Endothelin-1 activates mesangial cell ERK1/2 via EGF-receptortransactivation an - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:32 |只看该作者 |倒序浏览 |打印

作者：HongHua, SnezanaMunk,  Catharine I.Whiteside,作者单位：1 Institute of Medical Science, Department of Medicine, University of Toronto,Toronto, Ontario, Canada M5S 1A8 # ~' i: s- d& }1 T9 q
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      【摘要】
   Endothelin-1 (ET-1)stimulates glomerular mesangial cell proliferation and extracellularmatrix protein transcription through an ERK1/2-dependent pathway. Inthis study, we determined whether ET-1 activation of glomerularmesangial cell ERK1/2 is mediated through EGF receptor (EGF-R)transactivation and whether intact caveolae are required. We showedthat ET-1 stimulated tyrosine phosphorylation of the EGF-R in primarycultured, growth-arrested rat mesangial cells. In response to ET-1,ERK1/2 phosphorylation was increased by 27 ± 1-fold and attenuated byAG-1478, a specific EGF-R inhibitor, to 9 ± 1-fold. Moreover, filipinIII and -cyclodextrin, two cholesterol-depleting drugs known todisrupt caveolae, significantly reduced ET-1-induced phosphorylation ofERK1/2. In addition, preincubation of mesangial cells with amyristoylated peptide that binds to the caveolin-1 scaffolding domaindiminished ET-1 activation of ERK1/2. ET-1 caused interaction ofcaveolin-1 with phosphorylated ERK1/2 identified bycoimmunoprecipitation. Activation of ERK1/2 and its interaction withcaveolin-1 were reduced by AG-1478, -cyclodextrin, or inhibition ofPKC. Phosphorylated ERK1/2 localized at focal adhesion complexes alongwith phospho-caveolin-1, suggesting specific sites ofcompartmentalization of these signaling molecules. Hence, ET-1activates mesangial cell ERK1/2 predominantly through a pathway involving EGF-R transactivation, leading to a mechanism involving attachment to caveolin-1, presumably in caveolae. 8 L3 q) b- q/ s1 Z
      【关键词】 caveolae epidermal growth factor receptor transactivation/ ]1 ~- g  J  K5 B2 x
   INTRODUCTION

ENDOTHELIN-1 (ET-1) IS A POTENT vasomodulatory mitogeninvolved in the pathogenesis of glomerular disease ( 1, 3, 24, 27, 40, 45, 49 ). ET-1 induces mesangial cell proliferation ( 15, 20, 72 ), and expression of the predominantextracellular protein collagen IV is mediated in part through PKC andactivation of ERK1/2 ( 22 ). ET-1 signals through G-proteincoupled receptor (GPCR)-mediated PLC hydrolysis of phosphatidylinositol4,5-bisphosphate to generate inositol 1,4,5-trisphosphate anddiacylglycerol, releasing Ca 2  and activating PKC,respectively ( 11, 49 ). The exact molecular mechanismlinking ET-1 signal transactivation to ERK1/2 activation is notentirely known.
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Extensive research has focused on the transactivation of the EGFreceptor (EGF-R) in response to GCPR stimuli. Several GPCR agonists stimulate EGF-R transactivation, includinglysophosphatidic acid, thrombin ( 2, 25 ), ANG II ( 14, 37 ), arginine vasopressin ( 19 ), and ET-1( 10 ). More recently, a novel mechanism of EGF-R transactivation has been proposed whereby GPCR-mediated activation ofSrc, PYK2, and Ca 2  mobilization leads to a metalloproteaseproteolytic cleavage of the pro-heparin-binding (HB)-EGF precursor toyield the release of a mature ligand, which in turn activates the EGF-R( 9, 32 ). HB-EGF-like growth factor is expressed bymesangial cells and induces cellular proliferation ( 61 ).Hence, we postulated that ET-1 signaling and downstream activation ofERK1/2 involves EGF-R transactivation.; N# \9 D% }8 N2 p" D

Caveolin is an integral protein (21-24 kDa) and the principalcomponent of caveolae, which are flask-shaped cholesterol-enriched membrane invaginations ( 17, 51, 52 ). Caveolae function in vesicular and cholesterol trafficking as well as compartmentalization of specific signaling cascades ( 42, 50, 56, 60 ).Localization of signaling proteins has been identified within caveolaethrough immunoblotting of caveolae-enriched membrane fractions,colocalization by using confocal immunofluorescence, or ultrastructuralimmunogold labeling. These include heterotrimeric G-protein( 13 ), EGF-R ( 38 ), ET-1 receptor( 8 ), components of the ERK1/2 MAPK pathway ( 33-35, 38, 46, 74 ), and multiple PKC isozymes( 26, 39, 48 ).* D0 C) B/ X! L+ S
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Although ET-1 and EGF-Rs and their downstream effector kinases PKC andERK may be localized within caveolae, as identified in various celltypes, the roles of EGF-R transactivation and caveolae in ET-1stimulation of ERK1/2 have not been identified. In this study, weshowed that ET-1 activation of mesangial cell ERK1/2 is mediatedthrough EGF-R transactivation and requires intact caveolae. ET-1activation of ERK1/2, through the ET-A receptor, is mediated throughEGF-R transactivation as AG-1478, a specific EGF-R inhibitor,attenuated ET-1 activation of ERK1/2. Moreover, caveolae-disruptingagents significantly reduced ET-1 stimulation of phosphorylation ofERK1/2. In addition, a myristoylated peptide that binds to thecaveolin-1 scaffolding domain attenuated ET-1 activation of ERK1/2.Phosphorylated ERK1/2 interaction with caveolin-1, identified throughcoimmunoprecipitation, appears to be dependent on EGF-Rtransactivation, in part PKC dependent, and requiring intact caveolae.Finally, phospho-ERK1/2 localized at attachment complexes along withphospho-caveolin-1, suggesting specific sites of compartmentalizationof these signaling molecules./ V1 ^' y8 _2 M$ b8 B

MATERIALS AND METHODS, ]6 ~, l4 h0 o- h5 B0 v, y$ b
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Materials
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The following materials were used: DMEM (GIBCO Life Sciences),fetal bovine serum (Winsent), MatriGEL (BD Biosciences, Bedford, MA),ET-1, EGF, PMA, filipin III (Sigma, St. Louis, MO), calphostin C,AG-1478, -cyclodextrin (Calbiochem, San Diego, CA), enhanced chemiluminescene (KPL, Gaithersburg, MD), and immobilon polyvinylidine fluoride membranes (Millipore, Bedford, MA).9 L1 {& B- x& z
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Antibodies( F# {, _; D6 Q; A

The following antibodies were utilized for immunoblotting,immunoprecipitation, and immunofluorescence imaging: phosphorylated andtotal ERK1/2, phosphorylated EGF-R (Cell Signaling Technology, Beverly,MA), caveolin-1, phosphorylated caveolin-1 (BD Transduction Labs),A/G-agarose-conjugated caveolin-1 (Santa Cruz, Santa Cruz, CA),vinculin (Serotec, Oxford, UK), horseradish peroxidase-labeled goatanti-rabbit IgG (Bio-Rad, Hercules, CA), horseradish peroxidase-labeled goat anti-mouse IgG, and FITC-conjugated goat anti-mouse IgG, rhodamine-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch).
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Cell Culture8 X1 ?- p9 i( Y4 E0 s% H' g

Primary rat mesangial cells were isolated from Sprague-Dawleyrat kidney glomeruli as previously described ( 23 ). Passages 10-15 were used for all studies. Mesangialcells were grown on thin layer MatriGEL in DMEM containing 20% fetalbovine serum (FBS) to confluence and then growth arrested in 0.5% FBSfor 72 h.0 K, c& _$ C  n+ Z( E- D
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Western Blot Analysis
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Mesangial cells were growth arrested on six-well plates andstimulated with ET-1 (100 nM for 10 min), EGF (50-200 ng/ml for 10 min), and PMA (100 nM for 10 min) with or without indicated inhibitors.Cellular protein was extracted with 2× Laemmli sample buffer (0.13 mol/l Tris-base, pH 6.8, 20% glycerol, and 4% SDS). Aliquots weretaken for protein assay with Bradford Protein Assay (Bio-Rad). Theremaining cell extracts were denatured in 4× sample buffer (0.13 mol/lTris, 40% glycerol, 8% SDS, 4% -mercaptoethanol, and 0.02%bromophenol blue). Equal amounts of protein were separated by SDS-PAGEat 120 V for 1-2 h. The protein was transferred to Immobilonpolyvinylidine fluoride membranes (Millipore) overnight at 4°C intransfer buffer (25 mmol/l Tris-base, 192 mmol/l glycine, pH 8.3, and20% methanol). Membranes were blocked in 5% skim milk powder in Trisbuffer (pH 8) containing 0.05% Tween-20 or 5% BSA and probed with theindicated antibody.
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Immunoprecipitation

Mesangial cells were growth arrested in 10-cm plates andstimulated with ET-1, EGF, or PMA with or without inhibitors and lysedwith ice-cold radioimmunoprecipitation assay (RIPA) buffer containing50 mM HEPES, 5 mM EDTA, 50 mM NaCl, pH 7.4, 1% Triton X-100, 10 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, 10 µg/mlleupeptin, vanadate, 1 mmol/l benzamidine, and 50 mM sodium fluoride.Lysates were centrifuged at 14,000 rpm for 10 min, and 300 µg of thesupernatant were immunoprecipitated with 10 µg of the indicatedA/G-agarose-conjugated antibody overnight at 4°C. Immune complexeswere washed with cold RIPA buffer and denatured in Laemmli samplebuffer. Immunoprecipitated proteins were resolved by proteinelectrophoresis on 10-12% SDS-PAGE, transferred to polyvinylidinefluoride membrane, and probed with the indicated antibody.' J2 h* o) }2 n) o# a& _3 E' o

Cellular Fractions

To obtain membrane fractions, mesangial cells grown on 10-cmplates were lysed in ice-cold buffer A containing 50 mmol/lTris · HCl, pH 7.5, 10 mmol/l EGTA, 2 mmol/lEDTA, 1 mmol/l benzamidine, 1 mmol/l NaF, vanadate, 1 mMphenylmethylsulfonyl fluoride, and 25 µg/ml leupeptin. Cells weredisrupted by passage through a 26-gauge needle and centrifuged at100,000 g for 30 min at 4°C (TL-100, Bechman InstrumentsCanada, Mississauga, ON). The pellet was resuspended in bufferA plus 1% Triton X-100 and centrifuged at 100,000 g for 30 min. The supernatant was collected as the plasmamembrane-enriched fraction.1 p3 o2 e; |, O9 x# e

Confocal Immunofluorescence7 L% s7 j1 O8 V1 G4 B8 ?
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Mesangial cells were growth arrested on a glass coverslip coatedwith a thin-layer MatriGEL and fixed with 3.7% formaldehyde. Cellswere permeabilized with 0.1% Triton X-100, blocked in goat serumcontaining 0.1% BSA, and incubated with the indicated antibodies. FITC-conjugated goat anti-mouse IgG or rhodamine-conjugated goat anti-rabbit IgG were used as secondary antibodies. Cells were imagedwith a Zeiss confocal laser-scanning microscope (LSM 410, Dusseldorf,Germany), with excitation and emission wavelengths of 488 and 520 nm.
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Caveolin-1 Scaffold Domain Peptide

The caveolin-1 scaffolding domain peptide corresponding to aminoacids DGIWKASFTTFTVTKYWFYR ( 41, 42, 50, 52, 54, 59, 73 )with an additional myristoylated sequence was synthesized byBiotechnology Service Centre (The Hospital for Sick Children PeptideSynthesis Laboratory, Toronto, ON). The purity of the 95% as determined by high-pressure liquid chromatography and massspectroscopy. The cells were incubated with the peptide for 30 minbefore stimulation with ET-1 and lysed for immunoblotting or immunoprecipitation.3 p! S9 M6 C! i8 ~9 M, T

Statistical Analysis0 z1 ~- d5 s. m$ W0 c* c& x& s& y' i7 v
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All results are expressed as means ± SE. Statisticalanalysis was performed with InStat 2.01 statistics software (GraphPad, Sacramento, CA). The means of three or more groups were compared byone-way ANOVA. If significance of P the ANOVA, the Tukey multiple comparison posttest was applied.  [6 h, o5 w) o+ N
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RESULTS5 d# V; v2 |0 A: _

ET-1 Activation of ERK1/2' L6 }, b$ h4 Q$ ]7 l, J

EGF-R transactivation and function of caveolae. To determine whether ET-1 causes transactivation of EGF-R, primary ratmesangial cells were growth arrested (0.5% FBS for 72 h) andstimulated with ET-1 (100 nM for 10 min), and cell membrane isolateswere blotted with an antibody directed against an EGF-R-specific Srckinase phosphorylation site required for subsequent EGF-R activation.As a control, mesangial cells were also stimulated with EGF (100 ng/ml). Figure 1 shows acuteET-1-activated EGF-R phosphorylation (by 3.1 ± 0.2-fold) but not tothe same extent as EGF (12.2 ± 0.2-fold). Pretreatment with aspecific EGF-R inhibitor, AG-1478 (0.2 µM for 20 min), reducedET-1-induced phosphorylation of EGF-R to 0.6 ± 0.3-fold. Whenmesangial cells were preincubated with filipin III or -cyclodextrin, cholesterol-depleting agents that disruptcaveolae formation, ET-1 activation of EGF-R was also significantlyreduced to 2 ± 0.2 and 1.6 ± 0.05-fold, respectively (Fig. 1 ).Furthermore, inhibition of PKC with calphostin C (1 µM for 1 h)also inhibited EGF-R transactivation to 0.9 ± 0.3-fold (Fig. 1 ).

Fig. 1. Endothelin-1 (ET-1)-induced transactivation of the EGFreceptor (EGF-R) and effect of cholesterol depletion. Mesangial cellswere growth arrested for 72 h, pretreated with AG-1478 (AG; 0.2 µM for 20 min), a specific EGF-R inhibitor, filipin III (fil; 5 µg/ml for 1 h), -cyclodextrin (cyc; 10 mM for 1 h), orcalphostin C (Cal C; 1 µM for 1 h) stimulated with ET-1 or EGF,and cell membrane lysates were immunoblotted with ananti-phosphorylated EGF-R antibody. C, control. * P n = 4.

To ascertain the role of EGF-R transactivation in ET-1 activation ofERK1/2, mesangial cells were stimulated with ET-1 (100 nM for 10 min)in the absence or presence of AG-1478. Figure 2 illustrates that acute ET-1 stimulatedERK1/2 phosphorylation by 27 ± 1-fold. AG-1478 at 0.2 µM (20 min)significantly decreased ET-1 activation of ERK1/2 to 9 ± 1-fold. Theeffect of AG-1478 at 2 µM was not significantly different from 0.2 µM; therefore, the lower concentration was used throughout all theremaining experiments. As a control, mesangial cells werestimulated with EGF and total cell lysates were probed forphospho-ERK/12. EGF (50-200 ng/ml) stimulated ERK1/2phosphorylation by 20 ± 3-fold in mesangial cells. This responsewas completely abolished by AG-1478 (Fig. 2 B )., n4 I5 s+ q# {# m  _& m4 T

Fig. 2. Effect of AG-1478 on ET-1 and EGF activation of phospho-ERK1/2. A : mesangial cells were growth arrested and in some casespretreated with AG-1478 (0.2 and 2 µM for 20 min) and stimulated withET-1 (100 nM for 10 min). Shown is a representative blot of phospho-and total ERK1/2. The corresponding graph depicts densitometricanalysis of results of three experiments. B : mesangial cellswere pretreated with AG-1478 (0.2 µM) and stimulated with varyingconcentrations of EGF (50-200 ng/ml for 10 min).* P0 E& G8 e4 g& C3 e) @" H

To determine the role of intact caveolae in ET-1 activation of ERK1/2,mesangial cells were pretreated with -cyclodextrin (10 mM for 1 h) or filipin III (5 µg/ml for 1 h). ET-1 stimulated ERK1/2phosphorylation to 33 ± 3-fold, which was significantly inhibited by -cyclodextrin (17 ± 2-fold) (Fig. 3 )or filipin III (11 ± 3-fold). Interestingly, -cyclodextrin didnot statistically decrease EGF activation of ERK1/2 (Fig. 4 ). Thus ET-1 transactivation of EGF-Rleading to ERK activation may operate through a different signalingpathway compared with the mechanism activated by EGF binding to itsreceptor.

Fig. 3. Effect of cholesterol depletion on ET-1 activation ofphosphorylated ERK1/2. Mesangial cells were stimulated with ET-1 (100 nM for 10 min) with or without -cyclodextrin (10 mM for 1 h) orfilipin III (5 µg/ml for 1 h) and immunoblotted withphosphospecific and total ERK1/2 antibodies. Densitometric analysis ofat least 3 separate experiments was calculated. Values are foldincrease over control, means ± SE. * P
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Fig. 4. Effect of cholesterol depletion on EGF activation ofphosphorylated ERK1/2. Mesangial cells were stimulated with EGF (100 ng/ml for 10 min) with or without -cyclodextrin (10 mM for 1 h)and immunoblotted with phosphospecific and total ERK1/2 antibodies.Densitometric analysis of 5 separate experiments was calculated. Theresults are presented as fold increase over control.

We further show that ET-1 activation of ERK is most likely through theET-A receptor. BQ-123, the ET-A receptor blocker, reduced ET-1-inducedERK1/2 activation by 65% (0.1 µM) (Fig. 5 ). Conversely, the ET-B receptor blockerBQ-378 had insignificant effects on the phosphorylation of ERK by ET-1.0 y4 s) G# _) ?& |

Fig. 5. ET-A receptor regulates ET-1 stimulation of ERK1/2.Mesangial cells were pretreated with the ET-1 receptor inhibitor BQ-123(0.1 or 2 µM for 1 h) or BQ-378 and the ET-B receptor inhibitor(0.1 or 2 µM for 1 h) and stimulated with ET-1 (100 nM for 10 min). Cell lysates were immunoblotted for phospho- and total ERK1/2.Phospho-ERK1/2 is illustrated. * P0 V6 p: h. F, E$ Q

Direct binding of ERK1/2 with caveolin-1. Several studies have implicated caveolin-1 as a scaffolding proteinthat enables compartmentalization of specific signaling molecules. Totest whether ET-1-induced activation of ERK1/2 involved direct bindingof ERK1/2 to caveolin-1, we immunoprecipitated caveolin-1 and blottedfor phospho-ERK1/2. Figure 6 demonstrates that in the absence of ET-1 treatment, there was little binding ofphospho-ERK1/2 to caveolin-1. After acute exposure to ET-1, phospho-ERK1/2 coimmunoprecipiatated with caveolin-1 (32 ± 3-fold). Both AG-1478 and -cyclodextrin reduced this association to 6 ± 2 and 13 ± 3-fold, respectively. Furthermore, inhibition of PKC with calphostin C also attenuated phospho-ERK1/2 binding to caveolin-1 to 13 ± 3-fold in response to ET-1. In the same manner, PMAstimulated coimmunoprecipitation of phospho-ERK1/2 and caveolin-1. EGF(50 ng/ml) stimulated the association of phospho-ERK1/2 with caveolin-1by 51 ± 2-fold, which was markedly attenuated by AG-1478 to 5 ± 2-fold. As a further control, after ET-1 activation, cell lysates wereimmunoprecipitated with an anti-phospho-ERK1/2 antibody, and caveolin-1was found to colocalize with phospho-ERK1/2 on the immunoblot (data notshown).; v4 c) s  e* f+ _. [! `& m
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Fig. 6. ET-1-stimulated coimmunoprecipitation of phospho-ERK1/2and caveolin-1. Mesangial cells were cultured on 10-cm plates,stimulated with ET-1 or EGF, in some cases with specific inhibitors,and lysed in radioimmunoprecipitation assay buffer. Cell lysates wereimmunoprecipitated with caveolin-1 antibody (IP:CAV) and immunoblottedwith phospho-ERK1/2 (IB:pERK) and caveolin-1 antibodies (IB:CAV). Top : representative immunoblot of phospho-ERK1/2immunocomplex. Mesangial cells were treated with AG-1478 (0.2 µM for20 min), -cyclodextrin (10 mM for 1 h), or calphostin C (1 µMfor 1 h). In some cases, mesangial cells were stimulated with EGF(50 ng/ml for 10 min). Also shown is the representative blot of thesame membrane reprobed for caveolin-1. Bottom : densitometricanalysis of at least 3 separate experiments shown as means ± SE.* P P

To test whether the EGF-R localized with caveolin-1, cell lysates wereimmunoprecipitated with a caveolin-1 antibody and blotted with anantibody to phosphorylated EGF-R. Although the EGF-Rcoimmunoprecipitated with caveolin-1, the amount did not change withET-1 transactivation of the EGF-R (Fig. 7 ).
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Fig. 7. ET-1 does not increase coimmunoprecipitation ofcaveolin-1 and EGF-R. Mesangial cells were cultured on 10-cm plates,stimulated with ET-1 or EGF, in some cases with specific inhibitors,and lysed in radioimmunoprecipitation assay buffer. Cell lysates wereimmunoprecipitated with caveolin-1 antibody (IP: Cav) and immunoblottedwith phospho-EGF-R or caveolin antibody (IB: pEGF, Cav). Shown is arepresentative blot of 3 experiments.( W/ P( r8 K7 ?0 ~' w
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Caveolin-1 Interactions- Y( y8 i8 h# n; e0 I6 L% j

To study the functional consequences of ERK1/2 binding tocaveolin-1, we used a myristoylated synthetic peptide that specifically binds to the caveolin-1 scaffolding domain, designated as myristoylated caveolin-1 scaffolding domain (mCSD) peptide. This domain has been determined to be responsible for caveolin-1 interaction with signaling molecules ( 41, 42, 52, 54, 59, 73 ). Mesangial cells were treated with different concentrations of mCSD peptide for 30 min and stimulated with ET-1. Figure 8 illustrates that mCSD peptide attenuated ET-1 activation of ERK1/2 in aconcentration-dependent manner (ET-1 alone, 28 ± 2-fold;ET-1 mCSD, 5 µM, 22 ± 2-fold; 10 µM, 15 ± 3-fold; and 20 µM, 9 ± 1-fold). Furthermore, mCSD peptide also prevented thecoimmunoprecipitation of phospho-ERK1/2 with caveolin-1 in the presenceof ET-1 (ET-1, 25 ± 4-fold; ET-1 mCSD, 10 µM, 8 ± 2-fold;and ET-1 mCSD, 20 µM, 6 ± 1-fold) (Fig. 9 ).

Fig. 8. Effect of myristoylated caveolin-1 scaffolding domain(mCSD) peptide on ERK1/2 activation in response to ET-1. Mesangialcells were pretreated with different concentrations of mCSD peptide(5-20 µM) for 30 min and stimulated with ET-1, and total celllysates were probed for phospho- and total ERK1/2. Shown is arepresentative blot of mesangial cells treated with mCSD peptide andprobed for ERK1/2 with the densitometric analysis of at least 3 separate experiments presented as means ± SE fold increase overcontrol. * P3 P1 W9 W4 Z* F+ h8 n
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Fig. 9. The mCSD peptide attenuated ET-1-inducedERK1/2-caveolin-1 immunocomplex formation. Mesangial cells were treatedwith different concentrations of mCSD peptide and stimulated with ET-1,and cell lysates were immunoprecipitated with caveolin-1 antibody andprobed for phospho-ERK1/2. Shown is a representative blot and graphicpresented as means ± SE. * P; d% R+ U" T9 q- Y1 V

Immunolocalization of Caveolin-1 and Phospho-ERK1/2

Several reports have indicated that caveolin-1 is involved inintegrin-mediated signaling ( 7, 69, 71 ). We performed dual-channel confocal imaging to determine whether mesangial cell caveolin-1 is localized within focal adhesion sites. Figure 10 illustrates that caveolin-1,immunostained with a polyclonal primary antibody, is found dispersedthroughout the cell, with some localization within focal adhesionsites. However, we found that the intensity of immunofluorescence wasnot altered in response to ET-1. In contrast, a monoclonal antibody tothe phosphospecific caveolin-1 showed an immunoreactivity patternsimilar to the monoclonal antibody to vinculin (Fig. 11 ). Moreover, phospho-ERK1/2immunoreactivity was found in the cell nucleus as well as in regionsthat colocalized with vinculin (Fig. 11 ).' h/ R. |+ [# R
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Fig. 10. Caveolin-1 is immunolocalized near focal adhesion sites. Mesangialcells were cultured on glass coverslips and processed for confocalimmunofluorescence as described in MATERIALS AND METHODS. A-C : confocal immunofluorescence of vinculin visualizedwith FITC-conjugated secondary antibody and caveolin-1 visualized withrhodamine-conjugated secondary antibody in the absence of ET-1stimulation. D-F : ET-1 (10 min)stimulation.

Fig. 11. Phospho-caveolin-1 and phospho-ERK1/2 show similarimmunoreactivity to vinculin. A and B : confocalimage of mesangial cells treated with and without ET-1 and labeled withphospho-caveolin-1 antibody. C : confocal image ofmesangial cells in the absence of ET-1 and labeled withrhodamine-conjugated phospho-ERK1/2 antibody. D-F :confocal image of mesangial cells treated with ET-1 (10 min) and doublelabeled with rhodamine-conjugated phospho-ERK1/2 antibody andFITC-conjugated vinculin.

DISCUSSION
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In this study, we have demonstrated that ET-1 via ET-A receptoractivation of mesangial cell ERK1/2 is mediated predominantly through apathway requiring EGF-R transactivation and intact caveolae. ET-1-induced ERK1/2 phosphorylation was reduced significantly byAG-1478, a specific EGF-R inhibitor, as well as by twocholesterol-depleting agents ( -cyclodextrin and filipin III) knownto disrupt caveolae. This finding is supported by the recentobservation by Ushio-Fukai et al. ( 66 ), who observed thatcholesterol-depleting agents inhibit ANG II-induced transactivation ofEGF-R. We further showed that ET-1 stimulation of ERK1/2 requires thedirect association of the principal protein of caveolae, caveolin-1.This study is the first to demonstrate that a myristoylated peptide tothe scaffolding domain of caveolin-1 attenuates ET-1 activation ofERK1/2 in intact cells. Moreover, the sequence of EGF-Rtransactivation, caveolin-1 association, and ERK1/2 phosphorylation isPKC dependent. Finally, confocal imaging patterns ofphospho-caveolin-1, phosphorylated ERK1/2, and vinculin suggestlocalization of these signaling molecules at least in part within focaladhesion attachment sites., m% {7 }7 v4 X2 p/ |/ ?. ]: X

EGF-R transactivation is a novel mechanism to explain the activation ofgrowth-mediated signaling kinases such as ERK1/2 by GPCR. MultipleGPCR-dependent agonists have been shown to act through EGF-Rtransactivation, including ET-1 ( 10, 28, 64, 67 ). Severalstudies have indicated that ET-1 is a mitogenic stimulus that activatesERK1/2 in mesangial cells ( 15, 72 ). Here, we show thatET-1 activation of mesangial cell ERK1/2 depends in part on EGF-Rtransactivation. ET-1-stimulated phosphorylation of the EGF-R as wellas activation of ERK1/2, and both were attenuated by the EGF-Rinhibitor AG-1478. Takemura et al. ( 61 ) have described theexpression of HB-EGF in mesangial cells that was upregulated inexperimental glomerulonephritis, a model wherein ERK1/2 is alsoactivated. GPCR-induced transactivation of EGF-R is postulated to be inpart mediated through a PKC-dependent mechanism, because theproteolytic cleavage of HB-EGF was shown to be activated by PKC( 47 ). ANG II has been shown to phosphorylate EGF-R in a PKC- and metalloprotease-dependent manner in mesangial cells( 65 ). In cardiomyocytes, ET-1-stimulated EGF-Rtransactivation was solely dependent on PKC ( 28 ).Similarly, we previously showed that ET-1 activation of ERK1/2 was PKCdependent and, in this study, that EGF-R phosphorylation by ET-1 wasabrogated by inhibition of PKC. Thus the effect of ET-1 on EGF-Rtransactivation in mesangial cells may be through a PKC mechanism. Theattenuation by calphostin C of EGF-R phosphorylation and direct bindingof activated ERK1/2 to caveolin-1 is consistent with the action of PKCprincipally at the EGF-R transactivation event.

In the present study, AG-1478 partially inhibited ET-1 activation ofERK1/2, suggesting alternative pathways of ERK1/2 stimulation by ET-1in addition to EGF-R transactivation. Certainly, multiple cascades havebeen shown to stimulate ERK1/2 by ET-1 ( 36, 49, 57, 58 ).* I& G' v# D! ~0 f0 z

We have further extended the understanding of EGF transactivation by amesangial cell GPCR signal transduction by showing that intact caveolaemay be necessary to bring the relevant kinases within close proximityfor activation. In a number of cell types, the EGF-R is localizedwithin caveolae ( 5, 31, 70 ) along with its downstreamsignaling molecules, including PLC- ( 68 ). Caveolae areenriched with ET receptors ( 43, 63 ). As well, the ET-Areceptor and its bound ligand coimmunoprecipitated with caveolin( 8 ). We have employed cholesterol-depleting agents todisrupt caveolae to demonstrate that an intact caveola is necessary forET-1 transactivation of EGF-R. -Cyclodextrin has been shown todecrease total cellular cholesterol levels, caveolin mRNA, and protein( 21 ) as well as cause the loss of morphologically recognizable caveolae determined by electron microscopy in Madin-Darby canine kidney cells and vascular smooth muscle cells ( 6, 21, 53, 66 ). Both filipin III and -cyclodextrin significantly attenuate ET-1-induced tyrosine phosphorylation of EGF-R, ET-1-induced coimmunoprecipitaion of phosphorylated ERK1/2 with caveolin-1, and ET-1activation of phospho-ERK1/2.
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Biochemical and morphological studies have revealed that ERK1/2, aswell as its upstream kinases, localize within caveolae ( 34, 35, 38, 46, 74 ). Although caveolae disruption has been suggested tocause hyperactivation of ERK1/2 ( 12, 16, 18 ), we foundthat -cyclodextrin reduced ET-1-induced stimulation of mesangialcell ERK1/2. In addition, a peptide against the scaffolding domain ofcaveolin-1 attenuated ERK1/2 activation and binding to caveolin-1.Binding of signaling molecules to the caveolin-1 scaffolding domain iscritical for modifying signal transduction ( 41, 42, 52, 54, 59, 73 ). In vivo delivery of the caveolin-1 scaffolding domain canselectively regulate signaling in endothelial cells ( 4 ).Similarly, the caveolae-disrupting agents filipin III or cyclodextrinattenuate nerve growth factor-induced phosphorylation of ERK1/2 in PC12cells ( 46 ), and a neutralizing antibody to caveolin-1inhibits shear stress activation of ERK1/2 in vascular smooth musclecells ( 44 ). In the anti-Thy-1 nephritis rat model,increased mesangial caveolin-1 protein is accompanied by mesangialcellular proliferation ( 62 ). Cholesterol depletion ofcaveolae causes reduced association of Ras, Grb2, and ERK1/2 but doesnot prevent EGF-induced hyperactivation of ERK in the caveolae( 16 ). PDGF stimulates ERK1/2 activation in intact human fibroblasts or isolated caveolae with the kinetics of ERK activation inthe cytosol being slower than in the caveolae, suggesting cytosolic ERKmay originate from caveolae ( 34 ). Thus whether or notcaveolae subsequently sequester ERK1/2 activity, it appears that inresponse to certain agonists, localization of ERK1/2 within caveolae is required for its activation. Herein, we further demonstrate a requirement for a direct association of active ERK1/2 with caveolin-1.3 N; E4 T# l0 V7 o# G

Caveolin has been found to be positively involved in integrin-mediatedsignaling ( 55, 69, 71 ). In agreement with the study byUshio-Fukai et al. ( 66 ), we also found that phosphorylated caveolin-1 localized at focal adhesion complexes. Moreover, the downstream target of EGF transactivation, phosphorylated ERK1/2, colocalized with vinculin, suggesting a possible mechanism of caveolae-regulated ERK1/2 activation involving integrin binding. Phosphorylation of caveolin-1 on tyrosine 14 by Src kinase leads to itsinteraction with Grb7 as well as its localization near focal adhesions( 29, 30 ).2 n& l! C/ V7 \4 j9 n

In summary, we showed that ET-1 activation of ERK1/2 is mediatedthrough EGF-R transactivation, requires intact caveolae, and isregulated by caveolin-1 association through a mechanism that is PKCdependent. Phosphorylated ERK1/2 localized at attachment complexesalong with phospho-caveolin-1, suggesting a potential functional linkwith cell adhesion.

ACKNOWLEDGEMENTS" Z* R( f. o5 N  T
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This study was supported by the Juvenile Diabetes ResearchInternational and Canadian Institutes for Health Research and by theUniversity of Toronto Banting and Best Diabetes Centre NOVO Nordiskstudentship (H. Hua).
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