kjpp The Korean Journal of Physiology & Pharmacology Korean J Physiol Pharmacol 1226-4512 2093-3827 Korean J Physiol Pharmacol 10.4196/kjpp.2011.15.1.31 kjpp-15-31 Original Article Extracellular Nucleotides Can Induce Chemokine (C-C motif) Ligand 2 Expression in Human Vascular Smooth Muscle Cells KimJeung-Il 1 KimHye-Young 2 KimSun-Mi 2 LeeSae-A 2 SonYong-Hae 2 EoSeong-Kug 3 RhimByung-Yong 2 KimKoanhoi 2 Department of Orthopedic Surgery, Pusan National University Hospital, Busan 602-739, Korea Department of Pharmacology, School of Medicine, Pusan National University, Yangsan 626-870, Korea Laboratory of Microbiology, College of Veterinary Medicine and Bio-Safety Research Institute, Chonbuk National University, Jeonju 561-756, Korea Corresponding to: Koanhoi Kim, Department of Pharmacology, School of Medicine, Pusan National University, Beomeo-ri, Mulgeum-eup, Yangsan 626-770, Korea. (Tel) 82-51-510-8064, (Fax) 82-51-510-8068, (E-mail) koanhoi@pusan.ac.kr 022011 18022011 15 1 31 36 17012011 16022011 19022011 Copyright © 2011 Korean J Physiol Pharmacol 2011 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract

To understand the roles of purinergic receptors and cellular molecules below the receptors in the vascular inflammatory response, we determined if extracellular nucleotides up-regulated chemokine expression in vascular smooth muscle cells (VSMCs). Human aortic smooth muscle cells (AoSMCs) abundantly express P2Y1, P2Y6, and P2Y11 receptors, which all respond to extracellular nucleotides. Exposure of human AoSMCs to NAD+, an agonist of the human P2Y11 receptor, and NADP+ as well as ATP, an agonist for P2Y1 and P2Y11 receptors, caused increase in chemokine (C-C motif) ligand 2 gene (CCL2) transcript and CCL2 release; however, UPT did not affect CCL2 expression. CCL2 release by NAD+ and NADP+ was inhibited by a concentration dependent manner by suramin, an antagonist of P2-purinergic receptors. NAD+ and NADP+ activated protein kinase C and enhanced phosphorylation of mitogen-activated protein kinases and Akt. NAD+- and NADP+-mediated CCL2 release was significantly attenuated by SP6001250, U0126, LY294002, Akt inhibitor IV, RO318220, GF109203X, and diphenyleneiodium chloride. These results indicate that extracellular nucleotides can promote the proinflammatory VSMC phenotype by up-regulating CCL2 expression, and that multiple cellular elements, including phosphatidylinositol 3-kinase, Akt, protein kinase C, and mitogen-activated protein kinases, are involved in that process.

 CCL2 NAD+ P2 receptors Vascular smooth muscle cell References Aukrust P Halvorsen B Yndestad A Ueland T Øie E Otterdal K Gullestad L Damås JK Chemokines and cardiovascular risk Arterioscler Thromb Vasc Biol. 2008 2819091919 Hansson GK Libby P The immune response in atherosclerosis: a double-edged sword Nat Rev Immunol. 2006 6508519 Deshmane SL Kremlev S Amini S Sawaya BE Monocyte chemoattractant protein-1 (MCP-1): an overview J Interferon Cytokine Res. 2009 29313326 Ohtsuki K Hayase M Akashi K Kopiwoda S Strauss HW Detection of monocyte chemoattractant protein-1 receptor expression in experimental atherosclerotic lesions: an autoradiographic study Circulation. 2001 104203208 Takeya M Yoshimura T Leonard EJ Takahashi K Detection of monocyte chemoattractant protein-1 in human atherosclerotic lesions by an anti-monocyte chemoattractant protein-1 monoclonal antibody Hum Pathol. 1993 24534539 Boisvert WA Santiago R Curtiss LK Terkeltaub RA A leukocyte homologue of the IL-8 receptor CXCR-2 mediates the accumulation of macrophages in atherosclerotic lesions of LDL receptor-deficient mice J Clin Invest. 1998 101353363 Gosling J Slaymaker S Gu L Tseng S Zlot CH Young SG Rollins BJ Charo IF MCP-1 deficiency reduces susceptibility to atherosclerosis in mice that overexpress human apolipo-protein B J Clin Invest. 1999 103773778 Burnstock G The past, present and future of purine nucleotides as signalling molecules Neuropharmacology. 1997 3611271139 Communi D Janssens R Suarez-Huerta N Robaye B Boeynaems JM Advances in signalling by extracellular nucleotides. the role and transduction mechanisms of P2Y receptors Cell Signal. 2000 12351360 Burnstock G Purinergic regulation of vascular tone and remodelling Auton Autacoid Pharmacol. 2009 296372 Dawicki DD Chatterjee D Wyche J Rounds S Extracellular ATP and adenosine cause apoptosis of pulmonary artery endothelial cells Am J Physiol. 1997 273L485494 Malam-Souley R Seye C Gadeau AP Loirand G Pillois X Campan M Pacaud P Desgranges C Nucleotide receptor P2u partially mediates ATP-induced cell cycle progression of aortic smooth muscle cells J Cell Physiol. 1996 1665765 Zheng LM Zychlinsky A Liu CC Ojcius DM Young JD Extracellular ATP as a trigger for apoptosis or programmed cell death J Cell Biol. 1991 112279288 Choi KH Park JW Kim HY Kim YH Kim SM Son YH Park YC Eo SK Kim K Cellular factors involved in CXCL8 expression induced by glycated serum albumin in vascular smooth muscle cells Atherosclerosis. 2010 2095865 Wang L Karlsson L Moses S Hultgårdh-Nilsson A Andersson M Borna C Gudbjartsson T Jern S Erlinge D P2 receptor expression profiles in human vascular smooth muscle and endothelial cells J Cardiovasc Pharmacol. 2002 40841853 Sung SC Kim K Lee KA Choi KH Kim SM Son YH Moon YS Eo SK Rhim BY 7-Ketocholesterol upregulates interleukin-6 via mechanisms that are distinct from those of tumor necrosis factor-alpha, in vascular smooth muscle cells J Vasc Res. 2009 463644 Chung SW Park JW Lee SA Eo SK Kim K Thrombin promotes proinflammatory phenotype in human vascular smooth muscle cell Biochem Biophys Res Commun. 2010 396748754 Harper S Webb TE Charlton SJ Ng LL Boarder MR Evidence that P2Y4 nucleotide receptors are involved in the regulation of rat aortic smooth muscle cells by UTP and ATP Br J Pharmacol. 1998 124703710 Wilden PA Agazie YM Kaufman R Halenda SP ATP-stimulated smooth muscle cell proliferation requires independent ERK and PI3K signaling pathways Am J Physiol. 1998 275H12091215 Zhang W Liu HT MAPK signal pathways in the regulation of cell proliferation in mammalian cells Cell Res. 2002 12918 Hou M Möller S Edvinsson L Erlinge D Cytokines induce upregulation of vascular P2Y(2) receptors and increased mitogenic responses to UTP and ATP Arterioscler Thromb Vasc Biol. 2000 2020642069 Yu SM Chen SF Lau YT Yang CM Chen JC Mechanism of extracellular ATP-induced proliferation of vascular smooth muscle cells Mol Pharmacol. 1996 5010001009 Needleman P Minkes MS Douglas JR Jr Stimulation of prostaglandin biosynthesis by adenine nucleotides. Profile of prostaglandin release by perfused organs Circ Res. 1974 34455460 Taniyama Y Griendling KK Reactive oxygen species in the vasculature: molecular and cellular mechanisms Hypertension. 2003 4210751081 Figures and Tables

The effects of extracellular nucleotides on CCL2 gene transcripts in human VSMCs. (A) Total RNA was isolated from human AoSMCs, and transcripts of the indicated P2 purinergic receptors were identified by RT-PCR. (B) Human AoSMCs were treated with nucleotides for the indicated time periods, and CCL2 transcripts were amplified by RT-PCR. (C) Human AoSMCs were treated for 6 h with the indicated concentrations of NAD+ or NADP+, and induction of CCL2 gene transcripts was examined by RT-PCR.

The effects of nucleotides on NAD+- and NADP+-mediated CCL2 release. (A) Human AoSMCs (1×106 cells) were incubated in the absence (control) or presence of indicated nucleotides (10–4 M for each nucleotide). Culture medium was collected and the amount of secreted CCL2 was measured by ELISA. ∗p<0.001 vs. control. (B). Human AoSMCs were incubated with NAD+ or NADP+ (10–4 M) in the absence or presence of indicated concentrations of suramin. CCL2 released into the medium was measured by ELISA. ∗p<0.001 vs. control; #p<0.01 vs. NAD+ or NADP+, ##p<0.001 vs. NAD+ or NADP+.

The roles of MAPKs in NAD+- and NADP+-mediated CCL2 release. (A) Human AoSMCs were exposed to NAD+ or NADP+ for the indicated time periods, after which an equal amount of protein was subjected to Western blot analysis using antibodies for α-tubulin and phosphorylated and unphosphorylated forms of ERK, p38 MAPK, and JNK. (B) Human AoSMCs were incubated for an hour with SP600125, U0126, and SB202190 (10 μM each) and stimulated with NAD+ or NADP+. Culture media were collected to measure the amount of secreted CCL2. ∗p< 0.001 vs. control, #p<0.001 vs. NAD+ or NADP+.

The roles of Akt pathways in NAD+- and NADP+-mediated CCL2 release. (A) Human AoSMCs were exposed to NAD+ or NADP+ for the indicated time periods, after which an equal amount of protein was subjected to Western blot analysis using antibodies for Akt and phosphorylated Akt. (B) Human AoSMCs were incubated for 1 h with LY294002 and Akti IV (10 μM each) and stimulated with NAD+ or NADP+. The amount of secreted CCL2 was measured by ELISA. ∗p<0.001 vs. control, #p<0.001 vs. NAD+ or NADP+.

The roles of PKC in NAD+- and NADP+-mediated CCL2 release. (A) Human AoSMCs were exposed to NAD+ or NADP+ for the indicated time periods, after which PKC activity was determined. ∗p<0.01 vs. control. (B) Human AoSMCs were incubated for 1 h with GF109203X (3 μM) and RO318220 (1 μM) and stimulated with NAD+ or NADP+. The amount of secreted CCL2 was measured by ELISA. ∗p<0.001 vs. control, #p<0.001 vs. NAD+ or NADP+.

The roles of NADPH oxidase in NAD+- and NADP+ –mediated CCL2 release. Human AoSMCs were incubated for 1 h with DPI (10 μM) and stimulated with NAD+ or NADP+. CCL2 gene transcript was amplified by RT-PCR (A), and the amount of released CCL2 was measured by ELISA (B). ∗p<0.001 vs. control, #p<0.001 vs. NAD+ or NADP+.