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N. Sundarraj, J. Chen, E. Guerriero, P. Kinchington; Signaling Pathways in Downregulation of Keratan Sulfate Proteoglycans in Activated Corneal Keratocytes. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4541.
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To determine whether Rho GTPases or MAPKs regulate TGF-β1 or b-FGF- induced downregulation of KSPGs (lumican and keratocan) in corneal stromal keratocytes.
Keratocytes isolated from rabbit corneal stroma, plated in a serum free (SF) medium, were treated with b-FGF/heparin or TGF-β1 in the presence or absence of specific inhibitors of Rho, Rho kinase (ROCK) or MAPKs. In separate experiments keratocytes were infected with replication-defective inducible tet-off adenoviral vectors encoding cDNAs for dominant negative (DN) or constitutively active (CA) HA-tagged RhoA and expression of the recombinant proteins was induced during the activation of keratocytes with TGF-β1 or b-FGF. Specific phenotypic changes were analyzed by immunocytochemistry and western blotting, and the relative abundance of specific mRNAs was estimated by quantitative RT-PCR.
Immunocytochemical and western blot analyses indicated that in keratocytes activated with TGF-β1 and b-FGF, decreases in cell-associated and secreted KS were prevented by Rho or ROCK inhibition during the activation. While both TGF-β1 and b-FGF-induced downregulation of lumican mRNA levels was prevented by Rho and ROCK inhibition with C3 exoenzyme and Y27632, respectively, only b-FGF-induced downregulation of keratocan mRNA levels was prevented by Rho inhibition but not by ROCK-inhibition. Inhibition of Jun N-terminal kinase (JNK) with SP600125 during keratocyte activation prevented downregulation of lumican and keratocan mRNA. An overexpression of CA-RhoA during keratocyte activation had little effect on the downregulation of KSPGs. However, overexpression of DN-RhoA during TGF-β1 or b-FGF-induced activation prevented the downregulation of lumican.
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