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Graefe's Archive for Clinical and Experimental Ophthalmology

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Open Access 01-01-2012 | Basic Science

The PI3K/Akt pathway mediates the expression of type I collagen induced by TGF-β2 in human retinal pigment epithelial cells

Authors: Katsuhiko Yokoyama, Kenichi Kimoto, Yuji Itoh, Kazuo Nakatsuka, Noritaka Matsuo, Hidekatsu Yoshioka, Toshiaki Kubota

Published in: Graefe's Archive for Clinical and Experimental Ophthalmology | Issue 1/2012

Abstract

Purpose

Transforming growth factor (TGF)-β is a key mediator of proliferative vitreoretinopathy, but the cellular mechanisms by which TGF-β induces extracellular matrix protein (ECM) synthesis are not fully understood. This study examined whether the PI3K/Akt pathway is involved in TGF-β2-induced collagen expression in human retinal pigment epithelial cells.

Methods

Human retinal pigment epithelial cells ARPE-19 were cultured and stimulated with TGF-β2. The role of the PI3K/Akt pathway was evaluated using the biochemical inhibitor, wortmannin. The effect of wortmannin on the expression of type I collagen mRNA (COL1A1, COL1A2) induced by TGF-β2 was evaluated by real-time RT-PCR. The effect of wortmannin on the synthesis of type I collagen induced by TGF-β2 was assessed by an immunocytochemical analysis with anti-type I collagen antibody. Luciferase reporter assays were performed to examine the effect of wortmannin on the transcriptional activities of COL1A2. A luciferase assay using a mutation construct of the Smad binding site in COL1A2 promoter (Smad-mut/Luc) was also performed to examine the crosstalk between the Smad pathway and the PI3K/Akt pathway. The effects of wortmannin on the transcriptional activity of Smad3 were also examined using CAGA12-Luc. Moreover, the effect of wortmannin on TGF-β2-induced Smad7 mRNA expression was evaluated.

Results

The biochemical blockade of PI3K/Akt activation inhibited TGF-β2-induced type I collagen mRNA expression and type I collagen synthesis. The blockade of PI3K/Akt pathway inhibited the increase in COL1A2 promoter activities when induced by TGF-β2 and reduced TGF-β2 induction of Smad-mut/Luc promoter activity and CAGA12-Luc activity. Moreover, wortmannin increased the TGF-β2-induced Smad7 mRNA expression levels.

Conclusions

The PI3K/Akt pathway plays a role in relaying the TGF-β2 signal to induce type I collagen synthesis in the retinal pigment epithelium through Smad-dependent and Smad-independent pathways.

Machemer R (1988) Proliferative vitreoretinopathy (PVR): a personal account of its pathogenesis and treatment. Invest Ophthalmol Vis Sci 29:1771–1783PubMed

Pastor JC (1998) Proliferative vitreoretinopathy: an overview. Surv Ophthalmol 43:3–18PubMedCrossRef

Jerdan JA, Peose JS, Michels RG, Hayashi H, de Bustros S, Sebag M, Glaser BM (1989) Proliferative vitreoretinopathy membranes: an immunohistochemical study. Ophthalmology 96:801–810PubMed

Machemer R, Van Horn D, Aaberg TM (1978) Pigment epithelial proliferation in human retinal detachment with massive periretinal proliferation. Am J Ophthalmol 85:181–191PubMed

Scheiffarth OF, Kampik A, Günther H, von der Mark K (1988) Proteins of the extracellular matrix in vitreoretinal membranes. Graefes Arch Clin Exp Ophthalmol 226:357–361PubMedCrossRef

Hiscott P, Sheridan C, Magee RM, Grierson I (1999) Matrix and the retinal pigment epithelium in proliferative retinal disease. Prog Retin Eye Res 18:167–190PubMedCrossRef

Raymond MC, Thompson JT (1990) RPE-mediated collagen gel contraction. Inhibition by colchicine and stimulation by TGF-beta. Invest Ophthalmol Vis Sci 31:1079–1086PubMed

Grisanti S, Guidry C (1995) Transdifferentiation of retinal pigment epithelial cells from epithelial to mesenchymal phenotype. Invest Ophthalmol Vis Sci 36:391–405PubMed

Slack JL, Liska DJ, Bornstein P (1993) Regulation of expression of the type I collagen gene. Am J Med Genet 45:140–151PubMedCrossRef

10.

Border WA, Noble NA (1994) Transforming growth factor beta in tissue fibrosis. N Engl J Med 331:1286–1292PubMedCrossRef

11.

Saika S (2006) TGF-beta pathobiology in the eye. Lab Invest 86:106–115PubMedCrossRef

12.

Krupsky M, Fine A, Kuang PP, Berk JL, Goldstein RH (1996) Regulation of type I collagen production by insulin and transforming growth factor-beta in human lung fibroblasts. Connect Tissue Res 34:53–62PubMedCrossRef

13.

Liu X, Hu H, Yin JQ (2006) Therapeutic strategies against TGF-beta signaling pathway in hepatic fibrosis. Liver Int 26:8–22PubMedCrossRef

14.

Chen SJ, Yuan W, Mori Y, Levenson A, Trojanowska M, Varga J (1999) Stimulation of type I collagen transcription in human skin fibroblasts by TGF-beta: involvement of Smad3. J Invest Dermatol 112:49–57PubMedCrossRef

15.

Connor TB Jr, Roberts AB, Sporn MB, Danielpour D, Dart LL, Michels RG, de Bustros S, Enger C, Kato H, Lansing M, Hayashi H, Glaser BM (1989) Correlation of fibrosis and transforming growth factor-beta type 2 levels in the eye. J Clin Invest 83:1661–1666PubMedCrossRef

16.

Pfeffer BA, Flanders KC, Gúerin CJ, Danielpour D, Anderson DH (1994) Transforming growth factor-beta 2 is the predominant isoform in the neural retina, retinal pigment epithelium-choroid and vitreous of the monkey eye. Exp Eye Res 59:323–333PubMedCrossRef

17.

Cantley LC (2002) The Phosphoinositide 3-kinase pathway. Science 296:1655–1657PubMedCrossRef

18.

Runyan CE, Schnaper HW, Poncelet AC (2004) The phosphatidylinositol 3-kinase/Akt pathway enhances Smad3-stimulated mesangial cell collagen I expression in response to transforming growth factor-beta 1. J Biol Chem 279:2632–2639PubMedCrossRef

19.

Hubchak SC, Sparks EE, Hayashida T, Schnaper HW (2009) Rac1 promotes TGF-β-stimulated mesangial cell type I collagen expression through a PI3K/Akt-dependent mechanism. Am J Physiol Renal Physiol 297:F1316–F1323PubMedCrossRef

20.

Asano Y, Ihn H, Yamane K, Jinnin M, Mimura Y, Tamaki K (2004) Phosphatidylinositol 3-kinase is involved in alpha2(I) collagen gene expression in normal and scleroderma fibroblasts. J Immunol 172:7123–7135PubMed

21.

Yao K, Ye PP, Tang XJ, Shen Tu XC (2008) Involvement of PI3K/Akt pathway in TGF-β2-mediated epithelial mesenchymal transition in human lens epithelial cells. Ophthalmic Res 40:69–76PubMedCrossRef

22.

Kimoto K, Nakatsuka K, Matsuo N, Yoshioka H (2004) p38 MAPK mediates the expression of type I collagen induced by TGF-beta 2 in human retinal pigment epithelial cells ARPE-19. Invest Ophthalmol Vis Sci 45:2431–2437PubMedCrossRef

23.

Itoh Y, Kimoto K, Imaizumi M, Nakatsuka K (2007) Inhibition of RhoA/Rho-kinase pathway suppresses the expression of type I collagen induced by TGF-beta 2 in human retinal pigment epithelial cells. Exp Eye Res 84:464–472PubMedCrossRef

24.

Kita T, Hata Y, Arita R, Kawahara S, Miura M, Nakao S, Mochizuki Y, Enaida H, Goto Y, Shimokawa H, Hafezi-Moghadam A, Ishibashi T (2008) Role of TGF-beta in proliferative vitreoretinal diseases and ROCK as a therapeutic target. Proc Natl Acad Sci USA 45:17504–17509CrossRef

25.

Cassidy L, Barry P, Shaw C, Duffy J, Kennedy S (1998) Platelet derived growth factor and fibroblast growth factor basic levels in the vitreous of patients with vitreo-retinal disorders. Br J Ophthalmol 82:181–185PubMedCrossRef

26.

Kon CH, Occleston NL, Aylward GW, Khaw PT (1999) Expression of vitreous cytokines in proliferative vitreoretinopathy: a prospective study. Invest Ophthalmol Vis Sci 40:705–712PubMed

27.

Ignotz RA, Endo T, Massagué J (1987) Regulation of fibronectin and type I collagen mRNA levels by transforming growth factor-beta. J Biol Chem 262:6443–6446PubMed

28.

Derynck R, Zhang YE (2003) Smad-dependent and Smad-independent pathways in TGF-beta family signaling. Nature 425:577–584PubMedCrossRef

29.

Tsukada S, Westwick JK, Ikejima K, Sato N, Rippe RA (2005) SMAD and p38MAPK signaling pathways independently regulate alpha (I) collagen gene expression in unstimulated and transforming growth factor-beta-stimulated hepatic stellate cells. J Biol Chem 280:10055–10064PubMedCrossRef

30.

Poncelet AC, de Caestecker MP, Schnaper HW (1999) The transforming growth factor-beta/SMAD signaling pathway is present and functional in human mesangial cells. Kidney Int 56:1354–1365PubMedCrossRef

31.

Sato M, Shegogue D, Gore EA, Smith EA, McDermott PJ, Trojanowska M (2002) Role of p38 MAPK in transforming growth factor beta stimulation of collagen production by scleroderma and healthy dermal fibroblasts. J Invest Dermatol 118:704–711PubMedCrossRef

32.

Heldin CH, Miyazono K, Dijke P (1997) TGF-beta signaling from cell membrane to nucleus through Smad proteins. Nature 390:465–471PubMedCrossRef

33.

Derynck R, Zang Y, Feng XH (1998) Smad: transcriptional activators of TGF-beta responses. Cell 95:737–740PubMedCrossRef

34.

Nakao A, Afrakhte M, Morén A, Nakayama T, Christian JL, Heuchel R, Itoh S, Kawabata M, Heldin NE, Heldin CH, ten Dijke P (1997) Identification of Smad7, a TGFbeta-inducible antagonist of TGF-beta signaling. Nature 389:631–635PubMedCrossRef

35.

Wang B, Omar A, Angelovska T, Drobic V, Rattan SG, Jones SC, Dixon IM (2007) Regulation of collagen synthesis by inhibitory Smad7 in cardiac myofibroblasts. Am J Physiol Heart Circ Physiol 293:1282–1290CrossRef

36.

Saika S, Yamanaka O, Nishikawa-Ishida I, Kitano A, Flanders KC, Okada Y, Ohnishi Y, Nakajima Y, Ikeda K (2007) Effect of Smad7 gene overexpression on transforming growth factor beta-induced retinal pigment fibrosis in a proliferative vitreoretinopathy mouse model. Arch Ophthalmol 125:647–654PubMedCrossRef

37.

Bian ZM, Elner SG, Yoshida A, Elner VM (2004) Differential involvement of phosphoinositide 3-kinase/Akt in human RPE MCP-1 and IL-8 expression. Invest Ophthalmol Vis Sci 45:1887–1896PubMedCrossRef

38.

Bando H, Ikuno Y, Hori Y, Sayanagi K, Tano Y (2006) Mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3 kinase (PI3K) pathways differently regulate retinal pigment epithelial cell-mediated collagen gel contraction. Exp Eye Res 82:529–537PubMedCrossRef

39.

Cross MJ, Stewart A, Hodgkin MN, Kerr DJ, Wakelam MJ (1995) Wortmannin and its structural analogue demethoxyviridin inhibit stimulated phospholipase A2 activity in Swiss 3T3 cells. Wortmannin is not a specific inhibitor of phosphatidylinositol 3-kinase. J Biol Chem 270:25352–25355PubMedCrossRef

40.

Ferby I, Waga I, Kume K, Sakanaka C, Shimizu T (1996) PAF-induced MAPK activation is inhibited by wortmannin in neutrophils and macrophages. Adv Exp Med Biol 416:321–326PubMed

41.

Ferby IM, Waga I, Hoshino M, Kume K, Shimizu T (1996) Wortmannin inhibits mitogen-activated protein kinase activation by platelet-activating factor through a mechanism independent of p85/p110-type phosphatidylinositol 3-kinase. J Biol Chem 271:11684–11688PubMedCrossRef

Title: The PI3K/Akt pathway mediates the expression of type I collagen induced by TGF-β2 in human retinal pigment epithelial cells
Authors: Katsuhiko Yokoyama
Kenichi Kimoto
Yuji Itoh
Kazuo Nakatsuka
Noritaka Matsuo
Hidekatsu Yoshioka
Toshiaki Kubota
Publication date: 01-01-2012
Publisher: Springer-Verlag
Published in: Graefe's Archive for Clinical and Experimental Ophthalmology / Issue 1/2012
Print ISSN: 0721-832X
Electronic ISSN: 1435-702X
DOI: https://doi.org/10.1007/s00417-011-1766-x

Keynote webinar | Spotlight on medication adherence

Springer Medicine

The PI3K/Akt pathway mediates the expression of type I collagen induced by TGF-β2 in human retinal pigment epithelial cells

Abstract

Purpose

Methods

Results

Conclusions

Keynote webinar | Spotlight on medication adherence

Springer Medicine

Abstract

Purpose

Methods

Results

Conclusions

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