Activation of multiple signaling pathways is critical for fibroblast growth factor 2- and vascular endothelial growth factor-stimulated ovine fetoplacental endothelial cell proliferation

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.

FIG. 1

Total NOx production by OFPAE cells in response to FGF2 and VEGF. NOx concentrations in medium samples were determined by nitrite/nitrate chemiluminescence with an NO analyzer, based on a reaction of conversion of nitrite and nitrate to NO. Total NOx levels were calculated by a standard curve generated with sodium nitrate as the standard and normalized by the protein content of corresponding wells. Data are subtracted from those in medium controls and are expressed as means ± SEM from three independent experiments. Within each growth factor treatment, means with different letters (a, b, c) differ (P < 0.05) from the time 0 control. ^#, means differ (P < 0.07) from FGF2 treatment at the corresponding time point; *, means differ (P < 0.05) from FGF2 treatment at the corresponding time point.

FIG. 2

Effects of L-NMMA and PTIO on FGF2- and VEGF-stimulated OFPAE cell proliferation. After serum starvation for 16 h, cells were treated with 10 ng/ml of FGF2 or VEGF in the specially formulated DMEM supplemented with 25 μM L-arginine in the absence or presence of L-NMMA, D-NMMA, or PTIO (1-h pretreatment). Cells were counted after 48 h of treatment. Data for each point are averaged from six experiments and expressed as means ± SEM percentage of the controls. The numbers of cells per well in the control were 8665 ± 576.3 and 5593 ± 523.3, respectively, for the L/D-NMMA (A and B) and PTIO (C and D) treatments. Means with different letters (a, b, c, d) differ significantly (P < 0.05).

FIG. 3

Effects of PD98059 and LY294002 on FGF2- and VEGF-stimulated OFPAE cell proliferation. After 16 h of serum starvation, cells were treated with 10 ng/ml of FGF2 or VEGF in the absence or presence of PD98059 or LY294002 (1-h pretreatment). Cells were counted after 48 h of treatment. Data for each point are averaged from five experiments and expressed as means ± SEM percentage of the control. The number of cells per well in the control was 6111 ± 276.9 and 6194 ± 442.3, respectively, for the PD98059 (A and B) and LY294002 (C and D) treatments. Means with different letters (a, b, c, d, e, f) differ within each growth factor and kinase inhibitor treatment (P < 0.05).

FIG. 4

Effects of PD98059 and LY294002 combination treatments on FGF2-stimulated OFPAE cell proliferation. After 16 h of serum starvation, cells were treated with 10 ng/ml of FGF2 in the absence or presence of PD98059 (20 μM) and LY294002 (5 μM; 1-h pretreatment). Cells were counted after 48 h of treatment. Data for each point are averaged from five experiments and expressed as means ± SEM percentage of the control. The number of cells per well in control was 9704 ± 405.8. Means with different letters (a, b, c) differ significantly (P < 0.05).

FIG. 5

Phosphorylation of AKT1 induced by FGF2 and VEGF in OFPAE cells. Cells were treated with 10 ng/ml of FGF2 or VEGF for 0–60 min. Proteins were separated on SDS-PAGE gels and analyzed by immunoblotting with antibodies against phospho-specific (pAKT1) or total AKT1 (tAKT1). Data averaged from three independent experiments are expressed as means ± SEM fold of the control. Means with asterisks and number symbols differ significantly (P < 0.05) from the control for FGF2 and VEGF treatments, respectively.

FIG. 6

Effects of PD98059 and LY294002 on FGF2- and VEGF-induced phosphorylation of MAPK3/1 in OFPAE cells. After serum starvation, cells were treated with FGF2 or VEGF for 10 min in the absence or presence of PD98059 (20 μM) or LY294002 (5 μM) (1-h pretreatment). Proteins were separated on SDS-PAGE gels and were analyzed by immunoblotting with antibodies against phospho-specific (pMAPK3/1) or total MAPK3/1 (tMPAK3/1). Data averaged from four independent experiments are expressed as means ± SEM fold of the control. Means with different letters (a, b, c, d) differ significantly (P < 0.05).

FIG. 7

Effects of PD98059 and LY294002 on FGF2- and VEGF-induced phosphorylation of AKT1 in OFPAE cells. After serum starvation, cells were treated with FGF2 or VEGF for 10 min in the absence or presence of PD98059 (20 μM) or LY294002 (5 μM) (1-h pretreatment). Proteins were separated on SDS-PAGE gels and analyzed by immunoblotting with antibodies against phospho-specific AKT1 (pAKT1), or total (tAKT1). Data averaged from four independent experiments are expressed as means ± SEM fold of the control. Means with different letters (a, b) differ significantly (P < 0.05).

FIG. 8

Effects of L-NMMA on FGF2- and VEGF-induced MAPK3/1 phosphorylation in OFPAE cells. After cultured in DMEM containing 25 μM L-arginine, cells were treated with FGF2 (A) or VEGF (B) in the absence or presence of L-NMMA (5 mM) or D-NMMA (5 mM) (1-h pretreatment) for 10 min. MAPK3/1 phosphorylation was analyzed as described in Figure 7. Data averaged from four independent experiments are expressed as means ± SEM fold of the control. Means with different letters (a, b) differ significantly within each isoform of MAPK (P < 0.05).

FIG. 9

Effects of L-NMMA on FGF2- and VEGF-induced AKT1 phosphorylation in OFPAE cells. After cultured in DMEM containing 25 μM L-arginine, cells were treated with FGF2 or VEGF in the absence or presence of L-NMMA (5 mM) or D-NMMA (5 mM) (1-h pretreatment) for 10 min. AKT1 phosphorylation was analyzed as described in Figure 5. Data averaged from four independent experiments are expressed as means ± SEM fold of the control. Means with different letters (a, b) differ significantly (P < 0.05).