Endoplasmic reticulum stress-mediated inhibition of NSMase2 elevates plasma membrane cholesterol and attenuates NO production in endothelial cells Ruchi Chaube a, 1 , Vasantha Madhuri Kallakunta a, 1 , Michael Graham Espey b, 1 , Ryan McLarty a , Adam Faccenda a , Sirinart Ananvoranich a , Bulent Mutus a, ⁎ a Department of Chemistry & Biochemistry, University of Windsor, Windsor, ON, Canada b Molecular and Clinical Nutrition Section, NIDDK, National Institutes of Health Bethesda, MD 20892-1372, USA abstract article info Article history: Received 15 July 2011 Received in revised form 12 October 2011 Accepted 17 October 2011 Available online 25 October 2011 Keywords: Neutral sphingomyelinase 2 Nitric oxide synthase Nitric oxide ER stress Endothelial dysfunction Chronic exposure of blood vessels to cardiovascular risk factors such as free fatty acids, LDL-cholesterol, homocysteine and hyperglycemia can give rise to endothelial dysfunction, partially due to decreased syn- thesis and bioavailability of nitric oxide (NO). Many of these same risk factors have been shown to induce endoplasmic reticulum (ER) stress in endothelial cells. The objective of this study was to examine the mechanisms responsible for endothelial dysfunction mediated by ER stress. ER stress elevated both intra- cellular and plasma membrane (PM) cholesterols in BAEC by ~3-fold, indicated by epifluorescence and cholesterol oxidase methods. Increases in cholesterol levels inversely correlated with neutral sphingomyelinase 2 (NSMase2) activity, endothelial nitric oxide synthase (eNOS) phospho-activation and NO-production. To con- firm that ER stress-induced effects on PM cholesterol were a direct consequence of decreased NSMase2 activity, enzyme expression was either enhanced or knocked down in BAEC. NSMase2 over-expression did not signifi- cantly affect cholesterol levels or NO-production, but increased eNOS phosphorylation by ~1.7-fold. Molecular knock down of NSMase2 decreased eNOS phosphorylation and NO-production by 50% and 40%, respectively while increasing PM cholesterol by 1.7-fold and intracellular cholesterol by 2.7-fold. Furthermore, over- expression of NSMase2 in ER-stressed BAEC lowered cholesterol levels to within control levels as well as nearly doubled the NO production, restoring it to ~ 74% and 68% of controls using tunicamycin and palmitate, respectively. This study establishes NSMase2 as a pivotal enzyme in the onset of endothelial ER stress-mediated vascular dys- function as its inactivation leads to the attenuation of NO production and the elevation of cellular cholesterol. Crown Copyright © 2011 Published by Elsevier B.V. All rights reserved. 1. Introduction Endothelial dysfunction is a common occurrence in the pathogenesis of many cardiovascular diseases such as hypertension, coronary artery disease, chronic heart failure, diabetes, peripheral artery disease and chronic renal failure [1]. Endothelial dysfunction is also an initiating fac- tor in the pathogenesis of atherosclerosis [2]. The hallmark of endothe- lial dysfunction is the dominance of vasoconstriction over vasodilation, which is attributed to reduced NO generation by endothelial nitric oxide synthase (eNOS), increased endoplasmic reticulum (ER) stress and hypercholesterolemia. Endothelial dysfunction can be induced by a variety of vascular stresses that include both oxidative and nitrosative modifiers [3,4,5]. Stress within the ER, the organelle responsible for the synthesis and processing of numerous proteins, is an important early event in the progression of endothelial dysfunction. The cellular response to ER stress is accompanied by dysregulated cholesterol metabolism result- ing in increased intracellular cholesterol accumulation [6,7,8,9,10,11]. Golgi-resident proteases site-1-serine protease (S1P) and site-2-zinc metalloproteinase (S2P) enable restoration of ER homeostasis by facilitating nuclear translocation of activating transcription factor 6 (ATF6). S1P and S2P also cleave and activate the sterol-responsive element-binding protein (SREBP-2), a transcription factor that upregu- lates genes responsible for cholesterol synthesis and uptake [9,10]. In turn, hypercholesterolemia has been shown to contribute to endothelial Biochimica et Biophysica Acta 1821 (2012) 313–323 Abbreviations: NO, nitric oxide; ER, endoplasmic reticulum; BAEC, bovine aortic endothelial cells; eNOS, endothelial nitric oxide synthase; NSMase or NSM, neutral sphingomyelinase; SREBP-2, sterol-responsive element-binding protein; Tm, tunica- mycin; Pm, palmitate; RNAi, RNA interference; siRNA, small interfering RNA; AcCh, acetylcholine; NOA, nitric oxide analyzer; IP, immunoprecipitation; DAOS, N-ethyl-N- (2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline; 4-AAP, 4-aminoantipyrine; Carboxy- H 2 DCFDA, 5-(and-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate; DCF, dichloro- fluorescein; ROS, reactive oxygen species; TCA, trichloroacetic acid; OE, over-expression; KD, knock-down; S1P, site-1-serine protease; S2P, site-2-zinc metalloproteinase; ATF6, activating transcription factor 6; GRP78, glucose-regulated protein 78; CD36, cluster of dif- ferentiation 36; RONS, reactive oxygen/nitrogen species; PM, plasma membrane ⁎ Corresponding author: Department of Chemistry & Biochemistry, University of Windsor, Windsor, ON, Canada N9B 3P4. Tel.: +1 519 253 3000x3533; fax: +1 519 973 7098. E-mail address: mutusb@uwindsor.ca (B. Mutus). 1 Authors contributed equally. 1388-1981/$ – see front matter. Crown Copyright © 2011 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.bbalip.2011.10.015 Contents lists available at SciVerse ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbalip