GNA11 differentially mediates fibroblast growth factor 2- and vascular endothelial growth factor A-induced cellular responses in human fetoplacental endothelial cells

Abstract

Key points: Fetoplacental vascular growth is critical to fetal growth. Fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor A (VEGFA) are two major regulators of fetoplacental vascular growth. G protein α subunit 11 (GNA11) transmits signals from many external stimuli to the cellular interior and may mediate endothelial function. It is not known whether GNA11 mediates FGF2- and VEGFA-induced endothelial cell responses under physiological chronic low O₂ . In the present study, we show that knockdown of GNA11 significantly decreases FGF2- and VEGFA-induced fetoplacental endothelial cell migration but not proliferation and permeability. Such decreases in endothelial migration are associated with increased phosphorylation of phospholipase C-β3. The results of the present study suggest differential roles of GNA11 with respect to mediating FGF2- and VEGFA-induced fetoplacental endothelial function.

Abstract: During pregnancy, fetoplacental angiogenesis is dramatically increased in association with rapidly elevated blood flow. Any disruption of fetoplacental angiogenesis may lead to pregnancy complications such as intrauterine growth restriction. Fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor A (VEGFA) are crucial regulators of fetoplacental angiogenesis. G protein α subunits q (GNAq) and 11 (GNA11) are two members of the Gα_q/11 subfamily involved in mediating vascular growth and basal blood pressure. However, little is known about the roles of GNA11 alone with respect to mediating the FGF2- and VEGFA-induced fetoplacental endothelial function. Using a cell model of human umbilical cord vein endothelial cells cultured under physiological chronic low O₂ (3% O₂ ), we showed that GNA11 small interfering RNA (siRNA) dramatically inhibited (P < 0.05) FGF2- and VEGFA-stimulated fetoplacental endothelial migration (by ∼36% and ∼50%, respectively) but not proliferation and permeability. GNA11 siRNA also elevated (P < 0.05) FGF2- and VEGFA-induced phosphorylation of phospholipase C-β3 (PLCβ3) at S537 in a time-dependent fashion but not mitogen-activated protein kinase 3/1 (ERK1/2) and v-akt murine thymoma viral oncogene homologue 1 (AKT1). These data suggest that GNA11 mediates FGF2- and VEGFA-stimulated fetoplacental endothelial cell migration partially via altering the activation of PLCβ3.

Keywords: G-protein; growth factor; placental angiogenesis.

Figure 1. Effects of GNA11 siRNA on GNA11 and 14 protein levels in HUVECs

Subconfluent cells were treated with transfection reagent (vehicle), scrambled siRNA (ssiRNA, 20 nm) or GNA11 siRNA (siRNA, 20 nm) for up to 4 days. Cellular proteins (20–30 μg) were subjected to Western blotting to detect GNA11, GNA14 and β‐actin. Data are expressed as the mean ± SEM of the fold change relative to vehicle control. Means with different lowercase letters are significantly different (Bonferroni's all pairwise multiple comparison procedures; P < 0.05; n = 3 independent experiments).

Figure 2. Effects of GNA11 siRNA on FGF2‐ and VEGFA‐mediated cell migration, proliferation and permeability in HUVECs

Cells were transfected with vehicle, ssiRNA or GNA11 siRNA for 2 days. After serum‐starvation for 24 h (migration and proliferation) or 3 h (permeability), cells were treated with FGF2 and VEGFA (100 ng mL⁻¹) for (A) 16 h (cell migration, n = 6 independent experiments), (B) 48 h (cell proliferation, n = 4 independent experiments) or (C and D) 24 h (cell permeability, n = 3 independent experiments). Data are expressed as the mean ± SEM of the fold change relative to vehicle control or relative to time 0 for the control group. Means with different lowercase letters are significantly different (Bonferroni's all pairwise multiple comparison procedures; P < 0.05).

Figure 3. Effects of GNA11 siRNA on phosphorylation of ERK1/2 and AKT1 in response to FGF2 and VEGFA in HUVECs

Cells were transfected with ssiRNA or GNA11 siRNA for 2 days. After 24 h of serum‐starvation, cells were treated with 100 ng mL⁻¹ of (A) FGF2 or (B) VEGFA for up to 60 min. Cellular proteins (20–30 μg) were subjected to Western blotting. Data are expressed as the mean ± SEM of the fold change relative to the time 0 control. ^*Different from the respective time 0 control (Bonferroni's multiple comparison procedures vs. time 0 control; P < 0.05; n = 3 independent experiments).

Figure 4. Effects of GNA11 siRNA on mediating phosphorylation of PLCβ3 S537 and S1105 in response to FGF2 and VEGFA in HUVECs

Cells were transfected with ssiRNA or GNA11 siRNA. After 24 h of serum starvation, cells were treated with 100 ng mL⁻¹ of (A) FGF2 and (B) VEGFA for up to 60 min. Proteins were subjected to Western blotting. Data are expressed as the mean ± SEM of the fold change relative to the time 0 control. ^*Different from the respective time 0 control (Bonferroni's multiple comparison procedures vs. time 0 control; P < 0.05). ^#Different from the corresponding time point (Student's t test; P < 0.05; n = 4 independent experiments).

Figure 5. Verification of primary stock of Ad‐GNA11 and effects of Ad‐GNA11 on GNA11 and GNA14 protein levels

A, PCR was used to verify primary stock of Ad‐GNA11 and GNA14. Products of GNA11 and 14 primers are shown (∼100 and ∼98 bp, respectively). B, cells were transfected with Ad‐GNA11 or Ad‐GFP for 3 days. Proteins (20–30 μg) were subjected to Western blotting to detect the indicated proteins. C and D, quantitative data for Ad‐GFP‐ (C) and Ad‐GNA11‐ (D) induced changes in GNA11 and 14 protein levels. Data are expressed as the mean ± SEM of the fold change relative to 0 MOI. ^*Different from 0 MOI (Bonferroni's multiple comparison procedures vs. 0 MOI; P < 0.05; n = 4 independent experiments).

Figure 6. Hypothesized signalling model for FGF2‐ and VEGFA‐regulated fetal endothelial function via GNA11, PLCβ3 and ERK1/2 under physiological chronic low O₂

In this model, phosphorylation of PLCβ3 S537 is mediated via two pathways. First, FGF2‐ and VEGFA‐activated RTK and/or downstream signals including ERK1/2 phosphorylates GNA11, which blocks or does not affect (FGF2) or only partially increases (VEGFA) phosphorylation of PLCβ3 S537, depending on the degree and/or sites of GNA11 phosphorylation. Second, the activated downstream signal can also directly phosphorylate PLCβ3 S537; this phosphorylation is either blocked (FGF2) or only partially increased (VEGFA) by phospho‐GNA11. However, downregulation of GNA11 causes overphosphorylation of PLCβ3 S537, possibly by increasing and decreasing its sensitivity to FGF2‐ and VEGFA‐activated protein kinases and phosphatase, respectively. This overphosporylation, along with alternations in phosphorylation at other active and inhibitory sites of PLCβ3, partially inhibits FGF2‐ and VEGFA‐induced cell migration. In this model, the effect of ERK1/2 on endothelial cell proliferation and migration is well established, whereas its effect on cell permeability remains elusive.