The role of geranylgeranylated proteins in human
mesangial cell proliferation
A Khwaja
1, CC Sharpe
1, M Noor
1and BM Hendry
11Department of Renal Medicine, GKT School of Medicine, King’s College London, Bessemer Road, London, UK
The Rho family of guanine 50-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 lM) and lovastatin (5–10 lM) 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.
Kidney International (2006) 70, 1296–1304. doi:10.1038/sj.ki.5001713; published online 23 August 2006
KEYWORDS: geranylgeranyl; Rho; mesangial; proliferation; apoptosis
Aberrant human mesangial cell (HMC) proliferation plays an important role in the pathogenesis and progression of glomerular injury and glomerulosclerosis.1 HMC prolifera-tion is a hallmark of both immune-mediated and non-immune-mediated glomerular injury,2 and strategies to inhibit MC proliferation have been shown to reduce mesangial matrix production.3–5 Therefore, targeting key signaling proteins that underlie HMC proliferation may lead to the development of more effective therapies for mesan-gioproliferative disease.
The intracellular signaling pathways of many mesangial mitogens converge upon the Ras and Rho superfamily of small monomeric guanine 50-triphosphatases (GTPases),6,7 which act as molecular switches, transducing extracellular proliferative signals from the plasma membrane to the cell nucleus. Work from our group has shown that targeting Ras inhibits renal fibroblast proliferation in vitro8,9 and reduces glomerular injury in the Thy 1.1 nephritis model of mesangial proliferation.10 Rho GTPases play a key role in regulating biological processes that require a coordinated rearrangement of the actin cytoskeleton such as cell contraction, cell migration, phagocytosis, and cytokinesis.11 In addition to their effects on the actin cytoskeleton, Rho proteins also regulate a variety of other pathways including gene transcription, cell proliferation, and cellular transformation.11,12
Within the Rho superfamily of proteins, Rho itself regulates the formation of stress fibers, whereas Rac has been shown to induce lamellipodia and Cdc42 is involved in the development of actin spikes or filopodia.13Rho exists in three main isoforms, namely RhoA, RhoB, and RhoC. The functional differences between these isoforms remain poorly understood. Once activated, Rho cycles from an inactive, guanine diphosphate-bound form to an active, GTP-bound form. GTP-bound Rho is then able to activate a number of downstream effector cascades.14Rho proteins are activated by guanine nucleotide exchange factors and inactivated by GTPase-activating proteins. Further regulation of Rho occurs through guanine nucleotide dissociation inhibitors, which sequester guanine diphosphate-bound Rho in the cytosol, thereby inactivating them.11Rho proteins undergo a series of post-translational modifications to enable them to localize to cellular membranes and function as signaling molecules. Prenylation is a key modification that involves the binding of
Received 9 August 2005; revised 3 March 2006; accepted 8 March 2006; published online 23 August 2006
Correspondence: BM Hendry, Department of Renal Medicine, KCL School of Medicine, Kings’ College London, Bessemer Road, London SE5 9PJ, UK. E-mail: [email protected]
a hydrophobic, isoprenoid geranylgeranyl or farnesyl residue to the cysteine (C) in the Rho carboxy-terminal CAAX motif (where A is an aliphatic amino acid and X is methionine or serine).15 RhoA and RhoC undergo geranylgeranylation (GG), whereas RhoB can be either geranylgeranylated or farnesylated.
There is an increasing body of evidence to demonstrate that the antiproliferative effects of HMG-coacetyl A-reduc-tase inhibitors (statins) are mediated by the depletion of the prenylation substrate, geranylgeranyl pyrophosphate (GGPP).16–18 Furthermore, simvastatin has been shown to inhibit mesangial cell (MC) proliferation and matrix expansion in an in vivo model of mesangial proliferative nephritis.19However, in addition to cell proliferation, factors such as the release of profibrotic cytokines, influx of inflammatory cells, and glomerular hemodynamics are likely to be important in determining the response to glomerular injury. Recent work using simvastatin in rat MCs suggest that the antiproliferative effects of stains are mediated through inhibiting the prenylation of Rho pro-teins.20 In this study, we use lovastatin and GGTI 298 (a specific geranylgeranyl transferase inhibitor) to study the role GG plays in regulating HMC proliferation in primary culture and the effect of GG on RhoA activation and downstream signaling pathways.
RESULTS
HMC proliferation is dependent on geranylgeranylated proteins
Figure 1a demonstrates that 10 mM lovastatin inhibited serum-stimulated cell number curves after 72 h, causing a reduction in cell number of 44% compared to vehicle-treated cells. Lovastatin caused a similar reduction in cell number in platelet-derived growth factor (PDGF)-stimulated HMCs as shown in Figure 1b. The reduction in cell number was attenuated by coincubation with the prenylation substrate, GGPP. Specific inhibition of geranylgeranyl transferase with GGTI 298 (20 mM) caused a reduction in cell number of 49% at 72 h after serum stimulation as seen in Figure 1c. GGTI 298 (20 mM) also inhibited serum-stimulated bromodeoxyur-idine (BrdU) incorporation by 89% after 24 h as seen in Figure 1e. When HMC proliferation was stimulated by PDGF, GGTI 298 reduced cell number by 59% at 48 h (as seen in Figure 1d) and reduced BrdU incorporation by 79% when compared to vehicle-treated cells (Figure 1f).
Inhibiting GG promotes HMC apoptosis
Lovastatin also had a proapoptotic effect in serum-stimulated HMCs, with 13% of cells showing apoptotic features after 48 h compared with control values of less than 2%. This effect was partially reversed after coincubation with GGPP as shown in Figure 2a. GGTI 298 also induced apoptosis in 24% of serum-stimulated HMCs (Figure 2b). Figure 2c and d demonstrate that both lovastatin and GGTI 298 has similar proapoptotic effects in PDGF-stimulated HMCs.
Targeting Rho with C3 transferase has antiproliferative and proapoptotic effects in HMC
As Rho is a potential target of GG inhibitors, the effects of C3 transferase on HMC proliferation and apoptosis were studied. C3 transferase is a clostridium botulinum toxin, which specifically inactivates Rho through ADP-ribosylation of asparginine 41. Figure 3 demonstrates that C3 transferase (30 mg/ml) inhibited BrdU incorporation by 56% when compared to vehicle-treated HMCs. C3 transferase also induced apoptosis in 16% of HMCs as seen in Figure 3b.
Inhibiting GG disrupts the actin cytoskeleton
To investigate the hypothesis that RhoA is a target of GGTI 298, the effects of GGTI 298 on the actin cytoskeleton were studied by staining actin with tetramethyl rhodamine isothiocynate (TRITC)-conjugated phalloidin. As seen in Figure 4, untreated, fetal calf serum (FCS)-stimulated cells had marked stress fiber formation, whereas GGTI 298-treated cells lost proper actin polymerization. C3 transferase also caused a loss in stress fiber formation. Cells treated with GGTI 298 also appeared to lose contact sites with surround-ing cells.
RhoA is the predominantly expressed Rho isoform in HMC
Quantitative Western blotting was performed to assess the relative expression of Rho isoforms in HMC using standard recombinant Rho proteins and isoform-specific Rho anti-bodies as shown in Figure 5. These results show that the signal obtained with 40 mg of HMC lysate is equivalent to approximately 15 ng of RhoA recombinant protein (upper blot). Using an equal amount of cell lysate, the signal using the RhoB antibody appears to be less than 2 ng of RhoB recombinant protein (middle blot). Although the data using the RhoC antibody is less clear, the signal for 40 mg of cell lysate is less than 2 ng of RhoC recombinant protein RhoC (lower blot). These results clearly show RhoA to be the predominantly expressed Rho isoform in HMC.
GGTI 298 increases cytosolic expression of RhoA and prevents RhoA activation
Immunocytochemistry was performed to assess the effects of GG on the cellular localization of RhoA as shown in Figure 6a. As can be seen, in serum-starved cells RhoA appears to be diffusely expressed throughout the cell, presumably owing to sequestration of RhoA in the cytosol by Rho-GDI. However, stimulation with serum caused most of RhoA to be relocalized to perinuclear structures after 15 min. Pre-treatment of cells with GGTI inhibits this translocation of RhoA, with increased cytosolic staining of RhoA when compared to serum-stimulated cells that had no prior treatment with GGTI 298.
Figure 6b and c shows the effect of GGTI 298 on RhoA activation using the Rhotekin pull-down assay for activated, GTP-bound RhoA. There is little expression of active-RhoA in serum-starved cells. Stimulation with serum increases the activation of RhoA and this is partially inhibited by pre-treatment with GGTI 298.
Inhibiting GG attenuates activation of Erk1/2 and Akt
The effects of GGTI 298 on extracellular
signal-regulated protein kinase (Erk)1/2 and Akt activation were studied. Figure 7 shows a representative Western blot
analysis of this experiment. As seen in the upper panel, GGTI 298 partially inhibited serum-stimulated activation of Erk1/2, without affecting expression of total Erk1/2. GGTI 298 also partially inhibited the activation of the
1 2 3 0 0.5 1.0 1.5 1 2 3 0 0.5 1.0 1.5 1 2 3 0 0.5 1.0 1.5 0 10 20 30 40 50 0 10 20 30 40 0 1 2 3 0.5 1.0 1.5 ***
Cell number (a.u.)
after 10% FCS stimulation
Cell number (a.u.)
after 10% FCS stimulation
Cell number (a.u.)
after PDGF stimulation
Days
***
Cell number (a.u.)
after PDGF stimulation
Days Days
% cells BrdU +ve
after 10% FCS stimulation
% cells BrdU +ve
after PDGF stimulation
Vehicle GGTI 10 GGTI 20
Vehicle GGTI 298 10 GGTI 298 20 Vehicle Lovastatin Lovastatin + GGPP Vehicle Lovastatin Lovastatin + GGPP *** *** *** Days Vehicle GGTI 298 10 GGTI 298 20
Vehicle GGTI 10 GGTI 20
a b c d e f *** *** *** ***
Figure 1 | Statin and GGTI actions on cell proliferation. (a, b) Inhibitory effect 10 mMlovastatin on serum- and PDGF-stimulated
HMC cell number curves. Co-incubation of lovastatin with GGPP abrogates this antiproliferative effect. Cell number was estimated using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay by measuring the 490 nMabsorbance over
72 h. (c, d) Inhibitory effect of 10–20 mMGGTI 298 on serum- and PDGF-stimulated cell number curves. (e, f) Inhibitory effect of 10–20 mMGGTI 298 on serum- and PDGF-stimulated BrdU incorporation. The error bars are s.e.m., n¼ 3–6. ***Po0.001 with respect to vehicle-treated cells. 0 5 10 15 20 0 5 10 15 20 0 10 20 30 0 5 10 15 * *
% apoptotic cells after 10% FCS stim
ulation
% apoptotic cells after 10% FCS stim
ulation
Vehicle Lovastatin Lovastatin + GGPP Vehicle Lovastatin Lovastatin + GGPP
Vehicle GGTI 10 GGTI 20 Vehicle GGTI 10 GGTI 20
*
***
***
% apoptotic cells after
PDGF stim
ulation
% apoptotic cells after
PDGF stim ulation a c b d
Figure 2 | Study of apoptosis. (a, c) Proapoptotic effects of 10 mMlovastatin and (b, d) 10–20 mMGGTI 298 in serum- and
PDGF-stimulated HMCs. Apoptosis was determined by Hoechst 33342 and propidium iodide staining. Co-incubation of lovastatin with 7.5 mMGGPP reduces the proapoptoic effects of lovastatin. The error bars are s.e.m., n¼ 3. *Po0.05 and ***Po0.001 with respect to vehicle-treated cells.
survival protein Akt by serum without affecting the expression of total Akt.
DISCUSSION
We have shown that inhibiting GG with GGTI 298 and lovastatin in HMC inhibits cell proliferation and induces apoptosis. Although these effects have been observed in a number of cell types including smooth muscle cells21 and rat MCs,20 work in other cell models shows that
geranylgeranylated proteins (such as RhoB) may actually inhibit cell proliferation.22,23 Similarly, the proapoptotic effects of GGTI are not observed in all cell types, as in human macrophages GGTI 298 actually protects the cells from apoptosis.24 Taken together, these data suggest that the effect of inhibiting GG needs to be determined in individual cell types as the effects of geranylgeranylated proteins on the cell cycle not only vary between cell types but are also species-specific. Indeed, recent work from our lab has shown key differences in GTPase biology between rodent MCs and HMC.25
Although lovastatin also reduces the availability of both GGPP and farnesyl pyrophosphate, we have recently shown that inhibiting farnesylation with a potent, specific farnesyl-transferase inhibitor had no effect on cell proliferation or apoptosis.26 Thus, the antiproliferative and proapoptotic effects of lovastatin appear to be specifically mediated by the depletion of the isoprenyl substrate GGPP. Interestingly, although studies in rat MCs also suggest that farnesylated proteins do not regulate cell proliferation,16in murine MCs farnesyl substrates can ameliorate the antiproliferative effects of lovastatin.27Again, this suggests important species-specific differences in the role of prenylated proteins in regulating cell proliferation. The origins of such differences are unclear. Ras GTPases in human and mouse MCs appear to play quite distinct roles and, for example, Ha-Ras is antiapoptotic only in mouse cells.27 Other differences may be explained by different substrate availability in cells of different species.
GGTI 298 also inhibited the activation of phospho-Erk1/2 and phospho-Akt, but had no effect on Ras activation (data not shown). This suggests that the effects of GGTI 298 on Erk and Akt activation are directly owing to GG inhibition and not simply a secondary consequence of the effects of GGTI 298 on cell proliferation and apoptosis. These signaling 0 10 20 30 40 0 10 20 ** Vehicle C3 transferase Vehicle C3 transferase % cells apoptotic **
% cells BrdU positive
after 10% FCS stimulation
b
Figure 3 | Study of C3 transferase. (a) Inhibitory effect C3 transferase on BrdU incorporation in HMCs. HMCs were grown on 35 mm culture dishes until 70% confluent serum-starved for 24 h, before treatment with 30 mg/ml C3 transferase in 10% FCS. BrdU-positive cells and the total number of normal nuclei were identified as described in the Materials and methods section. Error bars are s.e.m. More than 300 nuclei were counted from two independent experiments. **Po0.01 with respect to vehicle-treated cells. (b) Proapoptotic effect of 30 mg/ml C3 transferase in 10% FCS after staining with Hoechst 33342 and propidium iodide as described previously. Error bars are s.e.m. More than 300 nuclei were counted from each of two independent experiments. **Po0.01 with respect to vehicle-treated cells.
Vehicle GGTI 298 C3 transferase
Figure 4 | The cytoskeleton. TRITC-phalloidin staining of the actin cytoskeleton. HMCs were grown on coverslips in 10% FCS and either left (a) untreated, (b) treated with 20 mMGGTI 298, or (c) 30 mg/ml C3
transferase for 24 h. Cells were fixed with 4% paraformaldehyde and stained with TRITC-phalloidin (1:500) for 30 min. Images were viewed with a confocal microscope at original magnification 400.
Cell lysate Recombinant RhoA
2 ng 5 ng 10 ng 15 ng 20 ng
Cell lysate Recombinant RhoB
2 ng 5 ng 10 ng 15 ng 20 ng
Cell lysate Recombinant RhoC
2 ng 5 ng 10 ng 15 ng 20 ng
Ab: RhoA
Ab: RhoB
Ab: RhoC
Figure 5 | Quantitative Western blot analysis of HMC lysate for Rho isoforms. These are compared to recombinant Rho proteins (2–20 ng) when detected with isoform-specific antibodies. The upper panel was detected with the anti-RhoA antibody, the middle panel was detected with the anti-RhoB antibody, and the lower panel was detected with the anti-RhoC antibody. These figures were represen-tative of three separate experiments.
effects of GGTI 298 are likely to be the result of inhibiting a range of geranylgeranylated proteins, including Rho, Rac, and Cdc42. This hypothesis is supported by the observation that both C3 transferase and GGTI 298 reduced HMC prolifera-tion, increased apoptosis, and inhibited stress fiber forma-tion. As C3 transferase specifically inactivates the Rho
isoforms and has no effect on Rac and Cdc42 activation, it is likely that some of the effects of GGTI 298 on cell-cycle progression and stress fiber formation are mediated by targeting of Rho isoforms. Furthermore, treatment with GGTI 298 is associated with altered RhoA distribution and activation. Taken together, these data strongly implicate RhoA as being a key target of GGTI 298.
The roles of RhoB and RhoC are harder to define. Quantitative Western blotting suggests that both RhoB and RhoC protein expression is less than 5% of total Rho expression in HMC. RhoB appears to actually inhibit cell proliferation and can be farnesylated,22 and therefore it is unlikely to be a target through which GGTI 298 mediates its antiproliferative effects. We are currently clarifying the role of RhoC using specific small interfering RNA. Owing to the minimal expression of RhoC in HMC and the relative insensitivity of the RhoC antibody, we were unable to ascertain the effect of GG inhibition on RhoC activation. However, its role in cell proliferation may be very important as there is evidence linking RhoC to malignant transforma-tion and metastasis.28At present, little is known about precise biological roles of the Rho isoforms in HMC. Preliminary unpublished work in our lab using small interfering RNA targeting each isoforms suggests that both RhoA and RhoC are important in regulating MC proliferation.
The mechanism by which Rho regulates cell proliferation is likely to involve a number of different pathways, including the regulation of key cell-cycle regulatory proteins such as cyclins and cyclin-dependent kinase inhibitors. For example, targeting Rho (using either GGTI 298, statins, or C3 transferase) results in increased expression of the cyclin-dependent kinase inhibitors, p21(WAF1)and p27 KIP1, thereby inhibiting cell-cycle progression.29–31 RhoA can also activate transcription factors such as serum response factor.13 Furthermore, RhoA appears to be required for sustained growth factor activation of the Ras–Raf—MAP kinase kinase–Erk pathway.32 This observation is consistent with 10% FCS RhoA-GTP Total RhoA 10% FCS + GGTI 298 Serum-starved 10% FCS CGTI 298 b c 150
% RhoA activation (a.u.)
100 50 10% FCS GGTI 298 0 + + − + − − + + + −
Figure 6 | RhoA activity. (a) Effect of GGTI 298 on RhoA localization. HMCs were plated on to coverslips and serum-starved for 24 h. They were then left in serum-free medium overnight with or without GGTI 298 20 mM. They were then fixed or stimulated with 10% FCS for 10 min as indicated followed by fixation. RhoA was detected with a specific anti-RhoA antibody and a fluorescein isothiocyanate-labeled secondary antibody. The cells were viewed at 200 original magnification with a fluorescent microscope. (b) Inhibitory effect of GGTI 298 on RhoA activation. HMCs were grown to 90% confluence, then serum starved for 24 h, incubated with either 20 mMGGTI 298,
vehicle alone, or serum-free media for a further 24 h. Untreated, serum-starved cells were then lysed, whereas vehicle- and GGTI 298-treated cells were stimulated with 10% FCS for 10 min before lysis. GTP-bound Rho was immediately recovered as described in the Materials and methods and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. The top panel shows a Western blot for GTP-bound Rho, whereas the bottom panel shows a Western blot for total cellular Rho taken from the same cell lysates. Data shown are representative of two separate experiments. (c) Effects of 20 mMGGTI 298 on RhoA activation as
determined by densitometric analysis.
Phospho-Erk1/2 Total Erk1/2 10% FCS − + + GGTI 298 − − + Phospho-Akt Total Akt
Figure 7 | Western blots for phospho-(Erk 1/2) and phospho-Akt activation in stimulated HMC after overnight treatment with GGTI 298 20 lMand stimulation with 10% FCS for 5 min were
shown. As can be seen, GGTI 298 20 mMinhibits the phosphorylation of both Erk1/2 and Akt by serum. Total Erk1/2 and Akt remained unchanged. Blots shown are representative of two separate experiments.
our data showing that GGTI 298 clearly inhibits Erk activation, suggesting that Rho has a permissive effect on the activation of the Ras-mediated cell proliferation. As both C3 and GGTI 298 promoted disruption of the actin cytoskeleton, it is also possible that an intact actin cytoskeleton is a pre-requisite for effective for growth-factor-stimulated cell proliferation and Erk signaling. Rho could also directly activate Erk, although the mechanism by which such activation could take place has not been elucidated.
It is important to note that a number of other geranylgeranylated proteins may mediate the effects of GGTI in HMC, including Rac, Cdc42, and Rap. For example, dominant-negative forms of Rac and Cdc42 have been shown to inhibit DNA synthesis in fibroblasts.12These effects may be mediated through the regulation of transcription factors such as serum response factor and nuclear factor-kappa B, as well as the activation of other mitogen-activated protein kinase cascades such as the p38 and janus kinase signaling pathways.14Rac and cdc42 may also regulate cell proliferation by modulating Erk activity. For example, constitutively activated Rac and cdc42 have been shown to increase Erk activation.33Furthermore, Rac appears to exert a permissive effect on Ras-dependent Erk activation via the serine/
threonine kinase PAK.33 Rac may also promote cell
proliferation by targeting cell-cycle regulatory proteins. For example, activated Rac has been shown to increase cyclin D expression, thereby promoting cell proliferation.14
The effects of Rap on the cell cycle are complex. There is evidence to suggest that Rap can antagonize Ras-stimulated cell proliferation, whereas in other cell types, Rap promotes mitogen-activated protein kinase activation.34
The effects of geranylgeranylated proteins on the actin cytoskeleton have been defined for some time. Microinjection studies using constitutively activated mutants demonstrated that Rho was able to induce the assembly of contractile actin and myosin filaments (stress fibers).35 Rac was shown to induce actin-rich curtain-like extensions (lamellipodia or ruffles) often formed along the edges of cells,36 whereas Cdc42 induced the formation of filopodia (actin-rich, finger-like membrane extensions) that probably regulate recognition of the extracellular environment.13 Thus, these proteins regulate three separate signal-transduction pathways that link plasma membrane receptors to the assembly of distinct actin structures.11There has been little work looking at the specific role of Rac and Cdc42 in HMC. However, connective tissue growth-factor-induced migration of MCs appears to be in part mediated by Cdc42.37
Immunocytochemistry for RhoA revealed some surprising results, suggesting that in HMC, RhoA may signal from intracellular compartments and not from the plasma membrane. There are very little data using immunocyto-chemistry to study endogenous RhoA localization. Some data suggest that RhoA may be activated at the plasma membrane,38,39 whereas others have shown little evidence for RhoA localizing to the plasma membrane.40 Thus, GG
may serve to localize RhoA to specific compartments within the cell. Recent work has shown that Ras can signal from the Golgi complex and not only from the plasma membrane as previously thought, suggesting that in certain cell types, Ras and Rho GTPases may signal from intracellular compart-ments.41
Our data show that geranylgeranylated proteins promote cell survival through the activation of the phosphatidylino-sitol 30-kinase/Akt signaling pathway. Recent work in human cancer epithelial cells demonstrates that GGTI may promote apoptosis by inhibiting the activation of Akt.42It is not clear which geranylgeranylated protein activates Akt, but both Rac and Cdc42 can interact specifically with phosphatidylinositol 30-kinase and activate Akt.43 Furthermore, Rac appears to play a key role in transducing integrin-mediated survival signals.44 RhoA may also protect against apoptosis by upregulating the expression of the survival protein Bcl-221 or increasing the expression of the tumor suppressor protein, p53.45 Although MC apoptosis may have a valuable role in counterbalancing excessive MC proliferation in vivo,46other work suggests that apoptosis may be harmful by promoting glomerular hypocellularity.47 MC proliferation may be seen in some situations as a reparative response to glomerular injury (e.g. post-streptococcal GN) and thus inhibiting repopulation of the glomerulus may prevent effective glomerular remodeling and repair. Therefore, inhibiting MC proliferation and promoting apoptosis per se may be a double-edged sword depending on the specific clinical context.
Rho plays a key role in regulating three distinct biological processes that are critical to the progression of many renal diseases, namely (i) cell proliferation and migration, (ii) tubulointerstitial fibrosis, and (iii) glomerular hemody-namics.48 Therefore, targeting Rho signaling could be a particularly attractive therapeutic strategy. The use of statins to target Rho and inhibit MC proliferation may be of therapeutic value. Clinical trials of statins in mesangiopro-liferative disease are required to determine whether they will be an effective therapeutic in the future. Future strategies to target RhoA signaling include the use of Rho kinase (ROCK) inhibitors. These have been shown to attenuate cell proliferation, fibrosis and macrophage infiltration in sub-totally-nephrectomized spontaneously hypertensive rats.49 Furthermore, these inhibitors promote vascular smooth muscle cell relaxation by increasing myosin light chain phosphatase activity and appear to have beneficial effects on the renal microcirculation.50,51ROCK inhibitors are currently in clinical trials for the treatment of cerebral vasospasm, but their beneficial effects in a variety of models of renal injury suggest they may be a promising development in the treatment of progressive renal diseases.48
Taken together, this work indicates that inhibiting Rho with either statins or GGTI may have beneficial effects in progressive renal diseases. Although statins are widely used clinically, GGTI have not yet been developed as a therapeutic because of worries about toxicity. However, GGTI 298 has
been given in a rat model of malignant glioma without evidence of in vivo toxicity,52 suggesting that specific targeting of geranylgeranylated proteins may be an alternative clinical strategy in proliferative renal diseases.
MATERALS AND METHODS Materials
All reagents and antibodies were purchased from Sigma-Aldrich (Gillingham, UK), unless otherwise stated. GGTI 298 was a kind gift from SM Sebti, University of Florida. Vehicle for GGTI 298 was 0.2% dimethylsulfoxide. We have recently shown that GGTI 298 inhibits the prenylation of Rap1a, an exclusively geranylgeranylated protein.26 Vehicle for lovasta-tin (Calbiochem, UK) was ethanol 25 mg/ml. RhoA, RhoB, and RhoC standard recombinant proteins were a kind gift from Shawn Ellerbroek, Department of Cell and Develop-mental Biology, University of North Carolina. Antibodies used were anti-Phospho-Erk1/2 and anti-phospho-Akt (Ser 473) antibodies (New England Biolabs, Hitchin, UK), anti-RhoA 418), anti-RhoB 8048) and anti-RhoC (sc-12116) antibodies were purchased from Santa Cruz Bio-technology (Santa Cruz, CA, USA). Dot blots were performed using RhoA, RhoB, and RhoC standard recombi-nant proteins and isoform-specific Rho antibodies. We and other workers53 have demonstrated that these isoform-specific antibodies were used at concentrations at which they did not crossreact with other Rho isoforms (data not shown).
Primary HMC culture
Primary culture HMC (Clonetics Biowhittaker, Wokingham, UK) were maintained in Rosewell Park Memorial Institute 1640 (Invitrogen Limited, Inchinnan, UK) medium with 10% FCS. All experiments were carried out between passages 6 and 9.
Cell proliferation
To study the effects of GGTI 298 and lovastatin on proliferation, HMCs were serum starved for 24 h, trypsinized, and seeded in triplicate into 96-well plates (5000 cells/well). GGTI 298 (0–20 mM), lovastatin (10 mM), and lovasta-tinþ GGPP (7.5 mM) were added and cells were grown in 10% FCS or PDGF (200 ng/ml). Viable cell numbers were determined by the Cell titer 96, Promega assay (3-(4,5- dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sul-fophenyl)-2H-tetrazolium) measuring absorbance at 490 nm. The correlation between viable cell number and absorbance at 490 nm is linear.
DNA synthesis was assessed by BrdU uptake. Cells were grown in 35 mm dishes and serum starved for 24 h before treatment with GGTI 298, lovastatin, or C3 transferase in the presence of 10% FCS. Six hours after the addition of drug and growth factor, BrdU (10 mM) was added to each dish for 16 h. Dishes were then washed with phosphate-buffered saline (PBS) and fixed for 45 min (three volume 50 mM glycine and seven volume ethanol). Cells were then incubated in 4M hydrochloric acid for 10 min, washed three times in
PBS, and blocked in 5% goat serum/0.05% Tween/PBS for 15 min before being incubated overnight in anti-BrdU antibody (1 in 100 dilution) at 41C. Cells were washed with PBS and incubated for 30 min with TRITC-labeled antimouse immunoglobulinG antibody. Cell nuclei were then counter-stained with Hoechst 33342 at a concentration of 5 mg/ml for 15 min. Cells were then visualized with a fluorescent microscope. BrdU-positive cells stain nuclei red, whereas all cell nuclei would stain blue with Hoechst 33342. A total of at least 150 cells were counted from at least five different fields. Nuclei that appeared apoptotic on Hoechst 33342 staining were not counted.
Apoptosis
HMCs were grown in serum in 35 mm dishes and treated with GGTI 298 or lovastatin as before. Hoechst 33342 was added (5 mg/ml) and then incubated at 371C for 15 min followed by propidium iodide (5 mM) for 5 min. Cell nuclei were then visualized under fluorescent microscopy. Apoptotic nuclei were then identified as showing characteristic signs of chromatin condensation and fragmentation. Late apoptotic or necrotic cells stained red. A total of at least 150 cells were counted from at least five different fields.
Immunocytochemistry and actin staining
Cells were grown on glass coverslips in 35 mm tissue culture dishes. Cells were seeded in normal growth medium and left for 24 h. The growth medium was then removed and the cells were left overnight either in serum-free medium alone or serum-free medium in the presence of GGTI 298. The medium was removed the next day and the cells were treated with 10% FCS for 10 min or left in serum-free medium. The coverslips were washed three times in PBS and then fixed in 4% paraformaldehyde for 10 min. Cells were washed again, permeabilized with 0.2% Triton X-100/PBS, and blocked with 5% goat serum/PBS. Coverslips were incubated with an anti-RhoA antibody at 41C overnight, washed three times in PBS before incubation with a fluorescein isothiocyanate-conjugated secondary antibody for 30 min at room tempera-ture. The coverslips were again washed three times in PBS and mounted onto glass slides with slow-fade mounting medium (Molecular Probes, Eugene, OR, USA; S7461). Cell staining for RhoA was then visualized by conventional fluorescent microscopy. To stain for the actin cytoskeleton, cells were fixed, permeabilized, and blocked as above before incubation with TRITC-conjugated phalloidin (1:500) for 30 min at room temperature. Coverslips were then washed and mounted, before viewing with the confocal microscope.
Western blotting
For Western blotting experiments, HMCs were grown to 80% confluence in 35- or 100-mm plates, in Rosewell Park Memorial Institute 1640 and 10% FCS. Cells were then serum-starved for 24 h and then treated for a further 24 h with 20 mMGGTI 298 in serum-free media containing either 10% FCS. Cells were stimulated for 5 min with 10% FCS, cell
lysates were extracted on ice, denatured in Western sample buffer, and 2-mercaptoethanol for 5 min at 1001C, before performing sodium dodecyl sulfate-polyacrylamide gel elec-trophoresis and Western blotting. Protein expression was detected by Amersham ECL chemiluminescence (Amersham Pharmacia Biotech, Little Chalfont, UK).
Rho activation
Affinity precipitation using the Rho-binding domain of Rhotekin (a downstream effector of Rho) was used to determine Rho activation. Nuclear material was removed by centrifugation and lysates were immediately precipitated with 60 ml of a 50% slurry of the Rhotekin–Rho-binding domain–glutathione-S-transferase–agarose, rotating at 41C for 45 min, to pull-down activated, GTP-bound Rho. The samples were then pelleted at 14 000 g at 41C for 2 min and washed twice in magnesium lysis buffer and denatured in Western sample buffer as before, before sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot-ting using an anti-RhoA antibody.
Statistical analysis
Statistical analysis was performed using analysis of variance followed by Dunnetts test of significance or an unpaired t-test using Prism software.
ACKNOWLEDGMENTS
AK was supported by a UK Medical Research Council Clinical Training Fellowship.
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