Critical role of the Rho-kinase pathway in TGF-β2-dependent collagen gel contraction by retinal pigment epithelial cells

Muneki Miura, Yasuaki Hata, Kumiko Hirayama, Takeshi Kita, Yoshihiro Noda, Kimihiko Fujisawa, Hiroaki Shimokawa, Tatsuro Ishibashi

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Abstract

Retinal pigment epithelial cells (RPEs) are thought to be one of the main components of fibrous membrane observed in eyes with proliferative vitreo-retinopathy. We investigated the signalling mechanisms of TGF-β2-dependent collagen gel contraction by RPEs.An in vitro type I collagen gel contraction assay was performed to evaluate the effect of TGF-β2 on gel contraction. The expression of alpha-smooth muscle actin (α-SMA) and the phosphorylation state of myosin light chain (MLC) were analyzed by Western blotting. The involvement of protein kinases such as p44/42 mitogen-activated protein kinase (MAPK), protein kinase C (PKC), p38 MAPK and phosphatidylinositol-3 kinase was investigated. The contribution of Rho-kinase and/or MLC-kinase was also evaluated using respective kinase inhibitors (Y27632, hydroxyfasudil and ML7). Additionally, RPEs were immunostained to examine whether the expression of α-SMA detected in our western blotting correlated to the stress fiber formation within the cells. TGF-β2 caused time (0-5 days)-and dose (0 10 ng ml-1)-dependent gel contraction associated with overexpression of α-SMA and phosphorylation of MLC (p<0.01, respectively). PKC inhibitor (GF109203X, 5 μM) and p38 MAPK inhibitor (SB203580, 10 μM) significantly attenuated TGF-β2-elicited gel contraction via partial downregulation of both α-SMA expression and MLC phosphorylation (p<0.01, respectively). The gel contraction was prominently inhibited in the presence of Y27632 (10 μM) or hydroxyfasudil (10 μM) with strong suppression of MLC phosphorylation but had no significant effect on α-SMA expression. Treatment with ML7, in contrast, resulted in a marginal inhibition of MLC phosphorylation and gel contraction. Finally, pretreatment of the cells with Y27632 or hydroxyfasudil prevented the formation of stress fiber within the cells. These results indicate that TGF-β2-dependent myofibroblastic transdifferentiation and MLC phosphorylation by RPEs involve both PKC and p38 MAPK pathways at least in part. Myofibroblastic transdifferentiation of RPEs appears to be independent of the Rho-kinase pathway, and the presence of α-SMA does not necessarily reflect the contractile potential of a cell. While Rho-kinase inhibitors are incapable of preventing myofibroblastic transdifferentiation itself, this pathway could be one of the critical targets of cell-mediated contraction of the tissue containing fibrillar collagens by transdifferentiatied RPEs.

Original languageEnglish
Pages (from-to)849-859
Number of pages11
JournalExperimental Eye Research
Volume82
Issue number5
DOIs
Publication statusPublished - May 1 2006

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rho-Associated Kinases
Retinal Pigments
Myosin Light Chains
Collagen
Gels
Epithelial Cells
Phosphorylation
p38 Mitogen-Activated Protein Kinases
Protein Kinase C
Stress Fibers
Protein Kinase Inhibitors
Western Blotting
Phosphatidylinositol 3-Kinase
Fibrillar Collagens
Myosin-Light-Chain Kinase
Protein C Inhibitor
Mitogen-Activated Protein Kinase Kinases
Collagen Type I
Protein Kinases
Smooth Muscle

All Science Journal Classification (ASJC) codes

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

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Critical role of the Rho-kinase pathway in TGF-β2-dependent collagen gel contraction by retinal pigment epithelial cells. / Miura, Muneki; Hata, Yasuaki; Hirayama, Kumiko; Kita, Takeshi; Noda, Yoshihiro; Fujisawa, Kimihiko; Shimokawa, Hiroaki; Ishibashi, Tatsuro.

In: Experimental Eye Research, Vol. 82, No. 5, 01.05.2006, p. 849-859.

Research output: Contribution to journalArticle

Miura, Muneki ; Hata, Yasuaki ; Hirayama, Kumiko ; Kita, Takeshi ; Noda, Yoshihiro ; Fujisawa, Kimihiko ; Shimokawa, Hiroaki ; Ishibashi, Tatsuro. / Critical role of the Rho-kinase pathway in TGF-β2-dependent collagen gel contraction by retinal pigment epithelial cells. In: Experimental Eye Research. 2006 ; Vol. 82, No. 5. pp. 849-859.
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