ABSTRACT. Glomerular capillary hypertension is an importantdeterminant of glomerulosclerosis in rats with subtotal renalablation. Dietary supplementation with L-arginine increasesrenal nitric oxide (NO) production and limits glomerular injuryin this model, and early benefits are seen without altered glomerularcapillary pressure. In an in vitro model of hemodynamicallymediated signaling, the authors have reported that subjectingMC to cyclic stretch/relaxation activates the mitogen-activatedprotein kinase p42/44 (Erk) cascade and that NO and cyclic GMPabrogate stretch-induced Erk activation by inducing actin cytoskeletaldisassembly. The actin cytoskeleton is regulated by the Rhofamily of GTPases, including RhoA; therefore, the authors examinedthe role of RhoA in stretch-induced Erk activation and as anNO target. In primary rat MC subjected to cyclic mechanicalstrain, RhoA activity was maximally increased (2.4-fold) after1 min of stretch, and Erk activation temporally followed. TheRho-kinase inhibitor Y-27632 attenuated Erk activation in adose-dependent manner and prevented stretch-induced actin stressfiber formation. The NO donors S-nitroso-N-acetylpenicillamineand cGMP both inhibited stretch-induced RhoA and Erk activationand stress fiber formation. Infection of MC with the RhoA mutantRhoA-Ala188, which is resistant to NO-dependent phosphorylation,abrogated the effects of NO and cGMP on stretch-induced Erkactivation and stress fiber formation. The authors concludethat the early activation of RhoA is essential for stretch-inducedactin stress fiber formation and Erk activation in MC, eventswhich are prevented by NO and cGMP through their action on RhoA.Inhibition of RhoA may thus be a new approach to the preventionof hemodynamically mediated glomerular injury. E-mail: krepinj@mcmaster.ca
The partially nephrectomized (Nx) rat is a well-characterizedmodel of chronic renal failure (CRF) in which glomerular capillaryhypertension is an important hemodynamic determinant of glomerulosclerosis(1). We and others have shown that L-arginine, an orally administeredprecursor for nitric oxide (NO) production, prevents the developmentand progression of renal lesions in this model (2,3). AlthoughNO is an intrarenal vasodilator (4), the early beneficial effectsseen with L-arginine occur in the absence of reductions in glomerularcapillary pressure (Pgc) (5).
Increased Pgc and wall tension are transmitted to resident glomerularcells. In other models of glomerulosclerosis associated withglomerular capillary hypertension such as the uninephrectomizedrat treated with deoxycortisone trimethyl acetate (DOCA) andthe fawn-hooded rat, podocyte injury is an important determinantof progressive glomerulosclerosis (6,7). However, the behaviorof mesangial cells (MC) is thought to play a central role inthe progression of glomerular disease in the remnant kidney.Although a rise in Pgc precedes MC proliferation and increasedextracellular matrix (ECM) production in the remnant glomerulus(1,8), the mechanisms responsible for linking glomerular capillaryhypertension and glomerular injury have not been fully elucidated.
Increased Pgc transmits to MC, which provide architectural supportfor the glomerular capillary tuft, as mechanical strain. Invitro, MC subjected to cyclic stretch/relaxation proliferate(9) and increase ECM protein synthesis (10), and we have utilizedthis model system to study mechanical strain-induced signalingin MC. As in vivo injury is initiated and promulgated by a 20to 30% increase in Pgc from the resting value of 35 to 55 mmHgin the Wistar rat, our initial work explored responses in MCstretched 20 to 30%. We noted MC proliferation at -28 kPa (average29% elongation of plates), but not at -14 kPa (average 20% elongationof plates) (9); accordingly, we have studied MC stretched at-28 kPa. Notably, podocytes also respond to mechanical strainin vitro, again indicating they may play a role in the responseto increased intraglomerular pressure in some models (11).
In cultured MC, activation of the mitogen-activated proteinkinase p42/44 (Erk) by mechanical strain mediates cell proliferation(9,12) and induces expression of Fos, a component of the transcriptionfactor activating protein 1 (AP-1) (13). In vivo, glomerularErk activation and AP-1 DNA-binding activity are seen in chronicallyhypertensive Dahl salt-sensitive rats (14). Transforming growthfactor beta-1 (TGF-) contains 2 AP-1 consensus sequences; therefore,Fos mediates its induction (15). Indeed, stretched MC displayincreased TGF- mRNA expression and protein secretion (16) andthe ECM protein synthesis observed in stretched MC is inhibitedby a neutralizing antibody to TGF- (16). Stretch-induced Erkactivation can thus be linked both to cellular proliferationand to TGF- induction and the accumulation of ECM in glomeruliexposed to elevated Pgc.
We have shown qualitatively that cyclic stretch induces theformation of F-actin stress fibers in MC (17) and that disruptionof the actin cytoskeleton with cytochalasin D inhibits stretch-inducedErk activation (18). The integrity of the actin cytoskeletonis thus essential to the activation of Erk by mechanical strain.We have further demonstrated that NO and cGMP both inhibit stretch-inducedErk activation in MC by disrupting the actin cytoskeleton (18).However, the mechanisms linking mechanical strain, Erk activation,NO, and the actin cytoskeleton have not been defined in MC.
The Rho family of GTPases, 20- to 30-kD proteins, which cyclebetween an active, GTP-bound form and an inactive, GDP-boundform, play an important role in the regulation of the cytoskeleton(19). Of this family, RhoA regulates the formation of F-actinstress fibers and focal adhesion complexes (20), with Rho-kinase(ROCK) as its primary effector in this regard (21). Erk activationin fibroblasts binding to fibronectin is blocked by RhoA inhibitionand accentuated by a constitutively active RhoA (22). In stretchedprimary rat cardiomyocytes, Erk activation is attenuated withvarious inhibitors of RhoA (23), and RhoA inhibition also preventsstress fiber formation and reduces AP-1 activity in endothelialcells under shear stress (24). Moreover, in other systems, NO,acting through its second messenger cGMP and the consequentactivation of cGMP-dependent protein kinase (cGK), can inhibitsignaling at several levels in the RhoA and Erk pathways (25,26).In vascular smooth muscle cells (VSMC) and HeLa cells, NO andcGMP disrupt the basal (unstimulated) actin fiber structure,an effect mediated by cGK-dependent phosphorylation of RhoAon Ser188 (27,28).
Accordingly, we first sought to determine if mechanical strain-inducedErk activation and stress fiber formation were dependent onRhoA in glomerular MC. We further sought to determine if NOprevents (1) stretch-induced actin stress fiber formation bya quantitative assay and (2) Erk activation by inhibiting RhoAactivation.
Cell Culture
Harlan Sprague-Dawley rat MC were cultured in Dulbecco modifiedEagle medium (DMEM) supplemented with 20% fetal calf serum (LifeTechnologies BRL), streptomycin (100 µg/ml), and penicillin(100 units/ml) at 37°C in 95% air/5% CO2. Experiments werecarried out using cells between passages 8 to 20.
Application of Strain/Relaxation
MC (5 x 104/well) were plated onto six-well plates with flexiblebottoms coated with bovine type I collagen (Flexcell InternationalCorp.). Cells were grown to confluence for 72 h and then renderedquiescent in serum-free medium for 24 h. Plates were exposedto continuous cycles of strain/relaxation generated by a cyclicvacuum produced by a computer-driven system (Flexercell StrainUnit 2000), each cycle consisting of 0.5 s of strain and 0.5s of relaxation. Experiments were performed at a vacuum pressureof -27 kPa, inducing 29% elongation in the diameter of the surface,as we have published (9,18).
Pharmacologic inhibitors were added at the indicated concentrationsand times before stretch: Y-27632 (Calbiochem) at varying concentrationsfor 30 min, cytochalasin D (Molecular Probes) at 200 ng/ml for60 min, jasplakinolide (Molecular Probes) at 50 nM for 60 min,C. botulinum exoenzyme (Cytoskeleton) at 16 µg/ml for48 h, 8-bromo cyclic guanosine monophosphate (8-bromo-cGMP,Sigma) at 1 mM for 10 min, and S-nitroso-N-acetylpenicillamine(SNAP, Sigma) at 70 µM for 10 min.
Activity Assays and Immunoblotting Rho Pull-Down Assay.
Cells were lysed in buffer containing 50 mM Tris pH 7.2, 1%Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 60 mM n-octylglucopyranoside, 500 mM NaCl, 10 mM MgCl2, 10 µg/ml leupeptin,10 µg/ml aprotinin, and 1 mM PMSF. Lysate was clearedof cellular debris by centrifugation at 15,000 rpm for 5 minat 4°C. To immunoprecipitate GTP-bound RhoA, lysate wasincubated with 30 µg of a glutathione-agarose bound GST-taggedrhotekin Rho binding domain (Upstate Biotechnology) at 4°Cfor 45 min with gentle rocking. Beads were collected by centrifugationat 15,000 rpm for 30 s at 4°C and washed 3 times in buffercontaining 50 mM Tris pH 7.2, 1% Triton X-100, 150 mM NaCl,10 mM MgCl2, 10 µg/ml leupeptin, 10 µg/ml aprotinin,and 0.1 mM PMSF. Beads were resuspended in 30 µl of 2xreducing sample buffer, boiled for 5 min and the supernatantresolved on 15% SDS-PAGE. Membranes were probed with anti-RhoAantibody 1 µg/ml (Upstate).
Immunoblotting.
Protein was isolated as described previously (18), boiled for5 min, and 50 µg was separated on a 10% SDS-PAGE gel.After transfer, nitrocellulose membranes were blocked for 1h at room temperature (TBS with 0.1% Tween [TBST] and 5% wt/volnonfat dry milk) and incubated with the appropriate primaryantibody overnight at 4°C in TBST with 5% wt/vol bovineserum albumin. Antibodies used included polyclonal phospho-Erk1/2(Thr202/Tyr204), 1:1000, polyclonal Erk1/2, 1:1000 (both CellSignaling), monoclonal -actin, 1:5000 (Sigma), and monoclonalFLAG 10 µg/ml (Sigma, M2). Membranes were washed threetimes with TBST and incubated with the appropriate horseradishperoxidase-conjugated antibody in TBST/5% milk for 1 h. Afterthree further washes, the membrane was incubated with LumiGloreagent and exposed to x-ray film.
F-Actin Staining and Quantification
MC were washed three times with PBS and fixed with 3.7% formaldehydefor 15 min at room temperature. After three further washes andpermeabilization with 0.1% Triton X-100 in PBS for 5 min, MCwere washed and blocked with 1% BSA in PBS for 30 min. Subsequently,rhodamine phalloidin (Molecular Probes) was applied for 20 min.Cells were then washed and incubated with Alexa Fluor 488 conjugatedDNase I (9 µg/ml) for 20 min to label G-actin (29). Afterfurther washing, the flexible base from each well was removed,placed on a glass slide and coverslipped with one drop of anti-fademounting medium (Slow Fade, Molecular Probes). Slides were storedat 4°C in the dark until analysis within 1 wk by confocallaser scanning microscopy (Zeiss 510). Imaging conditions wereoptimized for control cells for both phalloidin and Alexa 488,and kept constant for all subsequent imaging. The average pixelintensity was obtained for both fluorochromes using NIH ScionImage software for Macintosh, and their ratio taken to obtainthe F:G actin ratio. A minimum of 50 cells was assessed foreach experimental condition.
Infection of MC
A plasmid containing the Rho-Ala188 mutant in which Ser-188is replaced with Ala-188 was a kind gift of Dr. G. Loirand (27).The epitope FLAG was added N-terminal to the RhoA mutant andFLAG-RhoA-Ala188 was cloned into the pBabe retroviral vector.MC of passage 11 were infected with empty vector or RhoA-Ala188.In brief, 293 T cells were transiently transfected using thecalcium phosphate method with the following plasmids: 10 µgof pBabe-FLAG-RhoA-Ala188 or pBabe (empty vector), 10 µgof VSV-G (vesicular stomatitis virus G, encoding for the viralenvelope protein, Stratagene), and 10 µg of VSV-GP (encodingfor the gag and pol proteins, Stratagene). Medium was changedafter 12 h; 24 h later, medium was collected and virus concentratedby centrifugation for 90 min at 50,000 x g, 4°C. Virus wasresuspended in 1 ml of medium with 10 µg of polybrene.MC, plated the previous day, were exposed to concentrated virusfor 1 h with gentle rocking every 15 min, after which 9 ml ofmedium containing polybrene was added. Medium was changed after24 h, and selection began 48 h later using 1 µg/ml puromycin.MC were passaged once in puromycin, and experiments were thenperformed on the pooled passaged cells.
Fibronectin (FN) Detection by RT-PCR
After 24 h of stretch, MC RNA was extracted using TRIZOL (LifeTechnologies BRL), and 2 µg was reverse transcribed withSuperscript II (Life Technologies), both protocols accordingto product specifications. The resulting cDNA was used for semiquantitativePCR amplification of FN, using -actin as an internal control.These were detected as 190- and 240-bp products, respectively,on a 1.5% agarose gel. Primer sequences used were: rat FN sense,5'-GCAAGAGGCAGGCTCAGCAAATCG-3'; FN antisense, 5'-TTCAGGTTCAGGCTTGCTCTCGCA-3';rat -actin sense, 5'-AACCCTAAGGCCAACCGTGAAAAG-3'; -actin antisense,5'-TCATGAGGTAG-TCTGTCAG-3'. Reactions were carried out for 25to 28 cycles at 94°C for 30 s, 58°C for 30 s and 72°Cfor 60 s, with a 10-min final extension at 72°C using 5U Taq in PCR buffer (Life Technologies BRL), 1.5 mM MgCl2, 0.2mM dNTP mix, 0.3 uM each of sense and antisense primers, and2 µl of cDNA.
Enzyme-Linked Immunosorbent Assay (ELISA) for FN
After MC were stretched for 24 h, media were collected and debrisremoved by low-speed centrifugation. Ninety-six well microtiterplates were coated with media in a 1:6 dilution with ELISA CoatingBuffer (Sigma) overnight at 4°C. Each condition was coatedin triplicate. Plates were washed 3 times with wash buffer (0.25%BSA/0.05% Tween-20/0.05% sodium azide in PBS) and incubatedin blocking buffer (2% BSA/0.05% Tween-20/0.05% sodium azidein PBS) for 90 min at room temperature. After three washes,wells were incubated with a 1:5000 dilution of monoclonal anti-FNantibody (Transduction Labs) in blocking buffer for 5 h at roomtemperature followed by overnight incubation at 4°C. Afterthree further washes, wells were exposed to alkaline phosphatase-conjugatedgoat anti-mouse secondary antibody (Sigma), 1:30,000 dilutedin blocking buffer, for 2 h at room temperature. Following 3washes, p-nitrophenyl phosphate was added and after room temperatureincubation, reactions were read at 405 nm in a microplate autoreader.
Statistical Analyses
Statistical analyses were performed using ANOVA, with TukeyHSD used for post-hoc analysis to determine differences betweenindividual groups when ANOVA results were significant. P <0.05 (two-tailed) was considered significant. Data are representedas the mean ± SEM. All analyses used the statisticalpackage SPSS for Windows 11.0.
Stretch-Induced Activation of RhoA Is Required for Erk Activation
MC were exposed for various times to cyclic strain at 1 Hz (60cycles/min), -27 kPa for all experiments. Activation of RhoAwas determined by a pull-down assay (30) based on the much greateraffinity with which GTP-bound RhoA binds to its effector proteinrhotekin when compared with RhoA-GDP. Stretch induced a 2.4-foldincrease in RhoA activity at its peak of 1 min (Figure 1A).Total lysate was immunoblotted with the same RhoA antibody toillustrate equality in cellular RhoA across conditions. We nextexamined the early kinetics of Erk activation. Stretch resultedin phosphorylation of Erk 42/44 by 1 min as detected by Westernblot, which continued to increase through 5 min (Figure 1B).We have previously shown that cyclic strain at the above conditionsaffects maximal Erk activation at 10 to 20 min (31,32). Stretch-inducedRhoA activation temporally precedes that of Erk; we thereforeinvestigated the effects of inhibition of RhoA signaling onErk activation. Preincubation with the ROCK inhibitor Y-27632(33) dose-dependently inhibited maximal Erk phosphorylationat 20 min of stretch, with the greatest effect at 20 µM(Figure 2A). C3 exoenzyme from Clostridium botulinum is a RhoAinhibitor that acts through selective ADP-ribosylation at Asn-41,thus preventing exchange of GDP for GTP (34). C3 (8 to 16 µg/mlfor 48 h) also inhibited maximal stretch-induced Erk phosphorylation(Figure 2B). The effect was somewhat less than that observedwith Y-27632, likely since intracellular penetration of theC3 protein was less efficient than that of Y-27632 (33).
Figure 1. Kinetics of RhoA-Erk activation in stretched mesangial cells (MC). MC were exposed to 1 Hz, -27 kPa stretch as indicated. (A) Early activation of RhoA, as represented by immunoprecipitated RhoA-GTP, is seen maximally by 1 min of stretch, *P < 0.03 (n = 3). (B) Erk activation, as measured by Western blotting using a phospho-Erk specific antibody, temporally follows activation of RhoA, *P < 0.03 versus control and 30 s (n = 3).
Figure 2. RhoA signaling is required for stretch-induced Erk activation. (A) Preincubation for 30 min with the Rho-kinase (ROCK) inhibitor Y-27632 dose-dependently inhibits maximal Erk activation after 20 min of stretch. Densitometry corresponding to each representative gel lane is immediately below, *P < 0.02 versus control, #P 0.02 versus stretch (n = 3). (B) Inhibition of Erk activation is also seen after 48 h of incubation with the RhoA inhibitor C3 exoenzyme (16 µg/ml), *P < 0.02, #P < 0.03 (n = 3).
ROCK Inhibition Disrupts Basal and Stretch-Induced F-Actin Stress Fibers in MC
MC, in response to stretch, form intense F-actin stress fibersalong the long axis of the cell (17). This is shown in the leftpanels of Figure 3a, in which F-actin was visualized with rhodaminephalloidin after 20 min of stretch. To confirm the effects ofROCK inhibition on the actin cytoskeleton, we incubated MC withY-27632 at 20 µM for 30 min. This efficiently preventedstretch-induced stress fiber formation. The effects of cytochalasinD (60 min, 200 ng/ml), a cell-permeable agent that disruptsactin organization, are shown for comparison. We have previouslyshown that cytochalasin D abrogates stretch-induced Erk activation(18), and, as seen in Figure 3B, the inhibitory effects of Y-27632are comparable to those of cytochalasin D (60 min, 200 ng/ml).
Figure 3. Rho-kinase inhibition disrupts basal and stretch-induced stress fiber formation. (Left panels) MC exposed to 20-min cyclic stretch display substantially more intense actin stress fiber formation as visualized by confocal microscopy of rhodamine phalloidin-stained F-actin when compared with unstretched MC. (Middle panels) Preincubation with Cytochalasin D, 200 ng/ml profoundly interrupts stress fibers in both stretched and unstretched MC. (Right panels) The ROCK inhibitor Y-27632 at 20 µM also disrupts stress fibers in unstretched cells and prevents the formation of stretch-induced stress fibers. (B) Inhibition of stretch-induced Erk activation was seen after preincubation with either Y-27632 or cytochalasin D, *P < 0.01 versus control; #P < 0.01 versus stretch (n = 3).
Restoration of Cytoskeletal Integrity Prevents Y-27632-Induced Erk Inhibition
Jasplakinolide is a pharmacologic agent that stabilizes theactin cytoskeleton (35). We have previously shown that it canrescue Erk activation in stretched MC in the presence of NOdonors that disrupt the actin cytoskeleton (18). We thereforesought to cement the observation that ROCK inhibition preventedErk activation in response to mechanical stress through cytoskeletaldisruption by using jasplakinolide to stabilize the cytoskeleton.As shown in Figure 4, 50 nM jasplakinolide significantly restoresstretch-induced Erk activation in the presence of Y-27632, indicatingthat the effect of RhoA signaling on Erk is through the actincytoskeleton in this context.
Figure 4. The inhibitory effect of Y-27632 on stretch-induced Erk activation is reversed by cytoskeletal stabilization. MC were incubated with Y-27632, 20 µM for 30 min in the presence or absence of jasplakinolide, 50 nM for 60 min, before stretch for 20 min. Stretch-induced Erk activation is inhibited by Y-27632, and this effect is reversed by cytoskeletal stabilization with jasplakinolide, *P < 0.01 versus control, #P < 0.02 versus stretch and stretch+Y+jas (n = 3).
Cytoskeletal Disruption Does Not Affect Erk Activation in Response to Growth Factors
Y-27632 and cytochalasin D profoundly affected cytoskeletalorganization in stretched MC; we therefore sought to determinewhether inhibition of stretch-induced Erk activation by theseagents occurred in the context of other mitogenic stimuli. Platelet-derivedgrowth factor (PDGF)-BB activates the Erk cascade (36), and50 ng/ml PDGF potently induced Erk activation after 5 min (Figure 5),which was insensitive to Y-27632 (20 µM) and cytochalasinD (200 ng/ml). This supports the notion that signal transductionin response to mechanical strain specifically requires the presenceof an intact cytoskeleton.
Figure 5. Cytoskeletal disruption does not affect growth factor-induced Erk activation. Erk activation is seen in response to platelet-derived growth factor (PDGF)-BB, 50 ng/ml for 5 min in unstretched MC. Cytoskeletal disruption with either Y-27632 (20 µM for 30 min) or cytochalasin D (200 ng/ml for 60 min) has no inhibitory effect.
NO Prevents Stretch-Induced RhoA and Erk Activation
We have previously shown that NO and cGMP inhibit Erk activationthrough cytoskeletal disruption (18). RhoA plays a key rolein actin stress fiber formation (20). Since RhoA inhibitionprevents Erk activation in stretched MC, we hypothesized thatNO and cGMP might modulate the actin cytoskeleton through RhoAinhibition. Indeed, stretch-induced RhoA activation was abrogatedby preincubation of MC with either 8-Br-cGMP (1 mM, 10 min)or the NO donor SNAP (70 µM, 10 min) (Figure 6A). Erkactivation at 20 min was similarly inhibited (Figure 6B), aswe have observed previously (18).
Figure 6. NO and cGMP prevent stretch-induced RhoA and Erk activation. MC were incubated with either the NO donor S-nitroso-N-acetylpenicillamine (SNAP; 70 µM for 10 min) or 8-Br-cGMP (1 mM for 10 min) before stretch. (A) RhoA activity at 1 min is inhibited by both compounds, *P < 0.05 versus control; #P < 0.02 versus stretch (n = 3). (B) Similarly, at 20 min of stretch, Erk activity is attenuated by preincubation with SNAP or 8-Br-cGMP, *P < 0.01 versus control; #P < 0.01 versus stretch (n = 3).
Stretched MC Infected with RhoA-Ala188 Are Resistant to the Effects of NO and cGMP
NO, via cGMP, activates cGK, which may then phosphorylate RhoAon Ser-188 (27,28), thus inhibiting its activation (37). Mutationof the serine residue to alanine prevents cGK-dependent phosphorylationand renders RhoA resistant to the effects of NO (27,28). Weused the RhoA-Ala188 mutant, a kind gift of Dr. G. Loirand (27),to investigate whether the observed inhibition by NO and cGMPon stretch-induced RhoA activation was via a similar mechanism.FLAG was added to the N-terminus, and the construct introducedinto MC using a retroviral system to infect a pooled MC population.MC were infected in parallel with the empty retroviral vectorpBabe. Figure 7A shows successful expression of FLAG-RhoA-A188.MC infected with pBabe and exposed to stretch for 20 min showedactivation of Erk and its inhibition by SNAP and 8-Br-cGMP atthe previously used concentrations (Figure 7B). In contrast,MC expressing the RhoA mutant were resistant to the effectsof both compounds (Figure 7B).
Figure 7. NO and cGMP have no effect on stretch-induced Erk activation in MC infected with RhoA-Ala188. MC were infected with the empty vector pBabe or with the RhoA mutant RhoA-Ala188. This mutant is resistant to NO-cGMP-mediated phosphorylation by cGMP-dependent protein kinase. (A) Successful expression of RhoA-Ala188 is detected by immunoblotting for the epitope FLAG. (B) Treatment of RhoA-Ala188-infected MC with the NO donor SNAP (70 µM for 10 min) or 8-Br-cGMP (1 mM for 10 min) before 20 min of stretch has no effect on Erk activation (*P < 0.02 versus control). In contrast, Erk activation is significantly attenuated by both compounds in MC infected with only pBabe (*P < 0.01 versus control, #P < 0.01 versus stretch; n = 3).
Since RhoA activation by stretch was inhibited by NO and cGMPand RhoA-Ala188 restored stretch-induced Erk activation in thepresence of NO and cGMP, we next examined the effects of NOand cGMP on the actin cytoskeleton in MC infected with the RhoAmutant. The formation of stretch-induced actin stress fibersin empty vector-infected MC was significantly inhibited in thepresence of SNAP and cGMP (Figure 8). In contrast, MC infectedwith Rho-Ala188 displayed intense stress fiber formation inresponse to stretch even in the presence of SNAP and cGMP (Figure 9).
Figure 8. NO and cGMP inhibit stress fiber formation in stretched MC infected with empty vector pBabe. (A) Left panels: MC exposed to 20-min stretch show substantially increased actin stress fiber presence when compared with unstretched MC. Middle panels: 8-Br-cGMP (1 mM, 10 min) has little effect on actin stress fiber formation in quiescent MC, but it significantly inhibits the intense stress fibers formed after stretch. Right panels: Similar effects are seen with SNAP (70 µM, 10 min). (B) The change in stress fibers is quantified by measurement of the F:G actin ratio as outlined in Materials and Methods, *P < 0.01 versus control; #P < 0.01 versus stretch.
Figure 9. NO and cGMP do not inhibit stress fiber formation in stretched MC infected with RhoA-Ala188. (A) Left panels: MC exposed to 20-min stretch show substantially increased actin stress fiber presence when compared with unstretched MC. Middle panels: 8-Br-cGMP (1 mM, 10 min) has little effect on actin stress fiber formation in either stretched or quiescent MC infected with RhoA-Ala188. Right panels: Similarly, SNAP (70 µM, 10 min) also has little effect. (B) The change in stress fibers is quantified by measurement of the F:G actin ratio as outlined in Materials and Methods, *P < 0.01 versus control.
ECM Expression Is Inhibited by Y-27632 in Stretched MC
To relate our observations of RhoA signaling to a functionalevent in progressive glomerular disease, we studied FN messagelevels and protein production in stretched MC with or withoutY-27632. As shown in Figure 10, preincubation with Y-27632,20 µM for 30 min, abrogated the increase in both FN message(by RT-PCR) and protein (by ELISA) seen in MC and supernatantmedia, respectively, after 24 h of stretch. Thus, signalingthrough RhoA, presumably by permitting cytoskeletal organization,permits increased ECM production in stretched MC.
Figure 10. Rho-kinase inhibition abrogates stretch-induced fibronectin (FN) production. MC were stretched for 24 h with or without pretreatment with Y-27632 (20 µM, 30 min). (A) Stretch induces the expression of FN mRNA, normalized to -actin, as assessed by RT-PCR, *P < 0.03 (this increase is abrogated with ROCK inhibition), #P < 0.01 (n = 4). (B) ELISA was used to assay for FN present in the media. This was also increased by stretch at 24 h, *P < 0.01 (similarly decreased with ROCK inhibition), #P < 0.02 (n = 3 in triplicate).
In the subtotally Nx rat, the most well-characterized modelof CRF, increased Pgc is an important determinant of subsequentglomerulosclerosis (1,8). Several studies have shown that L-arginine,orally administered as a precursor for NO production, preventsthe development and progression of renal lesions in this model.We have shown that L-arginine decreased glomerular cell proliferationand endothelin-1 (ET-1) transcript abundance after 2 wk in subtotallyNx rats (2). ET-1 overexpression alone induces development ofglomerulosclerosis (38). Others observed that L-arginine administeredfor 6 wk maintained the GFR and decreased glomerulosclerosisand interstitial fibrosis without affecting BP in subtotallyNx animals (39). Although NO is an intrarenal vasodilator (4),further study indicated that these early beneficial effectswere seen in the absence of reductions in Pgc (5). The effectivenessof L-arginine was also evident when CRF was already established(3 wk after renal ablation) at a creatinine clearance (CrCl)60% of normal. Treatment for 4 wk normalized CrCl and decreasedproteinuria, effects which were maintained to 12 wk (3).
MC responses triggered by increased Pgc have been modeled invitro by the application of cyclic stretch/relaxation. MC exposedto mechanical strain proliferate (9) and produce ECM (10), andboth effects can be linked to stretch-induced activation ofErk (12,15). We have previously shown that NO and its secondmessenger cGMP inhibit stretch-induced Erk activation and consequentnuclear translocation, as well as AP-1 nuclear binding in MC,and demonstrated that these effects of NO occur through disruptionof the actin cytoskeleton (18). Consequently, we sought to elucidatethe mechanism whereby NO disrupts stretch-induced cytoskeletalreorganization and Erk activation.
Mitogenic responses in VSMC in response to stretch are mediatedby integrins (40). Engagement of integrins leads to clusteringand the formation of focal adhesions, which associate with cytoskeletalactin filaments; this may provide a scaffold for the aggregationof signaling molecules and consequent signal transduction (41).In NIH3T3 fibroblasts, integrin-mediated Erk activation inducedby cell adhesion to fibronectin is RhoA-dependent (22). RhoAmediates stress fiber formation through its effector ROCK (21);in stretched MC, actin filaments coalesce and orient themselvesalong the long axis to form intense stress fibers (17). We thushypothesized that RhoA activity mediates stretch-induced stressfiber formation in MC, and that NO disrupts cytoskeletal reorganizationthrough effects on RhoA, thus inhibiting Erk signaling.
In accord with this construct, we first showed that stretchincreases the amount of GTP-bound (active) RhoA at very earlytime points, maximally at 1 min of stretch. We subsequentlyobserved that stretch-induced Erk activation temporally followsand requires RhoA, because inhibition of RhoA with C3 exoenzymeor its downstream effector ROCK with Y-27632 abrogates Erk activation.Similarly, ROCK inhibition disrupts basal and stretch-inducedactin stress fiber formation, as measured in a quantitativeassay. Stabilization of the actin cytoskeleton by preincubationwith jasplakinolide prevented the inhibitory effects of Y-27632on Erk activation. Given that disruption of the actin cytoskeletonwith cytochalasin D also inhibits Erk activation in responseto stretch, the most parsimonious explanation for these dataare that the role of RhoA signaling is to enable Erk cascadeactivation by providing a scaffold for the assembly of signalingmolecules, allowing efficient interaction and initiation ofthese cascades. Indeed, Erk has been shown to interact withactin through a calponin homology domain (42). As we show herethat PDGF-induced Erk activation is not prevented by cytoskeletaldisruption, stimulus specificity in requirement for cytoskeletalscaffolding in Erk signaling exists.
Having shown stretch-induced RhoA activation and dependenceof Erk activation on the integrity of the RhoA-ROCK pathway,we addressed the potential effects of NO on RhoA signaling.We have previously observed that both SNAP and cGMP inhibitstretch-induced Erk activation through cytoskeletal disruption(18). Here, we observe that both SNAP and cGMP fully inhibitedRhoA activation by stretch. NO, through its second messengercGMP and the consequent activation of cGK, can inhibit signalingat several levels of both the RhoA and Erk pathways. Phosphorylationof Raf-1, an upstream kinase in the linear Erk cascade, at ser43by cGK, inhibits growth factor activation of Erk (26). Inhibitionof RhoA itself through phosphorylation on Ser188 (28), as wellas abrogation of downstream RhoA-mediated transcriptional eventsindependently of Ser188 modification are mediated by cGK (25).However, cGK-dependent phosphorylation of Ser188 on RhoA wasable to disrupt basal actin fiber structure in both VSMC andHeLa cells, an effect that could be abrogated by mutating Ser188to Ala188 in RhoA (27,28). Upon phosphorylation at this site,increased Rho-GDI (guanine nucleotide dissociation inhibitor)activity, decreased membrane-associated RhoA (37), and decreasedbinding of RhoA to ROCK are seen (43). We thus sought to furthercharacterize the mechanism whereby NO might inhibit stretch-inducedRhoA signaling by infecting MC with RhoA-Ala188. This mutantreversed the ability of NO and cGMP to prevent Erk activationin stretched MC and abrogated the inhibitory effects of thesecompounds on stretch-induced actin stress fiber formation. Phosphorylationat Ser-188 is thus important in mediating the effects of NOin stretched MC.
Finally, we relate RhoA signaling to an event of functionalimportance in models of increased intraglomerular pressure—ECM production. Most studies of mechanical stress show approximately20% increase in ECM production in MC. We show herein, at botha message and protein level, that induction of FN productionin stretched MC is abrogated by ROCK inhibition, attesting toan important functional role of RhoA.
The data presented here establish that RhoA-mediated actin cytoskeletalorganization is critical to mechanical strain-mediated Erk activation.Furthermore, the effects of NO on this pathway occur throughinhibition of RhoA activity and thus on cytoskeletal organization.Our data here are the first, to our knowledge, to carefullyquantify the magnitude of the stretch effect on actin stressfiber formation in MC.
Recent work has shown that in several hypertensive rat models,the aortic levels of activated RhoA are significantly elevated(44). Furthermore, the glomerulosclerosis produced in rats bychronic NO inhibition with L-NAME can be attenuated by ROCKinhibition with Y-27632 in the absence of BP effects (45). Inconcert with these data, the work presented here lends furtherimpetus to the initiation of studies seeking to establish theutility of NO donors and RhoA inhibitors in the treatment ofprogressive chronic renal disease.
Acknowledgments
J. Krepinsky is supported by a Research Fellowship from Bristol-Myers-Squibb(Canada). A. Ingram and J. Scholey are supported by the CanadianInstitutes of Health Research and Kidney Foundation of Canada(KFoC). D. Tang is supported by KFoC. We thank Dr. G. Loirand(France) for the Rho-Ala188 mutant and B. Calvieri and S. Doylefor invaluable technical assistance with imaging.
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Received for publication March 3, 2003.
Accepted for publication August 16, 2003.
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Top
Abstract
Introduction
) contains 2 AP-1 consensus sequences; therefore, Fos mediates its induction (15). Indeed, stretched MC display increased TGF-
-actin, 1:5000 (Sigma), and monoclonal FLAG 10 µg/ml (Sigma, M2). Membranes were washed three times with TBST and incubated with the appropriate horseradish peroxidase-conjugated antibody in TBST/5% milk for 1 h. After three further washes, the membrane was incubated with LumiGlo reagent and exposed to x-ray film. 
0.02 versus stretch (n = 3). (B) Inhibition of Erk activation is also seen after 48 h of incubation with the RhoA inhibitor C3 exoenzyme (16 µg/ml), *P < 0.02, #P < 0.03 (n = 3).
