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Activation of Multiple Signaling Pathways Is Critical for Fibroblast Growth Factor 2- and Vascular Endothelial Growth Factor-Stimulated Ovine Fetoplacental Endothelial Cell Proliferation¹

Department of Reproductive Medicine,⁷ Division of Maternal-Fetal Medicine, University of California, San Diego, California 92093 Department of Biochemistry,⁶ Dalian Medical University, Dalian, 116027, Liaoning, People's Republic of China Departments of Obstetrics and Gynecology,³ Pediatrics,⁴ and Animal Sciences,⁵ Perinatal Research Laboratories, University of Wisconsin-Madison, Madison, Wisconsin 53715

ABSTRACT

Fibroblast growth factor-2 (FGF2) and vascular endothelial growth factor (VEGF) are two key regulators of placental angiogenesis. The potent vasodilator nitric oxide (NO) could also act as a key mediator of FGF2- and VEGF-induced angiogenesis. However, the postreceptor signaling pathways governing these FGF2- and VEGF-induced placental angiogenic responses are poorly understood. In this study, we assessed the role of endogenous NO, mitogen-activated protein kinase 3/1 (MAPK3/1), and v-akt murine thymoma viral oncogene homolog 1 (AKT1) in FGF2- and VEGF-stimulated proliferation of ovine fetoplacental endothelial (OFPAE) cells. Both FGF2 and VEGF time-dependently stimulated (P < 0.05) NO production and activated AKT1. Both FGF2- and VEGF-stimulated cell proliferation was dose-dependently inhibited (P < 0.05) by N^G-monomethyl-L-arginine (L-NMMA; an NO synthase inhibitor), PD98059 (a selective MAPK3/1 kinase 1 and 2 [MAP2K1/2] inhibitor), or LY294002 (a selective phosphatidylinositol 3 kinase [PI3K] inhibitor) but not by phenyl-4,4,5,5 tetramethylimidazoline-1-oxyl 3-oxide (PTIO, a potent extracellular NO scavenger). At the maximal inhibitory dose without cytotoxicity, PD98059 and LY294002 completely inhibited VEGF-induced cell proliferation but only partially attenuated (P < 0.05) FGF2-induced cell proliferation. PD98059 and LY294002 also inhibited (P < 0.05) FGF2- and VEGF-induced phosphorylation of MAPK3/1 and AKT1, respectively. L-NMMA did not significantly affect FGF2- and VEGF-induced phosphorylation of either MAPK3/1 or AKT1. Thus, in OFPAE cells, both FGF2- and VEGF-stimulated cell proliferation is partly mediated via NO as an intracellular and downstream signal of MAPK3/1 and AKT1 activation. Moreover, activation of both MAP2K1/2/MAPK3/1 and PI3K/AKT1 pathways is critical for FGF2-stimulated cell proliferation, whereas activation of either one pathway is sufficient for mediating the VEGF-induced maximal cell proliferation, indicating that these two kinase pathways differentially mediate the FGF2- and VEGF-stimulated OFPAE cell proliferation.

AKT1, endothelial cell proliferation, FGF2, growth factors, kinases, MAPK3/1, nitric oxide, pregnancy, vascular endothelial growth factor

¹Supported, in part, by NIH grants HL64703 (to J.Z.), HD38843 (to R.R.M. and J.Z.), HL49210 (to R.R.M.), and HL74947 and HL70562 (to D.-B.C.).

Correspondence: ²Jing Zheng, Department of Obstetrics and Gynecology, University of Wisconsin-Madison, Perinatal Research Laboratories, PAB1 Meriter Hospital, 202 S. Park St., Madison, WI 53715. FAX: 608 257 1304; e-mail: jzheng{at}wisc.edu