Original article Endothelin-1 induces connective tissue growth factor expression in cardiomyocytes Anna Grazia Recchia a , Elisabetta Filice a , Daniela Pellegrino a , Aldo Dobrina b , Maria Carmela Cerra a , Marcello Maggiolini a, ⁎ a Department of Pharmaco-Biology, Cell Biology, University of Calabria, 87036 Rende (CS), Italy b Department of Physiology and Pathology, University of Trieste, Italy abstract article info Article history: Received 30 July 2008 Received in revised form 24 November 2008 Accepted 29 November 2008 Available online 10 December 2008 Keywords: Endothelin-1 Connective tissue growth factor Cardiomyocytes Signal transduction AP-1 Endothelin (ET)-1 is a vasoconstrictor involved in cardiovascular diseases. Connective tissue growth factor/ CCN2 (CTGF) is a fibrotic mediator overexpressed in human atherosclerotic lesions, myocardial infarction, and hypertension. In different cell types CTGF regulates cell proliferation/apoptosis, migration, and extracellular matrix (ECM) accumulation and plays important roles in angiogenesis, chondrogenesis, osteogenesis, tissue repair, cancer and fibrosis. In the present study, we investigated the ET-1 signaling which triggers CTGF expression in cultured adult mouse atrial-muscle HL-1 cells used as a model system. ET-1 activated the CTGF promoter and induced CTGF expression at both mRNA and protein levels. Real-time PCR analysis revealed CTGF induction also in isolated rat heart preparations perfused with ET-1. Several intracellular signals elicited by ET-1 via ET receptors and even Epidermal Growth Factor Receptor (EGFR) contributed to the up-regulation of CTGF, including ERK activation and induction of the AP-1 components c- fos and c-jun, as also evaluated by ChIP analysis. Moreover, in cells treated with ET-1 the expression of ECM component decorin was abolished by CTGF silencing, indicating that CTGF is involved in ET-1 induced ECM accumulation not only in a direct manner but also through downstream effectors. Collectively, our data indicate that CTGF could be a mediator of the profibrotic effects of ET-1 in cardiomyocytes. CTGF inhibitors should be considered in setting a comprehensive pharmacological approach towards ET-1 induced cardiovascular diseases. © 2008 Elsevier Inc. All rights reserved. 1. Introduction Endothelin-1(ET-1) is a potent vasoconstrictor peptide originally isolated from the conditioned medium of cultured vascular endothe- lial cells [1]. ET-1 plays a crucial role in several cardiovascular diseases, including chronic heart failure, ischemic heart disease, hypertension, atherosclerosis, pulmonary hypertension and chronic renal failure [2,3]. Although the endothelium is the principal source of vascular ET- 1 in humans, other tissues are capable of ET-1 production such as vascular smooth muscle cells (VSMCs), macrophages, leukocytes, fibroblasts, kidney cells and cardiomyocytes [3]. The effects of ET-1 are mediated by two main receptor subtypes, ET A and ET B , which although share approximately 60% sequence homology, bind ET-1 with similar affinities. ET A and ET B are co-expressed in a variety of human tissues, including the vasculature, whereas in endothelial cells and the renal medulla the ET B form has been reported to be more abundant [3]. Human cardiomyocytes predominantly express ET A , although ET B is present in the heart conducting tissue [3]. Activation of ET A and ET B elicits diverse biological responses depending on the identity of the receptor expressed in a particular tissue and the second messenger pathways activated [2,4]. Moreover, ET-1 receptor activation triggers signal transduction cascades that induce the transcription of genes like c- fos and c-jun, which are implicated in the mitogenic, hypertrophic and differentiation effects of ET-1 through the formation of the activator protein 1 (AP-1) complex [4]. In this regard, it is worth to note that different studies have demonstrated an increase in myocardial AP-1 DNA binding activity in experimental models of cardiac hypertrophy [5,6]. Moreover, the use of a dominant negative c- Jun (DN-Jun) which inhibits AP-1 activity has indicated a key role for c-jun as a mediator of cardiomyocyte hypertrophy [7]. Connective tissue growth factor/CCN2 (CTGF) is a heparin-binding 38-kDa member of CCN family proteins that regulates a wide-range of biological effects including cell proliferation, adhesion, angiogenesis, cell migration, extracellular matrix (ECM) production, fibrosis and apoptosis targeting primarily fibroblasts, endothelial cells, epithelial cells, chondrocytes, mesangial cells, neurons and as recently demon- strated also cardiac myocytes [8–11]. A significant number of studies have provided evidence that an increased expression of CTGF is associated with fibroproliferative disorders [9] and that CTGF knock- down or inhibition can prevent the progression of fibrotic lesions [12,13]. In the cardiovascular system, CTGF overexpression has been reported to be correlated with fibrosis in human atherosclerotic Journal of Molecular and Cellular Cardiology 46 (2009) 352–359 Abbreviations: ET-1, Endothelin-1; CTGF, Connective tissue growth factor/CCN2; ECM, extracellular matrix; VSMCs, vascular smooth muscle cells; AP-1, activator protein 1; EGFR, epidermal growth factor receptor; DCN, decorin. ⁎ Corresponding author. Tel.: +39 0984 493076; fax: +39 0984 493458. E-mail address: marcellomaggiolini@yahoo.it (M. Maggiolini). 0022-2828/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.yjmcc.2008.11.017 Contents lists available at ScienceDirect Journal of Molecular and Cellular Cardiology journal homepage: www.elsevier.com/locate/yjmcc