Original Contribution Neutral sphingomyelinase inhibition decreases ER stress-mediated apoptosis and inducible nitric oxide synthase in retinal pigment epithelial cells Ertan Kucuksayan a , Esma Kırımlıoglu Konuk b , Nejdet Demir b , Bülent Mutus c , Mutay Aslan a,n a Department of Medical Biochemistry, Akdeniz University Medical School, 07070 Antalya, Turkey b Department of Histology, Akdeniz University Medical School, 07070 Antalya, Turkey c Department of Chemistry & Biochemistry, University of Windsor, Windsor, ON, Canada article info Article history: Received 27 January 2014 Received in revised form 17 March 2014 Accepted 9 April 2014 Available online 15 April 2014 Keywords: Sphingomyelinase Endoplasmic reticulum stress Nitric oxide Retinal pigment epithelial cells Free radicals abstract Endoplasmic reticulum (ER) stress and excessive nitric oxide production via the induction of inducible nitric oxide synthase (NOS2) have been implicated in the pathogenesis of ocular diseases characterized by retinal degeneration. Previous studies have revealed the sphingomyelinase/ceramide pathway in the regulation of NOS2 induction. Thus, the objective of this study was to determine the activity of the sphingomyelinase/ceramide pathway, assess nitric oxide production, and examine apoptosis in human retinal pigment epithelial (RPE) cells undergoing ER stress. Sphingomyelinase (SMase) activity; nuclear factor κB (NF-κB) activation; NOS2, nitrite/nitrate, and nitrotyrosine levels; and apoptosis were determined in cultured human RPE cell lines subjected to ER stress via exposure to tunicamycin. Induction of ER stress was confirmed by increased intracellular levels of ER stress markers including phosphorylated PKR-like ER kinase, C/EBP-homologous protein, and 78-kDa glucose-regulated protein. ER stress increased nuclear translocation of NF-κB, NOS2 expression, nitrite/nitrate levels, and nitrotyrosine formation and caused apoptosis in RPE cell lines. Inhibition of neutral SMase (N-SMase) activity via GW 4869 treatment caused a significant reduction in nuclear translocation of NF-κB, NOS2 expression, nitrite/nitrate levels, nitrotyrosine formation, and apoptosis in ER-stressed RPE cells. In conclusion, N-SMase inhibition reduced nitrative stress and apoptosis in RPE cells undergoing ER stress. Obtained data suggest that NOS2 can be regulated by N-SMase in RPE cells experiencing ER stress. & 2014 Elsevier Inc. All rights reserved. The retinal pigment epithelium (RPE) is a monolayer of cells located outside the neural retina and is important in maintaining retinal function. The proximity of choroidal capillaries enables RPE cells to provide nutrients to maintain visual function and to play a key role in forming the outer blood–retinal barrier that prevents nonspecific diffusion and transport of material from the choroid [1]. Ischemic and hypoxic injuries as well as many retinal diseases affect RPE cells [2]. Apoptosis is a frequent type of cell death observed in RPE cells [3] and is an important feature of age-related macular degeneration, the most common cause of irreversible vision loss especially in the elderly population over 65 years of age [4]. Age-related alterations in the RPE include a reduction in cell density that can be caused by apoptosis resulting from accumula- tion of toxic substances [5]. Oxidative stress [6], hyperglycemia [7], mitochondrial dysfunction [8], and endoplasmic reticulum (ER) stress [9] are among the many studied proapoptotic factors in RPE cell lines. Protein folding, maturation, and trafficking; lipid synthesis; and intracellular calcium homeostasis are some of the major functions of the ER [10]. Endoplasmic reticulum stress results in the accumulation of unfolded proteins in the ER lumen leading to disturbed protein homeostasis. Cells activate an adaptive mechan- ism known as the unfolded protein response (UPR) to eliminate toxic protein components, which relieves ER stress and restores protein homeostasis [10]. However, extended periods of ER stress result in failed UPR and activate the apoptotic cascade [10]. Fundamental mechanisms resulting in the switch of the UPR from a prosurvival to a proapoptotic stimulus are still not clearly understood. Changes in sphingolipid metabolism and accumulation of cera- mide during ER stress have been demonstrated to induce apopto- sis [11]. Ceramide can be generated through de novo synthesis, Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/freeradbiomed Free Radical Biology and Medicine http://dx.doi.org/10.1016/j.freeradbiomed.2014.04.013 0891-5849/& 2014 Elsevier Inc. All rights reserved. n Corresponding author. Fax: 90 242 2496891. E-mail address: mutayaslan@akdeniz.edu.tr (M. Aslan). Free Radical Biology and Medicine 72 (2014) 113–123