G protein alpha inhibitory subunit 1/3 mediate Netrin-1-activated signaling and angiogenesis_Chinese Journal of Ocular Fundus Diseases

Authors：

Yao Yujia , Li Jiajun , Wang Suyu ,  Li Keran

Department of Ophthalmology, The Affiliated Eye Hospital of Nanjing Medical University, Nanjing 210029, China;

Corresponding author：

Li Keran, Email: kathykeran860327@126.com

Keywords：

G protein; Netrin-1; Diabetic retinopathy; Angiogenesis

DOI：

10.3760/cma.j.cn511434-20240222-00078

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Objective To observe the effect of G protein alpha inhibitory subunit (Gαi) 1 and Gαi3 on signal transduction and angiogenesis induced by Netrin-1 (NTN1) and explore the possible mechanisms. Methods Twenty male C57BL/6J mice aged 6 to 8 weeks were randomly assigned to a control group and a diabetic group, with 10 mice in each group. Diabete group mice were induced by streptozotocin to establish diabetes model. 12 weeks after modeling, quantitative real-time polymerase chain reaction and Western blot were performed to detect the expression of Ntn1, Gαi1 and Gαi3 in diabetic retinas. Additionally, 35 male C57BL/6J mice aged 2 weeks were randomly stratified into three groups: a control group, an intravitreal injection of NTN1 group (NTN1 group), and a retinal endothelial cell-specific Gαi1/Gαi3 knockdown coupled with intravitreal NTN1 injection group (Gαi1/Gαi3 eKD+NTN1 group), with 15 mice in each of the normal control and NTN1 groups, and 5 mice in the Gαi1/Gαi3 eKD+NTN1 group. Isolectin B4 staining was performed to observe retinal neovascularization. In vitro, human umbilical vein endothelial cells (HUVEC) were divided into four groups: negative control lentiviral transfection group (shC group), negative control lentiviral transfection+NTN1 treatment group (shC+NTN1 group), Gαi1/Gαi3 knockdown group (shGαi1/Gαi3 group), and Gαi1/Gαi3 knockdown+NTN1 treatment group (shGαi1/Gαi3+NTN1 group). The effects of NTN1, Gαi1, and Gαi3 on HUVEC proliferation were assessed using the EdU assay. Transwell assays were conducted to determine the effects on HUVEC migration, and Matrigel assays were used to evaluate the effects on HUVEC tube formation. Protein kinase B (Akt), phosphorylated Akt (p-Akt), ribosomal protein S6 kinase (S6K), phosphorylated S6K (p-S6K), extracellular regulatory protein kinase (Erk1/2), phosphorylated Erk1/2 (p-Erk1/2) protein expression on HUVEC were detected by Western blot. Results Compared with the control group, the relative expression levels of Ntn1, Gαi1, and Gαi3 mRNA and protein in the diabetic group retina were significantly increased, with statistically significant differences (t=11.800, 9.298, 10.620, 7.503, 3.432, 8.037; P＜0.000 1). Compared with the shC group, the relative expression levels of Gαi1 and Gαi3 mRNA and protein in the shGαi1/Gαi3 group in HUVEC were significantly reduced, showing statistically significant differences (t=16.310, 16.300, 13.600, 9.068; P＜0.000 1). HUVEC proliferation rate, migration number and lumen formation number: compared with shC group, shC+NTN1 group significantly increased, while shGαi1/Gαi3 group and shGαi1/Gαi3+NTN1 group significantly decreased, and the differences were statistically significant (F=62.750, 49.830, 54.900; P＜0.000 1). Compared with the control group, the relative expression levels of Gαi1 and Gαi3 mRNA and protein in retina were significantly decreased in the Gαi1/Gαi3 eKD group, showing statistically significant differences (t=10.920, 13.460, 9.219, 10.500; P＜0.000 1). Retinal neovasculogenesis area: compared with the normal control group, the area of retinal neovasculogenesis increased significantly in the NTN1 group, but decreased significantly in the Gαi1/Gαi3 eKD+NTN1 group, with statistical significance (F=24.010, P＜0.000 1). The protein expression of p-Akt relative to Akt, p-S6K relative to S6K, and p-Erk1/2 relative to Erk1/2: compared with shC group, the protein expression of shC+NTN1 group was significantly increased, while that of shGαi1/Gαi3 group and shGαi1/Gαi3+NTN1 group was significantly decreased, with statistical significance (F=78.610, 144.400, 77.010; P＜0.000 1). Conclusions NTN1 induces Gαi1/Gαi3 to mediate activation of downstream Akt-mammalian target proteins of rapamycin and Erk1/2, thereby promoting angiogenesis in vivo and in vitro environments. Knocking down Gαi1/Gαi3 significantly reduces the NTN1-induced angiogenesis effect.

Citation： Yao Yujia, Li Jiajun, Wang Suyu, Li Keran. G protein alpha inhibitory subunit 1/3 mediate Netrin-1-activated signaling and angiogenesis. Chinese Journal of Ocular Fundus Diseases, 2024, 40(10): 781-789. doi: 10.3760/cma.j.cn511434-20240222-00078 Copy

1.	Ruan Y, Jiang S, Musayeva A, et al. Oxidative stress and vascular dysfunction in the retina: therapeutic strategies[J]. Antioxidants (Basel), 2020, 9(8): 761. DOI: 10.3390/antiox9080761.
2.	Yang J, Liu Z. Mechanistic pathogenesis of endothelial dysfunction in diabetic nephropathy and retinopathy[J/OL]. Front Endocrinol (Lausanne), 2022, 13: 816400[2022-05-25]. https://pubmed.ncbi.nlm.nih.gov/35692405/. DOI: 10.3389/fendo.2022.816400.
3.	Serafini T, Kennedy TE, Galko MJ, et al. The netrins define a family of axon outgrowth-promoting proteins homologous to C. elegans UNC-6[J]. Cell, 1994, 78(3): 409-424. DOI: 10.1016/0092-8674(94)90420-0.
4.	Wilson BD, Ii M, Park KW, et al. Netrins promote developmental and therapeutic angiogenesis[J]. Science, 2006, 313(5787): 640-644. DOI: 10.1126/science.1124704.
5.	Larrivée B, Freitas C, Trombe M, et al. Activation of the UNC5B receptor by netrin-1 inhibits sprouting angiogenesis[J]. Genes Dev, 2007, 21(19): 2433-2447. DOI: 10.1101/gad.437807.
6.	Yu Y, Zou J, Han Y, et al. Effects of intravitreal injection of netrin-1 in retinal neovascularization of streptozotocin-induced diabetic rats[J]. Drug Des Devel Ther, 2015, 9: 6363-6377. DOI: 10.2147/DDDT.S93166.
7.	Tu T, Zhang C, Yan H, et al. CD146 acts as a novel receptor for netrin-1 in promoting angiogenesis and vascular development[J]. Cell Res, 2015, 25(3): 275-287. DOI: 10.1038/cr.2015.15.
8.	Marshall J, Zhou XZ, Chen G, et al. Antidepression action of BDNF requires and is mimicked by Gαi1/Gαi3 expression in the hippocampus[J/OL]. Proc Natl Acad Sci USA, 2018, 115(15): E3549-3558[2018-04-10]. https://pubmed.ncbi.nlm.nih.gov/29507199/. DOI: 10.1073/pnas.1722493115.
9.	Liu YY, Chen MB, Cheng L, et al. microRNA-200a downregulation in human glioma leads to Gαi1 over-expression, Akt activation, and cell proliferation[J]. Oncogene, 2018, 37(21): 2890-2902. DOI: 10.1038/s41388-018-0184-5.
10.	Zhang YM, Zhang ZQ, Liu YY, et al. Requirement of Gαi1/Gαi3-Gab1 signaling complex for keratinocyte growth factor-induced PI3K-AKT-mTORC1 activation[J]. J Invest Dermatol, 2015, 135(1): 181-191. DOI: 10.1038/jid.2014.326.
11.	Sun J, Huang W, Yang SF, et al. Gαi1 and Gαi3mediate VEGF-induced VEGFR2 endocytosis, signaling and angiogenesis[J]. Theranostics, 2018, 8(17): 4695-4709. DOI: 10.7150/thno.26203.
12.	Toque HA, Fernandez-Flores A, Mohamed R, et al. Netrin-1 is a novel regulator of vascular endothelial function in diabetes[J/OL]. PLoS One, 2017, 12(10): e0186734[2017-10-23]. https://pubmed.ncbi.nlm.nih.gov/29059224/. DOI: 10.1371/journal.pone.0186734.
13.	Yao J, Wu XY, Yu Q, et al. The requirement of phosphoenolpyruvate carboxykinase 1 for angiogenesis in vitro and in vivo[J/OL]. Sci Adv, 2022, 8(21): 6928[2022-05-27]. https://pubmed.ncbi.nlm.nih.gov/35622925/. DOI: 10.1126/sciadv.abn6928.
14.	Xu G, Qi LN, Zhang MQ, et al. Gαi1/Gαi3 mediation of Akt-mTOR activation is important for RSPO3-induced angiogenesis[J]. Protein Cell, 2023, 14(3): 217-222. DOI: 10.1093/procel/pwac035.
15.	Han Y, Shao Y, Lin Z, et al. Netrin-1 simultaneously suppresses corneal inflammation and neovascularization[J]. Invest Ophthalmol Vis Sci, 2012, 53(3): 1285-1295. DOI: 10.1167/iovs.11-8722.
16.	Castets M, Mehlen P. Netrin-1 role in angiogenesis: to be or not to be a pro-angiogenic factor?[J]. Cell Cycle, 2010, 9(8): 1466-1471. DOI: 10.4161/cc.9.8.11197.
17.	Wu W, Lei H, Shen J, et al. The role of netrin-1 in angiogenesis and diabetic retinopathy: a promising therapeutic strategy[J]. Discov Med, 2017, 23(128): 315-323.
18.	Xu H, Liu J, Xiong S, et al. Suppression of retinal neovascularization by lentivirus-mediated netrin-1 small hairpin RNA[J]. Ophthalmic Res, 2012, 47(3): 163-169. DOI: 10.1159/000331428.
19.	Zhang X, Liu J, Xiong S, et al. Expression of netrin-1 in diabetic rat retina[J]. Eye Sci, 2013, 28(3): 148-152.
20.	Yang X, Liu Y, Zhong W, et al. Netrin-1 attenuates cerebral ischemia/reperfusion injury by limiting mitochondrial ROS and Ca²⁺ levels via activation of AKT phosphorylation and mitochondrial m-AAA protease AFG3L2[J/OL]. FASEB J, 2023, 37(3): e22805[2023-03-01]. https://pubmed.ncbi.nlm.nih.gov/36786711/. DOI: 10.1096/fj.202201739R.
21.	Untiveros G, Raskind A, Linares L, et al. Netrin-1 stimulates migration of neogenin expressing aggressive melanoma cells[J]. Int J Mol Sci, 2022, 23(21): 1251. DOI: 10.3390/ijms232112751.
22.	Guo X, Ding S, Li T, et al. Macrophage-derived netrin-1 is critical for neuroangiogenesis in endometriosis[J]. Int J Biol Macromol, 2020, 148: 226-237. DOI: 10.1016/j.ijbiomac.2020.01.130.
23.	St Croix B. CD146: the unveiling of a pro-angiogenic netrin receptor[J]. Cell Res, 2015, 25(5): 533-534. DOI: 10.1038/cr.2015.42.
24.	Bai JY, Li Y, Xue GH, et al. Requirement of Gαi1 and Gαi3 in interleukin-4-induced signaling, macrophage M2 polarization and allergic asthma response[J]. Theranostics, 2021, 11(10): 4894-4909. DOI: 10.7150/thno.56383.
25.	Liu TT, Shi X, Hu HW, et al. Endothelial cell-derived RSPO3 activates Gαi1/Gαi3-Erk signaling and protects neurons from ischemia/reperfusion injury[J]. Cell Death Dis, 2023, 14(10): 654. DOI: 10.1038/s41419-023-06176-2.
26.	Wang W, Lo ACY. Diabetic retinopathy: pathophysiology and treatments[J]. Int J Mol Sci, 2018, 19(6): 1816. DOI: 10.3390/ijms19061816.

1. Ruan Y, Jiang S, Musayeva A, et al. Oxidative stress and vascular dysfunction in the retina: therapeutic strategies[J]. Antioxidants (Basel), 2020, 9(8): 761. DOI: 10.3390/antiox9080761.
2. Yang J, Liu Z. Mechanistic pathogenesis of endothelial dysfunction in diabetic nephropathy and retinopathy[J/OL]. Front Endocrinol (Lausanne), 2022, 13: 816400[2022-05-25]. https://pubmed.ncbi.nlm.nih.gov/35692405/. DOI: 10.3389/fendo.2022.816400.
3. Serafini T, Kennedy TE, Galko MJ, et al. The netrins define a family of axon outgrowth-promoting proteins homologous to C. elegans UNC-6[J]. Cell, 1994, 78(3): 409-424. DOI: 10.1016/0092-8674(94)90420-0.
4. Wilson BD, Ii M, Park KW, et al. Netrins promote developmental and therapeutic angiogenesis[J]. Science, 2006, 313(5787): 640-644. DOI: 10.1126/science.1124704.
5. Larrivée B, Freitas C, Trombe M, et al. Activation of the UNC5B receptor by netrin-1 inhibits sprouting angiogenesis[J]. Genes Dev, 2007, 21(19): 2433-2447. DOI: 10.1101/gad.437807.
6. Yu Y, Zou J, Han Y, et al. Effects of intravitreal injection of netrin-1 in retinal neovascularization of streptozotocin-induced diabetic rats[J]. Drug Des Devel Ther, 2015, 9: 6363-6377. DOI: 10.2147/DDDT.S93166.
7. Tu T, Zhang C, Yan H, et al. CD146 acts as a novel receptor for netrin-1 in promoting angiogenesis and vascular development[J]. Cell Res, 2015, 25(3): 275-287. DOI: 10.1038/cr.2015.15.
8. Marshall J, Zhou XZ, Chen G, et al. Antidepression action of BDNF requires and is mimicked by Gαi1/Gαi3 expression in the hippocampus[J/OL]. Proc Natl Acad Sci USA, 2018, 115(15): E3549-3558[2018-04-10]. https://pubmed.ncbi.nlm.nih.gov/29507199/. DOI: 10.1073/pnas.1722493115.
9. Liu YY, Chen MB, Cheng L, et al. microRNA-200a downregulation in human glioma leads to Gαi1 over-expression, Akt activation, and cell proliferation[J]. Oncogene, 2018, 37(21): 2890-2902. DOI: 10.1038/s41388-018-0184-5.
10. Zhang YM, Zhang ZQ, Liu YY, et al. Requirement of Gαi1/Gαi3-Gab1 signaling complex for keratinocyte growth factor-induced PI3K-AKT-mTORC1 activation[J]. J Invest Dermatol, 2015, 135(1): 181-191. DOI: 10.1038/jid.2014.326.
11. Sun J, Huang W, Yang SF, et al. Gαi1 and Gαi3mediate VEGF-induced VEGFR2 endocytosis, signaling and angiogenesis[J]. Theranostics, 2018, 8(17): 4695-4709. DOI: 10.7150/thno.26203.
12. Toque HA, Fernandez-Flores A, Mohamed R, et al. Netrin-1 is a novel regulator of vascular endothelial function in diabetes[J/OL]. PLoS One, 2017, 12(10): e0186734[2017-10-23]. https://pubmed.ncbi.nlm.nih.gov/29059224/. DOI: 10.1371/journal.pone.0186734.
13. Yao J, Wu XY, Yu Q, et al. The requirement of phosphoenolpyruvate carboxykinase 1 for angiogenesis in vitro and in vivo[J/OL]. Sci Adv, 2022, 8(21): 6928[2022-05-27]. https://pubmed.ncbi.nlm.nih.gov/35622925/. DOI: 10.1126/sciadv.abn6928.
14. Xu G, Qi LN, Zhang MQ, et al. Gαi1/Gαi3 mediation of Akt-mTOR activation is important for RSPO3-induced angiogenesis[J]. Protein Cell, 2023, 14(3): 217-222. DOI: 10.1093/procel/pwac035.
15. Han Y, Shao Y, Lin Z, et al. Netrin-1 simultaneously suppresses corneal inflammation and neovascularization[J]. Invest Ophthalmol Vis Sci, 2012, 53(3): 1285-1295. DOI: 10.1167/iovs.11-8722.
16. Castets M, Mehlen P. Netrin-1 role in angiogenesis: to be or not to be a pro-angiogenic factor?[J]. Cell Cycle, 2010, 9(8): 1466-1471. DOI: 10.4161/cc.9.8.11197.
17. Wu W, Lei H, Shen J, et al. The role of netrin-1 in angiogenesis and diabetic retinopathy: a promising therapeutic strategy[J]. Discov Med, 2017, 23(128): 315-323.
18. Xu H, Liu J, Xiong S, et al. Suppression of retinal neovascularization by lentivirus-mediated netrin-1 small hairpin RNA[J]. Ophthalmic Res, 2012, 47(3): 163-169. DOI: 10.1159/000331428.
19. Zhang X, Liu J, Xiong S, et al. Expression of netrin-1 in diabetic rat retina[J]. Eye Sci, 2013, 28(3): 148-152.
20. Yang X, Liu Y, Zhong W, et al. Netrin-1 attenuates cerebral ischemia/reperfusion injury by limiting mitochondrial ROS and Ca²⁺ levels via activation of AKT phosphorylation and mitochondrial m-AAA protease AFG3L2[J/OL]. FASEB J, 2023, 37(3): e22805[2023-03-01]. https://pubmed.ncbi.nlm.nih.gov/36786711/. DOI: 10.1096/fj.202201739R.
21. Untiveros G, Raskind A, Linares L, et al. Netrin-1 stimulates migration of neogenin expressing aggressive melanoma cells[J]. Int J Mol Sci, 2022, 23(21): 1251. DOI: 10.3390/ijms232112751.
22. Guo X, Ding S, Li T, et al. Macrophage-derived netrin-1 is critical for neuroangiogenesis in endometriosis[J]. Int J Biol Macromol, 2020, 148: 226-237. DOI: 10.1016/j.ijbiomac.2020.01.130.
23. St Croix B. CD146: the unveiling of a pro-angiogenic netrin receptor[J]. Cell Res, 2015, 25(5): 533-534. DOI: 10.1038/cr.2015.42.
24. Bai JY, Li Y, Xue GH, et al. Requirement of Gαi1 and Gαi3 in interleukin-4-induced signaling, macrophage M2 polarization and allergic asthma response[J]. Theranostics, 2021, 11(10): 4894-4909. DOI: 10.7150/thno.56383.
25. Liu TT, Shi X, Hu HW, et al. Endothelial cell-derived RSPO3 activates Gαi1/Gαi3-Erk signaling and protects neurons from ischemia/reperfusion injury[J]. Cell Death Dis, 2023, 14(10): 654. DOI: 10.1038/s41419-023-06176-2.
26. Wang W, Lo ACY. Diabetic retinopathy: pathophysiology and treatments[J]. Int J Mol Sci, 2018, 19(6): 1816. DOI: 10.3390/ijms19061816.

Chinese Journal of Ocular Fundus Diseases

G protein alpha inhibitory subunit 1/3 mediate Netrin-1-activated signaling and angiogenesis

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