UNCORRECTED PROOF 1 Endothelin-1 activates extracellular signal-regulated kinases 1/2 via 2 transactivation of platelet-derived growth factor receptor in rat 3 L6 myoblasts 4 Takuya Q1 Harada, Takahiro Horinouchi ⁎, Tsunaki Higa, Akimasa Hoshi, Tsunehito Higashi, Koji Terada, 5 Yosuke Mai, Prabha Nepal, Mika Horiguchi, Chizuru Hatate, Soichi Miwa 6 Department of Cellular Pharmacology, Hokkaido University Graduate School of Medicine, Hokkaido 060-8638, Japan abstract 7 article info 8 Article history: 9 Received 23 November 2013 10 Accepted 3 April 2014 11 Available online xxxx 12 Keywords: 13 endothelin receptor 14 endothelin-1 15 extracellular signal-regulated kinases 1/2 16 transactivation 17 platelet-derived growth factor receptor 18 skeletal muscle 19 phosphorylation 20 Aims: Endothelin (ET) system plays a critical role in the development of insulin resistance and type 2 diabetes. In 21 skeletal muscle, differentiation of myoblasts to myotubes is accompanied by the development of insulin sensitiv- 22 ity. Activation of extracellular signal-regulated kinase (ERK) 1/2 inhibits the differentiation of myoblasts, leading 23 to insulin resistance. Although ET receptor (ETR) stimulation generally activates ERK1/2, the mechanism for ETR- 24 mediated ERK1/2 activation in skeletal muscle is unknown. The purpose of this study was to determine the signal 25 transduction pathway involved in ET-1-stimulated ERK1/2 phosphorylation in L6 myoblasts derived from rat 26 skeletal muscle. 27 Main methods: Changes in phosphorylation levels of ERK1/2 following stimulation with ET-1 were analyzed by 28 Western blot in L6 myoblasts. To inhibit receptor internalization, dominant-negative dynamin (K44A) was 29 overexpressed in L6 myoblasts using adenovirus-mediated gene transfer. 30 Key findings: ET-1 induced phosphorylation of ERK1/2 in L6 myoblasts. The ERK1/2 phosphorylation was 31 abolished by BQ123 (a selective ET type A receptor (ET A R) antagonist), YM-254890 (a G αq/11 protein inhibitor), 32 and AG370 (a platelet-derived growth factor receptor (PDGFR) kinase inhibitor), while U-73122 (a phospholi- 33 pase C (PLC) inhibitor) was less potent. The ERK1/2 phosphorylation was inhibited by overexpression of domi- 34 nant-negative dynamin (K44A). These results suggest that ET A R stimulation induces ERK1/2 phosphorylation 35 in L6 myoblasts through G q/11 protein-dependent, PLC-independent PDGFR transactivation which requires 36 dynamin-dependent ET A R internalization. 37 Significance: Because activation of ERK1/2 is considered to inhibit differentiation of myoblasts with the develop- 38 ment of insulin sensitivity, the ET A R-mediated PDGFR transactivation and subsequent ERK1/2 activation play an 39 important role in ET-1-induced insulin resistance. 40 © 2014 Published by Elsevier Inc. 41 42 43 44 45 Introduction 46 Endothelin (ET) system consists of three endogenous ligands, ET-1, 47 ET-2 and ET-3, and two homologous receptors, ET type A (ET A R) and 48 type B receptor (ET B R) (Horinouchi et al., 2013). The ET A R is located 49 on vascular smooth muscle cells (VSMCs), and participates in ET-1- 50 induced contraction of VSMCs. The ET B R is expressed on endothelial 51 cells, and produces vasodilatation by accelerating the release of vasodi- 52 lators such as nitric oxide. Excessive production of ET-1 induces a 53 prolonged and powerful vasoconstriction mediated through ET A R and 54 endothelial dysfunction by downregulation of endothelial ET B R, 55 resulting in enhanced peripheral vasoconstriction (Rubin, 2012). The 56 peripheral vasoconstriction causes attenuation of insulin delivery to pe- 57 ripheral tissues, leading to the development of insulin resistance 58 (Kubota et al., 2011). 59 Notably, recent studies have implied another mechanism for ET-1- 60 induced insulin resistance, i.e. a direct action on skeletal muscle cells. Life Sciences xxx (2014) xxx–xxx Abbreviations: AM, acetoxymethyl ester; BSA, bovine serum albumin; EGFR, epidermal growth factor receptor; ERK1/2, extracellular signal-regulated kinases 1/2; ET-1, endothelin-1; ET A R, endothelin type A receptor; ET B R, endothelin type B receptor; FCS, fetal calf serum; GFP, green fluorescent protein; GLUT4, glucose transporter 4; GPCR, G protein-coupled receptor; HRP, horseradish peroxidase; IRS, insulin receptor substrate; 2-ME, 2-mercaptoethanol; MEK, mitogen-activated protein kinase kinase; MOI, multiplic- ity of infection; PBS, phosphate-buffered saline; PDGFR, platelet-derived growth factor re- ceptor; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; PLC, phospholipase C; PMSF, phenylmethylsulfonyl fluoride; RFP, red fluorescent protein; RT-PCR, reverse transcription-polymerase chain reaction; SDS, sodium dodecyl sulfate; TBS-T, Tris- buffered saline-Tween 20; TKR, tyrosine kinase receptor; VSMC, vascular smooth muscle cell. ⁎ Corresponding author at: Department of Cellular Pharmacology, Hokkaido University Graduate School of Medicine, North 15, West 7, Sapporo City, Hokkaido 060-8638, Japan. Tel.: +81 11 706 6921; fax: +81 11 706 7824. E-mail address: horinouc@med.hokudai.ac.jp (T. Horinouchi). LFS-13989; No of Pages 8 http://dx.doi.org/10.1016/j.lfs.2014.04.002 0024-3205/© 2014 Published by Elsevier Inc. Contents lists available at ScienceDirect Life Sciences journal homepage: www.elsevier.com/locate/lifescie Please cite this article as: Harada T, et al, Endothelin-1 activates extracellular signal-regulated kinases 1/2 via transactivation of platelet-derived growth factor receptor i..., Life Sci (2014), http://dx.doi.org/10.1016/j.lfs.2014.04.002