Endothelin-1 promotes myofibroblast induction through the ETA receptor via a rac/PI3 kinase/Akt-dependent pathway and is essential for the enhanced contractile phenotype of fibrotic fibroblasts

Xu Shi-wen¹, Yunliang Chen¹, Christopher P. Denton¹, Mark Eastwood², Elisabetta A. Renzoni³, George Bou-Gharios⁴, Jeremy D. Pearson⁵, Michael Dashwood⁶, Roland M. duBois³, Carol M. Black¹, Andrew Leask¹^*, and David J. Abraham¹

¹ Centre for Rheumatology, Royal Free and University College Medical School, London NW3 2PF, UK
² Centre for Tissue Engineering Research, University of Westminster, 115 New Cavendish St. London W1W 6UM
³ Interstitial Lung Disease Unit, Royal Brompton Hospital, Imperial College School of Medicine, London SW3 6LR, U.K.
⁴ Dept. of Medicine, Imperial College London, Hammersmith Campus, Du Cane Road, London W12 0NN
⁵ Centre for Cardiovascular Biology & Medicine, GKT School of Biomedical Sciences, King’s College London, Guy’s Campus, London, SE1 1UL, UK
⁶ Department of Molecular Pathology, Royal Free and University College Medical School, London NW3 2PF, UK

^* Corresponding author. E-mail address: a.leask{at}rfc.ucl.ac.uk.

The endothelins are a family of endothelium-derived peptidesthat possess a variety of functions, including vasoconstriction.Endothelin-1 (ET-1) is up-regulated during tissue repair, andpromotes myofibroblast contraction and migration, hence contributingto matrix remodeling during tissue repair. Here, we show thataddition of ET-1 to normal lung fibroblasts induces expressionof proteins that contribute to a contractile phenotype, including ${alpha}$ -smooth muscle actin ( ${alpha}$ -SMA), ezrin, moesin and paxillin. Weconfirm that ET-1 enhances the ability of lung fibroblasts tocontract extracellular matrix, a function essential for tissuerepair, through induction of de novo protein synthesis. Blockadeof the Akt/PI-3 kinase pathway with Ly294002 and wortmanninprevents the ability of ET-1 to induce ${alpha}$ -SMA, ezrin, paxillinand moesin, and to promote matrix contraction. Dominant negativerac and Akt blocked the ability of ET-1 to promote formationof ${alpha}$ -SMA stress fibers. Using specific ET-1 receptor inhibitors,we show that ET-1 induces collagen matrix contraction throughthe ETA, but not the ETB, receptor. Relative to normal pulmonaryfibroblasts, fibroblasts cultured from scars of patients withthe fibrotic disease systemic sclerosis (scleroderma; SSc) showenhanced ET-1 expression and binding. SSc lung fibroblasts showincreased ability to contract a collagen matrix and elevatedexpression of the procontractile proteins ${alpha}$ -SMA, ezrin, paxillinand moesin, which are greatly reduced by antagonizing endogenousET-1 signaling. Thus, blocking ET-1 or the PI-3-kinase/Akt cascadesmight be beneficial in reducing scar formation in pulmonaryfibrosis.