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J. E. Chatterton, D. Sun, D. D. Donohue, D. L. Fleenor, I.-H. Pang, M. B. Wax, A. F. Clark; Effect of siRNA-Mediated ROCK Inhibition on TGFβ2-Induced PAI-1 Secretion. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3818.
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© ARVO (1962-2015); The Authors (2016-present)
Treatment with small molecule Rho kinase (ROCK) inhibitors increases aqueous humor outflow facility in organ culture, and lowers IOP in vivo, but also causes moderate to severe hyperemia. Most of the well-characterized ROCK inhibitors (e.g., H1152) affect both the ROCK1 and ROCK2 isoforms, as well as multiple other kinases. ROCK1- and ROCK2-specific cell-based assays are required for the development of isoform-specific ROCK inhibitors. We have used RNAi to further characterize one of these cell-based assays.
We used ROCK1- and ROCK2-specific siRNAs to disrupt secretion of plasminogen activator inhibitor-1 (PAI-1), a TGFβ2-induced response that is inhibited by H1152. We transfected GTM-3 cells with ROCK siRNAs, and challenged them at 48 h post-transfection with serum-withdrawal for 24 h followed by 5 ng/ml TGFβ2 for 24 h. Secreted PAI-1 levels were determined by ELISA; knockdown of ROCK1 and ROCK2 mRNA and protein were verified by qRT-PCR and western blot, respectively.
Transfection with the ROCK1 siRNA reduced ROCK1 mRNA and protein expression by 96 ± 4% and ~95%, respectively, but had essentially no effect on ROCK2. Similarly, transfection with the ROCK2 siRNA reduced ROCK2 mRNA and protein expression by 70 ± 4% and ~90%, respectively, but had little effect on ROCK1. Knockdown of ROCK1 decreased TGFβ2-induced PAI-1 secretion by 69 ± 16% relative to control siRNA transfected cells. In contrast, knockdown of ROCK2 had little, if any, effect. Co-transfection with both the ROCK1 and ROCK2 siRNAs inhibited expression of both targets, but caused no further reduction in TGFβ2-induced PAI-1 secretion relative to the ROCK1 siRNA alone. Surprisingly, addition of H1152 to ROCK1 siRNA-transfected cells reduced PAI-1 secretion by an additional 22 ± 6%, suggesting that at least a portion of the H1152 activity in this assay is independent of ROCK suppression.
TGFβ2-induced PAI-1 secretion appears to be ROCK1-dependent, but not ROCK2-dependent, reinforcing the non-redundancy of these highly similar kinases. This cell-based assay may be suitable for screening ROCK inhibitors for ROCK1 activity and for verifying the specificity of putative ROCK2-specific inhibitors.
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