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  1. 1. Lysophosphatidic Acid Dependent-Redox Signaling in Proliferation and Atherosclerosis Keren Pereda , LeAnn Rogers*, Chananat Klomsiri*, Leslie Poole*, and Larry W. Daniel* Excellence In Cardiovascular Science Summer Research Program and Department of Biochemistry* Wake Forest University Introduction Lysophosphatidic acid (LPA) is a small signaling lipid which is able to promote a variety of cellular responses including growth, differentiation, survival and motility.1,2 LPA binds to its receptor, and is internalized as an endosome containing the NADPH oxidase complex. NADPH oxidase produces ROS in response to LPA, which also signals the recruitment of specific proteins to the endosome. ROS is release from the endosome and oxidizes these proteins.3 Many proteins need to be oxidized in order for them to be phosphorylated leading to the corresponding pathway activations.4 Akt and ERK are two of many specific proteins activated by LPA that may be phosphorylated/oxidized in response to ROS, other proteins may also be phosphorylated/oxidized downstream or upstream of Akt and ERK. The Akt and ERK pathways are important because they promote cell proliferation and activation of molecules that contribute to atherosclerosis. We examined how LPA-dependent ROS production affects the Akt and ERK pathways in HUVEC and SKOV3 cells. Methods ROS Production HUVEC cells were cultured and plated in EBM-2 media from Lonza containing 2% FBS. SKOV3 cells were cultured and plated with RPMI-1640 media containing 10% FBS. All cells were maintained at 37C and 5% CO2 . For imaging, cells were treated with 1 M LPA for 30 minutes. Cells were incubated with H2DCF-DA (DCF) for the final 10 minutes of LPA stimulation, then washed three times with media before visualizing with an Olympus inverted epi-fluorescence microscope with FITC filters. Cells were pretreated with DPI for 10 minutes where indicated. Lines on the graph (Fig.2B) represent individual cells monitored over time for DCF fluorescence. Western Blot SKOV3 cells were cultured, plated, and maintained as described. Where indicated, cells were challenged with DPI or EUK-134 30 min prior to LPA stimulation. The cells were harvested by detergent lysis, sonicated, and centrifuged to remove cellular debris. Protein concentration was determined using Pierce BSA Protein Assay Reagent in order to control for protein loading amounts, and 40 g of protein was resolved by SDS-PAGE. The proteins were then transferred onto nitrocellulose, and blocked in 5% milk in TBS-T. The blot was incubated overnight at 4C with primary antibodies specific for phospho- or total-MAPK p44/42(ERK) or phospho(Ser473)- or total-Akt, followed by secondary HRP-linked antibodies for 2 hrs at room temperature. The blot was developed using Perkin Elmer Western Lightning Chemiluminescence Reagent. References 1. Rivera-Lopez, C.M., Tucker, A.L. & Lynch, K.R. Lysophosphatidic acid (LPA) and angiogenesis. Angiogenesis 11, 301-10 (2008). 2. Teo, S.T., Yung, Y.C., Herr, D.R. & Chun, J. Lysophosphatidic acid in vascular development and disease. IUBMB Life 61, 791-9 (2009). 3. Daniel, et.al. unpublished. 4. Poole LB, Nelson KJ. Discovering mechanisms of signaling-mediated cysteine oxidation. Curr Opin Chem Biol.1, 1218-24 (2008). Results A. Control B. +LPA Figure1. ROS production in HUVEC Cells. Control (left panel) and LPA treated (right panel). 0 10 120 240 36030 60Time (min) LPA - - + - + - + - + - + - + p-ERK p-Akt Figure 3. LPA stimulates the phosphorylation of Akt and ERK. Objectives To evaluate the production of ROS in response to LPA in HUVEC and SKOV3 cells To determine the effect of blocking the ROS component of LPA signaling on the phosphorylation of Akt and ERK Conclusions LPA stimulates ROS production in both HUVEC and SKOV3 cells LPA stimulates phosphorylation of proteins in pathways important in cancer cell proliferation and atherosclerosis Phosphorylation of Akt and ERK in response to LPA is dependent on ROS production and protein oxidation Acknowlegments Research supported by NHLBI R25 HL 092618 Figure 4. DPI, an inhibitor of NADPH oxidase an dflavin containing proteins, and EUK-134, a SOD and catalase mimic, inhibit LPA stimulated phorphorylation of Akt and ERK p-Akt p-ERK Akt ERK 50 500 100 0 100 10 60Time (min.) Euk-134(M)10 M DPI Time (min.) + + + + +- - - - - 5 10 30 60 120 A. B. A. 0 min 10 min 20 min 30 min ControlLPA Figure 2. A. ROS production in SKOV3 cells. B. DPI inhibits LPA-dependent ROS production. B. Time(min)