1. ¹Department of Pathology, University of Western Ontario, London, Ontario, Canada
2. ²Department of Microbiology and Immunology, University of Western Ontario, London, Ontario, Canada

Correspondence: Professor S Chakrabarti, PhD, Department of Pathology, 4011 Dental Sciences Building, University of Western Ontario, London, Ontario, Canada. E-mail: schakrab@uwo.ca

Received 31 March 2004; Revised 20 July 2004; Accepted 27 July 2004; Published online 27 September 2004.

Abstract

Increased extracellular matrix protein deposition and basement membrane thickening are important features of diabetic angiopathy. One key matrix protein that has been shown to be instrumental in basement membrane thickening is fibronectin (FN). We have previously demonstrated that glucose-induced increased expression of endothelin-1 (ET-1), may in part, be responsible for increased FN expression via nuclear factor-B (NF-B) and activating protein (AP-1) activation. The present study was aimed at elucidating the mechanism of ET-1 with respect to mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway activation and glucose-induced FN upregulation. Human endothelial cells were exposed to either low (5 mM) or high (25 mM) glucose levels. Cells in low glucose were also treated with ET-1 peptide (5 nM). In addition, we treated cells exposed to high glucose levels with specific MAPK/ERK inhibitor PD098059 (50 M), dual ET-receptor antagonist, bosentan (10 M), and PKC blocker, chelerythrine (1 M). Following incubation period, RNA and total proteins were extracted for RT-PCR for FN and immunoblot analysis of MAPK/ERK activation. Confocal microscopy was performed for analysis of FN protein and nuclear localization of activated Elk. Electrophoretic mobility shift assay was carried out to detect NF-B and AP-1 activation. Our data demonstrates that high glucose-induced upregulation of FN messenger RNA and protein levels occur via activation of MAPK/ERK pathway, which was prevented by treatment of cells with bosentan, PD098059 and PKC blocker chelerythrine. Confocal microscopy demonstrated nuclear localization of phospho-Elk protein. Glucose-induced FN expression was also associated with protein kinase C, NF-B, and AP-1 activation. These results suggested that glucose-induced, ET- and PKC-dependent, upregulation of FN is, in part, mediated via MAPK/ERK activation.