Effects of bone morphogenetic protein 4 on migration and reactive oxygen species production of Müller cells by regulating SMAD9 expression_Chinese Journal of Ocular Fundus Diseases

Authors：

Wang Yong , Cao Jingjing , Kou Zhenyu , Han Feifei , Liu Aihua ,  Dong Lijie

Tianjin Key Laboratory of Retinal Functions and Diseases, Tianjin Branch of National Clinical Research Center for Ocular Disease, Eye Institute and School of Optometry, Tianjin Medical University Eye Hospital, Tianjin 300384, China;

Corresponding author：

Dong Lijie, Email: aitaomubang@126.com

Keywords：

Bone morphogenetic protein 4; SMAD9; Reactive oxygen species; Vascular endothelial growth factor; Müller cell; Cell migration; Cell experiment

DOI：

10.3760/cma.j.cn511434-20220818-00462

Video：

Export PDF Favorites Scan Get Citation

Abstract Full text Figures/Tables Video References Cited by

Objective To investigate the effects of targeted regulation of SMAD9 expression by bone morphogenetic protein 4 (BMP4) on Müller cell migration, reactive oxygen species (ROS) generation and vascular endothelial growth factor (VEGF) expression. Methods Müller cells cultured in vitro were divided into normal control group, BMP4 group, BMP4+ no-load plasmid group (BMP4+NC group) and BMP4+SMAD9 small interference plasmid group (BMP4+siSMAD9). Cells in BMP4 group, BMP4+NC group and BMP4+siSMAD9 group were induced by adding 100 ng/ml BMP4 into cell medium for 24 h. Subsequently, BMP4+NC group was transfected with empty plasmid. BMP4+siSMAD9 group was transfected with SMAD9 small interference plasmid for 48 h. The effect of BMP4 on Müller cell migration was determined by cell scratch test. The effect of BMP4 on the production of ROS in Müller cells was detected by flow cytometry. Western blots and real-time quantitative fluorescence polymerase chain reaction (qPCR) were used to detect the relative mRNA expression levels of glutamine synthetase (GS) and glial fibrinoacidic protein (GFAP) in Müller cells. VEGF expression in Müller cells was detected by immunofluorescence. One-way analysis of variance was used to compare groups. Results The results of cell scratch test showed that the cell mobility of BMP4+siSMAD9 group was significantly lower than that of BMP4 and BMP4+NC group, and the difference was statistically significant (F=68.319, P＜0.001). Flow cytomethods showed that the level of ROS in BMP4+siSMAD9 group was significantly lower than that in BMP4 and BMP4+NC group, and the difference was statistically significant (F=52.158, P＜0.001). Western blot and qPCR results showed that the protein levels of GS and GFAP (F=42.715, 36.618) and mRNA relative expression levels (F=45.164, 43.165) in BMP4+siSMAD9 group were significantly lower than those in BMP4 and BMP4+NC group. The difference was statistically significant (P＜0.01). The results of immunofluorescence detection showed that the intracellular VEGF fluorescence intensity in BMP4 group and BMP4+NC group was significantly higher than that in BMP4+siSMAD9 group, and the difference was statistically significant (F=46.384, P＜0.05). Conclusion Targeted regulation of SMAD9 expression by BMP4 can up-regulate VEGF expression and promote the migration and ROS production of Müller cells.

Citation： Wang Yong, Cao Jingjing, Kou Zhenyu, Han Feifei, Liu Aihua, Dong Lijie. Effects of bone morphogenetic protein 4 on migration and reactive oxygen species production of Müller cells by regulating SMAD9 expression. Chinese Journal of Ocular Fundus Diseases, 2023, 39(9): 754-759. doi: 10.3760/cma.j.cn511434-20220818-00462 Copy

1.	Dong L, Cheng R, Ma X, et al. Regulation of monocyte activation by PPARα through interaction with the cGAS-STING pathway[J]. Diabetes, 2023, 72(7): 958-972. DOI: 10.2337/db22-0654.
2.	Antonetti DA, Barber AJ, Bronson SK, et al. Diabetic retinopathy: seeing beyond glucose-induced microvascular disease[J]. Diabetes, 2006, 55(9): 2401-2411. DOI: 10.2337/db05-1635.
3.	Nian S, Lo ACY, Mi Y, et al. Neurovascular unit in diabetic retinopathy: pathophysiological roles and potential therapeutical targets[J]. Eye Vis (Lond), 2021, 8(1): 15. DOI: 10.1186/s40662-021-00239-1.
4.	Vecino E, Rodriguez FD, Ruzafa N, et al. Glia-neuron interactions in the mammalian retina[J]. Prog Retin Eye Res, 2016, 51: 1-40. DOI: 10.1016/j.preteyeres.2015.06.003.
5.	Mrugacz M, Bryl A, Zorena K. Retinal vascular endothelial cell dysfunction and neuroretinal degeneration in diabetic patients[J]. J Clin Med, 2021, 10(3): 458. DOI: 10.3390/JCM10030458.
6.	邢小丽, 黄亮瑜, 苏睿虹, 等. 丁基苯酞对过氧化氢诱导下视网膜Müller细胞凋亡的影响[J]. 中华眼底病杂志, 2018, 34(5): 481-486. DOI: 10.3760/cma.j.issn.1005-1015.2018.05.014.Xing XL, Huang LY, Su RH, et al. Effect of dl-3-n-Butylphthalide on apoptosis of retinal müller cells induced by hydrogen peroxide[J]. Chin J Ocul Fundus Dis, 2018, 34(5): 481-486. DOI: 10.3760/cma.j.issn.1005-1015.2018.05.014.
7.	Schmitt JM, Hwang K, Winn SR, et al. Bone morphogenetic proteins: an update on basic biology and clinical relevance[J]. J Orthop Res, 1999, 17(2): 269-278. DOI: 10.1002/jor.1100170217.
8.	Rothhammer T, Poser I, Soncin F, et al. Bone morphogenic proteins are overexpressed in malignant melanoma and promote cell invasion and migration[J]. Cancer Res, 2005, 65(2): 448-456. DOI: 10.1158/0008-5472.448.65.2.
9.	He C, Chen X. Transcription regulation of the vegf gene by the BMP/Smad pathway in the angioblast of zebrafish embryos[J]. Biochem Biophys Res Commun, 2005, 329(1): 324-330. DOI: 10.1016/j.bbrc.2005.01.133.
10.	Dyer LA, Pi X, Patterson C. The role of BMPs in endothelial cell function and dysfunction[J]. Trends Endocrinol Metab, 2014, 25(9): 472-480. DOI: 10.1016/j.tem.2014.05.003.
11.	Dong L, Zhang Z, Liu X, et al. RNA sequencing reveals BMP4 as a basis for the dual-target treatment of diabetic retinopathy[J]. J Mol Med (Berl), 2021, 99(2): 225-240. DOI: 10.1007/s00109-020-01995-8.
12.	Vogt RR, Unda R, Yeh LC, et al. Bone morphogenetic protein-4 enhances vascular endothelial growth factor secretion by human retinal pigment epithelial cells[J]. J Cell Biochem, 2006, 98(5): 1196-1202. DOI: 10.1002/JCB.20831.
13.	Wu L, Li J, Zhao F, et al. MiR-340-5p inhibits Müller cell activation and pro-inflammatory cytokine production by targeting BMP4 in experimental diabetic retinopathy[J/OL]. Cytokine, 2022, 149: 155745[2021-10-22]. https://pubmed.ncbi.nlm.nih.gov/34689058/. DOI: 10.1016/j.cyto.2021.155745.
14.	Deng G, Zeng S, Qu Y, et al. BMP4 promotes hepatocellular carcinoma proliferation by autophagy activation through JNK1-mediated Bcl-2 phosphorylation[J]. J Exp Clin Cancer Res, 2018, 37(1): 156. DOI: 10.1186/s13046-018-0828-x.
15.	步绍翀, 张哲, 王琼, 等. 结缔组织生长因子刺激后视网膜Müller细胞基因表达谱的转录组学分析及验证[J]. 中华眼底病杂志, 2020, 36(12): 964-970. DOI: 10.3760/cma.j.cn511434-20200116-00026.Bu SC, Zhang Z, Wang Q, et al. Transcriptome profile analysis and validation of differential gene expression of retinal Müller cells stimulated by connective tissue growth factor[J]. Chin J Ocul Fundus Dis, 2020, 36(12): 964-970. DOI: 10.3760/cma.j.cn511434-20200116-00026.
16.	Toft-Kehler AK, Skytt DM, Svare A, et al. Mitochondrial function in Muller cells-does it matter?[J]. Mitochondrion, 2017, 36: 43-51. DOI: 10.1016/j.mito.2017.02.002.
17.	李辉, 洪亚茹, 刘勃实, 等. 骨形成蛋白4对人视网膜微血管内皮细胞糖酵解水平的影响[J]. 中华眼底病杂志, 2022, 38(10): 840-845. DOI: 10.3760/cma.j.cn511434-20210714-00379.Li H, Hong YR, Liu BS, et al. Effect of bone morphogenetic protein 4 on glycolysis of human retinal vascular endothelial cells[J]. Chin J Ocul Fundus Dis, 2022, 38(10): 840-845. DOI: 10.3760/cma.j.cn511434-20210714-00379.
18.	Dong L, Zhang X, Fu X, et al. PTB-associated splicing factor (PSF) functions as a repressor of STAT6-mediated Ig epsilon gene transcription by recruitment of HDAC1[J]. J Biol Chem, 2011, 286(5): 3451-3459. DOI: 10.1074/JBC.M110.168377.
19.	Cheng KH, Ponte JF, Thiagalingam S. Elucidation of epigenetic inactivation of SMAD8 in cancer using targeted expressed gene display[J]. Cancer Res, 2004, 64(5): 1639-1646. DOI: 10.1158/0008-5472.can-03-2688.
20.	Katakawa Y, Funaba M, Murakami M. Smad8/9 is regulated through the BMP pathway[J]. J Cell Biochem, 2016, 117(8): 1788-1796. DOI: 10.1002/JCB.25478.

1. Dong L, Cheng R, Ma X, et al. Regulation of monocyte activation by PPARα through interaction with the cGAS-STING pathway[J]. Diabetes, 2023, 72(7): 958-972. DOI: 10.2337/db22-0654.
2. Antonetti DA, Barber AJ, Bronson SK, et al. Diabetic retinopathy: seeing beyond glucose-induced microvascular disease[J]. Diabetes, 2006, 55(9): 2401-2411. DOI: 10.2337/db05-1635.
3. Nian S, Lo ACY, Mi Y, et al. Neurovascular unit in diabetic retinopathy: pathophysiological roles and potential therapeutical targets[J]. Eye Vis (Lond), 2021, 8(1): 15. DOI: 10.1186/s40662-021-00239-1.
4. Vecino E, Rodriguez FD, Ruzafa N, et al. Glia-neuron interactions in the mammalian retina[J]. Prog Retin Eye Res, 2016, 51: 1-40. DOI: 10.1016/j.preteyeres.2015.06.003.
5. Mrugacz M, Bryl A, Zorena K. Retinal vascular endothelial cell dysfunction and neuroretinal degeneration in diabetic patients[J]. J Clin Med, 2021, 10(3): 458. DOI: 10.3390/JCM10030458.
6. 邢小丽, 黄亮瑜, 苏睿虹, 等. 丁基苯酞对过氧化氢诱导下视网膜Müller细胞凋亡的影响[J]. 中华眼底病杂志, 2018, 34(5): 481-486. DOI: 10.3760/cma.j.issn.1005-1015.2018.05.014.Xing XL, Huang LY, Su RH, et al. Effect of dl-3-n-Butylphthalide on apoptosis of retinal müller cells induced by hydrogen peroxide[J]. Chin J Ocul Fundus Dis, 2018, 34(5): 481-486. DOI: 10.3760/cma.j.issn.1005-1015.2018.05.014.
7. Schmitt JM, Hwang K, Winn SR, et al. Bone morphogenetic proteins: an update on basic biology and clinical relevance[J]. J Orthop Res, 1999, 17(2): 269-278. DOI: 10.1002/jor.1100170217.
8. Rothhammer T, Poser I, Soncin F, et al. Bone morphogenic proteins are overexpressed in malignant melanoma and promote cell invasion and migration[J]. Cancer Res, 2005, 65(2): 448-456. DOI: 10.1158/0008-5472.448.65.2.
9. He C, Chen X. Transcription regulation of the vegf gene by the BMP/Smad pathway in the angioblast of zebrafish embryos[J]. Biochem Biophys Res Commun, 2005, 329(1): 324-330. DOI: 10.1016/j.bbrc.2005.01.133.
10. Dyer LA, Pi X, Patterson C. The role of BMPs in endothelial cell function and dysfunction[J]. Trends Endocrinol Metab, 2014, 25(9): 472-480. DOI: 10.1016/j.tem.2014.05.003.
11. Dong L, Zhang Z, Liu X, et al. RNA sequencing reveals BMP4 as a basis for the dual-target treatment of diabetic retinopathy[J]. J Mol Med (Berl), 2021, 99(2): 225-240. DOI: 10.1007/s00109-020-01995-8.
12. Vogt RR, Unda R, Yeh LC, et al. Bone morphogenetic protein-4 enhances vascular endothelial growth factor secretion by human retinal pigment epithelial cells[J]. J Cell Biochem, 2006, 98(5): 1196-1202. DOI: 10.1002/JCB.20831.
13. Wu L, Li J, Zhao F, et al. MiR-340-5p inhibits Müller cell activation and pro-inflammatory cytokine production by targeting BMP4 in experimental diabetic retinopathy[J/OL]. Cytokine, 2022, 149: 155745[2021-10-22]. https://pubmed.ncbi.nlm.nih.gov/34689058/. DOI: 10.1016/j.cyto.2021.155745.
14. Deng G, Zeng S, Qu Y, et al. BMP4 promotes hepatocellular carcinoma proliferation by autophagy activation through JNK1-mediated Bcl-2 phosphorylation[J]. J Exp Clin Cancer Res, 2018, 37(1): 156. DOI: 10.1186/s13046-018-0828-x.
15. 步绍翀, 张哲, 王琼, 等. 结缔组织生长因子刺激后视网膜Müller细胞基因表达谱的转录组学分析及验证[J]. 中华眼底病杂志, 2020, 36(12): 964-970. DOI: 10.3760/cma.j.cn511434-20200116-00026.Bu SC, Zhang Z, Wang Q, et al. Transcriptome profile analysis and validation of differential gene expression of retinal Müller cells stimulated by connective tissue growth factor[J]. Chin J Ocul Fundus Dis, 2020, 36(12): 964-970. DOI: 10.3760/cma.j.cn511434-20200116-00026.
16. Toft-Kehler AK, Skytt DM, Svare A, et al. Mitochondrial function in Muller cells-does it matter?[J]. Mitochondrion, 2017, 36: 43-51. DOI: 10.1016/j.mito.2017.02.002.
17. 李辉, 洪亚茹, 刘勃实, 等. 骨形成蛋白4对人视网膜微血管内皮细胞糖酵解水平的影响[J]. 中华眼底病杂志, 2022, 38(10): 840-845. DOI: 10.3760/cma.j.cn511434-20210714-00379.Li H, Hong YR, Liu BS, et al. Effect of bone morphogenetic protein 4 on glycolysis of human retinal vascular endothelial cells[J]. Chin J Ocul Fundus Dis, 2022, 38(10): 840-845. DOI: 10.3760/cma.j.cn511434-20210714-00379.
18. Dong L, Zhang X, Fu X, et al. PTB-associated splicing factor (PSF) functions as a repressor of STAT6-mediated Ig epsilon gene transcription by recruitment of HDAC1[J]. J Biol Chem, 2011, 286(5): 3451-3459. DOI: 10.1074/JBC.M110.168377.
19. Cheng KH, Ponte JF, Thiagalingam S. Elucidation of epigenetic inactivation of SMAD8 in cancer using targeted expressed gene display[J]. Cancer Res, 2004, 64(5): 1639-1646. DOI: 10.1158/0008-5472.can-03-2688.
20. Katakawa Y, Funaba M, Murakami M. Smad8/9 is regulated through the BMP pathway[J]. J Cell Biochem, 2016, 117(8): 1788-1796. DOI: 10.1002/JCB.25478.

Previous Article
Study on adaptive optics fundus imaging in pre-clinical hydroxychloroquine retinopathy
Next Article
Preparation and evaluation of animal model of diabetic microvascular complications

Chinese Journal of Ocular Fundus Diseases

Effects of bone morphogenetic protein 4 on migration and reactive oxygen species production of Müller cells by regulating SMAD9 expression

Abstract Full text Figures/Tables Video References Cited by

Previous Article

Next Article

Format

Content