The involvement of the rho-kinase pathway and its regulation in cytokine-induced collagen gel contraction by hyalocytes.
OBJECTIVE
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-beta2. 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 alpha-smooth muscle actin (alphaSMA) 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-beta2, whose effect was time dependent, at least up to 5 days. Although transient and maximum MLC phosphorylation by PDGF-BB was observed at approximately 4 hours after stimulation (180.8%, P <0.01), TGF-beta2-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 microM) prohibited the phosphorylation of MLC, and inhibited collagen gel contraction at a concentration corresponding to that which inhibited MLC phosphorylation. TGF-beta2, but not PDGF-BB, also caused myofibroblast-like transdifferentiation with alphaSMA 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-beta2. Whereas PDGF-BB initiates collagen gel contraction by transient activation of the Rho-kinase pathway, sustained activation of the Rho-kinase pathway and myofibroblast-like transdifferentiation appears to be involved in the TGF-beta2-dependent contractile properties of hyalocytes.
Hirayama
Kumiko
K
Departments of Ophthalmology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
Hata
Yasuaki
Y
Noda
Yoshihiro
Y
Miura
Muneki
M
Yamanaka
Ichiro
I
Shimokawa
Hiroaki
H
Ishibashi
Tatsuro
T
D000199:Actins / Q000378:metabolism; D000818:Animals; D000077214:Becaplermin; D015153:Blotting, Western; D002417:Cattle; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005782:Gels; D047908:Intracellular Signaling Peptides and Proteins; D008264:Macrophages / Q000187:drug effects* / Q000378:metabolism; D018994:Myosin Light Chains / Q000378:metabolism; D010766:Phosphorylation; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020574:Proto-Oncogene Proteins c-sis; D011994:Recombinant Proteins / Q000494:pharmacology; D015398:Signal Transduction; D013997:Time Factors; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D053781:Transforming Growth Factor beta2; D014822:Vitreous Body / Q000166:cytology*; D054460:rho-Associated Kinases
D000199:Actins; D004791:Enzyme Inhibitors; D005782:Gels; D047908:Intracellular Signaling Peptides and Proteins; D018994:Myosin Light Chains; D010982:Platelet-Derived Growth Factor; D020574:Proto-Oncogene Proteins c-sis; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; D053781:Transforming Growth Factor beta2; D000077214:Becaplermin; D003094:Collagen; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases