The band intensity of 3 independent blots were measured by densitometrics quantitation. in the human VSMCs were measured by Western blot analysis using anti phospho-ERK1/2 (Thr202/Tyr204) and anti CHSY1 antibodies. Results ET-1 (100 nM) and EGF (100 ng/ml) stimulated ERK1/2 phosphorylation and inhibited in the presence of bosentan (ET receptor inhibitor), AG1478 (EGFR inhibitor), and OSI-420 DPI (NOX antagonist). Also, ET-1 treatment increased CHSY1 enzyme level; this response was suppressed by bosentan, AG1478, DPI, and SB431542, TGF- receptor antagonist. This study revealed that ET-1 increases expression of CHSY1 through transactivation of EGF and TGF- receptors. Conclusion Transactivation through the EGF receptor mediated by phospho-ERK1/2 leads to expression of CHSY1 protein. EGF receptor transactivation by ET-1 is shown for the first time, to be dependent on NOX enzymes. strong class=”kwd-title” Keywords: CHSY1 Enzyme, Endothelin-1, Epidermal Growth Factor, NADPH Oxidase Introduction Based on the “response to retention hypothesis”stating, atherosclerosis is commenced by atherogenic lipoprotein entrapment by proteoglycan Rabbit Polyclonal to TEP1 in the vessel wall. This is one of the crucial causes of atherosclerosis early stage. Proteoglycans are highly glycosylated proteins, consisting of core protein, which is covalently linked to glycosaminoglycan (GAG) chain. GAG chain is negatively charged due to the sulfate and carboxylic acid groups of chondroitin/dermatan sulfate (CS/ DS) chains. Therefore low density lipoprotein (LDL) particle (apo B100 has positively charged amino acid residues) interact with negative GAG chain results in retention of LDL in intima space of artery wall. Due to interactions with extracellular matrix (ECM) proteoglycan, LDL retention in the extracellular space of the arterial increases the chance of its oxidation and accumulation, promoting atherosclerosis (1, 2). Studies have shown that an increasing GAG chain length association with lipid binding affinity results in LDL retention in vascular wall and atherosclerosis development (3, 4). Chondroitin sulfate synthase 1 (CHSY1) is one of the family of GAG synthesizing enzymes involved in hyper elongation of GAG chain (5, 6). Previous atherosclerosis mouse model studies have demonstrated that GAG synthesizing enzymes genes elevated expression is correlated with increase in the GAG chain length and lipid deposition in vascular wall (3, 7). Several growth factors have been shown to mediate alteration and modification (hyper-elongation) of proteoglycan structure via regulated expression of GAG bio-synthesis enzymes in the vascular smooth muscle cells (VSMCs) (8, 9). Endothelin-1 (ET-1) is a novel vasoconstrictor peptide, a 21 amino acid peptide that was produced by endothelial cells in the vascular wall (10, 11). Studies have revealed that ET-1 level increases in cardiovascular diseases such as atherosclerosis (12). Ballinger et al. (13) reported that ET-1 mediated elevation in radiosulfate incorporation secreted proteoglycan and induced proteoglycan synthesis in the VSMCs. ET-1 receptors OSI-420 (type A and type B) belong to G protein coupled receptors (GPCRs) family. GPCRs are seven transmembrane cell surface receptors which mediate pathophysiological cellular response through three signaling pathways. First, in classical pathway, GPCR agonists cause conformational changes in receptor that leads to downstream signaling activation. Second is -arrestin scaffold pathway (14, 15) and third is transactivation pathway; transactivation signaling pathway was initially described by Daub et al. (16) who reported that GPCR agonists such as angiotensin II (Ang II) lead to activation and phosphorylation of epidermal growth factor receptor (EGFR) resulting in phosphorylation of downstream signaling mediators such as extracellular signal-regulated kinases (ERKs). GPCR agonists such as ET-1 and thrombin mediated TGF- receptor type I (TRI) activation by C-terminal phosphorylation of smad2 (pSmad2) induction, which is immediately TRI downstream regulation that also, followed by increasing proteoglycan synthesis (14, 17, 18). Chen et al. (19) reported that ET-1 mediated EGFR transactivation stimulated NADPH Oxidase (NOX), which produced reactive oxygen species (ROS) in different cell types such as cardiac fibroblasts. ROS generation is one of the critical components of the ET-1 signaling pathway. ROS activates and regulates numerous signal transduction cascades and gene expression in VSMC stimulated by ET-1, Thrombin, and Ang II. NOX enzymes especially produce ROS through acting as an electron donor to oxygen molecule in the superoxide anion formation. NOX1 is important in atherosclerosis pathogenesis. And, it is expressed in endothelial cells and VSMCs (20-22). In this study, we tested hypothesis that ET-1 could increase CHSY1 enzyme level through transactivation of EGF receptor and whether NOX is involved in this transactivation signaling pathway. The results revealed that ET-1 mediated p-ERK1/2 via EGFR transactivation. ET-1 from transactivation of two receptors OSI-420 (EGFR and TGFR) increased the expression of CHSY1 protein and NOX as an important mediator involved in this signaling pathway. Materials and Methods This study was approved by the Research Committee and The Ethical Committee of The Ahvaz Jundishapur University of Medical.