The role of geranylgeranylated proteins in human mesangial cell proliferation.
The Rho family of guanine 5'-triphosphatases (GTPases) play a key role in regulating cell proliferation, tubulointerstitial fibrosis, and glomerular hemodynamics. The post-translational prenylation of RhoGTPases by the addition of a geranylgeranyl moiety is critical for cellular localization and signaling activity. This study investigates the effects of (i) inhibiting geranylgeranylation (GG) in human mesangial cell (HMC) proliferation and apoptosis, using GGTI 298, a specific inhibitor of GG and (ii) lovastatin, an HMG-coacetyl A-reductase inhibitor, which depletes the availability of prenylation substrates. HMC proliferation was assessed using an assay of viable cell number and measuring bromodeoxyuridine (BrdU) incorporation. Hoechst 33342 staining was used to determine apoptosis. Extracellular signal-regulated protein kinase (Erk)1/2 and Akt activation were analysed by Western blotting. Rho activation was determined using the Rhotekin pull-down assay. Immunocytochemistry was performed to study the effects on the actin cytoskeleton and RhoA localization. GGTI 298 (10-20 muM) and lovastatin (5-10 muM) potently inhibited platelet-derived growth factor and serum-stimulated HMC proliferation and induced apoptosis. These effects of lovastatin were attenuated by co-incubation with geranylgeranylpyrophosphate. C3 exoenzyme, a clostridial toxin that specifically targets Rho also inhibited BrdU incorporation and promoted apoptosis. GGTI 298 increased cytosolic expression of RhoA, prevented RhoA activation, and inhibited the activation of Erk1/2 and the survival protein Akt. GGTI 298, lovastatin, and C3 exoenzyme inhibit HMC proliferation and promote apoptosis. Inhibiting GG increases cytosolic RhoA expression, disrupts the actin cytoskeleton, and inhibits RhoA activation. These results suggest that targeting geranylgeranylated proteins with statins or GGTI 298 is a promising therapeutic strategy in human mesangioproliferative renal disease.
Khwaja
A
A
Department of Renal Medicine, GKT School of Medicine, King's College London, Bessemer Road, London, UK.
Sharpe
C C
CC
Noor
M
M
Hendry
B M
BM
D019883:Alkyl and Aryl Transferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017209:Apoptosis / Q000187:drug effects; D001549:Benzamides / Q000494:pharmacology*; D015153:Blotting, Western; D001973:Bromodeoxyuridine / Q000378:metabolism; D049109:Cell Proliferation; D002478:Cells, Cultured; D003470:Culture Media; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors / Q000494:pharmacology*; D007150:Immunohistochemistry; D008148:Lovastatin / Q000494:pharmacology*; D050527:Mesangial Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D017368:Protein Prenylation; D015398:Signal Transduction; D013194:Staining and Labeling; D020741:rho GTP-Binding Proteins / Q000378:metabolism* / Q000502:physiology
D001549:Benzamides; D003470:Culture Media; C102521:GGTI 298; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D008148:Lovastatin; D019883:Alkyl and Aryl Transferases; C083291:geranylgeranyltransferase type-I; D020741:rho GTP-Binding Proteins; D001973:Bromodeoxyuridine