The regulation of PTB-associated splicing factor on phosphatidylinositol 3 kinase/Akt signaling pathway in retinal pigment epithelial cells_Chinese Journal of Ocular Fundus Diseases

Authors：

QiChen ,  DongLijie , LeYi , 张晓敏 , 李筱荣 , 郭如如 , 茹玉莎 , 苏畅

Tianjin Medical University Hospital, Tianjin Medical University Eye Institute, College of Optometry and Ophthalmology, Tianjin Medical University, Tianjin 300384, China;

Corresponding author：

DongLijie, Email: aitaomubang@126.com

Keywords：

Polypyrimidine tract-binding protein; Retinal pigment epithelium/enzymology; Phosphatidylinositol 3-kinases; p21-Activated kinases; Signal transduction

DOI：

10.3760/cma.j.issn.1005-1015.2015.04.013

Video：

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Objective To observe the regulation of PTB-associated splicing factor (PSF) exerts on phosphatidylinositol 3 kinase (PI3K)/Akt signaling pathway in cultured retinal pigment epithelial (RPE) cells. Methods ARPE-19 RPE cells were divided into five groups including PSF overexpression (0.25, 0.50, 1.00 μg of pEGFP-C2-PSF plasmid DNA), PSF overexpression control (pEGFP-C2 empty vector DNA), PSF inhibition (0.25, 0.50, 1.00 μg of pGenesil-PSF-RNAi plasmid DNA), PSF inhibition control (pGenesil-scramble-siRNA empty vector) and sham transfected group (treated with lipofactamine 2000 reagent, but without adding plasmid DNA) groups. After transfecting with plasmid DNA, the cells were stimulated with insulin-like growth factor-1 (IGF-1). IGF-1-stimulated ARPE-19 cells were also treated with Wortmannin and /or PSF over-expression. WST-1 assay was used to detect the proliferation rates, the VEGF mRNA levels were analyzed using real time polymerase chain reaction (PCR), the levels of phosphorylation Akt and total Akt expression were measured by western blotting. Results After IGF-1 stimulation, the difference of the cell proliferation rates between PSF overexpression group, PSF overexpression control group and sham transfected group was statistically significant (F=29.728, P<0.05). The difference of the cell proliferation rates between PSF inhibition group, PSF inhibition control group and sham transfected was also statistically significant (F=14.121, P<0.05). Compared with control group, the VEGF mRNA levels was decreased in PSF overexpression group (P=0.000 3), but increased in PSF low expression group (P=0.030 9). Furthermore, overexpression of PSF could down-regulate the activation of pAkt after IGF-1 stimulation. When combined with Wortmannin treatment, the VEGF mRNA levels in PSF overexpression group was significantly lower than the control group (P<0.05). Conclusions After IGF-1 treatment, PSF plays a role in suppressing the proliferation and VEGF expression in RPE cells by inactivating PI3K/Akt signaling pathway.

Citation： QiChen, DongLijie, LeYi. The regulation of PTB-associated splicing factor on phosphatidylinositol 3 kinase/Akt signaling pathway in retinal pigment epithelial cells. Chinese Journal of Ocular Fundus Diseases, 2015, 31(4): 363-367. doi: 10.3760/cma.j.issn.1005-1015.2015.04.013 Copy

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2.	Slomiany MG, Rosenzweig SA. IGF-1-induced VEGF and IGFBP-3 secretion correlates with increased HIF-1 alpha expression and activity in retinal pigment epithelial cell line D407[J]. Invest Ophthalmol Vis Sci, 2004, 45(8):2838-2847.
3.	Takagi H, Yoshimura N, Tanihara H, et al. Insulin-like growth factor-related genes, receptors, and binding proteins in cultured human retinal pigment epithelial cells[J]. Invest Ophthalmol Vis Sci, 1994, 35(3):916-923.
4.	Schwesinger C, Yee C, Rohan RM, et al. Intrachoroidal neovascularization in transgenic mice overexpressing vascular endothelial growth factor in the retinal pigment epithelium[J]. Am J Pathol, 2001, 158(3):1161-1172.
5.	Spilsbury K, Garrett KL, Shen WY, et al. Overexpression of vascular endothelial growth factor (VEGF) in the retinal pigment epithelium leads to the development of choroidal neovascularization[J]. Am J Pathol, 2000, 157(1):135-144.
6.	Urban RJ, Bodenburg Y, Kurosky A, et al. Polypyrimidine tract-binding protein-associated splicing factor is a negative regulator of transcriptional activity of the porcine p450scc insulin-like growth factor response element[J]. Mol Endocrinol, 2000, 14(6):774-782.
7.	Urban RJ, Bodenburg Y. PTB-associated splicing factor regulates growth factor-stimulated gene expression in mammalian cells[J]. Am J Physiol Endocrinol Metab, 2002, 283(4):794-798.
8.	田芳,东莉洁,周玉,等. 重组腺相关病毒多聚嘧啶序列结合蛋白相关剪接因子对氧诱导视网膜新生血管形成的抑制作用[J]. 中华眼底病杂志, 2014, 30(5):504-508.
9.	Qin D, Zhang GM, Xu X, et al. The PI3K/Akt signaling pathway mediates the high glucose-induced expression of extracellular matrix molecules in human retinal pigment epithelial cells[J/OL]. J Diabetes Res, 2015, 2015:920280[2015-01-28]. http://www.hindawi.com/journals/jdr/2015/920280/.
10.	Hsiung J, Zhu D, Hinton DR. Polarized human embryonic stem cell-derived retinal pigment epithelial cell monolayers have higher resistance to oxidative stress-induced cell death than nonpolarized cultures[J]. Stem Cells Transl Med, 2015, 4(1):10-20.
11.	Slomiany MG, Rosenzweig SA. Hypoxia-inducible factor-1-dependent and -independent regulation of insulin-like growth factor-1-stimulated vascular endothelial growth factor secretion[J]. J Pharmacol Exp Ther, 2006, 318(2):666-675.
12.	Lukong KE, Huot ME, Richard S. BRK phosphorylates PSF promoting its cytoplasmic localization and cell cycle arrest[J]. Cell Signal, 2009, 21(9):1415-1422.
13.	Dong L, Nian H, Shao Y, et al. PTB-associated splicing factor inhibits IGF-1-induced VEGF upregulation in a mouse model of oxygen-induced retinopathy[J]. Cell Tissue Res, 2015, 360(2):233-243.
14.	Weng CY, Kothary PC, Verkade AJ, et al. MAP kinase pathway is involved in IGF-1-stimulated proliferation of human retinal pigment epithelial cells (hRPE)[J]. Curr Eye Res, 2009, 34(10):867-876.
15.	Kokona D, Thermos K. Synthetic and endogenous cannabinoids protect retinal neurons from AMPA excitotoxicity in vivo, via activation of CB1 receptors:involvement of PI3K/Akt and MEK/ERK signaling pathways[J]. Exp Eye Res, 2015, 136:45-58.
16.	Wilhelm F, Kassner F, Schmid G, et al. Phosphatidylinositol 3-kinase (PI3K) signalling regulates insulin-like-growth factor binding protein-2(IGFBP-2) production in human adipocytes[J]. Growth Horm IGF Res, 2015, 25(3):115-120.

1. Sall JW, Klisovic DD, O'Dorisio MS, et al. Somatostatin inhibits IGF-1 mediated induction of VEGF in human retinal pigment epithelial cells[J]. Exp Eye Res, 2004, 79(4):465-476.
2. Slomiany MG, Rosenzweig SA. IGF-1-induced VEGF and IGFBP-3 secretion correlates with increased HIF-1 alpha expression and activity in retinal pigment epithelial cell line D407[J]. Invest Ophthalmol Vis Sci, 2004, 45(8):2838-2847.
3. Takagi H, Yoshimura N, Tanihara H, et al. Insulin-like growth factor-related genes, receptors, and binding proteins in cultured human retinal pigment epithelial cells[J]. Invest Ophthalmol Vis Sci, 1994, 35(3):916-923.
4. Schwesinger C, Yee C, Rohan RM, et al. Intrachoroidal neovascularization in transgenic mice overexpressing vascular endothelial growth factor in the retinal pigment epithelium[J]. Am J Pathol, 2001, 158(3):1161-1172.
5. Spilsbury K, Garrett KL, Shen WY, et al. Overexpression of vascular endothelial growth factor (VEGF) in the retinal pigment epithelium leads to the development of choroidal neovascularization[J]. Am J Pathol, 2000, 157(1):135-144.
6. Urban RJ, Bodenburg Y, Kurosky A, et al. Polypyrimidine tract-binding protein-associated splicing factor is a negative regulator of transcriptional activity of the porcine p450scc insulin-like growth factor response element[J]. Mol Endocrinol, 2000, 14(6):774-782.
7. Urban RJ, Bodenburg Y. PTB-associated splicing factor regulates growth factor-stimulated gene expression in mammalian cells[J]. Am J Physiol Endocrinol Metab, 2002, 283(4):794-798.
8. 田芳,东莉洁,周玉,等. 重组腺相关病毒多聚嘧啶序列结合蛋白相关剪接因子对氧诱导视网膜新生血管形成的抑制作用[J]. 中华眼底病杂志, 2014, 30(5):504-508.
9. Qin D, Zhang GM, Xu X, et al. The PI3K/Akt signaling pathway mediates the high glucose-induced expression of extracellular matrix molecules in human retinal pigment epithelial cells[J/OL]. J Diabetes Res, 2015, 2015:920280[2015-01-28]. http://www.hindawi.com/journals/jdr/2015/920280/.
10. Hsiung J, Zhu D, Hinton DR. Polarized human embryonic stem cell-derived retinal pigment epithelial cell monolayers have higher resistance to oxidative stress-induced cell death than nonpolarized cultures[J]. Stem Cells Transl Med, 2015, 4(1):10-20.
11. Slomiany MG, Rosenzweig SA. Hypoxia-inducible factor-1-dependent and -independent regulation of insulin-like growth factor-1-stimulated vascular endothelial growth factor secretion[J]. J Pharmacol Exp Ther, 2006, 318(2):666-675.
12. Lukong KE, Huot ME, Richard S. BRK phosphorylates PSF promoting its cytoplasmic localization and cell cycle arrest[J]. Cell Signal, 2009, 21(9):1415-1422.
13. Dong L, Nian H, Shao Y, et al. PTB-associated splicing factor inhibits IGF-1-induced VEGF upregulation in a mouse model of oxygen-induced retinopathy[J]. Cell Tissue Res, 2015, 360(2):233-243.
14. Weng CY, Kothary PC, Verkade AJ, et al. MAP kinase pathway is involved in IGF-1-stimulated proliferation of human retinal pigment epithelial cells (hRPE)[J]. Curr Eye Res, 2009, 34(10):867-876.
15. Kokona D, Thermos K. Synthetic and endogenous cannabinoids protect retinal neurons from AMPA excitotoxicity in vivo, via activation of CB1 receptors:involvement of PI3K/Akt and MEK/ERK signaling pathways[J]. Exp Eye Res, 2015, 136:45-58.
16. Wilhelm F, Kassner F, Schmid G, et al. Phosphatidylinositol 3-kinase (PI3K) signalling regulates insulin-like-growth factor binding protein-2(IGFBP-2) production in human adipocytes[J]. Growth Horm IGF Res, 2015, 25(3):115-120.

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The regulation of PTB-associated splicing factor on phosphatidylinositol 3 kinase/Akt signaling pathway in retinal pigment epithelial cells

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