HDAC Inhibitors as Epigenetic Regulators

Summary == These results suggest that local high-salt may contribute to the intraocular inflammatory response by promoting proinflammatory cytokine production. == Acknowledgments == This work was supported by the Natural Science Foundation Major International (Regional) Joint Research Project (81320108009), Key Project of Natural Science Foundation (81130019), National Natural BAY885 Science Foundation Project (31370893), Research Fund intended for the Doctoral Program of Higher Education of China (20115503110002), Basic Research Program of Chongqing (cstc2013jcyjC10001), Chongqing Key Laboratory of Ophthalmology (CSTC, 2008CA5003), Chongqing Science & Technology Platform and Base Construction Program (cstc2014pt-sy10002), National Important Clinical Specialties Construction Program of China, and Account for PAR-EU Scholars Program. == Conflict of Interests == The authors declare that there is BAY885 no conflict of interests regarding the publication of this paper. == Authors’ Contribution == Dike Zhang and Chaokui Wang contributed equally to the work presented here and should therefore be regarded as equivalent authors. == References ==. resulted in significant increases in the phosphorylation of p38 MAPK, Akt, and NF-B and an upregulation of the transcription factors NFAT5 and SGK1. High-salt significantly promotes IL-6 and MCP-1 production by ARPE-19 cells and is associated with activation of the p38 MAPK, Akt, and NF-B pathway and NFAT-SGK1 pathways. == 1 . Introduction == The retinal pigment epithelium (RPE), situated on the outer side of the retina, is a monolayer of cells connected by tight junctions with important functions for the visual system [1]. ARPE-19, a spontaneously arising cell line of RPE, has been extensively used in the past decades to investigate the role of this cell layer in the pathogenesis of a number of diseases including age-related macular degeneration (AMD), vitreoretinopathy, and uveitis [25]. The major inflammatory cytokines produced by ARPE-19 in response to various stimuli are interleukin-6 (IL-6), interleukin-8 (CXCL8, IL-8), and monocyte chemoattractant protein-1 (CCL2, MCP-1) Rabbit polyclonal to MAP2 [6, 7]. IL-6 is a proinflammatory cytokine that plays an important role in intensifying the intraocular immune and inflammatory response [811]. IL-8 and MCP-1 are important chemoattractants of neutrophils, lymphocytes, and monocytes, causing these cells to infiltrate into intraocular tissues during inflammatory retinal disease [1214]. Both genetic [15] and environmental [16, 17] factors are considered to play a role in the pathogenesis of intraocular inflammation. The role of environmental factors has mainly focused on a role of infections in the development of autoimmune or autoinflammatory uveitis and as yet little attention has been paid to dietary factors [18]. Evidence is now emerging that a high-salt diet may be involved in the pathogenesis of autoimmune disease [19]. Recent studies have shown that high-salt could aggravate the severity of experimental autoimmune encephalomyelitis (EAE) by inducing a Th17 cell immune response, whereby the effect of high-salt on Th17 cell polarization was mediated by activating the p38/MAPK pathway via nuclear factor of activated T cells 5 (NFAT5) and serum/glucocorticoid-regulated kinase 1 (SGK1) [19, 20]. Case-control studies showed that high-salt intake in smokers was associated with the risk of rheumatoid arthritis and a higher salt intake is also associated with increased clinical and radiological disease activity in patients with multiple sclerosis [21]. Whether high-salt affects autoimmune diseases of the eye such as uveitis is not yet known. Since RPE cells play an important role as cellular mediators of the intraocular inflammatory response we decided to investigate the effect BAY885 of high-salt on the cytokine release by these cells. Our results revealed that high-salt significantly promoted the secretion of IL-6 and MCP-1 by ARPE-19 cells. The high-salt induced release of IL-6 and MCP-1 by ARPE-19 cells was associated with the phosphorylation of p38 MAPK, Akt, and NF-B and an upregulation of the transcription factors NFAT5 and SGK1. == 2 . Material and Methods == == 2 . 1 . Reagents == Sodium chloride (Chuandong Chemical Group Co., Ltd., Chongqing, China) with a purity of 99. 5% was dissolved in deionized water as a stock solution at a concentration of 4000 mM (234 mg/mL). Either 5L or 10L of this stock solution was added per mL to the cell cultures described below (20 mM or 40 mM additional NaCl). LPS was obtained from Sigma-Aldrich (St. Louis, MO). The ELISA kits to measure IL-8, IL-6, and MCP-1 were purchased from R&D Systems. The annexin V-FITC apoptosis/necrosis detection kit was from KeyGEN Biotech (Nanjing, China). RNA was isolated from ARPE-19 cells using an RNeasy Mini Kit which was purchased from QIAGEN (Valencia, CA); the reverse transcription system and SYBR Green master mix for real-time PCR were from TAKARA (Dalian, China). Antibodies against p38, NF-B, Akt, JNK, and ERK1/2 were obtained from BD Biosciences (Sunnyvale, CA). A cell counting kit (CCK-8) was from Dojindo (Kumamoto, Japan). Mannitol was obtained from Kelun Pharmaceutical Co., Ltd. (Sichuan, China). == 2 . 2 . Cell Culture == ARPE-19 was obtained from the American Type Culture Collection (ATCC). Cells were cultured in medium (Dulbecco’s modified Eagle’s medium: nutrient mixture F12 (DMEM/F12), 1: 1; Invitrogen, Carlsbad, CA) with 10% fetal bovine serum (FBS, Invitrogen), 100 U/mL penicillin, and 100 ng/mL streptomycin. This medium contains an average of 120 mM NaCl according to the manufacturer. The cells were incubated in a humidified 5% CO2atmosphere at BAY885 37C and passaged every 5 to 7 days. After reaching confluence, the cells were detached with trypsin-EDTA solution, diluted 1: 3 to 1: 4, and plated into Corning flask (Corning, Lowell, MA) for subculture at 1 . 2 106cells/flask and cultured in DMEM/F12 with 10% FBS. The ARPE-19 cells used in the experiments were confluent. == 2 . 3. Cell Apoptosis Array == Before stimulation, ARPE-19 cells were serum-starved for 24 hours in serum-free DMEM/F12. Cells were stimulated with 100 ng/mL LPS in the presence or absence of 20 mM.