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The Journal of Physiology logoLink to The Journal of Physiology
. 2018 May 12;596(12):2333–2344. doi: 10.1113/JP275677

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

Qing‐yun Zou 1, Ying‐jie Zhao 1,2, Hua Li 1,3, Xiang‐zhen Wang 1,4, Ai‐xia Liu 1,5, Xin‐qi Zhong 1,6, Qin Yan 1,7, Yan Li 1, Chi Zhou 1, Jing Zheng 1,8,✉
PMCID: PMC6002203  PMID: 29659033

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 Gαq/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

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.

Introduction

During pregnancy, the fetoplacental vasculature undergoes tremendous growth and remodelling to support normal fetal growth (Magness & Zheng, 1996). In addition, impaired fetoplacental vascular growth and function are associated with several pregnancy complications, including pre‐eclampsia and intrauterine growth restriction (Zygmunt et al. 2003). These complications not only jeopardize maternal and fetal health, but also increase the risk of cardiovascular disease onset in adult offspring (Wang et al. 2002; Powe et al. 2011; Boeldt et al. 2014; Brodowski et al. 2017; Zhou et al. 2017). Thus, a better understanding of the regulation of fetoplacental endothelial growth and function is a prerequisite for the design of therapeutic controls for fetoplacental vascular function.

Two major regulators of fetoplacental angiogenesis, fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor A (VEGFA), activate high‐affinity receptor tyrosine kinases (RTKs) (Klein et al. 1997; Ferrara et al. 2003; Wang & Zheng, 2012). Subsequently, they induce the phosphorylation of a cascade of protein kinases, such as mitogen‐activated protein kinase 3/1 (ERK1/2) and v‐akt murine thymoma viral oncogene homologue 1 (AKT1), thus leading to various cell responses (Ferrara et al. 2003; Podar & Anderson, 2008; Turner & Grose, 2010). These two protein kinases are actively involved in mediating fetal endothelial proliferation, migration and survival (Wang & Zheng, 2012; Jiang et al. 2013a,b).

Besides RTKs, peptide growth factors may also interact with G protein and mediate a vast array of cell function (Pyne & Pyne, 2011). G protein is composed of α, β and γ subunits, among which the α subunit is a major functional component. G protein α subunits are classified into four subfamilies: Gαs, Gαi/o, Gαq/11 and Gα12/13. Gαq/11 consists of four members: Gαq, Gα11, Gα14 and Gα15/16 (GNAq, 11, 14 and 15/16, respectively) (Wettschureck & Offermanns, 2005; Hubbard & Hepler, 2006). Among these four members, GNAq and 11 are key mediators of cardiovascular function. For example, double knockout of GNAq and 11 in mice results in embryonic death as a result of malformation of the heart (Offermanns et al. 1998). Mice carrying only one intact allele of GNAq or GNA11 also die shortly after birth (Offermanns et al. 1998). In addition, both GNAq and 11 are necessary for maintaining basal blood pressure and developing salt‐induced hypertension (Wirth et al. 2008). We have reported the expression of GNA11 in human placentas and in human umbilical cord vein endothelial cells (HUVECs) (Zhao et al. 2014). In HUVECs, double knockdown of GNAq and 11 impairs VEGFA‐induced cell migration (Zeng et al. 2002) and inhibits VEGFA‐ but not FGF2‐induced cell proliferation (Zeng et al. 2003). Our knowledge of the roles of GNA11 with respect to mediating endothelial function is limited. Sivaraj et al. (2015) have demonstrated that knockout of GNA11 in endothelial cells does not affect postnatal angiogenesis in mice retina, whereas knockout of GNAq or both of GNAq and 11 in endothelial cells significantly decreases postnatal angiogenesis in mice. More recently, Couto et al. (2017) reported that a somatic mutation of GNA11 is associated with capillary malformation in human. These data suggest the importance of GNA11 with respect to mediating endothelial function, possibly depending on the origins of endothelial cells.

Members of the Gαq/11 subfamily can activate phospholipase C (PLC), predominantly PLCβ3 (Offermanns, 1999; Rhee, 2001). In HUVECs, knockdown of PLCβ3 inhibits VEGFA‐stimulated cell migration, whereas it enhances VEGFA‐stimulated cell proliferation in association with increased phosphorylation of PLCβ3 at serine 537 and 1105 (S537 and S1105) (Bhattacharya et al. 2009).

Fetoplacental endothelial cells in vivo reside under low oxygen environments (3–8% O2) relative to the ambient O2 level (∼ 21%) at sea level throughout pregnancy (Rodesch et al. 1992; Jauniaux et al. 2001; Meschia, 2013). The O2 level is 3.7% (range 2.3–5.1%) in the umbilical vein at the end of gestation (Meschia, 2013). These low O2 levels are assumed to be important for normal endothelial function (Burton et al. 1996; Mayhew, 2003; Zamudio, 2007; Meschia, 2013), including gene expression and FGF2‐ and VEGFA‐induced fetoplacental endothelial responses (Jiang et al. 2013a,b). As such, HUVECs cultured under physiological low O2 were used in the present study because physiological low O2 may more closely mimic in vivo conditions.

To date, little is known regarding the roles of GNA11 with respect to mediating FGF2‐ and VEGFA‐induced fetal endothelial function under physiological low O2 during pregnancy. In the present study, we tested the hypothesis that GNA11 alone mediates FGF2‐ and VEGFA‐induced fetal endothelial cell proliferation, migration and permeability by altering the phosphorylation of ERK1/2, AKT1 and PLCβ3 using HUVECs constantly cultured under physiological low O2 as a cell model.

Methods

Ethical approval

The umbilical cord collection protocol was approved by the Institutional Review Board of UnityPoint Meriter Hospital (Madison, WI, USA) and the Health Sciences Institutional Review Boards of the University of Wisconsin–Madison (Protocol number 2004–006). The subjects provided their written, informed consent before participating. All procedures were conducted in accordance with the Declaration of Helsinki, except for registration in a database.

Primary HUVECs and culture conditions

HUVECs were isolated from umbilical cord veins of normal pregnancy using a collagenase enzyme protocol described previously (Jiang et al. 2013a, b, 2014; Li et al. 2015; Zhou et al. 2017). After isolation, cells were cultured in endothelial culture medium (ECM) (catalog number 1001; Sciencell, Carlsbad, CA, USA) supplemented with 5% fetal bovine serum (FBS), 1% penicillin/streptomycin (p/s), 1% amphotericin B (AB) (catalog number 15290018; Thermo Fisher Scientific, Waltham, MA, USA) and 1% endothelial cell growth supplement (ECGS) under physiological chronic normoxia (PCN) (37°C, 5% CO2, 3% O2, ∼92% N2).

Cells were purified and verified for their endothelial phenotypes as described previously (Jiang et al. 2013a, b, 2014). After verification, cells from five individual cell preparations were pooled and cells at passages 4–5 were used in the present study.

All media used for cell culture and experiments were pre‐purged with N2 and equilibrated in a hypoxia incubator adjusted to the PCN condition before addition to cells. Dissolved O2 in media was monitored using a dissolved O2 meter. All experiments were conducted either in a hypoxia incubator adjusted to the PCN condition or a 3% O2 heated O2‐controlled glovebox (Coy Laboratory Products, Grass Lake, MI, USA) as described previously (Jiang et al. 2013a, b, 2014).

GNA11 small interfering RNA (siRNA) transfection

To study roles of GNA11 in FGF2‐ and VEGFA‐induced cell responses in HUVECs, siRNA transfection was performed as described previously (Wang et al. 2008; Jiang et al. 2014; Li et al. 2015). A pool of four siRNAs specifically targeting human GNA11 (catalogue number L‐010860‐00‐0005; GenBank number NM_002067.4) and a pool of four scrambled siRNAs (ssiRNA) (catalog number D‐001810‐10‐05) were purchased from Dharmacon (Lafayette, CO, USA). The siRNA and ssiRNA were pre‐mixed with Lipofectamine RNAiMAX transfection reagent (vehicle; catalog number 13778030; Invitrogen, Carlsbad, CA, USA) at room temperature. Subconfluent cells were cultured in antibiotic‐ and serum‐free media (ECMb) (catalog number 1001; Sciencell) containing GNA11 siRNA (20 nm) or ssiRNA (20 nm). After 4 h of culture (transfection), an equal amount of ECM supplied with 10% FBS, 2% p/s, 2% AB and 2% ECGS was added. Cells were harvested and subjected to Western blotting 2, 3 and 4 days post transfection.

Cell migration

For cell migration assays, after 2 days of transfection with the vehicle, ssiRNA and GNA11 siRNA, cells were serum‐starved and placed (30,000 per insert) into each insert using a transwell system (catalog number 351158, Corning, Corning, NY, USA) as described previously (Jiang et al. 2013a, b, 2014; Li et al. 2015, 2017). FGF2 or VEGFA was added into the bottom wells (final concentration of 100 ng mL−1; catalog number 10014‐HNAE and catalog number 80006‐RNAB, respectively; Sino Biologic Inc., Beijing, China). After 16 h of culture, calcein AM (catalog number C3100MP; Invitrogen) was added into the bottom wells (final concentration of 0.2 μg mL−1). Five images were taken at random sites using a TE2000U inverted microscope (Nikon, Tokyo, Japan). Cell number was quantified using Metamorph imaging analysis software (Molecular Devices, Sunnyvale, CA, USA). ECMb supplemented with 2% heated inactivated FBS, 1% p/s and 1% AB was used for preparing control media and final growth factor solutions.

Cell proliferation

Cell proliferation was evaluated using the crystal violet method as described previously (Wang et al. 2008; Song et al. 2009; Li et al. 2015, 2017). Briefly, after 2 days of transfection, cells were inoculated into 96‐well plates (5 ‐ 8000 cells per well) and cultured overnight. After serum starvation for 16–24 h, cells were treated with control media, FGF2 or VEGFA (100 ng mL−1) for 48 h. Cells were rinsed with PBS, fixed in a methanol and stained with 0.1% crystal violet solution (Sigma, St Louis, MO, USA). After solubilization, the optical density value of each well was measured using a microplate reader at 570 nm (Bio‐Tek, Winooski, VT, USA). ECMb supplemented with 0.2% heated inactivated FBS, 1% p/s and 1% AB was used for preparing control media, as well as the final growth factor solutions.

Cell permeability

Cell permeability was examined using an electric cell‐substrate impedance sensing system (ECIS Zθ; Applied Biophysics, Troy, NY, USA) as described previously (Zhou et al. 2017). Briefly, HUVECs were treated with vehicle, ssiRNA or GNA11 siRNA as described above. After 2 days of transfection, cells (50,000 cells per well) were inoculated into 96W10E+ ECIS array plates (Applied Biophysics) pre‐coated with 10 nm cysteine and 0.1% gelatin. The resistance of each electrode was monitored at 4000 Hz. After culturing for 16–20 h, the resistance reached a plateau, indicating 100% confluence. Cells were serum‐starved for 3–4 h in ECMb containing 1% p/s and 1% AB. FGF2 or VEGFA was added (final concentration of 100 ng mL−1). Changes in resistance were monitored for up to 24 h.

Adenoviral transduction

To further explore the roles of GNA11 in FGF2‐ and VEGFA‐induced cell responses in HUVECs, GNA11 was overexpressed using adenoviruses carrying GNA11 with a green fluorescent protein (GFP) reporter (Ad‐GNA11; Vector Biolabs, Malvern, PA, USA) as described previously (Jiang et al. 2014). Adenoviruses carrying GFP (Ad‐GFP) were used as a control vector (Ren et al. 2014). Amplification and transduction of adenovirus were performed as described previously (Liao et al. 2009; Jiang et al. 2014). We first confirmed the specificity of Ad‐GNA11 using a PCR with TaqMan Universal Master Mix II (Applied Biosystems, Foster City, CA, USA). TaqMan primers for GNA11 (assay number: Hs00976153_m1, amplicon length 103 bp) and GNA14 (assay number: Hs01030246_m1, amplicon length 98 bp) were purchased (Applied Biosystems). PCR products were separated in a 2% agarose gel using the E‐Gel system in accordance with the manufacturer's instructions (Thermo Fisher Scientific).

After confirmation of the specificity of Ad‐GNA11, HUVECs at 50–60% confluency were transfected with Ad‐GNA11 or Ad‐GFP at a different multiplicity of infection (MOI). Cells were harvested and subjected to Western blotting after 3 days of transfection.

Western blotting

Western blot analysis was conducted as described (Dai et al. 2011; Jiang et al. 2013a, b; Li et al. 2015, 2017). Cells were lysed in the lysis buffer [50 mm Hepes, 0.1 m NaCl, 10 mm EDTA, 4 mm sodium pyrophosphate, 10 mm sodium fluoride, 2 mm sodium orthovanadate (pH 7.5), 1 mm phenylmethylsulphonylfluoride, 1% Triton X‐100, 5 μg mL−1 leupeptin, 5 μg mL−1 aprotinin]. Cell lysates were collected and centrifuged. Protein samples (20–30 μg per sample) were separated on 10% SDS‐PAGE gels, and electrically transferred to polyvinylidene difluoride membranes. Membranes were probed by primary antibodies (Table 1). Proteins were visualized by enhanced chemiluminescence (ECL) or ECL2 (Thermo Fisher Scientific). Signals were recorded using a Epson Perfection 4990 Photo Scanner (Epson America, Long Beach, CA, USA, USA). Data were analysed using ImageJ (NIH, Bethesda. MD, USA). Polyclonal rabbit GNA14 antibody (GeneTel Laboratory, Madison, WI, USA) was used for detecting GNA14 protein in a GNA11 siRNA knockdown verification. We have previously verified and used this GNA14 antibody in human placental tissues and HUVECs (Zhao et al. 2014). Monoclonal mouse GNA14 antibody (Abnova, Walnut, CA, USA) was used to examine GNA14 protein in the Ad‐GNA11 overexpression assay.

Table 1.

Antibodies used in the Western blot analysis

Antibody Vendor Catalogue number Dilution
β‐actin Cell Signaling Technology (Beverly, MA, USA) 4967 1: 1000
GAPDH Novus Biologicals (Littleton, CO, USA) H00002597‐M01 1: 10000
GNA11 Abgent (Suzhou, China) AP19441a 1: 1000
GNA14 Abnova (Taipei, Taiwan) H00009630‐M06A 1: 500
GNA14 GeneTel Laboratories (Madison, WI, USA) Rb139‐Rb140 1: 500
Phospho‐p44/42 MAPK T202/Y204 (p‐ERK1/2) Cell Signaling Technology 9101 1: 2000
p44/42 MAPK (t‐ERK1/2) Cell Signaling Technology 9102 1: 2000
Phospho‐AKT1 S473 (p‐AKT1) Santa Cruz Biotechnology (Santa Cruz, CA, USA) sc‐7985‐R 1: 2000
AKT1 (t‐AKT1) Cell Signaling Technology 9272 1: 2000
Phospho‐PLCβ3 S537 (p‐PLCβ3 S537) Cell Signaling Technology 2481 1: 500
Phospho‐PLCβ3 S1105 (p‐PLCβ3 S1105) Thermo Fisher Scientific (Waltham, MA, USA) PA5‐38089 1: 1000
PLCβ3 (t‐PLCβ3) Millipore (Billerica, MA, USA) ABS‐512 1: 1000
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Statistical analysis

Data were analysed using one‐way ANOVA in SigmaStat (Jandel Co., San Rafael, CA, USA). When an F test was significant, data were compared using Bonferroni's method (all pairwise or vs. control multiple comparison procedures) or Student's t test. P < 0.05 was considered statistically significant.

Results

GNA11 siRNA suppresses GNA11 protein levels in PCN‐HUVECs

To investigate the role of GNA11 in mediating FGF2‐ and VEGFA‐induced cell responses, human‐specific GNA11 siRNA was used to knockdown GNA11 expression (Fig. 1). Western blotting revealed that GNA11 siRNA (but not ssiRNA) significantly (P < 0.05) suppressed protein levels of GNA11 by ∼70% compared to vehicle after 3 and 4 days of transfection (Fig. 1). As a demonstration of the specificity of GNA11 siRNA, GNA11 siRNA transfection did not significantly alter protein levels of GNA14 for up to 4 days. GNA14 is another member of Gαq/11 subfamily and shares ∼70% of identity with GNA11 in amino acid sequences in humans (Hubbard & Hepler, 2006). We also observed that GNA11 protein levels in the vehicle group were slightly decreased after 3 and 4 days (0.7 ± 0.17‐fold of day 2 after data were normalized to β‐actin) of culture compared to day 2 (1.0 ± 0.15‐fold of day 2 mean), although this decrease did not reach statistical significance.

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

Figure 1

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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).

GNA11 siRNA differentially mediates endothelial migration, proliferation and monolayer integrity in response to FGF2 and VEGFA

Compared with the control (serum‐free media), FGF2 and VEGFA stimulated (P < 0.05) cell migration of HUVECs by ∼2.4‐ and ∼3.5‐fold, respectively (Fig. 2 A). Compared with vehicle, GNA11 siRNA, significantly (P < 0.05) attenuated FGF2‐ and VEGFA‐stimulated cell migration by ∼36% and ∼50%, respectively (Fig. 2 A), whereas ssiRNA had no effect.

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

Figure 2

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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).

Both FGF2 and VEGFA promoted (P < 0.05) proliferation of HUVECs (∼2.7‐ and ∼2.5‐fold of control, respectively; Fig. 2 B). However, GNA11 siRNA did not significantly change FGF2‐ and VEGFA‐stimulated cell proliferation (Fig. 2 B).

Compared with the control, VEGFA (but not FGF2) decreased (P < 0.05) the resistance of the cell monolayer in a time‐dependent fashion (Fig. 2 C and D), suggesting that VEGFA increases cell permeability. After 2.5 h of treatment, VEGFA maximally decreased (P < 0.05) the resistance of the cell monolayer by ∼28%, which was maintained for up to 24 h. However, GNA11 siRNA had no significant effects on cell monolayer integrity in response to FGF2 and VEGFA.

GNA11 siRNA does not alter FGF2‐ and VEGFA‐induced phosphorylation of ERK1/2 and AKT1

To explore the signalling mechanisms by which GNA11 mediates FGF2‐ and VEGFA‐induced cell migration, phosphorylation of ERK1/2 (T202/Y204) and AKT1 (S473) was examined (Fig. 3). FGF2 and VEGFA time‐dependently elevated (P < 0.05) phosphorylation of ERK1/2 (∼3.22‐ and ∼3.05‐fold at 5 min vs. time 0, respectively). However, GNA11 siRNA did not significantly alter phosphorylation patterns of ERK1/2 compared to ssiRNA control (Fig. 3).

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

Figure 3

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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).

Both FGF2 and VEGFA slightly induced phosphorylation of AKT1, although neither induction reached statistical significance compared to time 0, nor did GNA11 siRNA significantly change the phosphorylation patterns of AKT1 (Fig. 3). GNA11 siRNA did not significantly change protein levels of total ERK1/2 and AKT1 (Fig. 3; quantitative data not shown).

GNA11 siRNA increases phosphorylation of PLCβ3 at S537 in response to FGF2 and VEGFA

In the ssiRNA group, FGF2 treatment did not induce phosphorylation of PLCβ3 S537 for up to 60 min (Fig. 4). VEGFA rapidly increased (P < 0.05) phosphorylation of S537, starting at 5 min; this stimulatory effect was maintained through the final 30 min time point (Fig. 4). Interestingly, GNA11 siRNA time‐dependently increased (P < 0.05) phosphorylation of S537 when cells were treated with FGF2 and VEGFA (Fig. 4). Specifically, compared to the ssiRNA control at the corresponding time point, GNA11 siRNA elevated (P < 0.05) FGF2‐inducd phosphorylation of S537 by ∼1.5‐fold at 10, 20 and 30 min, whereas it increased VEGFA‐induced phosphorylation of S537 by ∼15.2‐ and ∼12.0‐fold, at 10 and 20 min, respectively. Nonetheless, in both ssiRNA and siRNA groups, neither FGF2, nor VEGFA induced significant phosphorylation of S1105 for up to 60 min (Fig. 4). GNA11 siRNA did not significantly change protein levels of total PLCβ3 (Fig. 4; quantitative data not shown).

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

Figure 4

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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).

Elevation of GNA11 and 14 protein levels by Ad‐GNA11

We also attempted to further dissect roles of GNA11 by overexpressing GNA11 using Ad‐GNA11 in HUVECs. We confirmed the specificity of Ad‐GNA11 by PCR assay (Fig. 5 A). GNA11 (but not GNA14) primers generated a single band at ∼100 bp from the primary Ad‐GNA11 stock. Similarly, in the primary Ad‐GNA14 stock, GNA14 (but not GNA11) primers produced a single band at ∼98 bp (Fig. 5 A). These two bands correspond to the predicted amplicon lengths of GNA11 and 14, indicating the specificity of Ad‐GNA11.

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

Figure 5

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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).

After confirmation of Ad‐GNA11 specificity, HUVECs were transfected with Ad‐GNA11 (Fig. 5 B). Surprisingly, after 3 days of transfection, Ad‐GNA11 increased (P < 0.05) GNA11 and GNA14 protein levels comparably, whereas Ad‐GFP had no significant effect on either GNA11 or GNA14 protein levels in HUVECs (Fig. 5 B–D). This Ad‐GNA11‐induced overexpression started at 5 MOI of Ad‐GNA11 (∼1.8‐ vs. ∼1.5‐fold for GNA11 and 14 protein, respectively) and was maintained at 10 MOI (∼5.2‐ vs. ∼2.7‐fold, respectively) and also further elevated at 20 MOI (∼18.2‐ vs. ∼8.6‐fold, respectively). Because we were unable to exclusively overexpress GNA11 in HUVECs using Ad‐GNA11, no further gain of function assay was performed.

Discussion

In the present study, we demonstrate that knockdown of GNA11 alone inhibits FGF2‐ and VEGFA‐stimulated fetoplacental endothelial cell migration but not proliferation and permeability under physiological chronic low O2. Such inhibition is associated with elevated phosphorylation of PLCβ3 S537, although without any change in phosphorylation of PLCβ3 S1105, ERK1/2 and AKT1 in response to FGF2 and VEGFA. These data indicate that GNA11 differentially mediates different FGF2‐ and VEGFA‐induced fetoplacental endothelial responses. It is also suggested that GNA11 plays an important role in mediating FGF2‐ and VEGFA‐induced fetoplacental cell migration, one major step of angiogenesis, possibly in part via enhanced phosphorylation of PLCβ3 S537.

To date, only a few studies have explored the roles of the GNAq/11 subfamily with respect to mediating endothelial angiogenic responses. Specifically, recent evidence has implicated the potential importance of GNAq (Sivaraj et al. 2015) and GNA11 (Couto et al. 2017) in mediating angiogenesis. In addition, double knockdown of GNAq and 11 in HUVECs blocks VEGFA‐induced cell migration (Zeng et al. 2002; Sivaraj et al. 2015) and inhibits VEGFA‐ but not FGF2‐induced cell proliferation (Zeng et al. 2003). The present study provides novel evidence that GNA11 alone critically mediates not only VEGFA‐, but also FGF2‐induced migration in HUVECs under physiological chronic low O2. However, in contrast to the inhibitory effects of double knockdown of GNAq and GNA11 on VEGFA‐stimulated proliferation and permeability (Zeng et al. 2003; Sivaraj et al. 2015), knockdown of GNA11 alone did not significantly affect either FGF2‐ or VEGFA‐induced cell proliferation and permeability. Collectively, these data suggest that, although GNAq is a major mediator for endothelial proliferation and permeability, GNA11 critically mediates endothelial cell migration in response to FGF2 and VEGFA during placental angiogenesis.

Alternatively, different O2 levels (3% vs. presumably 21% O2) used to culture cells in the present study and the studies by Zeng et al. (2003) and Sivaraj et al. (2015) might explain such discrepancies. Particularly, we have recently shown that physiological chronic low O2 (3% O2) robustly alters gene expression profiles and promotes cell responses (migration, proliferation, and signalling pathway activation) to FGF2 and VEGFA in HUVECs (Jiang et al. 2013a, b). In addition, we developed the primary cell line used in the present study by pooling five individual cell preparations, which might more closely represent the HUVEC phenotype because each individual cell preparation could vary highly in their response to stimuli. Given that the GNA11 siRNA only partially suppressed the GNA11 protein expression, it is possible that the residual GNA11 protein is sufficient to mediate cellular responses such as cell proliferation and permeability in HUVECs.

Both ERK1/2 and AKT1 critically mediate fetal endothelial function including proliferation, migration and survival (Wang & Zheng, 2012; Jiang et al. 2013a, b). However, our data from the present study show that GNA11 had no effect on activation of these two protein kinases, in agreement with the previous study when both GNAq and GNA11 are knocked down in HUVECs (Sivaraj et al. 2015). These data indicate that GNAq and GNA11 do not play an important role in mediating FGF2‐ and VEGFA‐mediated ERK1/2 and AKT1 activation in HUVECs.

Upon activation, PLC can trigger the release of intracellular calcium and further enhances protein kinase C activity, leading to modulation of various cell function (Offermanns, 1999; Hubbard & Hepler, 2006). In the PLCβ subfamily, PLCβ3 has the highest affinity for the Gαq/11 subfamily (PLCβ4 and β3 ≥ PLC‐β1 ˃˃ PLC‐β2; Offermanns, 1999) and is a key mediator of the action of VEGFA in endothelial cells (Mukhopadhyay & Zeng, 2002; Bhattacharya et al. 2009). For example, knockdown of PLCβ3 inhibits VEGFA‐stimulated cell migration, yet promotes VEGFA‐stimulated cell proliferation in HUVECs (Bhattacharya et al. 2009). The phosphorylation of PLCβ3 at different sites is considered to be one of major mechanisms by which the activity of PLCβ3 is regulated (Yue et al. 1998; Xia et al. 2001; Yue & Sanborn, 2001; Bhattacharya et al. 2009). In this regard, phosphorylation of PLCβ3 S1105 is considered to inhibit PLCβ3 activity (Yue et al. 1998; Xia et al. 2001), whereas phosphorylation of S537 appears to be active (Yue & Sanborn, 2001). However, VEGFA can increase phosphorylation of PLCβ3 S537 and S1105 in HUVECs in association with increased cell migration and proliferation (Bhattacharya et al. 2009), suggesting that both sites could be active for VEGFA‐induced endothelial function in HUVECs. In the present study, we also observed that, in the control (ssiRNA) group, VEGFA (but not FGF2) elevated phosphorylation of PLCβ3 S537; however, neither VEGFA, nor FGF2 altered phosphorylation of PLCβ3 S1105. Thus, increasing phosphorylation of PLCβ3 S537 to certain levels might be important for VEGFA‐ but not FGF2‐stimulated cell function in HUVECs. Nonetheless, the finding in the present study that GNA11 knockdown robustly elevated FGF2‐ and VEGFA‐mediated phosphorylation of PLCβ3 S537 implies that GNA11 is a negative regulator of PLCβ3 S537 phosphorylation in response to FGF2 and VEGFA in HUVECs. More importantly, because GNA11 knockdown‐increased PLCβ3 S537 phosphorylation is associated with decreases in FGF2‐ and VEGFA‐stimulated cell migration, the excessive phosphorylation of PLCβ3 S537 could adversely affect cell migration in HUVECs under physiological chronic low O2, as we have proposed (Fig. 6). Further studies are needed to define the exact interaction of GNA11 and PLCβ3, as well as the role of PLCβ3 S537 phosphorylation in regulating the activity of PLCβ3 because GNA11 and phosphorylation of PLCβ3 could be potential targets for therapeutic intervention for aberrant angiogenesis, which is associated with many human diseases such as fetal growth restriction.

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

Figure 6

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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.

Surprisingly, we observed that overexpression of GNA11 also elevated protein levels of GNA14, even at a low MOI of Ad‐GNA11, although the specificity of the Ad‐GNA11 expression vector was confirmed. To date, the mechanisms underlying such cross‐over overexpression are yet to be determined.

In conclusion, our data from the present study clearly indicate differential roles of GNA11 in mediating FGF2‐ and VEGFA‐induced fetoplacental endothelial function in association with different regulation of phosphorylation of PLCβ3 under a physiological low O2 condition, suggesting the importance of GNA11 in fetoplacental angiogenesis.

Additional information

Competing interests

The authors declare that they have no competing interests.

Author contributions

QYZ, YJZ and JZ conceived the study. QYZ, YJZ, CZ and JZ were responsible for the design of the study. QYZ, YJZ, HL, AXL, XZW, XQZ, YL, QY collected and assembled data. QYZ, HL, AXL, XZW, XQZ, YL, QY, CZ and JZ were responsible for data analysis. QYZ, CZ and JZ were responsible for data interpretation. QYZ drafted the manuscript. QYZ, YJZ, CZ and JZ critically revised the manuscript. QYZ, YJZ, HL, AXL, XZW, XQZ, YL, QY, CZ and JZ approved the final version of the manuscript submitted for publication. JZ was responsible for financial support, administrative support and the provision of study materials. All authors agree to be accountable for all aspects of the work. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed.

Funding

This work was supported in part by National Institutes of Health grant PO1 HD38843 to JZ. The project was also supported by the Clinical and Translational Science Award program, through the NIH National Centre for Advancing Translational Sciences, grant UL1TR002373. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. The present study is in partial fulfilment for a PhD degree (QYZ) in the Endocrinology and Reproductive Physiology Training Program (http://www.erp.wisc.edu).

Translational perspective

G proteins are a family of proteins mediating many cellular functions that may affect human health. Fetoplacental vascular growth is critical to fetal growth and normal pregnancy outcome. Little is known about the role of G proteins in mediating growth factor‐induced endothelial functions. We tested the hypothesis that GNA11 (one of the G proteins) significantly affects fetoplacental endothelial responses induced by fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor A (VEGFA), which are two potent growth factors. We observed that knockdown of GNA11 significantly decreased FGF2‐ and VEGFA‐induced fetoplacental endothelial cell migration but not proliferation and permeability in association with increased phosphorylation of phospholipase C‐β3 (PLCβ3). These data suggest that GNA11 may play an important role in mediating fetoplacental angiogenesis via altering the activation of PLCβ3 and also that GNA11 might be used as a target of intervention for abnormal placental angiogenesis.

Acknowledgements

We thank Laura H. Hogan, PhD, a Science Writer/Newsletter Editor from the Institute for Clinical and Translational Research, University of Wisconsin School of Medicine and Public Health, for critically reading and editing the manuscript submitted for publication.

Biography

Qing‐yun Zou received his MBBS in 2011 from the Harbin Medical University, China. Currently, Qing‐yun is a PhD candidate of the Endocrinology and Reproductive Physiology Program, University of Wisconsin‐Madison, Wisconsin, Madison, USA, under the guidance of Professor Jing Zheng. Qing‐yun's research focuses on understanding the roles of G protein α subunits with respect to mediating human fetoplacental endothelial function in responses to peptide growth factors

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Edited by: Laura Bennet & Kathleen Morgan

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