Transcriptional regulation of protease-activated receptor-4 (PAR-4) by high glucose in human vascular smooth muscle cells

Dangwal, Seema , Dr. Rauch, Bernhard , Prof. Dr. Schrör, Karsten , Dr. Rosenkranz, Anke. Universitaetsklinikum der Heinrich-Heine Universitaet Duesseldorf Institut fuer Pharmakologie und Klinische Pharmakologie, Universitaetsstrasse 1, 40225 Duesseldorf, Germany.

Background and purpose: Diabetes is strongly associated with vascular proliferation and remodelling, inflammation and enhanced thrombogenicity. Thrombin stimulates vascular smooth muscle cell (SMC) proliferation, migration and inflammatory gene expression, independently of its coagulatory actions. These effects are mediated via protease-activated receptors PAR-1, PAR-3 and PAR-4. We have examined if PAR expression is regulated by high glucose in human vascular SMCs.

Experimental approach: Human saphenous vein SMC maintained in normal glucose (5.5mM) were serum-deprived prior to stimulation with high glucose (25 mM) ± study drugs. PAR expression levels were quantified by real-time PCR, western blotting and immunofluorescence; PAR promoter activity was measured by luciferase reporter assay. SMC migration was determined by wound scratch assay.

Key results: High glucose induced a rapid and sustained increase in PAR-4 mRNA expression (3.5±0.6 fold at 1.5h; 2.7±0.4 fold at 96h; n=7, p<0.05). Neither PAR-1 nor PAR-3 were regulated by high glucose. PAR-4 promoter activity was significantly increased by 6h (to 642±80%, p<0.05) and this increase was sustained to 48h. PAR-4 immunofluorescence and total protein expression were also increased over 48-96h. Accordingly, high glucose pretreatment (48h) enhanced the stimulatory effect of a PAR-4 activating peptide (AYPGKF) on SMC migration and TNFα expression. The osmolar control mannitol did not influence PAR-4 expression or function. Transcriptional effects of high glucose on PAR-4 were prevented by inhibition of protein kinase C or NFκB, and were associated with enhanced NFκB binding to the PAR-4 promoter in preliminary chromatin immunoprecipitation assays.

Conclusions and implications: High glucose evokes a rapid and sustained induction of PAR-4 in human SMC. This mechanism involves protein kinase C and NFκB, and enhances the inflammatory and migratory effects of PAR-4 activation. Such actions might contribute to the enhanced thrombotic and proliferative responses of diabetic vessels upon injury.