inhibitory kappa B kinases α and β in human endothelial cells

Ritchie, E., Saka, M., Kanke T., Drummond R & Plevin, R. Department of Physiology & Pharmacology, University of Strathclyde, Glasgow, UK.

Proteinase activated receptor-2 (PAR-2) is a G-protein coupled receptor that is activated by cleavage of the N-terminus by serine proteases with trypsin like selectivity (Nystedt et al., 1995; Molino et al., 1997), or by activating peptides based on the tethered ligand sequence SLIGKV (Hollenberg et al., 1997). The receptor is expressed in multiple tissues by a wide variety of cells where it has an important role in both mitogenesis and inflammation (Macfarlane et al., 2001). Interestingly, studies in PAR-2 knockout homozygous and heterozygous mice suggest that the severity of swelling following inflammatory stimuli is dependent on the level of PAR-2 expression (Ferrel et al., 2003). Indeed, upregulation of PAR-2 appears to be an important factor in a number of disease states. Therefore, in this study we examined the signalling pathways involved in the functional expression of the receptor following cytokine pre-treatment.

Human umbilical vein endothelial cells (HUVECs) were used in this study. Changes in PAR gene expression was assessed by quantitative and semi-quantitative RT-PCR, while enhanced f unctional expression of PAR-2 was indirectly assessed by [3H]inositol phosphate accumulation and Ca2+ fluorescence. Statistical analysis was performed by one way analysis of variance using a Dunnets post test.

Increased PAR-2 gene expression was obtained for LPS (200ng/ml) (2.857 +/-0.204-fold, n=3), Il-1β (10ng/ml) (3.11 +/-0.156-fold, n=3) and PMA (100nM) (3.027 +/- 0.413-fold, n=3). Receptor upregulation by TNFα enhanced [3H]-inositol phosphate accumulation (from 2.18 +/-0.456-fold to 4.489 +/-0.891-fold, p<0.05 n=4) and increased intracellular Ca2+ mobilisation following PAR-2 activation. Quantitative RT-PCR revealed that following TNFα treatment PAR-4 expression was upregulated concomitantly with PAR-2 (PAR-2: 3.19 +/-0.483-fold, PAR-4: 3.568 +/-0.896 fold, p<0.05, n=3). Increased PAR-2 and PAR-4 expression was significantly abrogated by pre-treatment with 10 µM of the p38 MAP kinase inhibitor SB203580 (PAR-2: TNF α = 3.846 +/- 0.541-fold, TNF α + SB=1.063 +/-0.25-fold. PAR-4: TNF α = 3.568 +/-0.517, TNFα + SB = 0.156 +/-0.06 fold) but not by inhibition of ERK (PD98059) or JNK (SP600125). Infection of cells with adenovirus constructs (300pfu/cell) encoding dominant negative isoforms of IKKα and IKKβ also significantly inhibited PAR-2 and PAR-4 expression (PAR-2: Il-1β= 3.753 +/-0.623-fold, Ad.IKK α+/- + Il-1β= 1.742 +/-0.579-fold, IKK β+/- + Il-1β= 0.733 +/-0.165-fold. PAR-4: Il-1β= 3.69 +/-0.823-fold, IKK α+/- + Il-1β = 2.24 +/-0.296-fold, IKK β+/- + Il-1β= 0.733 +/-0.165-fold). Taken together these results implicate p38 MAP kinase and IKK in the regulation of PAR-2 and PAR-4 expression in human endothelial cells.

Ferrell et al., (2003) J Clin Invest, 111(1): 35-41.
Hollenberg et al., (1997) Can J Physiol Pharmacol 75:832-841
Macfarlane et al., (2001) Pharmacol Rev. 53(2): 245-82.
Molino et al., (1997) J Biol Chem, 272(7): 4043-49.
Nystedt et al., (1995) J Biol Chem, 270(11): 5950-55.

This work was supported by a BHF studentship to ER .