1 Institute of Medical Science, 2 Department of Medicine, University of Toronto, Toronto, Ontario, Canada M5S 1A8
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ABSTRACT |
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Endothelin-1 (ET-1)
stimulates glomerular mesangial cell proliferation and extracellular
matrix protein transcription through an ERK1/2-dependent pathway. In
this study, we determined whether ET-1 activation of glomerular
mesangial cell ERK1/2 is mediated through EGF receptor (EGF-R)
transactivation and whether intact caveolae are required. We showed
that ET-1 stimulated tyrosine phosphorylation of the EGF-R in primary
cultured, growth-arrested rat mesangial cells. In response to ET-1,
ERK1/2 phosphorylation was increased by 27 ± 1-fold and attenuated by
AG-1478, a specific EGF-R inhibitor, to 9 ± 1-fold. Moreover, filipin
III and -cyclodextrin, two cholesterol-depleting drugs known to
disrupt caveolae, significantly reduced ET-1-induced phosphorylation of
ERK1/2. In addition, preincubation of mesangial cells with a
myristoylated peptide that binds to the caveolin-1 scaffolding domain
diminished ET-1 activation of ERK1/2. ET-1 caused interaction of
caveolin-1 with phosphorylated ERK1/2 identified by
coimmunoprecipitation. Activation of ERK1/2 and its interaction with
caveolin-1 were reduced by AG-1478,
-cyclodextrin, or inhibition of
PKC. Phosphorylated ERK1/2 localized at focal adhesion complexes along
with phospho-caveolin-1, suggesting specific sites of
compartmentalization of these signaling molecules. Hence, ET-1
activates mesangial cell ERK1/2 predominantly through a pathway involving EGF-R transactivation, leading to a mechanism involving attachment to caveolin-1, presumably in caveolae.
caveolae; epidermal growth factor receptor transactivation
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INTRODUCTION |
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ENDOTHELIN-1 (ET-1) IS A POTENT vasomodulatory mitogen involved 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 predominant extracellular protein collagen IV is mediated in part through PKC and activation of ERK1/2 (22). ET-1 signals through G-protein coupled receptor (GPCR)-mediated PLC hydrolysis of phosphatidylinositol 4,5-bisphosphate to generate inositol 1,4,5-trisphosphate and diacylglycerol, releasing Ca2+ and activating PKC, respectively (11, 49). The exact molecular mechanism linking ET-1 signal transactivation to ERK1/2 activation is not entirely known.
Extensive research has focused on the transactivation of the EGF receptor (EGF-R) in response to GCPR stimuli. Several GPCR agonists stimulate EGF-R transactivation, including lysophosphatidic 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 of Src, PYK2, and Ca2+ mobilization leads to a metalloprotease proteolytic cleavage of the pro-heparin-binding (HB)-EGF precursor to yield the release of a mature ligand, which in turn activates the EGF-R (9, 32). HB-EGF-like growth factor is expressed by mesangial cells and induces cellular proliferation (61). Hence, we postulated that ET-1 signaling and downstream activation of ERK1/2 involves EGF-R transactivation.
Caveolin is an integral protein (21-24 kDa) and the principal component 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 caveolae through immunoblotting of caveolae-enriched membrane fractions, colocalization by using confocal immunofluorescence, or ultrastructural immunogold 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).
Although ET-1 and EGF-Rs and their downstream effector kinases PKC and ERK may be localized within caveolae, as identified in various cell types, the roles of EGF-R transactivation and caveolae in ET-1 stimulation of ERK1/2 have not been identified. In this study, we showed that ET-1 activation of mesangial cell ERK1/2 is mediated through EGF-R transactivation and requires intact caveolae. ET-1 activation of ERK1/2, through the ET-A receptor, is mediated through EGF-R transactivation as AG-1478, a specific EGF-R inhibitor, attenuated ET-1 activation of ERK1/2. Moreover, caveolae-disrupting agents significantly reduced ET-1 stimulation of phosphorylation of ERK1/2. In addition, a myristoylated peptide that binds to the caveolin-1 scaffolding domain attenuated ET-1 activation of ERK1/2. Phosphorylated ERK1/2 interaction with caveolin-1, identified through coimmunoprecipitation, appears to be dependent on EGF-R transactivation, in part PKC dependent, and requiring intact caveolae. Finally, phospho-ERK1/2 localized at attachment complexes along with phospho-caveolin-1, suggesting specific sites of compartmentalization of these signaling molecules.
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MATERIALS AND METHODS |
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Materials
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,Antibodies
The following antibodies were utilized for immunoblotting, immunoprecipitation, and immunofluorescence imaging: phosphorylated and total 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 goat anti-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).Cell Culture
Primary rat mesangial cells were isolated from Sprague-Dawley rat kidney glomeruli as previously described (23). Passages 10-15 were used for all studies. Mesangial cells were grown on thin layer MatriGEL in DMEM containing 20% fetal bovine serum (FBS) to confluence and then growth arrested in 0.5% FBS for 72 h.Western Blot Analysis
Mesangial cells were growth arrested on six-well plates and stimulated 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 were taken for protein assay with Bradford Protein Assay (Bio-Rad). The remaining cell extracts were denatured in 4× sample buffer (0.13 mol/l Tris, 40% glycerol, 8% SDS, 4%Immunoprecipitation
Mesangial cells were growth arrested in 10-cm plates and stimulated with ET-1, EGF, or PMA with or without inhibitors and lysed with ice-cold radioimmunoprecipitation assay (RIPA) buffer containing 50 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/ml leupeptin, vanadate, 1 mmol/l benzamidine, and 50 mM sodium fluoride. Lysates were centrifuged at 14,000 rpm for 10 min, and 300 µg of the supernatant were immunoprecipitated with 10 µg of the indicated A/G-agarose-conjugated antibody overnight at 4°C. Immune complexes were washed with cold RIPA buffer and denatured in Laemmli sample buffer. Immunoprecipitated proteins were resolved by protein electrophoresis on 10-12% SDS-PAGE, transferred to polyvinylidine fluoride membrane, and probed with the indicated antibody.Cellular Fractions
To obtain membrane fractions, mesangial cells grown on 10-cm plates were lysed in ice-cold buffer A containing 50 mmol/l Tris · HCl, pH 7.5, 10 mmol/l EGTA, 2 mmol/l EDTA, 1 mmol/l benzamidine, 1 mmol/l NaF, vanadate, 1 mM phenylmethylsulfonyl fluoride, and 25 µg/ml leupeptin. Cells were disrupted by passage through a 26-gauge needle and centrifuged at 100,000 g for 30 min at 4°C (TL-100, Bechman Instruments Canada, Mississauga, ON). The pellet was resuspended in buffer A plus 1% Triton X-100 and centrifuged at 100,000 g for 30 min. The supernatant was collected as the plasma membrane-enriched fraction.Confocal Immunofluorescence
Mesangial cells were growth arrested on a glass coverslip coated with a thin-layer MatriGEL and fixed with 3.7% formaldehyde. Cells were permeabilized with 0.1% Triton X-100, blocked in goat serum containing 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 imaged with a Zeiss confocal laser-scanning microscope (LSM 410, Dusseldorf, Germany), with excitation and emission wavelengths of 488 and 520 nm.Caveolin-1 Scaffold Domain Peptide
The caveolin-1 scaffolding domain peptide corresponding to amino acids DGIWKASFTTFTVTKYWFYR (41, 42, 50, 52, 54, 59, 73) with an additional myristoylated sequence was synthesized by Biotechnology Service Centre (The Hospital for Sick Children Peptide Synthesis Laboratory, Toronto, ON). The purity of the peptide was >95% as determined by high-pressure liquid chromatography and mass spectroscopy. The cells were incubated with the peptide for 30 min before stimulation with ET-1 and lysed for immunoblotting or immunoprecipitation.Statistical Analysis
All results are expressed as means ± SE. Statistical analysis was performed with InStat 2.01 statistics software (GraphPad, Sacramento, CA). The means of three or more groups were compared by one-way ANOVA. If significance of P < 0.05 was obtained in the ANOVA, the Tukey multiple comparison posttest was applied. ![]() |
RESULTS |
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ET-1 Activation of ERK1/2
EGF-R transactivation and function of caveolae.
To determine whether ET-1 causes transactivation of EGF-R, primary rat
mesangial cells were growth arrested (0.5% FBS for 72 h) and
stimulated with ET-1 (100 nM for 10 min), and cell membrane isolates
were blotted with an antibody directed against an EGF-R-specific Src
kinase phosphorylation site required for subsequent EGF-R activation.
As a control, mesangial cells were also stimulated with EGF (100 ng/ml). Figure 1 shows acute
ET-1-activated EGF-R phosphorylation (by 3.1 ± 0.2-fold) but not to
the same extent as EGF (12.2 ± 0.2-fold). Pretreatment with a
specific EGF-R inhibitor, AG-1478 (0.2 µM for 20 min), reduced
ET-1-induced phosphorylation of EGF-R to 0.6 ± 0.3-fold. When
mesangial cells were preincubated with filipin III or
-cyclodextrin, cholesterol-depleting agents that disrupt
caveolae formation, ET-1 activation of EGF-R was also significantly
reduced 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).
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Direct binding of ERK1/2 with caveolin-1.
Several studies have implicated caveolin-1 as a scaffolding protein
that enables compartmentalization of specific signaling molecules. To
test whether ET-1-induced activation of ERK1/2 involved direct binding
of ERK1/2 to caveolin-1, we immunoprecipitated caveolin-1 and blotted
for phospho-ERK1/2. Figure 6 demonstrates that in the absence of ET-1 treatment, there was little binding of
phospho-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, PMA
stimulated coimmunoprecipitation of phospho-ERK1/2 and caveolin-1. EGF
(50 ng/ml) stimulated the association of phospho-ERK1/2 with caveolin-1
by 51 ± 2-fold, which was markedly attenuated by AG-1478 to 5 ± 2-fold. As a further control, after ET-1 activation, cell lysates were
immunoprecipitated with an anti-phospho-ERK1/2 antibody, and caveolin-1
was found to colocalize with phospho-ERK1/2 on the immunoblot (data not
shown).
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Caveolin-1 Interactions
To study the functional consequences of ERK1/2 binding to caveolin-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 a concentration-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 the coimmunoprecipitation of phospho-ERK1/2 with caveolin-1 in the presence of 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).
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Immunolocalization of Caveolin-1 and Phospho-ERK1/2
Several reports have indicated that caveolin-1 is involved in integrin-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 dispersed throughout the cell, with some localization within focal adhesion sites. However, we found that the intensity of immunofluorescence was not altered in response to ET-1. In contrast, a monoclonal antibody to the phosphospecific caveolin-1 showed an immunoreactivity pattern similar to the monoclonal antibody to vinculin (Fig. 11). Moreover, phospho-ERK1/2 immunoreactivity was found in the cell nucleus as well as in regions that colocalized with vinculin (Fig. 11).
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DISCUSSION |
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In this study, we have demonstrated that ET-1 via ET-A receptor
activation of mesangial cell ERK1/2 is mediated predominantly through a
pathway requiring EGF-R transactivation and intact caveolae. ET-1-induced ERK1/2 phosphorylation was reduced significantly by
AG-1478, a specific EGF-R inhibitor, as well as by two
cholesterol-depleting agents (-cyclodextrin and filipin III) known
to disrupt caveolae. This finding is supported by the recent
observation by Ushio-Fukai et al. (66), who observed that
cholesterol-depleting agents inhibit ANG II-induced transactivation of
EGF-R. We further showed that ET-1 stimulation of ERK1/2 requires the
direct association of the principal protein of caveolae, caveolin-1.
This study is the first to demonstrate that a myristoylated peptide to
the scaffolding domain of caveolin-1 attenuates ET-1 activation of
ERK1/2 in intact cells. Moreover, the sequence of EGF-R
transactivation, caveolin-1 association, and ERK1/2 phosphorylation is
PKC dependent. Finally, confocal imaging patterns of
phospho-caveolin-1, phosphorylated ERK1/2, and vinculin suggest
localization of these signaling molecules at least in part within focal
adhesion attachment sites.
EGF-R transactivation is a novel mechanism to explain the activation of growth-mediated signaling kinases such as ERK1/2 by GPCR. Multiple GPCR-dependent agonists have been shown to act through EGF-R transactivation, including ET-1 (10, 28, 64, 67). Several studies have indicated that ET-1 is a mitogenic stimulus that activates ERK1/2 in mesangial cells (15, 72). Here, we show that ET-1 activation of mesangial cell ERK1/2 depends in part on EGF-R transactivation. ET-1-stimulated phosphorylation of the EGF-R as well as activation of ERK1/2, and both were attenuated by the EGF-R inhibitor AG-1478. Takemura et al. (61) have described the expression of HB-EGF in mesangial cells that was upregulated in experimental glomerulonephritis, a model wherein ERK1/2 is also activated. GPCR-induced transactivation of EGF-R is postulated to be in part mediated through a PKC-dependent mechanism, because the proteolytic 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-R transactivation was solely dependent on PKC (28). Similarly, we previously showed that ET-1 activation of ERK1/2 was PKC dependent and, in this study, that EGF-R phosphorylation by ET-1 was abrogated by inhibition of PKC. Thus the effect of ET-1 on EGF-R transactivation in mesangial cells may be through a PKC mechanism. The attenuation by calphostin C of EGF-R phosphorylation and direct binding of activated ERK1/2 to caveolin-1 is consistent with the action of PKC principally at the EGF-R transactivation event.
In the present study, AG-1478 partially inhibited ET-1 activation of ERK1/2, suggesting alternative pathways of ERK1/2 stimulation by ET-1 in addition to EGF-R transactivation. Certainly, multiple cascades have been shown to stimulate ERK1/2 by ET-1 (36, 49, 57, 58).
We have further extended the understanding of EGF transactivation by a
mesangial cell GPCR signal transduction by showing that intact caveolae
may be necessary to bring the relevant kinases within close proximity
for activation. In a number of cell types, the EGF-R is localized
within caveolae (5, 31, 70) along with its downstream
signaling molecules, including PLC- (68). Caveolae are
enriched with ET receptors (43, 63). As well, the ET-A
receptor and its bound ligand coimmunoprecipitated with caveolin
(8). We have employed cholesterol-depleting agents to
disrupt caveolae to demonstrate that an intact caveola is necessary for
ET-1 transactivation of EGF-R.
-Cyclodextrin has been shown to
decrease 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-1
activation of phospho-ERK1/2.
Biochemical and morphological studies have revealed that ERK1/2, as
well as its upstream kinases, localize within caveolae (34, 35,
38, 46, 74). Although caveolae disruption has been suggested to
cause hyperactivation of ERK1/2 (12, 16, 18), we found
that -cyclodextrin reduced ET-1-induced stimulation of mesangial
cell ERK1/2. In addition, a peptide against the scaffolding domain of
caveolin-1 attenuated ERK1/2 activation and binding to caveolin-1.
Binding of signaling molecules to the caveolin-1 scaffolding domain is
critical for modifying signal transduction (41, 42, 52, 54, 59,
73). In vivo delivery of the caveolin-1 scaffolding domain can
selectively regulate signaling in endothelial cells (4).
Similarly, the caveolae-disrupting agents filipin III or cyclodextrin
attenuate nerve growth factor-induced phosphorylation of ERK1/2 in PC12
cells (46), and a neutralizing antibody to caveolin-1
inhibits shear stress activation of ERK1/2 in vascular smooth muscle
cells (44). In the anti-Thy-1 nephritis rat model,
increased mesangial caveolin-1 protein is accompanied by mesangial
cellular proliferation (62). Cholesterol depletion of
caveolae causes reduced association of Ras, Grb2, and ERK1/2 but does
not 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 in
the cytosol being slower than in the caveolae, suggesting cytosolic ERK
may originate from caveolae (34). Thus whether or not
caveolae subsequently sequester ERK1/2 activity, it appears that in
response 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.
Caveolin has been found to be positively involved in integrin-mediated signaling (55, 69, 71). In agreement with the study by Ushio-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 its interaction with Grb7 as well as its localization near focal adhesions (29, 30).
In summary, we showed that ET-1 activation of ERK1/2 is mediated through EGF-R transactivation, requires intact caveolae, and is regulated by caveolin-1 association through a mechanism that is PKC dependent. Phosphorylated ERK1/2 localized at attachment complexes along with phospho-caveolin-1, suggesting a potential functional link with cell adhesion.
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ACKNOWLEDGEMENTS |
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This study was supported by the Juvenile Diabetes Research International and Canadian Institutes for Health Research and by the University of Toronto Banting and Best Diabetes Centre NOVO Nordisk studentship (H. Hua).
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FOOTNOTES |
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Address for reprint requests and other correspondence: C. I. Whiteside, Rm. 7302, Medical Sciences Bldg., 1 King's College Circle, Toronto, Ontario, Canada M5S 1A8 (E-mail: catharine.whiteside{at}utoronto.ca).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published September 17, 2002;10.1152/ajprenal.00127.2002
Received 1 April 2002; accepted in final form 15 September 2002.
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