The effect of Rac1 inhibition to retinal pigment epithelial cellular behavior change induced by transforming growth factorβ_Chinese Journal of Ocular Fundus Diseases

Authors：

MaHaizhi ,  ZhangShaochong , HuangXionggao , 魏雁涛 , 蒋欣桐

State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center of Sun Yat-sen University, Guangzhou 510060, China;

Corresponding author：

ZhangShaochong, Email: zhshaochong@163.com

Keywords：

Retinal pigment epithelium; Cell transdifferentiation; Transforming growth factor beta; rac1 GTP-binding protein

DOI：

10.3760/cma.j.issn.1005-1015.2015.01.014

Video：

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Abstract Full text Figures/Tables Video References Cited by

Objective To study the role of Rac1 in the epithelial-mesenchymal transition (EMT) process of retinal pigment epithelial cells (RPE) induced by transforming growth factorβ(TGF-β). Methods Human ARPE-19 cells were divided into 4 groups including control group, TGF-βgroup, TGF-β+NSC23766 group, NSC23766 group. NSC23766 was added to medium 2 hours before TGF-βtreatment to block the Rac1 receptors.α-smooth muscle actin (α-SMA) expression was measured by immunofluorescence and Western blot. Cell scratch assay, invasion assay and gel contraction experiments were used to measure cell migration, invasion, cell contraction. Results The expression ofα-SM A was higher in TGF-βgroup, compared with the control group, TGF-β+NSC23766 group (F=825.314, P < 0.05). Cell scratch assay showed that the cellular gap was less in GF-βgroup, compared with the control group, TGF-β+NSC23766 group, NSC23766 group (F=177.351, P < 0.05). Cell invasion assay showed that, the number of cells pass through the fiber membrane was the same in TGF-βgroup and other 3 groups (F=0.371, P=0.055). Gel contraction assay showed that TGF-βcan promote the cellular contraction, compare to the control group, TGF-β+NSC23766 group, NSC23766 group, the difference was statistically significant (F=40.473, P < 0.05). Conclusion Rac1 play a role in TGF-β-induced behavioral changes of RPE cells; NSC23766 inhibit RPE cellular behavior change by regulating Rac1 activation.

Citation： MaHaizhi, ZhangShaochong, HuangXionggao. The effect of Rac1 inhibition to retinal pigment epithelial cellular behavior change induced by transforming growth factorβ. Chinese Journal of Ocular Fundus Diseases, 2015, 31(1): 52-56. doi: 10.3760/cma.j.issn.1005-1015.2015.01.014 Copy

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2.	Pastor JC, de la Rúa ER, Martín F, et al.Proliferative vitreoretinopathy:risk factors and pathobiology[J]. Prog Retin Eye Res, 2002, 21(1):127-144.
3.	Zhu J, Nguyen D, Ouyang H, et al.Inhibition of RhoA/Rho-kinase pathway suppresses the expression of extracellular matrix induced by CTGF or TGF-βin ARPE-19[J].Int J Ophthalmol, 2013, 6(1):8-14.
4.	Hirayama K, Hata Y, Noda Y, et al. The involvement of the rho-kinase pathway and its regulation in cytokine-induced collagen gel contraction by hyalocytes[J].Invest Ophthalmol Vis Sci, 2004, 45(11):3896-3903.
5.	Saika S. TGFbeta pathobiology in the eye[J].Lab Invest, 2006, 86(2):106-115.
6.	Nassar K, Grisanti S, Tura A, et al. A TGF-βreceptor 1 inhibitor for prevention of proliferative vitreoretinopathy[J]. Exp Eye Res, 2014, 123:72-86.
7.	Hoerster R, Hermann MM, Rosentreter A, et al.Profibrotic cytokines in aqueous humour correlate with aqueous flare in patients with rhegmatogenous retinal detachment[J]. Br J Ophthalmol, 2013, 97(4):450-453.
8.	闫慧娟, 勿呢尔, 莫日根, 等. Rho小G蛋白相关激酶的结构与功能Rho小G蛋白相关激酶的结构与功能[J].中国细胞生物学学报, 2014, 36(7):998-1008.
9.	Gao Y, Dickerson JB, Guo F, et al. Rational design and characterization of a Rac GTPase-specific small molecule inhibitor[J]. Proc Natl Acad Sci, 2004, 18(20):7618-7623.
10.	Cancelas J, Williams DA, Zheng J, et al. Rational design and applications of a Rac GTPase-specific small molecule inhibitor[J]. Methods Enzymol, 2006, 406:554-565.
11.	Lee J, Ko M, Joo CK, et al. Rho plays a key role in TGF-beta1-induced cytoskeletal rearrangement in human retinal pigment epithelium[J].J Cell Physiol, 2008, 216(2):520-526.
12.	黄雄高, 张少冲, 魏雁涛, 等.玻璃体液诱导视网膜色素上皮细胞间质转分化作用[J].中华跟底病杂志, 2013, 29(2):166-170.
13.	Connor TB, Roberts AB, Sporn MB, et al. Correlation of fibrosis and transforming growth factor-beta type 2 levels in the eye[J]. J Clin Invest, 1989, 83(5):1661-1666.
14.	Zhu Y, Liu Y, Qian Y, et al. Research on the efficacy of Celastrus Orbiculatus in suppressing TGF-β₁-induced epithelial-mesenchymal transition by inhibiting HSP27 and TNF-α-induced NF-κB/Snail signaling pathway in human gastric[J]. BMC Complement Altern Med, 2014, 14:433.
15.	Parapuram SK, Chang B, Li L, et al. Differential effects of TGFbeta and vitreous on the transformation of retinal pigment epithelial cells[J]. Invest Ophthalmol Vis Sci, 2009, 50(12):5965-5974.
16.	Kirchhof B, Kirchhof E, Ryan SJ, et al.Vitreous modulation ofmigration and proliferation of retinal pigment epithelial cells in vitro[J]. Invest Ophthalmol Vis Sci, 1989, 30(9):1951-1957.
17.	Huang XG, Chen YZ, Zhang ZT, et al.Racl modulates the vitreous-induced plasticity of mesenchymal movement in retinal pigment epithelial cells[J]. Clin Experiment Ophthalmol, 2013, 41(8):779-787.

1. Hoerster R, Muether PS, Vierkotten S, et al.Upregulation of TGF-β₁ in experimental proliferative vitreoretinopathy is accompanied by epithelial to mesenchymal transition[J]. Graefe's Arch Clin Exp Ophthalmol, 2014, 252(1):11-16.
2. Pastor JC, de la Rúa ER, Martín F, et al.Proliferative vitreoretinopathy:risk factors and pathobiology[J]. Prog Retin Eye Res, 2002, 21(1):127-144.
3. Zhu J, Nguyen D, Ouyang H, et al.Inhibition of RhoA/Rho-kinase pathway suppresses the expression of extracellular matrix induced by CTGF or TGF-βin ARPE-19[J].Int J Ophthalmol, 2013, 6(1):8-14.
4. Hirayama K, Hata Y, Noda Y, et al. The involvement of the rho-kinase pathway and its regulation in cytokine-induced collagen gel contraction by hyalocytes[J].Invest Ophthalmol Vis Sci, 2004, 45(11):3896-3903.
5. Saika S. TGFbeta pathobiology in the eye[J].Lab Invest, 2006, 86(2):106-115.
6. Nassar K, Grisanti S, Tura A, et al. A TGF-βreceptor 1 inhibitor for prevention of proliferative vitreoretinopathy[J]. Exp Eye Res, 2014, 123:72-86.
7. Hoerster R, Hermann MM, Rosentreter A, et al.Profibrotic cytokines in aqueous humour correlate with aqueous flare in patients with rhegmatogenous retinal detachment[J]. Br J Ophthalmol, 2013, 97(4):450-453.
8. 闫慧娟, 勿呢尔, 莫日根, 等. Rho小G蛋白相关激酶的结构与功能Rho小G蛋白相关激酶的结构与功能[J].中国细胞生物学学报, 2014, 36(7):998-1008.
9. Gao Y, Dickerson JB, Guo F, et al. Rational design and characterization of a Rac GTPase-specific small molecule inhibitor[J]. Proc Natl Acad Sci, 2004, 18(20):7618-7623.
10. Cancelas J, Williams DA, Zheng J, et al. Rational design and applications of a Rac GTPase-specific small molecule inhibitor[J]. Methods Enzymol, 2006, 406:554-565.
11. Lee J, Ko M, Joo CK, et al. Rho plays a key role in TGF-beta1-induced cytoskeletal rearrangement in human retinal pigment epithelium[J].J Cell Physiol, 2008, 216(2):520-526.
12. 黄雄高, 张少冲, 魏雁涛, 等.玻璃体液诱导视网膜色素上皮细胞间质转分化作用[J].中华跟底病杂志, 2013, 29(2):166-170.
13. Connor TB, Roberts AB, Sporn MB, et al. Correlation of fibrosis and transforming growth factor-beta type 2 levels in the eye[J]. J Clin Invest, 1989, 83(5):1661-1666.
14. Zhu Y, Liu Y, Qian Y, et al. Research on the efficacy of Celastrus Orbiculatus in suppressing TGF-β₁-induced epithelial-mesenchymal transition by inhibiting HSP27 and TNF-α-induced NF-κB/Snail signaling pathway in human gastric[J]. BMC Complement Altern Med, 2014, 14:433.
15. Parapuram SK, Chang B, Li L, et al. Differential effects of TGFbeta and vitreous on the transformation of retinal pigment epithelial cells[J]. Invest Ophthalmol Vis Sci, 2009, 50(12):5965-5974.
16. Kirchhof B, Kirchhof E, Ryan SJ, et al.Vitreous modulation ofmigration and proliferation of retinal pigment epithelial cells in vitro[J]. Invest Ophthalmol Vis Sci, 1989, 30(9):1951-1957.
17. Huang XG, Chen YZ, Zhang ZT, et al.Racl modulates the vitreous-induced plasticity of mesenchymal movement in retinal pigment epithelial cells[J]. Clin Experiment Ophthalmol, 2013, 41(8):779-787.

Chinese Journal of Ocular Fundus Diseases

The effect of Rac1 inhibition to retinal pigment epithelial cellular behavior change induced by transforming growth factorβ

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