Requirement Of Gαi1/3 Protein On Vascular Endothelial Growth Factor-induced Ocular Neovascularization

Background&Objective:Guanine nucleotide binding protein(G protein)have been reported to exert the function mainly by cooperating with G protein-coupled receptors(GPCRs)in signal transduction.In previous study,we found Gαi1/3,an inhibitory subunit of G protein,participated in signal transduction by a new way—Gαi1/3 are required for epidermal growth factor(EGF)and keratinocyte growth fact(KGF)-mediated activation of the downstream Akt-mTORC1 pathway,which Gail/3 formed a complex with growth factor receptor binding protein 2(Grb2)-associated binding protein 1(Gab1)to orderly phosphorylate Gab1,PI3K and other related proteins in combination with EGF or KGF receptor.Vascular endothelial growth factor(VEGF),a glycoprotein composed of homodimers,is a key factor in neovascularization.We aim to investigate whether Gαi1/3 plays an important role in VEGF-VEGFR-mediated downstream signaling pathways and explore whether Gαi3 involve in ocular neovascularization.Methods:(1)Several mouse embryonic fibroblasts(MEFs)were treated with VEGF:MEFs derived from wild type(WT);MEFs deficient in Gαi1,Gα,Gαi3,Gab1 or transfected with Gαi1/Gαi3 RNA interference(RNAi);Gαi1,Gai3 doubly deficient(DKO)MEFs and reconstitution of either Gαi1 or Gαi3 in DKO MEFs.Western Blot assay was used to detect the activation level of related proteins(Akt,S6,mTOR,GSK3α/3β,Erk)in MAPK/ERK and AKT-mTORC1 signaling pathways.(2)Umbilical vein endothelial cells(HUVECs)silenced scramble,Gαi1 or Gαi3 genes were respectively treated with VEGF.Western blot was performed to test the activation of related proteins.MTT assay,clonogenicity assay and tube formation assay in vitro were applied to detect HUVECs survival,proliferationand tube formation respectively.(3)The OIR mice models were built up.The retinal vascular leakages in OIR mice were detected by Evans-blue-albumin method to compare groups of OIR mice neovascularizations after intraocular injection of Scr-shRNA or Gai3-shRNA lentiviruses.Result:(1)The results showed that compared with WT,MEFs lacking Gail and Gai3[termed double-knockout(DKO)cells]or Gab 1 were severely impaired in VEGF-induced phosphorylation of Gab1,AktT308,AktS473,GSK-3a,GSK-3β,and S6,as well as Erkl/2.In contrast,loss of either Gail or Gai3 led to a small but considerable decrease in the phosphorylation of Gab1,AktT308,AktS473,S6 and Erkl/2,whereas Gai2-deficient MEFs did not show a clear defect in phosphorylation of the targets in response to VEGF.Moreover,reconstitution of either Gail or Gai3 in DKO MEFs restored phosphorylation of AktT308,AktS473,S6 and Erkl/2 in response to VEGF.(2)The results revealed that compared with Scr-shRNA HUVECs,Gail-shRNA or Gai3-shRNA HUVECs were severely impaired in VEGF-induced phosphorylation of Gab1,AktT308,AktS473,S6,and Erkl/2 as well as cellular functions.(3)Compared with OIR model mice injected with Scr-shRNA lentiviruses,mice lacking Gai3 had fewer retinal vessel leakages in OIR model.CONCLUSION:Gabl and Gail/3 play vital roles in VEGF-induced downstream PI3K-AKT-mTORCl and MAPK/ERK signaling.Gail/3 are involved in survival,proliferationand tube formation of vascular endothelial cells.Gai1/3 and Gab1 are expected to be new targets in ocular neovascular therapy.