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C. L. Von Zee, M. P. Richards, P. Bu, J. I. Perlman, E. B. Stubbs, Jr.; Lovastatin Increases Rho Expression in Human Trabecular Meshwork Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1628. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To determine the effect of lovastatin on expression of Rho G-proteins in human trabecular meshwork (TM) cells.
Primary human TM cells were harvested from discarded corneoscleral rims and cultured to confluency in serum-supplemented Eagle's MEM. SV40-transformed TM cells from a male glaucomatous patient (GTM3) were previously established (Alcon Laboratories) and cultured in serum-containing Dulbecco's MEM. At confluency, cells were cultured for 24h in the presence of vehicle (0.01% ethanol) or activated lovastatin (10 µM). Mevalonate, isoprenoid pyrophosphate intermediates, or transcriptional or translational inhibitors were used to determine mechanism of action. Rho mRNA- and protein-expression were quantitated by real-time RT-PCR and Western blotting, respectively. Stress fiber formation was determined using AlexaFluor488-conjugated phalloidin.
Lovastatin, compared with vehicle controls, elicited marked increases in RhoA (12-fold) and RhoB (54-fold) protein expression in GTM3 cells. Similarly, lovastatin enhanced expression of RhoA (51-fold) and RhoB (70-fold) proteins in primary human TM cells. Interestingly, in GTM3 cells lovastatin increased mRNA expression of RhoB (46-fold), but not RhoA. Actinomycin D, an inhibitor of gene transcription, prevented lovastatin increases in RhoB, but not RhoA, proteins. By comparison, inhibition of translation with cycloheximide prevented lovastatin increases in both RhoA and RhoB proteins. Supplementation of mevalonate or geranylgeranyl (GGPP), but not farnesyl, pyrophosphate prevented lovastatin increases in Rho proteins, implicating GGPP-specific isoprenylation as a mechanism of action. GTM3 cells treated with GGTI-298, a transferase inhibitor, showed increases in both RhoA and RhoB proteins. Lovastatin markedly altered GTM3 and primary TM cell morphology and decreased F-actin organization.
Lovastatin markedly increases Rho G-protein expression in transformed and primary human TM cultures. Selective changes by lovastatin in Rho protein isoform levels occur by differential mechanisms involving, in part, inhibition of GGPP-specific isoprenylation of Rho proteins.
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