1 Institut de recherches cliniques de Montréal, Montreal H2W 1R7; 2 Department of Pharmacology, Faculty of Medicine, University of Montreal; and 3 Faculty of Pharmacy, University of Montreal, Montreal Quebec, Canada H3C 3J7
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ABSTRACT |
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The epidermal growth factor receptor (EGFR) was recently identified as a signal transducer of G protein-coupled receptors (GPCRs). In this study, we have examined the contribution of EGFR transactivation to the growth-promoting effect of GPCRs on vascular smooth muscle cells. Activation of the Gq-coupled ANG II receptor or Gi-coupled lysophosphatidic acid receptor resulted in increased tyrosine phosphorylation and activation of EGFR. Specific inhibition of EGFR kinase activity by tyrphostin AG-1478 or expression of a dominant-negative EGFR mutant abolished this response. Importantly, inhibition of EGFR function strongly attenuated the global stimulation of protein synthesis by GPCR agonists in vitro in cultured aortic smooth muscle cells and in vivo in the rat aorta and in small resistance arteries. The growth inhibition was associated with a marked reduction of extracellular signal-regulated kinase and phosphoinositide 3-kinase pathway activity and the resulting suppression of eukaryotic translation initiation factor 4E and 4E binding protein 1 phosphorylation. Our results demonstrate that EGFR transactivation is a physiologically relevant action of GPCRs linked to translational control and protein synthesis.
smooth muscle cell; translation; signal transduction; epidermal growth factor
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INTRODUCTION |
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IN RECENT YEARS, THE ROLE of G protein-coupled receptors (GPCRs) as important mediators of cellular growth and proliferation has been clearly recognized (reviewed in Refs. 5, 39). Several agonists that signal via GPCRs can stimulate mitogenic signaling pathways, expression of growth-associated genes, protein synthesis, and cell proliferation. Ectopic expression of wild-type or constitutively activated GPCRs is sufficient to induce transformation of rodent fibroblasts (2, 16, 19). Naturally occurring gain-of-function mutations of both GPCRs and heterotrimeric G proteins have been identified in endocrine tumors and hyperplastic diseases (reviewed in Refs. 7, 29). GPCRs may also contribute to human cancers by stimulating cell proliferation via autocrine or paracrine release of mitogenic neuropeptides (5).
The molecular mechanisms that couple GPCRs to the regulation of cell growth and division have been subject to intense investigation. It has been shown that GPCR agonists induce tyrosine phosphorylation of multiple substrates in target cells (10, 22, 43) and that broad-spectrum tyrosine kinase inhibitors can block the stimulation of DNA (34, 42) or protein (21) synthesis by these factors. It also became apparent that GPCRs and receptor tyrosine kinases (RTKs) share common signaling intermediates in the pathway leading to activation of the extracellular signal-regulated kinase (ERK) subfamily of mitogen-activated protein (MAP) kinases (25). Interestingly, Daub et al. (4) reported that epidermal growth factor receptor (EGFR) is rapidly tyrosine phosphorylated upon stimulation of Rat1 cells with GPCR agonists and that inhibition of EGFR kinase activity suppresses ERK activation and c-fos induction. Subsequent studies demonstrated that such cross talk occurs in other cell types and may involve distinct RTKs (see Ref. 44 and references therein). These findings support the notion that transactivation of EGFR and possibly other RTKs can contribute to GPCR growth signaling.
Although several studies have documented the cross communication between GPCRs and EGFR, little is known about the biological significance of this mechanism in the normal physiological context. To address this question, we have examined the contribution of EGFR transactivation to the growth-promoting effects of GPCR agonists on rat vascular smooth muscle cells (VSMC). These cells represent a good model to study growth responses as they can be cultured in vitro in a normal, nonimmortalized state and are amenable to in vivo analysis. We report here that EGFR activity is necessary for GPCR-stimulated protein synthesis in VSMC in vitro and in the intact animal. We show that the EGFR signals to the ERK MAP kinase and the phosphoinositide 3-kinase (PI3K) pathways to modulate the phosphorylation and activity of eukaryotic translation initiation factor 4E (eIF-4E).
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EXPERIMENTAL PROCEDURES |
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Reagents, antibodies, and plasmids. ANG II was purchased from Hukabel Scientific (St. Laurent, QC). Human recombinant EGF and lysophosphatidic acid (LPA) were obtained from Sigma (Oakville, ON). Platelet-derived growth factor (PDGF)-BB, tyrphostin AG-1478, and LY-294002 were from Calbiochem (Mississauga, ON). Pertussis toxin (PTX) was from List Biological Laboratories (Campbell, CA). Commercial antibodies were from the following suppliers: anti-EGFR (SC-03; Santa Cruz Biotechnology, Santa Cruz, CA); anti-phospho-EGFR (Tyr845; New England BioLabs, Mississauga, ON); anti-phosphotyrosine monoclonal antibody (MAb) 4G10 and anti-Akt1 (06-558; Upstate Biotechnology, Lake Placid, NY); anti-eIF-4E MAb (Transduction Laboratories, Mississauga, Canada); anti-phospho-eIF-4E (Ser209; BioSource International). The EGFR dominant-negative mutant HERCD533 (31) was subcloned into the expression vector pcDNA3.
Cell culture and generation of VSMC lines. Rat aortic smooth muscle cells (SMC) were cultured and synchronized as described previously (12). The pcDNA-HERCD533 vector was transfected into aortic SMC using FuGene (Roche Diagnostics, Laval, QC), and stably transfected cells were selected in complete medium that contained 0.4 mg/ml of G418 (Life Technologies, Burlington, ON). The results presented were obtained with a representative population of clones (BN-HERCD533).
Immunoprecipitation and immunoblot analysis. Cells or vascular tissues were lysed and immunoprecipitated as described (13) by using 4 µl of anti-EGFR antibody. Immunoprecipitated proteins were resolved by SDS-gel electrophoresis and transferred to nitrocellulose membranes. The blots were probed with anti-phosphotyrosine MAb 4G10 and revealed by enhanced chemiluminescence (13). Activation of the EGFR was monitored by immunoblotting with a phospho-EGFR (Tyr845) antibody according to the manufacturer's specifications. Immunoblotting analysis of 4E binding protein 1 (4E-BP1) phosphorylation has been described previously (8). Immunoblotting results were quantitated by densitometry analysis using NIH Image software, and the phosphorylation data were corrected for the amount of total protein.
Protein kinase and PI3K assays.
The phosphotransferase activity of ERK1 and p70S6K was
measured by specific immune complex kinase assays using myelin basic
protein and S6 peptide as substrates, respectively (12).
For Akt1 assays, cellular extracts (500 µg of protein) prepared in
Akt lysis buffer [50 mM Tris · HCl, pH 7.4, 50 mM NaF, 50 mM
Na-pyrophosphate, 1 mM EGTA, 1 mM EDTA, 10 mM -glycerophosphate, 1 mM Na-orthovanadate, 1 µM okadaic acid, 0.1%
-mercaptoethanol,
0.1 mM phenylmethylsulfonyl fluoride (PMSF), 1 µg/ml leupeptin, 1 µM pepstatin A, and 0.1% Triton X-100] were incubated for 3 h
at 4°C with 2 µg of anti-Akt1 antibody preadsorbed to protein
G-Sepharose beads. The immune complexes were washed three times with
Akt lysis buffer with 0.5 M NaCl and twice with kinase buffer [20 mM
HEPES, pH 7.4, 10 mM MgCl2, 10 mM MnCl2, 1 mM
dithiothreitol (DTT), 10 mM
-glycerophosphate, 1 mM
Na-orthovanadate, and 17 µM protein kinase A (PKA) inhibitor peptide]. Akt1 activity was assayed by resuspending the beads in 25 µl of kinase buffer containing 5 µg of histone H2B, 50 µM ATP,
and 5 µCi of [
-32P]ATP. The reaction was incubated
at 30°C for 20 min and stopped by addition of 2× Laemmli's sample
buffer. The samples were analyzed by SDS-gel electrophoresis and the
band corresponding to histone H2B was excised and counted.
Analysis of eIF-4E phosphorylation. The eIF-4E was isolated by incubation of cellular extracts with m7-GTP-agarose beads (8). Isoelectric focusing was performed as described by Flynn and Proud (9) by using Pharmalyte carrier ampholytes (Amersham Pharmacia Biotech, Baie d'Urfé, QC) in the pH range 3-10. Immunoblotting analysis of eIF-4E protein was as previously described (8). Alternatively, phosphorylation of eIF-4E on regulatory Ser209 residue was monitored by immunoblotting with a phospho-specific antibody according to the manufacturer's specifications.
Protein synthesis measurements in vitro. Quiescent aortic SMCs in triplicate wells of 24-well plates were stimulated for 2 or 24 h with 100 nM ANG II in serum-free medium containing 0.5-2.0 µCi/ml [3H]leucine. After 24 h of stimulation, the radioactivity incorporated into TCA-precipitable material was measured as previously described (12).
In vivo measurement of protein synthesis in blood vessels.
For determination of in vivo protein synthesis rates, male
Sprague-Dawley rats weighing 300-325 g (Charles River
Laboratories, St. Constant, QC) were anesthetized with pentobarbital
sodium for insertion of a polyethylene catheter into the femoral artery and vein. The catheters were tunneled subcutaneously, exteriorized at
the back of the neck, and protected by a tethering system
(28). In some animals, an osmotic Alzet pump (model 1003D)
was simultaneously implanted subcutaneously in the subcostal region to
release a constant dose of 400 ng · kg1 · min
1 of ANG II.
Rats were allowed to recover unrestrained until drug injection and had
free access to food and water. After 22 h of ANG II infusion, a
saline solution containing [3H]leucine was infused
intravenously during 4 h at a rate of 12 µCi/h according to the
methods of McNulty et al. (26). Control rats received
leucine infusion alone 22 h after catheter implantation. One hour
before leucine infusion, a group of animals received an intravenous
bolus of 0.1 or 0.5 mg/kg AG-1478 (suspended in 0.2% carboxymethyl
cellulose and sonicated). Arterial pressure was continuously monitored
in freely moving rats 15 min before and after drug administration,
including during leucine infusion. Mean arterial pressure was averaged
over the 5-h period. At the end of treatments, the thoracic aorta and
the mesenteric arteries (small ramifications of first order and
smaller) were collected, freed from surrounding tissue, and frozen in
liquid nitrogen.
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RESULTS |
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EGFR transactivation by Gq- and Gi-coupled
receptors in VSMCs.
We first examined the ability of two GPCRs known to be coupled to
Gq (ANG II) and Gi (LPA) to induce tyrosine
phosphorylation of the EGFR in normal rat VSMCs. As shown in Fig.
1, addition of ANG II or LPA resulted in
increased tyrosine phosphorylation of endogenous EGFR. The
phosphorylation of the receptor reached a maximum within 1 min and then
returned to near-basal levels at 30 min (data not shown). As expected,
PTX strongly attenuated LPA-stimulated EGFR phosphorylation but had no
effect on the ANG II response (Fig. 1A). Pretreatment of
cells with AG-1478, a selective EGFR kinase inhibitor
(23), or expression of the dominant-negative EGFR mutant
HERCD533 (31) markedly inhibited EGFR tyrosine
phosphorylation induced by GPCR ligands (Fig. 1, B and
C).
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EGFR activation is necessary for GPCR-stimulated protein synthesis
in vascular smooth muscle.
ANG II and LPA stimulate protein synthesis and induce cellular
hypertrophy in cultured VSMCs. To assess the role of EGFR activation in
this growth response, we used a combination of pharmacological and
genetic approaches to block EGFR signaling. Incubation with AG-1478
significantly inhibited the stimulatory effect of ANG II (30%) and LPA
(38%) on the global rate of protein synthesis (Fig.
3A). The effect of AG-1478 was
dose dependent with half-maximal inhibition of ANG II-induced protein
synthesis observed at 11.8 nM (Fig. 3B). Expression of
HERCD533 also markedly attenuated the induction of protein synthesis by
ANG II or LPA (Fig. 3C). Similar results were observed when
the cells were stimulated for a short period of time (2 h) with GPCR
agonists (Fig. 3D). As predicted, AG-1478 also inhibited the
stimulatory effect of mitogenic GPCR agonists on DNA synthesis (data
not shown).
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EGFR activation contributes to the regulation of translation
initiation factor eIF-4E.
We next sought to characterize the downstream effectors that couple
EGFR activation to the increase in global protein synthesis. We have
previously shown that activation of the ERK MAP kinase (36) and p70S6K (12) pathways are
necessary for maximal ANG II-stimulated protein synthesis in VSMCs. We
thus examined the effects of EGFR kinase inhibition on the enzymatic
activation of the ERK module by ANG II and LPA. In agreement with
previous findings, we found that incubation with AG-1478 or expression
of HERCD533 strongly attenuated ERK1 activation by both ligands (Fig.
7, A and B). The
inhibition of ERK1 activation was associated with inhibition of the
upstream kinases MEK1 and Raf-1 (data not shown). Consistent with the
results of Fig. 1A, treatment of cells with PTX completely
blocked LPA-stimulated ERK1 activation but did not affect the ANG II
response (not shown).
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DISCUSSION |
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In addition to the role as receptors for their own specific
ligands, RTKs can also serve as signal transducers for a variety of
extracellular stimuli (reviewed in Ref. 44). Exposure of cells to stresses such as hyperosmotic shock or UV radiation or to
membrane depolarization rapidly induces tyrosine phosphorylation of
numerous RTKs. Besides these nonphysiological stimuli, the EGFR was
also identified as an essential signaling effector of GPCRs in rat
fibroblasts (4). Subsequent work showed that the cross
talk between GPCRs and EGFR can be generalized to several GPCRs and
diverse cell types including VSMCs (6). The observation that LPA, which induces tyrosine phosphorylation of EGFR in COS-7 and
Rat1 cells, can activate the PDGF receptor in L cells that lack EGFR
suggests that GPCRs link to several RTKs (18).
Transactivation of distinct RTKs occurs in a cell type-specific manner,
which may reflect the relative abundance of these RTKs. In cultured VSMCs, ANG II was also reported to induce tyrosine phosphorylation of
the PDGF receptor- (17, 24). Furthermore, induction of vascular injury by balloon catheterization of the rat carotid artery
(1) and chronic infusion of ANG II in stroke-prone
spontaneously hypertensive rats (20) were found to
increase the extent of PDGF receptor phosphorylation. However, other
investigators failed to detect any significant change in the
phosphotyrosine content of the PDGF receptor-
upon treatment of
VSMCs with ANG II (6) or after acute ANG II infusion in
spontaneously hypertensive rats (20). Whereas the
importance of PDGF receptor-
phosphorylation remains unclear, the
results presented here strongly argue for a critical role of the EGFR
as a signaling intermediate of GPCRs in vascular cells.
The most important finding of this study is the demonstration that EGFR activation is necessary for maximal stimulation of protein synthesis by GPCR agonists in VSMCs. We found that inhibition of EGFR kinase activity significantly inhibits GPCR-induced protein synthesis in cultured aortic SMCs and almost completely blocks ANG II-stimulated protein synthesis in vivo in both the aorta and mesenteric vessels. Similar effects on protein synthesis were observed by using two independent strategies to block EGFR function, although expression of the dominant-negative EGFR mutant led to a more robust inhibition. It is therefore possible that overexpression of HERCD533 interferes with the action of other members of the EGFR family or other signaling proteins that link GPCRs to growth control. It is interesting to note that the inhibitory effect of AG-1478 was more pronounced in vivo than in cultured cells. The reason for this difference is not known, but it may suggest either that EGFR transactivation is relatively more important in VSMCs in vivo or, alternatively, that downstream effectors of the EGFR contribute more to the regulation of global protein synthesis. Very little is known about the signaling pathways that control VSMC growth in intact animals.
The results presented here also further our understanding of the role of EGFR transactivation in the growth-promoting action of GPCRs by linking EGFR function to the translational machinery of the cell. The initiation step of mRNA translation is generally rate limiting in protein synthesis and is a major target of regulation by signal-transduction pathways (32). A key step in translation initiation is the recognition of the mRNA 5' cap structure by the initiation factor eIF-4F (reviewed in Refs. 15, 33). The eIF-4F is a trimeric complex that is composed of the cap-binding subunit eIF-4E, the ATP-dependent RNA helicase eIF-4A, and the scaffolding protein eIF-4G, which binds eIF-4E, eIF-4A, and eIF-3, another initiation factor associated with the 40S ribosomal subunit. The eIF-4F complex together with eIF-4B unwinds the secondary structure in the 5' untranslated region of the mRNA to create a binding site for the ribosome and allow assembly of a functional translation initiation complex. The eIF-4E is the least abundant of all initiation factors and is considered to be rate limiting for translation initiation. The activity of eIF-4E is primarily regulated by phosphorylation and its reversible association with the 4E binding proteins (15). Treatment of cells with growth factors stimulates phosphorylation of eIF-4E on Ser209, which results in stabilization of the interaction between the cap and eIF-4E and enhanced translation. One good candidate for the eIF-4E kinase is Mnk1, a Ser/Thr kinase that is activated by ERK and p38 MAP kinases and is able to phosphorylate eIF-4E on Ser209 in vitro and in vivo (41). The interaction of eIF-4E with 4E binding proteins is also regulated by phosphorylation. Under resting conditions, 4E binding proteins associate strongly with eIF-4E and repress cap-dependent translation. Exposure of cells to growth factors induces the hyperphosphorylation of 4E binding proteins and the release of eIF-4E, which is then free to associate with eIF-4G to form the eIF-4F complex. The pathway leading to phosphorylation of 4E-BP1 involves PI3K, its downstream effector Akt, and the kinase mammalian target of rapamycin, mTOR (3, 14). We showed that EGFR inactivation significantly attenuates the stimulation of ERK1, PI3K, and Akt1 activity by GPCR agonists, which results in inhibition of eIF-4E and 4E-BP1 phosphorylation. We also found that EGFR inactivation markedly inhibits the activation of p70S6K, another major effector of the PI3K signaling pathway. p70S6K phosphorylates the 40S ribosomal protein S6, which leads to the selective translational upregulation of a class of mRNAs that contains a polypyrimidine stretch in the 5' untranslated region (40). These mRNAs encode components of the translational machinery such as ribosomal proteins and translation elongation factors. Thus attenuation of the ERK and PI3K signaling pathways would be expected to have an additive inhibitory effect on long-term global protein synthesis.
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ACKNOWLEDGEMENTS |
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We thank Dr. A. Ullrich for the EGFR mutant, J. Noël and L. Grondin for technical assistance, and S. Pelletier for discussions.
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FOOTNOTES |
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* L. Voisin and S. Foisy contributed equally to this work.
This work was supported by grants from the Canadian Institutes of Health Research (CIHR; to S. Meloche and P. Moreau) and the Heart and Stroke Foundation of Canada (to S. Meloche). L. Voisin is recipient of a fellowship from the Heart and Stroke Foundation of Canada. P. Moreau is a New Investigator and S. Meloche is an Investigator of the CIHR.
Address for reprint requests and other correspondence: S. Meloche, Institut de recherches cliniques de Montréal, Montreal, Quebec H2W 1R7, Canada (E-mail: melochs{at}ircm.qc.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.
March 27, 2002;10.1152/ajpcell.00261.2001
Received 12 June 2001; accepted in final form 20 March 2002.
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