The involvement of the Rho-kinase pathway and its regulation in cytokine-induced collagen gel contraction by hyalocytes

Kumiko Hirayama, Yasuaki Hata, Yoshihiro Noda, Muneki Miura, Ichiro Yamanaka, Hiroaki Shimokawa, Tatsuro Ishibashi

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Abstract

PURPOSE. To investigate the involvement of the Rho-kinase pathway in collagen gel contraction by hyalocytes. METHODS. An in vitro type I collagen gel contraction assay using cultured bovine hyalocytes was performed to evaluate the effect of PDGF-BB and TGF-β2. The effect of both cytokines on the phosphorylation of myosin light chain (MLC) was analyzed by Western blot analysis. To confirm the involvement of the Rho-kinase pathway in the collagen gel contraction, the effects of Y27632, a specific Rho-kinase inhibitor were examined. The effect of hydroxyfasudil, another potent Rho-kinase inhibitor, was also evaluated. The expression of α-smooth muscle actin (αSMA) was analyzed by Western blot analysis to examine the myofibroblast-like transdifferentiation of the hyalocytes. RESULTS. Maximum collagen gel contraction was observed within 24 hours after treatment with PDGF-BB and much stronger contraction with TGF-β2, whose effect was time dependent, at least up to 5 days. Although transient and maximum MLC phosphorylation by PDGF-BB was observed at ∼4 hours after stimulation (180.8%, P < 0.01), TGF-β2-elicited MLC phosphorylation occurred in a time-dependent manner at least up to 24 hours (220.0%, P < 0.01) and was maintained up to 5 days. Y27632 demonstrated significant inhibition of collagen gel contraction induced by both cytokines. Hydroxyfasudil dose-dependently (0.03-20.00 μM) prohibited the phosphorylation of MLC, and inhibited collagen gel contraction at a concentration corresponding to that which inhibited MLC phosphorylation. TGF-β2, but not PDGF-BB, also caused myofibroblast-like transdifferentiation with αSMA overexpression, which was downregulated by hydroxyfasudil in part (P < 0.01). CONCLUSIONS. The hyalocytes have a contractile property in the presence of PDGF-BB and TGF-β2. Whereas PDGF-BB initiates collagen gel contraction by transient activation of the Rho-kinase pathway, sustained activation of the Rhokinase pathway and myofibroblast-like transdifferentiation appears to be involved in the TGF-β2-dependent contractile properties of hyalocytes.

Original languageEnglish
Pages (from-to)3896-3903
Number of pages8
JournalInvestigative Ophthalmology and Visual Science
Volume45
Issue number11
DOIs
Publication statusPublished - Nov 1 2004

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rho-Associated Kinases
Collagen
Gels
Myosin Light Chains
Cytokines
Phosphorylation
Myofibroblasts
Smooth Muscle
Actins
Western Blotting
Collagen Type I
platelet-derived growth factor BB
Down-Regulation

All Science Journal Classification (ASJC) codes

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

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The involvement of the Rho-kinase pathway and its regulation in cytokine-induced collagen gel contraction by hyalocytes. / Hirayama, Kumiko; Hata, Yasuaki; Noda, Yoshihiro; Miura, Muneki; Yamanaka, Ichiro; Shimokawa, Hiroaki; Ishibashi, Tatsuro.

In: Investigative Ophthalmology and Visual Science, Vol. 45, No. 11, 01.11.2004, p. 3896-3903.

Research output: Contribution to journalArticle

Hirayama, Kumiko ; Hata, Yasuaki ; Noda, Yoshihiro ; Miura, Muneki ; Yamanaka, Ichiro ; Shimokawa, Hiroaki ; Ishibashi, Tatsuro. / The involvement of the Rho-kinase pathway and its regulation in cytokine-induced collagen gel contraction by hyalocytes. In: Investigative Ophthalmology and Visual Science. 2004 ; Vol. 45, No. 11. pp. 3896-3903.
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abstract = "PURPOSE. To investigate the involvement of the Rho-kinase pathway in collagen gel contraction by hyalocytes. METHODS. An in vitro type I collagen gel contraction assay using cultured bovine hyalocytes was performed to evaluate the effect of PDGF-BB and TGF-β2. The effect of both cytokines on the phosphorylation of myosin light chain (MLC) was analyzed by Western blot analysis. To confirm the involvement of the Rho-kinase pathway in the collagen gel contraction, the effects of Y27632, a specific Rho-kinase inhibitor were examined. The effect of hydroxyfasudil, another potent Rho-kinase inhibitor, was also evaluated. The expression of α-smooth muscle actin (αSMA) was analyzed by Western blot analysis to examine the myofibroblast-like transdifferentiation of the hyalocytes. RESULTS. Maximum collagen gel contraction was observed within 24 hours after treatment with PDGF-BB and much stronger contraction with TGF-β2, whose effect was time dependent, at least up to 5 days. Although transient and maximum MLC phosphorylation by PDGF-BB was observed at ∼4 hours after stimulation (180.8{\%}, P < 0.01), TGF-β2-elicited MLC phosphorylation occurred in a time-dependent manner at least up to 24 hours (220.0{\%}, P < 0.01) and was maintained up to 5 days. Y27632 demonstrated significant inhibition of collagen gel contraction induced by both cytokines. Hydroxyfasudil dose-dependently (0.03-20.00 μM) prohibited the phosphorylation of MLC, and inhibited collagen gel contraction at a concentration corresponding to that which inhibited MLC phosphorylation. TGF-β2, but not PDGF-BB, also caused myofibroblast-like transdifferentiation with αSMA overexpression, which was downregulated by hydroxyfasudil in part (P < 0.01). CONCLUSIONS. The hyalocytes have a contractile property in the presence of PDGF-BB and TGF-β2. Whereas PDGF-BB initiates collagen gel contraction by transient activation of the Rho-kinase pathway, sustained activation of the Rhokinase pathway and myofibroblast-like transdifferentiation appears to be involved in the TGF-β2-dependent contractile properties of hyalocytes.",
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T1 - The involvement of the Rho-kinase pathway and its regulation in cytokine-induced collagen gel contraction by hyalocytes

AU - Hirayama, Kumiko

AU - Hata, Yasuaki

AU - Noda, Yoshihiro

AU - Miura, Muneki

AU - Yamanaka, Ichiro

AU - Shimokawa, Hiroaki

AU - Ishibashi, Tatsuro

PY - 2004/11/1

Y1 - 2004/11/1

N2 - PURPOSE. To investigate the involvement of the Rho-kinase pathway in collagen gel contraction by hyalocytes. METHODS. An in vitro type I collagen gel contraction assay using cultured bovine hyalocytes was performed to evaluate the effect of PDGF-BB and TGF-β2. The effect of both cytokines on the phosphorylation of myosin light chain (MLC) was analyzed by Western blot analysis. To confirm the involvement of the Rho-kinase pathway in the collagen gel contraction, the effects of Y27632, a specific Rho-kinase inhibitor were examined. The effect of hydroxyfasudil, another potent Rho-kinase inhibitor, was also evaluated. The expression of α-smooth muscle actin (αSMA) was analyzed by Western blot analysis to examine the myofibroblast-like transdifferentiation of the hyalocytes. RESULTS. Maximum collagen gel contraction was observed within 24 hours after treatment with PDGF-BB and much stronger contraction with TGF-β2, whose effect was time dependent, at least up to 5 days. Although transient and maximum MLC phosphorylation by PDGF-BB was observed at ∼4 hours after stimulation (180.8%, P < 0.01), TGF-β2-elicited MLC phosphorylation occurred in a time-dependent manner at least up to 24 hours (220.0%, P < 0.01) and was maintained up to 5 days. Y27632 demonstrated significant inhibition of collagen gel contraction induced by both cytokines. Hydroxyfasudil dose-dependently (0.03-20.00 μM) prohibited the phosphorylation of MLC, and inhibited collagen gel contraction at a concentration corresponding to that which inhibited MLC phosphorylation. TGF-β2, but not PDGF-BB, also caused myofibroblast-like transdifferentiation with αSMA overexpression, which was downregulated by hydroxyfasudil in part (P < 0.01). CONCLUSIONS. The hyalocytes have a contractile property in the presence of PDGF-BB and TGF-β2. Whereas PDGF-BB initiates collagen gel contraction by transient activation of the Rho-kinase pathway, sustained activation of the Rhokinase pathway and myofibroblast-like transdifferentiation appears to be involved in the TGF-β2-dependent contractile properties of hyalocytes.

AB - PURPOSE. To investigate the involvement of the Rho-kinase pathway in collagen gel contraction by hyalocytes. METHODS. An in vitro type I collagen gel contraction assay using cultured bovine hyalocytes was performed to evaluate the effect of PDGF-BB and TGF-β2. The effect of both cytokines on the phosphorylation of myosin light chain (MLC) was analyzed by Western blot analysis. To confirm the involvement of the Rho-kinase pathway in the collagen gel contraction, the effects of Y27632, a specific Rho-kinase inhibitor were examined. The effect of hydroxyfasudil, another potent Rho-kinase inhibitor, was also evaluated. The expression of α-smooth muscle actin (αSMA) was analyzed by Western blot analysis to examine the myofibroblast-like transdifferentiation of the hyalocytes. RESULTS. Maximum collagen gel contraction was observed within 24 hours after treatment with PDGF-BB and much stronger contraction with TGF-β2, whose effect was time dependent, at least up to 5 days. Although transient and maximum MLC phosphorylation by PDGF-BB was observed at ∼4 hours after stimulation (180.8%, P < 0.01), TGF-β2-elicited MLC phosphorylation occurred in a time-dependent manner at least up to 24 hours (220.0%, P < 0.01) and was maintained up to 5 days. Y27632 demonstrated significant inhibition of collagen gel contraction induced by both cytokines. Hydroxyfasudil dose-dependently (0.03-20.00 μM) prohibited the phosphorylation of MLC, and inhibited collagen gel contraction at a concentration corresponding to that which inhibited MLC phosphorylation. TGF-β2, but not PDGF-BB, also caused myofibroblast-like transdifferentiation with αSMA overexpression, which was downregulated by hydroxyfasudil in part (P < 0.01). CONCLUSIONS. The hyalocytes have a contractile property in the presence of PDGF-BB and TGF-β2. Whereas PDGF-BB initiates collagen gel contraction by transient activation of the Rho-kinase pathway, sustained activation of the Rhokinase pathway and myofibroblast-like transdifferentiation appears to be involved in the TGF-β2-dependent contractile properties of hyalocytes.

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