Transcript No Slide Title

Geranylgeranylation Limits Rho G-Protein Expression
in Human Trabecular Meshwork Cells
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2
C.L. Von Zee and E.B. Stubbs, Jr.
Departments of 1Cell Biology, Neurobiology, and Anatomy and 2Ophthalmology, Loyola University Chicago, Maywood,
IL; Department of Veterans Affairs, Edward Hines, Jr. VA Hospital, Hines, IL
RhoB
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Vehicle
Lovastatin
L + FPP
L + GGPP
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Vehicle
Lovastatin
FTI-277
GGTI-298
Relative Fold-change
(Normalized to GAPDH)
Relative Fold-change
(Normalized to GAPDH)
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0.25
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Lovastatin
FTI-277
F
GGTI-298
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Figure 6. Geranylgeranyl pyrophosphate facilitates restoration of F-actin stress fiber
organization. Confocal microscopy images of F-actin stress fibers. GTM3 cells were pretreated as described in Fig. 4. (A) Vehicle (0.01% ethanol) or (B-F) lovastatin (10 µM) pre-treated
cells were washed once and cultured in media supplemented with GGPP (10 µM) for 0h (B), 1h (C),
2h (D), 4h (E), or 6h (F). F-actin was visualized using AlexaFluor488-conjugated phalloidin. Data
shown are representative images (n = 2-3 cultures).
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10 12 14 16 18 20 22 24
Time (h)
Base
Base
GAPDH
RhoA
GAPDH
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Lovastatin +
GGPP
Lovastatin
Lovastatin +
GGPP Lovastatin
RhoA
CHX (h):
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 Lovastatin does not alter the stability of RhoA or RhoB mRNA
GAPDH
 Geranylgeranyl pyrophosphate markedly destabilizes RhoA and RhoB proteins
RhoB
 Geranylgeranyl pyrophosphate selectively facilitates subcellular re-distribution of RhoA and RhoB
proteins
GAPDH
 Geranylgeranyl pyrophosphate restores expression of GTP-bound (active) RhoA
RhoB
 Geranylgeranyl pyrophosphate facilitates F-actin stress fiber re-organization
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CONCLUSIONS
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Cytosol
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Membrane
Filamentous Actin Staining: GTM3 cells grown to confluence on chambered coverslips were treated as described above,
fixed (buffered 4% paraformaldehyde), and filamentous actin was stained with AlexaFluor488-conjugated phalloidin.
Stained cells were visualized by confocal microscopy.
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Geranylgeranyl pyrophosphate selectively facilitates restoration of Rho membrane localization and
signaling.
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RhoB
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GAPDH
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REFERENCES
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1. Liao JK. Isoprenoids as Mediators of the Biological Effects of Statins. J Clin Invest 2002;110(3):285288.
0.75
RhoA
Figure 4. Geranylgeranyl pyrophosphate facilitates subcellular redistribution of Rho proteins. Immunoblots of RhoA or RhoB
proteins. GTM3 cell (n=3) were pre-treated for 24h with vehicle (0.01%
ethanol, V) or lovastatin (10 μM, time 0). Pre-treated cells were washed
to remove residual statin and cultured in media supplemented with
GGPP (10 μM, upper panel) or FPP + GGTI-298 (10 μM, lower panel)
for the times as indicated.
Enhancement of Rho G-protein expression by lovastatin or GGTI-298 likely occurs as a compensatory
response to disruption of endogenous geranylgeranylation and Rho-dependent signaling.
RhoA
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O.D. (490 nm)
GAPDH
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Statin-dependent inhibition of geranylgeranylation elicits marked increases in Rho G-protein content in
human TM cells through enhanced mRNA synthesis and protein stability.
Membrane
RhoA
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 Inhibition of protein geranylgeranylation with lovastatin or GGTI-298 enhances RhoA and RhoB mRNA
expression in human GTM3 cells
RhoB
Figure 3. Geranylgeranyl pyrophosphate destabilizes RhoA and RhoB protein stability. Immunoblots of RhoA or RhoB
proteins. GTM3 cells (n=2 per time point) were pre-treated for 24h with lovastatin (10 μM, time 0). Control cells were washed and
treated with vehicle (0.01% ethanol, base) or remained in lovastatin-containing media (lovastatin) without or with GGPP (10 μM)
supplementation. Treated cells were incubated for an additional 0-24h as indicated in the presence of cycloheximide (CHX, 1.4 μg/ml).
Proteins (20 µg per lane) remaining in cell lysates were resolved by SDS-PAGE and immunostained for the presence of RhoA (left
panel) or RhoB (right panel). Levels of GAPDH, a housekeeping enzyme, are shown for comparison as loading controls.
Cytosol
SUMMARY
GAPDH
RhoB
ACKNOWLEDGEMENTS: This work was supported, in part, by grants from the Department of
Veterans Affairs (RR&D C3638R, EBS, Jr.), RR&D VA pre-doctoral fellowship (CVZ), Illinois Society
for the Prevention of Blindness, and the Richard A. Peritt Charitable Foundation.
Vehicle
GAPDH
Withdrawal (h):
E
10
1.00
10 12 14 16 18 20 22 24
Time (h)
RhoA
Withdrawal (h):
D
20
L + GGPP
0.00
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Subcellular Fractionation: Lysates of treated TM cells were centrifuged at 100,000g x 60 min and the resulting
supernatant (soluble cytosolic fraction) was collected and stored at -80°C until use. Membrane pellets were solubilized by
gentle homogenization in Triton X-100 containing buffer and centrifuged at 15,000g x 60 min to obtain a clarified
supernatant. The resulting membrane soluble fraction was stored at -80°C until use. Protein concentrations in cell lysates
and prepared subcellular fractions were measured by the BCA method, using bovine serum albumin as a standard.
Rho Activation Assay: A commercially available G-LISA™ activation assay kit was used according to package
instructions. GTM3 cell lysates were incubated as described above. Results were quantified spectrophotometrically at 490
nm.
L + FPP
Figure 2. Lovastatin does not alter the half-life of expressed RhoA or RhoB mRNA. GAPDH-normalized fold changes in RhoA
or RhoB mRNA stability. GTM3 cells (n=2 per time point) were pre-treated for 24h with lovastatin (10 µM). Control cells were washed
and treated with vehicle (0.01% ethanol, closed symbol) or remained in lovastatin-containing media (open symbol). Treated cells were
incubated for an additional 0-24h as indicated in the presence of actinomycin D (0.5 µg/ml). Fold-decreases in mRNA content
compared with baseline (time 0) are shown.
0
C
RhoB
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0
CHX (h):
B
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Rho mRNA or Protein Stability: GTM3 cells were pre-treated with lovastatin (10 mM) for 24h. Actinomycin D (0.5 µg/ml;
mRNA stability) or cycloheximide (CHX, 1.4 µg/ml; protein stability) was added to pre-treated cultures and cells incubated
for an additional 0-24h. In control experiments, lovastatin pre-treated cells were washed once to remove residual
lovastatin. Total RNA was extracted from treated TM cell cultures and 5 µg reverse-transcribed. Forward and reverse
primer sequences specific for RhoA, RhoB, or GAPDH (reference control) were used. Sequences were amplified (iQ SYBR
Green Supermix) using a Mini-Opticon qPCR detection system. The specificity of the reaction product was determined by
melting curve analysis. For determination of Rho protein stability, proteins (20 µg per lane) from lysates were analyzed by
Western immunoblot (1:200-10,000 dilution of mouse anti-RhoA, rabbit anti-RhoB, or rabbit anti-GAPDH primary antibody;
1:2,500-10,000 dilution of peroxidase-conjugated goat anti-mouse or anti-rabbit secondary antibody). Immunostained
proteins were visualized by enhanced chemiluminescence (ECL).
Lovastatin
RhoA
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METHODS
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Vehicle
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In this study, we determined the mechanism by which post-translational geranylgeranylation limits
expression of Rho G-proteins in human TM cells. The functional consequence of lovastatindependent inhibition of geranylgeranylation is discussed.
Drug Treatments: Prior to use, lovastatin pro-drug was chemically activated by alkaline hydrolysis. Confluent cultures
were treated with vehicle (0.01% ethanol) or activated lovastatin (10 µM) for 24h. In some cases, GTM3 cells were
incubated in the presence of lovastatin and farnesyl pyrophosphate or geranylgeranyl pyrophosphate, inhibitors of farnesyl
transferase or geranylgeranyl transferase-I.
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Figure 1. Inhibition of protein geranylgeranylation increases RhoA and RhoB mRNA expression in GTM3 cells. GAPDHnormalized fold changes in RhoA or RhoB mRNA content. GTM3 cells (n=3 per treatment) were incubated for 24h with vehicle
(0.01% ethanol), and equal concentrations (10 µM) of the following: lovastatin, lovastatin plus farnesyl-PP (L + FPP) or geranylgeranylPP (L + GGPP), farnesyl transferase inhibitor (FTI-277), or geranylgeranyl transferase inhibitor (GGTI-298) as indicated. Data shown
are mean ± SD. *, p < 0.05; **, p < 0.01 (one-way ANOVA with Dunnett's post-hoc analysis).
Recently, we have reported that inhibition of Rho geranylgeranylation limits Rho-dependent
signaling, and concurrently enhances RhoA and RhoB mRNA and protein content in human TM
cells.2 However, the specific mechanism of how this occurs remains unknown.
Cell Culture: SV40-transformed human TM cells from a male glaucomatous patient (GTM3) were previously established
and provided by Alcon Laboratories. Cell lines were grown to confluence in Dulbecco's Modified Eagle's Medium (DMEM)
containing L-glutamine (GlutaMAX-I), 10% FBS, 100 U penicillin and 0.1 mg streptomycin at 37°C under an atmosphere of
5% CO2/95% air.
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Relative Fold-change
(Normalized to GAPDH)
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4.0
3.5
Relative Fold-change
(Normalized to GAPDH)
Statins have been shown to inhibit downstream Rho signaling, including actin stress fiber formation,
in human TM cells or porcine anterior segments2,3 resulting in increased outflow in vitro.3
Pharmacologic inhibition of geranylgeranylation with specific geranylgeranyl transferase-I inhibitors
similarly results in actin stress fiber depolymerization2 and enhanced anterior segment outflow.4 A
preliminary clinical trial suggests that long-term statin use may offer a protective effect in
glaucoma.5
RhoA
**
4.0
Relative Fold-change
(Normalized to GAPDH)
Increased contractility of trabecular meshwork (TM) cells can chronically elevate IOP, contributing to
the pathogenesis of blinding diseases such as primary open-angle glaucoma. Activation of the Rho
subfamily of GTP-binding proteins, including RhoA and RhoB, is associated with enhanced actin
stress fiber polymerization and increased cellular contractility. Statins, a group of potent cholesterollowering drugs, function as indirect Rho kinase inhibitors through inhibition of Rho isoprenylation.1
RESULTS
Relative Fold-change
(Normalized to GAPDH)
INTRODUCTION
**
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0
1
2
Withdrawal time (h)
4
6
Figure 5. Geranylgeranyl pyrophosphate facilitates
restoration of GTP-bound (active) RhoA content. GLISA quantification of GTP-bound RhoA. GTM3 cell
cultures (n=3) were pre-treated as described in Fig. 4,
washed and subsequently cultured in media
supplemented with GGPP (10 μM) for the times
indicated. Data shown are expressed as the mean ± SD
from a single experiment performed in triplicate. **, p <
0.01 compared with vehicle control (one-way ANOVA
with Dunnett’s post-hoc analysis)
2. Von Zee CL, Richards MP, Bu P, Perlman JI, and Stubbs, Jr. EB. Lovastatin Increases RhoA and
RhoB Protein Accumulation in Cultured Human Trabecular Meshwork Cells. Invest. Ophthal. Vis.
Sci. (In press, 2009).
3. Song J, Deng PF, Stinnett SS, Epstein DL, Rao PV. Effects of Cholesterol-Lowering Statins on the
Aqueous Humor Outflow Pathway. Invest Ophthal. Vis. Sci. 2005;46(7):2424-2432.
4. Rao PV, Peterson YK, Inoue T, Casey PJ. Effects of Pharmacologic Inhibition of Protein
Geranylgeranyl Transferase Type I on Aqueous Humor Outflow Through the Trabecular Meshwork.
Invest. Ophthal. Vis. Sci. 2008;49(6):2464-71.
5. McGwin G, McNeal S, Owsley C, Girkin C, Epstein DL, Lee PP. Statins and Other CholesterolLowering Medications and the Presence of Glaucoma. Arch Ophthalmol 2004;122(6):822-826.