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Vascular endothelial growth factor acts through novel, pregnancy-enhanced receptor signalling pathways to stimulate endothelial nitric oxide synthase activity in uterine artery endothelial cells.

ABSTRACT: During pregnancy, VEGF (vascular endothelial growth factor) regulates in part endothelial angiogenesis and vasodilation. In the present study we examine the relative roles of VEGFRs (VEGF receptors) and associated signalling pathways mediating the effects of VEGF(165) on eNOS (endothelial nitric oxide synthase) activation. Despite equal expression levels of VEGFR-1 and VEGFR-2 in UAECs (uterine artery endothelial cells) from NP (non-pregnant) and P (pregnant) sheep, VEGF(165) activates eNOS at a greater level in P- compared with NP-UAEC, independently of Akt activation. The selective VEGFR-1 agonist PlGF (placental growth factor)-1 elicits only a modest activation of eNOS in P-UAECs compared with VEGF(165), whereas the VEGFR-2 kinase inhibitor blocks VEGF(165)-stimulated eNOS activation, suggesting VEGF(165) predominantly activates eNOS via VEGFR-2. Although VEGF(165) also activates ERK (extracellular-signal-regulated kinase)-1/2, this is not necessary for eNOS activation since U0126 blocks ERK-1/2 phosphorylation, but not eNOS activation, and the VEGFR-2 kinase inhibitor inhibits eNOS activation, but not ERK-1/2 phosphorylation. Furthermore, the inability of PlGF to activate ERK-1/2 and the ability of the VEGFR-2 selective agonist VEGF-E to activate ERK-1/2 and eNOS suggests again that both eNOS and ERK-1/2 activation occur predominantly via VEGFR-2. The lack of VEGF(165)-stimulated Akt phosphorylation is consistent with a lack of robust phosphorylation of Ser(1179)-eNOS. Although VEGF(165)-stimulated eNOS phosphorylation is observed at Ser(617) and Ser(635), pregnancy does not significantly alter this response. Our finding that VEGF(165) activation of eNOS is completely inhibited by wortmannin but not LY294002 implies a downstream kinase, possibly a wortmannin-selective PI3K (phosphoinositide 3-kinase), is acting between the VEGFR-2 and eNOS independently of Akt.

SUBMITTER: Grummer MA

PROVIDER: S-EPMC2680709 | biostudies-literature | 2009 Jan

REPOSITORIES: biostudies-literature

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Vascular endothelial growth factor acts through novel, pregnancy-enhanced receptor signalling pathways to stimulate endothelial nitric oxide synthase activity in uterine artery endothelial cells.

Grummer Mary A MA Sullivan Jeremy A JA Magness Ronald R RR Bird Ian M IM

The Biochemical journal 20090101 2

During pregnancy, VEGF (vascular endothelial growth factor) regulates in part endothelial angiogenesis and vasodilation. In the present study we examine the relative roles of VEGFRs (VEGF receptors) and associated signalling pathways mediating the effects of VEGF(165) on eNOS (endothelial nitric oxide synthase) activation. Despite equal expression levels of VEGFR-1 and VEGFR-2 in UAECs (uterine artery endothelial cells) from NP (non-pregnant) and P (pregnant) sheep, VEGF(165) activates eNOS at a ...[more]

PMID: 18816248

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Project description:Despite extensive studies have shown that increased endothelial nitric oxide synthase (NOS3) expression in the uterine artery endothelial cells (UAEC) plays a key role in uterine vasodilatation, the molecular mechanism controlling NOS3 expression in UAEC is unknown. According to the sheep NOS3 promoter sequence isolated in our laboratory, we hypothesize that the activator protein-1 (AP-1) site in the proximal sheep NOS3 promoter (TGAGTCA, -682 to -676) is important for NOS3 expression. We developed a c-Jun adenoviral expression system to overexpress c-Jun protein into UAEC to investigate the effects of c-Jun/AP-1 on NOS3 expression. Basal levels of c-Jun protein and mRNA were detected in UAEC. c-Jun protein was overexpressed in a concentration and time-dependent fashion in UAEC infected with sense c-Jun (S-c-Jun), but not sham and antisense c-Jun (A-c-Jun) adenoviruses. Infection with S-c-Jun adenovirus (25 MOI, multiplicity of infection) resulted in efficient c-Jun protein overexpression in UAEC up to 3 days. In S-c-Jun, but not sham and A-c-Jun adenovirus infected UAEC, NOS3 mRNA and protein levels were increased (P<0.05) compared to noninfected controls. Increased NOS3 expression was associated with increased total NOS activity. Transient transfections showed that c-Jun overexpression augmented the transactivation of the sheep NOS3 promoter-driven luciferase/reporter constructs with the AP-1 site but not of deletion constructs without the AP-1 site. When the AP-1 site was mutated, c-Jun failed to trans-activate the sheep NOS3 promoter. AP-1 DNA binding activity also increased in c-Jun overexpressed UAEC. Lastly, the pharmacological AP-1 activator phorbol myristate acetate increased AP-1 binding, trans-activated the wild-type but not the AP-1 mutant NOS3 promoter and dose-dependently stimulated UAEC NOS3 and c-Jun protein expression. Hence, our data show that c-Jun/AP-1 regulates NOS3 transcription involving the proximal AP-1 site in the 5'-regulatory region of the sheep NOS3 gene.

Dataset Information

Vascular endothelial growth factor acts through novel, pregnancy-enhanced receptor signalling pathways to stimulate endothelial nitric oxide synthase activity in uterine artery endothelial cells.

Publications

Vascular endothelial growth factor acts through novel, pregnancy-enhanced receptor signalling pathways to stimulate endothelial nitric oxide synthase activity in uterine artery endothelial cells.

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