Differential activation of multiple signalling pathways dictates eNOS upregulation by FGF2 but not VEGF in placental artery endothelial cells

Abstract

Fibroblast growth factor (FGF2), but not vascular endothelial growth factor (VEGF), upregulates endothelial nitric oxide synthase (eNOS) protein expression, at least partially, via activation of extracellular signal-regulated kinase 2/1 (ERK2/1) in ovine fetoplacental artery endothelial (oFPAE) cells. Herein we further investigated the temporal effects of FGF2 and VEGF on other signalling pathways including members (Jun N-terminal kinase JNK1/2 and p38MAPK) of mitogen-activated protein kinases (MAPK), phosphatidylinositol-3 kinase/v-akt murine thymoma viral oncogene homologue 1 (PI3K/AKT1), and the tyrosine kinase c-SRC, and examined if either one or more of these pathways play a role in the differential regulation of eNOS by FGF2 and VEGF. We first confirmed that in oFPAE cells, FGF2, but not VEGF, increased eNOS protein. FGF2 stimulated eNOS protein in a time- and concentration-dependent manner, which also depended on cell density. FGF2 provoked sustained (5min to 12h) whereas VEGF only stimulated transient (5min) ERK2/1 phosphorylation. FGF2 was 1.7-fold more potent in stimulating ERK2/1 phosphorylation than VEGF. FGF2 and VEGF only transiently activated JNK1/2 and AKT1 within 5min; however, FGF2 was a stronger stimulus than VEGF. FGF2 and VEGF did not significantly activate p38MAPK at 5min; however, VEGF stimulated p38MAPK phosphorylation at 60min. VEGF but not FGF2 significantly stimulated c-SRC phosphorylation. Inhibitors of MEK-ERK2/1 (PD98059), JNK1/2 (SP600125) and PI3K (wortmannin), but not p38MAPK (SB203580) and SRC (PP2), decreased the FGF2-increased eNOS protein expression. Thus, the FGF2-induced eNOS protein expression requires activation of multiple signalling pathways including ERK2/1, JNK1/2 and PI3K/AKT1. Differences in intensity and temporal patterns of activation of these pathways by FGF2 and VEGF may account for their differential effects on eNOS expression in OFPAE cells.

Fig 1. Effects of cell density on basal, FGF2- and VEGF-induced eNOS protein expression in oFPAE cells

Cells were grown to ∼50%, ∼70% and ∼90% confluence as described in ‘Methods’ prior to growth factor stimulation. A. Serum starved cells were treated with 10 ng/ml growth factor for 24 h and used to analyze eNOS protein by immunoblotting. Representative blots of one experiment are shown for each growth factor. B. Cells were treated with 10 ng/ml growth factor for analyzing mitogenic effects at 24 h. For both A and B panels, data are summarized as Mean ± SEM (n=4). Bars with different superscripts differ significantly, p<0.05.

Fig 2. Temporal Thr202/Thr204 phosphorylation of ERK2/1 by FGF2 and VEGF in oFPAE cells

Subconfluent cells were serum starved and stimulated with FGF2 (10 ng/ml) or VEGF (10 ng/ml) for the indicated times (up to 12 h). Total protein extracts were harvested for determining phosphorylated and total ERK2/1 via immunoblotting. Representative blots of phosphorylated ERK2/1 (upper) and total ERK2/1 (lower) of one typical experiment are shown. Bar graph summarize data (Mean ± SEM, n=4) relative to the untreated controls. *p< 0.05 vs. control.

Fig 3. Temporal Thr183/Tyr185 phosphorylation of JNK1/2 and Thr180/Tyr182 phosphorylation of p38MAPK by FGF2 and VEGF in oFPAE cells

Subconfluent cells were serum starved and stimulated with FGF2 (10 ng/ml) or VEGF (10 ng/ml) for up to 1h. Total protein extracts were harvested for determining phosphorylated and total JNK1/2 and p38MAPK via immunoblotting. A: Representative blots of phosphorylated JNK1/2 (upper) and total JNK1/2 (lower) are shown. B: Representative blots of phosphorylated p38MAPK (upper) and total p38MAPK (lower) of one typical experiment are shown. For both JNK and p38MAPK, bar graphs summarize data (Mean ± SEM, n=4) relative to the untreated controls.*p< 0.05 vs. control.

Fig 4. Temporal Ser473 phosphorylation of AKT1 and Tyr416 phosphorylation of c-SRC by FGF2 and VEGF in oFPAE cells

Subconfluent cells were serum starved and stimulated with FGF2 (10 ng/ml) or VEGF (10 ng/ml) for the indicated times. Total protein extracts were harvested for determining phosphorylated and total AKT1 and c-Src via immunoblotting. A: Representative blots of phosphorylated AKT1 (upper) and total AKT1 (lower) are shown for one typical experiment. B: Representative blots of phosphorylated c-SRC (upper) and total c-SRC (lower) of one typical experiment are shown. For both AKT1 and c-SRC, lower panels of bar graphs summarize data (Mean ± SEM, n=4) relative to the untreated controls. *p< 0.05 vs. control.

Fig 5. Role of ERK2/1, JNK1/2 and PI3K/AKT1 in FGF2 upregulation of eNOS in oFPAE cells

Panels A, C and E - Pharmacological inhibition of FGF2-induced kinase activation: Cells were pretreated with increasing concentrations of the specific inhibitors PD98059 (MEK1/ERK2/1), SP600125 (JNK1/2) and Wortmannin (PI3K/AKT1) for 60 min, followed by treatment with 10 ng/ml FGF2 for 10 min. Protein samples were prepared for analyzing kinase phosphorylation as described in Fig. 2-4. Representative immunoblots (A, C and E) of one typical experiment show dose-dependent inhibition of ERK2/1, JNK1/2, and AKT1 by each inhibitor to block FGF2 induced phosphorylation of its corresponding kinase. Panels B, D, and F – Involvement of kinase pathways in FGF upregulation of eNOS protein: Serum-starved cells were pretreated with or without increasing concentrations of each inhibitor for 60 min, followed by treatment with or without 10 ng/ml FGF2 for 24 h. Bar graphs summarize data (Means ± SEM, n=3) of eNOS/β-actin protein levels relative to controls. Bars with different superscripts differ significantly (p<0.05).

Fig 6. Sustained activation of ERK2/1 is required for FGF2 upregulation of eNOS protein expression in oFPAE cells

Serum starved subconfluent cells were either pretreated with the MEK1/ERK2/1 inhibitor PD98058 (10 μM) for 30 min followed by FGF2 (10 ng/ml), or treated with FGF2 in combination with additions of PD98058 at 5, 30 and 60 min post-FGF treatment. Protein samples were then harvested for analyzing ERK2/1 phosphorylation at 2h (A) or eNOS/β-actin protein levels at 24 h (B) post-FGF2 treatment. Bar graphs summarize data (Means ± SEM, n=3) relative to controls. Bars with different superscripts differ significantly (P<0.05).