Akt Activation Induced by Lysophosphatidic Acid and Sphingosine-1-phosphate Requires Both Mitogen-Activated Protein Kinase Kinase and p38 Mitogen-Activated Protein Kinase and Is Cell-Line Specific LINNEA M. BAUDHUIN, KELLY L. CRISTINA, JUN LU, and YAN XU Departments of Cancer Biology (L.M.B., K.L.C., J.L., Y.X.) and Gynecology and Obstetrics (Y.X.), Cleveland Clinic Foundation, Cleveland, Ohio; and Department of Chemistry, Cleveland State University, Cleveland, Ohio (L.M.B., Y.X.) Received February 27, 2002; accepted May 28, 2002 This article is available online at http://molpharm.aspetjournals.org ABSTRACT The signaling pathways that lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) use to activate Akt in ovarian cancer cells are investigated here. We show for the first time, with the use of both pharmacological and genetic inhibitors, that the kinase activity and S473 phosphorylation of Akt in- duced by LPA and S1P requires both mitogen-activated protein (MAP) kinase kinase (MEK) and p38 MAP kinase, and MEK is likely to be upstream of p38, in HEY ovarian cancer cells. The requirement for both MEK and p38 is cell type- and stimulus- specific. Among 12 cell lines that we tested, 11 respond to LPA and S1P and all of the responsive cell lines require p38 but only nine of them require MEK. Among different stimuli tested, plate- let-derived growth factor stimulates S473 phosphorylation of Akt in a MEK- and p38-dependent manner. However, epidermal growth factor, thrombin, and endothelin-1–stimulated Akt S473 phosphorylation require p38 but not MEK. Insulin, on the other hand, stimulates Akt S473 phosphorylation independent of both MEK and p38 in HEY cells. T308 phosphorylation stimu- lated by LPA/S1P requires MEK but not p38 activation. MEK and p38 activation were sufficient for Akt S473 but not T308 phosphorylation in HEY cells. In contrast to S1P and PDGF, LPA requires Rho for Akt S473 phosphorylation, and Rho is upstream of phosphatidylinositol 3-kinase (PI3-K). LPA/S1P- induced Akt activation may be involved in cell survival, because LPA and S1P treatment in HEY ovarian cancer cells results in a decrease in paclitaxel-induced caspase-3 activity in a PI3-K/ MEK/p38-dependent manner. LPA and S1P are bioactive lysolipids that exert many of their effects and signaling activities through G protein-cou- pled receptors (GPCRs) (Goetzl and An, 1998; Moolenaar, 1999; Spiegel, 1999). We have reported previously that both LPA and S1P are important signaling molecules in ovarian cancer, regulating both growth and metastatic potentials of ovarian cancer cells (Xu et al., 1995a,b, 1998, 2001; Hong et al., 1999; Schwartz et al., 2001). We have detected both of these lysolipids in ascitic fluids in patients with ovarian cancer (Xiao et al., 2000, 2001). Moreover, we have reported that LPA is elevated in the plasma of patients with ovarian cancer but not in that of patients with breast cancer or leukemia, indicating its potential as a marker for ovarian cancer (Xu et al., 1998). LPA has been reported to have a cytoprotective effect in HEY ovarian cancer cells exposed to cis-diamminedichloroplatinum (Frankel and Mills, 1996). Furthermore, under certain conditions in vitro, ovarian can- cer cells produce LPA (Shen et al., 1998; Eder et al., 2000), suggesting that LPA, and potentially S1P, function as auto- crine growth factors in ovarian cancer. LPA and/or S1P have been shown to activate extracellular signal regulated kinase (ERK) and PI3-K and/or Akt (PKB) via a PTX-sensitive pathway in a number of cell types (Marte and Downward, 1997; Weiner and Chun, 1999; Fang et al., This work was supported in part by an American Cancer Society Grant RPG-99-062-01-CNE, U.S. Army Medical Research grant DAMD 17-99-1-9563, and National Institutes of Health Grant R21-CA84038 – 01 (to Y.X.). ABBREVIATIONS: LPA, lysophosphatidic acid; S1P, sphingosine-1-phosphate; GPCR, G protein-coupled receptor; ERK, extracellular signal- regulated kinase; PI3-K, phosphatidylinositol 3-kinase; PKB, protein kinase B; PTX, pertussis toxin; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase kinase; MK2, mitogen-activated protein kinase-activated protein kinase-2; PDK, 3-phosphoinositide-dependent kinase; ILK, integrin-linked kinase; PIP 3 , phosphatidylinositol-3,4,5-trisphosphate; PDGF, platelet-derived growth factor; EGF, epidermal growth factor; Et-1, endothelin-1; PBS, phosphate-buffered saline; LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; PD98059, 2'-amino- 3'-methoxyflavone; SB203580, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole; FBS, fetal bovine serum; RT-PCR, reverse transcriptioon-polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; p-ERK, phospho-specific extracellular signal- regulated kinase; p-p38, phospho-specific p38 mitogen-activated protein kinase. 0026-895X/02/6203-660 –671$7.00 MOLECULAR PHARMACOLOGY Vol. 62, No. 3 Copyright © 2002 The American Society for Pharmacology and Experimental Therapeutics 1705/1003848 Mol Pharmacol 62:660–671, 2002 Printed in U.S.A. 660 at ASPET Journals on May 13, 2016 molpharm.aspetjournals.org Downloaded from