Increased RhoA and RhoB protein accumulation in cultured human trabecular meshwork cells by lovastatin.
OBJECTIVE
This study aimed to determine the effect of lovastatin on Rho G-protein expression and activation in human trabecular meshwork (TM) cells.


METHODS
Confluent cultures of low-passage (primary) or transformed (GTM3) human TM cells were incubated overnight with vehicle (0.01% ethanol) or activated lovastatin (10 microM). Changes in Rho mRNA, protein content, and activation were quantified by qRT-PCR, immunoblotting, and ELISA, respectively. F-actin organization was determined using Alexa Fluor 488-conjugated phalloidin.


RESULTS
Low-passage or transformed TM cells treated with lovastatin exhibited marked increases in RhoA and RhoB mRNA and protein content. Actinomycin D prevented lovastatin-dependent increases in RhoB, but not RhoA, protein accumulation. In contrast, cycloheximide prevented lovastatin from increasing both RhoA and RhoB. Supplementation with mevalonate or geranylgeranyl pyrophosphate prevented, whereas inhibition of geranylgeranyl transferase mimicked, the effects of lovastatin on RhoA and RhoB accumulation. The effect of lovastatin was dose dependent, with newly synthesized protein accumulating in the cytosol. The amount of functionally active (GTP-bound) RhoA in cell lysates was significantly reduced by lovastatin. Lovastatin altered the morphology of TM cells by disrupting F-actin organization.


CONCLUSIONS
Lovastatin enhances the accumulation of RhoA and RhoB in human TM cells, in part, by limiting geranylgeranyl isoprenylation of these G-proteins. We propose that post-translational geranylgeranylation serves as a regulator of both RhoA and RhoB protein expression and processing in human TM cells. Increased accumulation of unprenylated forms of RhoA and RhoB may disrupt Rho-dependent regulation of TM cell cytoskeletal organization.
Von Zee
Cynthia L
CL
Department of Veterans Affairs, Edward Hines Jr VA Hospital, Hines, Illinois 60141, USA.
Richards
Michael P
MP
Bu
Ping
P
Perlman
Jay I
JI
Stubbs
Evan B
EB
D000199:Actins / Q000378:metabolism; D002461:Cell Line, Transformed; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D003609:Dactinomycin / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D019084:Fluorescent Antibody Technique, Indirect; D005786:Gene Expression Regulation / Q000502:physiology; D006801:Humans; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors / Q000494:pharmacology*; D015151:Immunoblotting; D008148:Lovastatin / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D011106:Polyisoprenyl Phosphates / Q000494:pharmacology; D054876:Prenylation / Q000187:drug effects; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013347:Subcellular Fractions; D014129:Trabecular Meshwork / Q000187:drug effects* / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*; D020743:rhoB GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
D000199:Actins; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D011106:Polyisoprenyl Phosphates; D012333:RNA, Messenger; C066737:RHOA protein, human; D003609:Dactinomycin; D003513:Cycloheximide; D008148:Lovastatin; D020742:rhoA GTP-Binding Protein; D020743:rhoB GTP-Binding Protein; C002963:geranylgeranyl pyrophosphate