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 in part, 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 signaling 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 homolog 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 (5 min to 12 h) whereas VEGF only stimulated transient (5 min) 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 5 min; however, FGF2 was a stronger stimulus than VEGF. FGF2 and VEGF did not significantly activate p38MAPK at 5 min; however, VEGF stimulated p38MAPK phosphorylation at 60 min. 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 signaling 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.

Placental development during early pregnancy in sheep: cell proliferation, global methylation, and angiogenesis in the fetal placenta

“…NOS are recognized as regulators of implantation and pregnancy maintenance, and angiogenesis in the fetal and maternal placenta (Gagioti et al 2000, Maul et al 2003; however, the mechanism of NOS effects on these processes remains to be elucidated. Furthermore, it has been demonstrated that NOS3 expression is regulated by FGF2 and VEGF in ovine placental artery endothelial cells (Mata-Greenwood et al 2008). These interactions seem to be reflected in our study by significant correlations between the mRNA expression of NOS3 and expression of members of the VEGF and FGF2 systems.…”

Activation of AP-1 Transcription Factors Differentiates FGF2 and Vascular Endothelial Growth Factor Regulation of Endothelial Nitric-oxide Synthase Expression in Placental Artery Endothelial Cells

“…Cell Culture, Experimental Conditions, and Total Cell Extracts-Three primary oFPAEC lines were isolated by collagenase digestion from second degree placental arteries obtained from late pregnant (day 120 -130 of gestation, term ϳ145 days) sheep placentas and validated and were cultured and used as described (7). Uterine artery EC (UAEC) were prepared from the same animals as described previously (15).…”

“…For example, in ovine fetoplacental artery endothelial cells (oFPAEC), we have shown that, although both acutely stimulate NO production, VEGF does so with greater potency than FGF2 (8); surprisingly, FGF2, but not VEGF, stimulates NOS3 protein expression (6,7). Both activate similar signaling pathways, including phosphatidylinositol 3-kinase/AKT1 (protein kinase B) and mitogen-activated protein kinase (MAPK) members, such as extracellular-signal regulated kinase 2/1 (ERK2/1) and Jun N-terminal kinase 1/2 (JNK1/2); however, differences in intensity and temporal patterns of the activation signals may explain the differential effects of FGF2 and VEGF on NOS3 expression in oFPAEC (7). Of note, sustained ERK2/1 activation apparently plays a key role in differentiating why FGF2, but not VEGF, stimulates NOS3 protein expression (7).…”

“…Increased NO production during pregnancy is derived, at least in part, from activation of placental endothelial NOS3 by FGF2 and VEGF (6,7). However, significant differences exist in the effects of FGF2 and VEGF on the placental endothelial NOS3-NO system.…”

“…Both activate similar signaling pathways, including phosphatidylinositol 3-kinase/AKT1 (protein kinase B) and mitogen-activated protein kinase (MAPK) members, such as extracellular-signal regulated kinase 2/1 (ERK2/1) and Jun N-terminal kinase 1/2 (JNK1/2); however, differences in intensity and temporal patterns of the activation signals may explain the differential effects of FGF2 and VEGF on NOS3 expression in oFPAEC (7). Of note, sustained ERK2/1 activation apparently plays a key role in differentiating why FGF2, but not VEGF, stimulates NOS3 protein expression (7). The mechanism of such regula-tion is unknown; however, it is known that activated ERK2/1 translocate into the nucleus, where they activate various transcription factors to regulate gene transcription (9,10).…”