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Circulation Research. 2004;95:989-997
Published online before print October 14, 2004, doi: 10.1161/01.RES.0000147962.01036.bb
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(Circulation Research. 2004;95:989.)
© 2004 American Heart Association, Inc.

Molecular Medicine

Transactivation of Epidermal Growth Factor Receptor Mediates Catecholamine-Induced Growth of Vascular Smooth Muscle

Hua Zhang, Dan Chalothorn, Leslie F. Jackson, David C. Lee, James E. Faber

From the Departments of Cell and Molecular Physiology (H.Z., D.C., J.E.F.) and Biochemistry and Biophysics (L.F.J., D.C.L.), School of Medicine, University of North Carolina, Chapel Hill.

Correspondence to James Faber, Department of Cell and Molecular Physiology, School of Medicine, University of North Carolina, Chapel Hill, NC 27599-7545. E-mail jefaber{at}med.unc.edu


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Stimulation of {alpha}1-adrenoceptors induces proliferation of vascular smooth muscle cells (SMCs) and contributes to arterial remodeling. Although activation of NAD(P)H oxidase and generation of reactive oxygen species (ROS) are required, little is known about this pathway. In this study, we examined the hypothesis that epidermal growth factor receptor (EGFR) transactivation and extracellular regulated kinases (ERK) are involved in {alpha}1-adrenoceptor–mediated SMC growth. Phenylephrine increased protein synthesis in association with a rapid (≤5 minutes) and sustained (≥60 minutes) doubling of phosphorylation of EGFR and ERK1/2, but not p38 or JNK in the media of rat aorta maintained in organ culture. Antagonists of EGFR phosphotyrosine activity (AG-1478) and ERK phosphorylation (PD-98059, U-0126) abolished phenylephrine-induced protein synthesis, whereas antagonists of p38 or JNK phosphorylation had no specific effect. A competitive antagonist (P22) for heparin binding EGF-like growth factor (HB-EGF) blocked phenylephrine-induced protein synthesis, as did downregulation of pro-HB-EGF (CRM197). Phenylephrine-induced protein synthesis was inhibited by neutralizing antibody to HB-EGF and absent in HB-EGF–/– SMCs. Inhibitors of metalloproteinases (BiPS, KB-R7785) also blocked adrenergic growth. The neutralizing antibody against HB-EGF had no effect on the two-fold increase in ROS generation induced by phenylephrine (DCF fluorescence), suggesting that stimulation of NAD(P)H oxidase by {alpha}1-adrenoceptor occupation precedes HB-EGF release. Cell culture studies confirmed and extended these findings. These data suggest that {alpha}1-adrenoceptor–mediated SMC growth requires ROS-dependent shedding of HB-EGF, transactivation of EGFR, and activation of the MEK1/2-dependent MAP kinase pathway. This trophic pathway may link sympathetic activity to arterial wall growth in adaptive remodeling and hypertrophic disease.


Key Words: {alpha}-adrenergic receptor • vascular smooth muscle cell proliferation • signal transduction • reactive oxygen species • metalloproteinase


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Vascular smooth muscle cell (SMC) proliferation, hypertrophy, and migration are central to development of vascular disease such as restenosis after vessel injury, atherosclerosis, and wall hypertrophy. In addition to evidence that prolonged elevation of plasma catecholamines is a risk factor for vascular diseases,1,2 recent reports have shown that catecholamines directly induce hypertrophy of the arterial wall by stimulation of {alpha}1-adrenoceptors ({alpha}1-ARs), which are G-protein–coupled receptors (GPCRs). Catecholamine stimulation in cell and organ culture induces dose-dependent proliferation, protein synthesis, and migration of SMCs and adventitial fibroblasts and promotes dedifferentiation of the SMC phenotype.3–8 Furthermore, the potency of these effects is strongly augmented in injured arteries.7 Similar effects are seen in vivo, where endogenous vascular wall catecholamines contribute to hypertrophy, fibrosis, and lumen loss after balloon injury of rat and mouse carotid6,9,10 and chronic catecholamine infusion.11 Although stimulation of NAD(P)H oxidase and generation of reactive oxygen species (ROS) are required for the trophic actions of catecholamines,12 little additional is known about this trophic pathway.

Like receptor tyrosine kinases (RTKs), certain GPCRs stimulate mitogen-activated protein kinases (MAPK) and thereby induce cellular growth.13 The MAPK family includes three subfamilies with multiple members: the extracellular signal-regulated kinases (ERKs), the c-Jun N-amino-terminal/stress-activated protein kinases (JNKs/SAPKs), and the p38 MAPKs. Each MAPK is a member of a three-protein kinase cascade, consisting of a MAPK kinase kinase (MKKK), a MAPK kinase (MKK), and the final MAPK that are induced to organize into signaling complexes by upstream effectors. Similar to non-GPCR growth factor RTKs such as the epidermal growth factor receptor (EGFR), several GPCRs signal growth factor-like activity through one or more of these MAPK cascades.13 Although {alpha}1-AR stimulation has been shown to activate ERK1/2 (also denoted p42/p44 MAPK) in cultured SMCs,4,14,15 no studies have determined which MAPK pathways are required for {alpha}1-AR–induced growth in the intact artery.

The proximal signals linking GPCRs to downstream MAPK pathways and cell growth are less well defined than those for RTKs. Stimulation of some GPCRs (eg, the angiotensin AT1 receptor) in specific cell types studied in cell culture induces ectodomain shedding, by proteolytic cleavage, of membrane-anchored proheparin binding-EGF–like growth factor (pro-HB-EGF).16,17 Soluble HB-EGF then binds to and activates EGFR, leading to recruitment of adaptor and regulatory proteins that activate ERK1/2. This mechanism, denoted EGFR transactivation, mediates part of the mitogenic signaling in cultured SMCs by the GPCRs ligands, endothelin, angiotensin, and thrombin.16–19 Certain other growth factors, cytokines (eg, TGF{alpha} and TNF{alpha}), growth factor receptors, cell-adhesion molecules, and membrane proteins are proteolytically processed to yield soluble signaling proteins.16,20 Pro-HB-EGF, which contains a signal peptide domain, heparin-binding site, EGF-like domain, transmembrane domain, and cytoplasmic domain, complexes with CD9 and integrin {alpha}3ß1 at cell-cell attachment sites.16 In contrast to proliferative actions of soluble HB-EGF, membrane-anchored pro-HB-EGF acts in a juxtacrine manner to inhibit cell proliferation.21 Pro-HB-EGF also serves as the receptor for diphtheria toxin.22 No studies have determined whether {alpha}1-AR stimulation activates HB-EGF-dependent transactivation of EGFR in SMCs.

Soluble HB-EGF is involved in regulation of tissue development and growth. HB-EGF is a potent mitogen of fibroblasts, keratinocytes, and SMCs.23 In SMCs, HB-EGF and PDGF-B have similar mitogenic potency, whereas EGF is much less potent.24 Immunoreactivity for HB-EGF and EGFR are strongly induced in the intimal thickening that precedes atheroma development in human coronary arteries and in SMCs and macrophages in and around core lesions.25 After balloon injury, HB-EGF is induced in rat carotid media and neointimal.26 Moreover, neutralizing antibody to EGFR inhibits SMC proliferation and intimal hyperplasia induced by balloon injury.27

Certain metalloproteinase-disintegrins, which are a family of more than 33 transmembrane glycoproteins, denoted "a disintegrin and metalloproteinase" (ADAM) or "metalloproteinase-disintegrin/cysteine-rich" (MDC) proteins, mediate ectodomain shedding.28 ADAM/MDC proteins possess a prodomain and domains with metalloprotease, disintegrin, cysteine-rich, EGF-like, transmembrane, and cytoplasmic activities or homologies.29 The first ADAM shown to induce protease-dependent shedding was TNF{alpha} converting enzyme (TACE, ADAM-17).29 ADAM-17 also mediates shedding of TGF{alpha} and HB-EGF in mice.30,31 Evidence suggests that ADAM-12 is required for {alpha}1-AR and AT1 receptor-induced HB-EGF shedding and transactivation in murine cardiomyocytes.32

In the present study, we tested the hypothesis that stimulation of medial SMC growth by {alpha}1-ARs is mediated by cleavage of membrane-bound pro-HB-EGF that transactivates EGFR. We also sought to determine whether this pathway activates specific downstream MAPK pathways. Experiments were conducted in cultured SMCs and the rat aorta organ culture model to simulate in vivo conditions.3,4,7,12


*    Materials and Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Rat thoracic aortae (Charles River Laboratories, Wilmington, Mass) received balloon injury7 and four days later were placed into organ culture with wall tension set to 0.45 g per mm vessel length to simulate normal circumferential stress.3,4,7,12 Cultured SMCs were dispersed from the media of thoracic aortae5 from rats or mice (HB-EGF+/+ and HB-EGF–/–).33 Protein synthesis was measured at 48 hours with [35S] methionine.

For immunohistochemistry and immunoblot studies, 4-day balloon-injured aortae were maintained in organ culture and exposed to the {alpha}1-AR agonist phenylephrine (PE) ±AG1478 for 5 to 60 minutes, followed by removal to 4°C PBS containing proteinase inhibitors. After extirpation of the cut ends, vessels were sectioned or extracted. Generation of reactive oxygen species (ROS) in response to PE was measured with 10 µmol/L 2',7'-dichlorofluoroscein diacetate (DCFDA).34 In-cell Western assays were conducted on cultured SMCs.

The following reagents were used: P22 (a gift from Dr Yohki Hieda, Osaka University, Toyonaka, Japan),35,36 PD 98059, U-0126, SB203580, and SP600125 (Biomol), BiPS [(2R)-[(4-biphenylylsulfonyl) amino]-N-hydroxy-3-phenylpropionamide], thrombin, GM6001 (Calbiochem), AG1478, and CRM197 (Sigma). KB-R7785 ([4-(N-hydroxyamino)-2R-isobutyl-3S-methylsuccinyl]-L-phenylglycine-N-methylamide) was a gift from Dr Hiroshi Ishiguro (Organon, Osaka). Neutralizing antibody to HB-EGF was from R&D Systems.

Unless noted, data are expressed as mean±SE, and significance is given as P<0.05 for Student t tests.

An expanded Materials and Methods section can be found in the online data supplement available at http://circres.ahajournals.org.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
{alpha}1-Adrenoceptor–Induced Protein Synthesis in SMCs of Aorta Media Is Dependent on ERK1/2 Phosphorylation
We previously found that {alpha}1-AR–mediated protein synthesis and hypertrophy of cultured rat aorta SMCs are associated with phosphorylation of Raf1 and ERK1/2 and are inhibited by the MEK1/2 antagonist, PD98059, with an IC50 of 2 µmol/L.4 To determine whether this MAPK pathway is also required for PE growth of SMCs in situ, rat aorta was injured by balloon and placed 4 days later into organ culture before a neointima forms.7,12 Injured aorta was used because we have previously shown, ex vivo and in vivo, that injury increases {alpha}1-AR–dependent trophic activity in media and adventitia and contributes to restenosis and wall hypertrophy.6,7,9,10

Treatment of aorta with PE for 48 hours increased protein synthesis in the medial layer by 36% to 44% (Figures 1 and 4Down). This was blocked by PD98059 and the more selective MEK inhibitor, U-0126 (Figure 1). In the absence of PE, 2 µmol/L U-0126, which has no effect on big MAPK-1 (ERK5) activity,37 had no effect (Figure 1). By contrast, the p38 and JNK inhibitors SB203580 and SP600125 cause the same absolute amount of inhibition when given alone as when given in the presence of PE (Figure 1). PD98059 was not tested alone because U-0126 alone had no significant effect in another experiment (103±6%), PD98059 alone had no effect on basal protein synthesis,4 and both U-0126 and PD had identical actions against PE-induced wall growth, ie, complete inhibition (Figure 1).



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  		Figure 1. Phenylephrine (PE)-induced protein synthesis in rat aorta media, in situ, is inhibited by MEK antagonists. Rat aortae were isolated 4 days after balloon injury and maintained in organ culture under tangential wall tension (for the data in this figure and also data for figure 2 to 4DownDown). Vessels were exposed for 48 hours to PE alone or PE plus the MEK (MAPK-kinase) inhibitors U-O126 (UO), PD98059 (PD), SB203580 (SB), and SP600125 (SP). Final concentrations of drugs in Figure 1 and other figures are given in µmol/L unless indicated otherwise. Protein synthesis was measured by [35S]-methionine incorporation, after separation of media from intima and adventitia. Data in this and all subsequent figures are mean±SE for "n" number of vessels per group, where data are given as percent of vehicle (Veh)-treated vessels. Vehicle was DMSO (1:1000, v/v) and ascorbate (250 µmol/L) (final concentrations) in this and all figures, unless indicated otherwise.

These data suggest that PE induces ERK1/2 activation and augments growth in smooth muscle, in parallel with p38 and JNK pathways that remain activated by injury, itself, 4 days after injury. To test this hypothesis, we examined phosphorylation of ERK1/2, p38, and JNK with immunoblot and immunohistochemistry. In contrast to the above experiment where protein synthesis was measured in the medial layer, in this experiment media could not be separated from the endothelium and adventitia. This was because the enzyme treatment and dissection required for separation (see online data supplement) would activate MAPK pathways directly. Treatment of aorta to PE caused rapid (≤5 minutes) and sustained (≥60 minutes) phosphorylation of ERK1/2, but not p38 and JNK (Figure 2), whereas total ERK1/2 did not change. Immunohistochemistry showed that activation of ERK1/2 was induced in both media and adventitia (Figure 3).



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  		Figure 2. Phenylephrine-induced phosphorylation of ERK1/2 and EGFR (A) and P38 and JNK (B) in rat aorta media. Aortae were maintained in organ culture for 5 to 60 minutes (min) in the presence of vehicle (same as in Figure 1), PE, or the EGFR tyrosine kinase inhibitor AG1478 (AG, 250 nmol/L). Whole aortae (with intima, media, and adventitia intact) were lysed and 20 to 30 µg of protein was added to each lane of 6% to 10% SDS-PAGE gels.



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  		Figure 3. Phenylephrine-induced phosphorylation of ERK1/2 (A) and EGFR (B) in medial and adventitial layers of rat aorta, as determined by immunohistochemistry. PE, 10 µmol/L; AG1478, 250 nmol/L; vehicle, same as in Figure 1. A and B, Representative 6-micron thick sections of rat thoracic aorta showing PE-induced phosphorylation of ERK1/2, as determined by HRP-diaminobenzidine immunohistochemistry (brown reaction product). See Materials and Methods for additional details. Aorta was treated with vehicle (A) or 10 µmol/L PE (B) for 48 hours in organ culture. Light hematoxylin counter stain. Magnification, 200x. Data in C and D are from images quantified by standard thresholding methods using Scion Image software. Same threshold was used for all sections.

{alpha}1-Adrenoceptor–Induced Protein Synthesis Is Mediated by EGFR Transactivation
Previous studies have reported that stimulation of certain GPCRs induces growth of cultured SMCs, in part, by transactivation of EGFR.16–19,38 In this study, stimulation of protein synthesis by PE was completely inhibited by the EGFR tyrosine kinase inhibitor AG1478, whereas AG1478 alone had no effect (Figure 4). Immunoblotting showed that PE caused phosphorylation of EGFR, where the time course and magnitude were similar to ERK1/2 (Figure 2A). This effect was also inhibited by AG1478. Like ERK1/2 activation, immunohistochemistry showed that PE caused phosphorylation of EGFR in both media and adventitia that was inhibited by AG1478 (Figure 3).



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  		Figure 4. Phenylephrine-induced protein synthesis in rat aorta media is mediated by HB-EGF. Vessels were pretreated with inhibitors for 30 minutes before addition of PE, and protein synthesis was measured 48 hours later. P22 is a competitive peptide antagonist of HB-EGF binding. CRM197 (CRM) stimulates internalization of proHB-EGF. BiPS and KB-R7785 (KB) are inhibitors of metalloproteinases. Vehicle same as in Figure 1.

These data suggest that {alpha}1-AR–dependent growth is coupled by EGFR transactivation to downstream ERK1/2 activation. However, at variance with this was the absence of effect of AG1478 on PE-mediated phosphorylation of ERK1/2 (Figure 2A, last bar-pair). Because these immunoblot experiments were necessarily performed on protein extracted from intact aorta without separation of layers, immunohistochemistry was used to differentiate activation within the media versus adventitia. Whereas AG1478 prevented PE-induced EGFR phosphorylation in both media and adventitia, it inhibited PE-induced ERK1/2 phosphorylation of cells in media but not in adventitial (Figure 3, hatched bars). These data suggest a hypothesis for the differential effect of AG1478 obtained in the Western blot assays, ie, PE-induced growth of medial cells relies on EGFR-transactivation for ERK1/2 phosphorylation, whereas PE-induced growth of adventitial cells, although accompanied by EGFR phosphorylation, does not require this for activation of the downstream ERK1/2 pathway.

Phenylephrine-Induced Growth Is Dependent on HB-EGF
There is evidence that EGFR transactivation is elicited either by direct induction of EGFR tyrosine kinase activity or by release (shedding) of EGFR ligands such as HB-EGF.38–40 Several experiments were conducted in aorta media to differentiate between these possibilities. Stimulation of protein synthesis by PE was blocked by AG1478 (Figure 4), confirming the data obtained in Figure 1 and 2Up with AG1478. P22 is a 22-residue peptide (spanning the heparin-binding domain of HB-EGF) that interferes with high-affinity binding of HB-EGF to EGFR.35,36 Phenylephrine-induced protein synthesis was blocked by P22, whereas P22, alone, had no effect (Figure 4). CRM197, which is a nontoxic mutant of diphtheria toxin that binds and internalizes pro-HB-EGF in human and rat,16,17,22,39,41 had similar effects. In addition, protein synthesis that was induced by PE treatment of cultured rat aorta SMCs for 48 hours was partially inhibited by a neutralizing antibody to human HB-EGF (Figure 5A). This experiment was conducted in cultured SMCs because the amount of antibody needed for the large volumes of medium used in organ culture was prohibitive. In a fourth experiment, PE-induced protein synthesis in aorta SMCs isolated from HB-EGF wild-type mice was abolished in pro-HB-EGF–/– SMCs (Figure 5B). Thrombin-induced protein synthesis was also inhibited, which served as a positive control.39,42 In contrast, serum-induced protein synthesis was similar in HB-EGF–/– and wild-type cells, which indicates that HB-EGF–/– cells were responsive to the multiple growth factors present in serum. We conducted this experiment in cultured SMCs because the system for maintaining wall tension in rat aorta is too large for mouse aorta.



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  		Figure 5. Phenylephrine-induced protein synthesis in cultured aorta SMCs is dependent on HB-EGF release. A, Two-days after confluence cultured rat aorta SMCs were placed into serum-free defined medium for 24 hours and treated with PE (10 µmol/L) or pretreated for 30 minutes with neutralizing antibody (Ab) to HB-EGF (or isotype control antibody) before PE treatment for 48 hours. Protein synthesis was measured by [35S]-methionine incorporation. B, Aorta SMCs from HB-EGF–/– (knockout) or wild-type mice were grown to 50% confluence and placed into serum-free defined medium for 12 hours, followed by PE exposure for 36 hours. Vehicle, 250 µmol/L ascorbate.

Collectively, the above findings suggest that PE-induced transactivation of EGFR and subsequent induction of protein synthesis in SMCs is mediated by shedding of HB-EGF. To further test this hypothesis, we examined two inhibitors of metalloproteinases that reportedly possess selectivity for inhibition of HB-EGF shedding.32,43 BiPS and KB-R7785 both inhibited PE-induced protein synthesis by an amount in excess of that produced by the agents alone (Figure 4).

{alpha}1-Adrenoceptor–Induced Generation of ROS Precedes HB-EGF Shedding
We previously found that norepinephrine stimulates SMC growth by {alpha}1-AR–induced activation of NAD(P)H oxidase and generation of ROS (ie, H2O2).12 In that study, we also found that PE-induced ROS generation was unaffected by AG1478, PD98059, or U-O126, whereas these agents inhibited PE-induced protein synthesis. Together with the present study, these findings suggest that the oxidase and HB-EGF shedding are "upstream" of EGFR activation in the signaling pathway. To test this hypothesis, we measured ROS production with DCFDA, an H2O2-sensitive fluoroprobe, in SMCs. The increase in ROS induced by PE was unaffected by pretreatment with HB-EGF neutralizing antibody or the IgG isotype control antibody (Figure 6). Phenylephrine-induced increase in DCF fluorescence was inhibited by the antioxidants apocynin and n-acetyl cysteine (NAC) (Figure 7A) and the glutathione peroxidase-mimetic antioxidant ebselen (2-phenyl-1,2-benzisoselenazol-3-(2H)-one) (Figure 7B), but not by CRM197 that causes downregulation of pro-HB-EGF or by the metalloproteinase inhibitor (putative ADAM-12 inhibitor) KB-R7785 (Figure 7C) that did, however, inhibit PE-induced growth (Figure 4). Neutralizing antibody to HB-EGF, CRM197, NAC, ebselen, GM6001 (metalloproteinase inhibitor), and KB-R7785 inhibited PE-induced EGFR phosphorylation, using in-cell Western assays (Figure 7D), whereas agents alone had no effect (online data supplement). We also tested ROS production in aorta SMCs from HB-EGF–/– mice. Despite that PE-induced protein synthesis was abolished (Figure 5), the increase in DCF fluorescence (203±10% of vehicle, P<0.001, n=6; data not shown), which was abolished by 10 µmol/L ebselen (64±5%, P<0.001, n=6; ebselen alone, 43±5%, n=6; data not shown), was similar to that seen in SMCs with the HB-EGF gene intact (Figure 7). Together with other data, these results strengthen our hypothesis that ROS generation is upstream of HB-EGF shedding in the SMC adrenergic trophic pathway. A recent publication reached a similar conclusion for PE-induced constriction of rat mesenteric arteries.41



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  		Figure 6. Phenylephrine-induced generation of ROS precedes HB-EGF shedding. Cultured rat aorta SMCs were grown in 24-well plates to 95% to 100% confluence. After maintenance in serum-free defined medium for 24 hours, cells were treated with neutralizing antibody (nAb) to HB-EGF for 30 minutes before PE treatment for 20 minutes, followed by incubation with DCFDA (10 µmol/L) for 30 minutes and fluorescence measurement. Vehicle, 10 µmol/L ascorbate.



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  		Figure 7. Phenylephrine-induced ROS generation precedes HB-EGF shedding and EGF receptor phosphorylation. Studies were conducted as described in Figure 6 legend (see also online data supplement for additional details). Vehicle, 10 µmol/L ascorbate.

Phenylephrine-Induced Protein Synthesis in Aorta Adventitia
Although the focus of this study was on vascular smooth muscle, we also examined adventitia in several experiments. Like medial SMCs, {alpha}1-AR stimulation induces proliferation and protein synthesis of rat aorta adventitial fibroblasts studied in cell culture, organ culture, and in vivo.3,5,7,9,10 Also like SMCs, this is associated with stimulation of ROS generation that was not inhibited by PD98059, U-O126, and AG1478.12 In the present study, PE (10 µmol/L, 48 hour) increased adventitial protein synthesis by 197±18 (in percent of vehicle and with n=6 here and for each of the following group). This was inhibited by (P<0.05 for each group): PD98059 (77±10%), SB203580 (158±8%), and SP600125 (104±9%), whereas SB203580 and SP600125, alone, had no significant effect (116±4%, 98±13%). In contrast to media, CRM197 had no effect on PE-induced protein synthesis in adventitia (208±22%); CRM197 had no effect alone (88±16%). In cultured rat adventitial fibroblasts (passage 3, two days postconfluent, two days in serum-free defined medium), PE (100 µmol/L) increased DCF fluorescence to 156±2% of vehicle (P<0.05, n=6). This was inhibited by (P<0.05, n=6 for each group) NAC (94±2%; 1mmol/L) and ebselen (125+4%; 40 µmol/L), but not by CRM197 (157±3%; 10µmol/L). NAC, ebselen, and CRM197 had small inhibitory effects alone (86±4%, 87±3%, 88±4%, respectively).

These data suggest that, in distinction to media, PE-induced growth of adventitia involves HB-EGF–independent transactivation of EGFR and activation of all three MAP kinases. The fact that adventitia has approximately two-thirds fewer cells than media5 may underlie why activation of p38 and JNK in adventitia, as suggested by these inhibitor experiments, was not detected in immunoblots of whole aorta (Figure 2B). These data also demonstrate that PE’s trophic action on adventitial fibroblasts, like SMCs, is associated with ROS generation that is reversed by ROS and H2O2 scavengers, but unaffected by pro-HB-EGF downregulation with CRM197.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
Endogenous norepinephrine exerts growth factor-like activity, through {alpha}1A-ARs on medial SMCs and {alpha}1B-ARs on adventitial fibroblasts, that contributes to wall hypertrophy and restenosis of balloon-injured rat aorta and carotid.6,7,10 This adrenergic trophic activity has been confirmed in carotid arteries of mice with genetic deletion of either catecholamine synthesis or {alpha}1-AR subtypes.9 Prolonged elevation of wall norepinephrine also causes hypertrophy of uninjured arteries.10,11 Catecholamine-induced growth of medial SMCs and adventitial fibroblasts requires generation of NAD(P)H oxidase–dependent ROS.12 The present study identified additional steps in this pathway in rat aorta studied ex vivo. In media, these steps consist of ROS generation, followed by HB-EGF shedding, EGFR activation, and ERK1/2 activation (online Figure 1 in the online data supplement provides a model of the pathway in SMCs). This trophic pathway differs from that described in cell culture for angiotensin, thrombin, and endothelin-1, wherein their trophic actions do not rely solely on EGFR transactivation or ERK1/2.17–19,38,44,45

In contrast to the medial layer of aorta, adrenergic stimulation of protein synthesis in adventitia appears to be mediated by EGFR-independent activation of ERK1/2, p38, and JNK. Moreover, the results obtained with AG1478 and CRM197 suggest that {alpha}1-AR stimulation causes "direct" EGFR transactivation in adventitia, compared with HB-EGF release in media. However, this direct EGFR transactivation appears not to be involved in adrenergic-induced protein synthesis. Although a detailed study of adventitia is required to confirm these findings, such differences between media and adventitia could result from differences in cell type or {alpha}1-AR subtypes mediating the growth response in SMCs and adventitial fibroblasts.7 The differences also provided controls in the present study for the specificity of the concentrations used of PD98059, U-O126, SB203580, SP600125, AG1478, and CRM197.

Although beyond the purpose of the present study, additional work is required to determine how {alpha}1-AR stimulation activates NAD(P)H oxidase and to identify the protease responsible for pro-HB-EGF cleavage. The major matrix-bound metalloproteinases (MMP) and membrane-type (anchored) metalloproteinases (MT-MMP) expressed in arteries are MMP-1, -2, -9, and MT1-MMP.46,47 The particular metalloproteinase(s) responsible for pro-HB-EGF cleavage in tissues remains unclear. For example, BiPS, which inhibits MMP-2 and -9 (but may also inhibit other metalloproteinases), blocked angiotensin-induced transactivation of EGFR in rat aortic SMCs; however, other inhibitors of MMP-2 or MMP-9 were without effect.43 In a study of cardiac hypertrophy, the MMP inhibitors OSU7–6 and OSU9–6 had similar potency against EGFR transactivation, yet OUS7–6 is a more potent inhibitor of MMP-1, -3, and -9 than OSU9–6.32 These results suggest that MMPs other than MMP-1, -2, -3, or -9 are involved in EGFR transactivation.

Members of the ADAM family of membrane-anchored glycoproteins have recently been implicated in ectodomain shedding. Compared with MMPs, ADAMs have different peptide cleavage sites, potencies, and are modulated by different tissue inhibitors of matrix metalloproteinases.29,48 Although many ADAMs have a relatively well-conserved metalloproteinase domain, only approximately 15 of them contain the catalytic consensus site predicting proteolytic activity.29 Of these, ADAM-1, -12, -15, and -17 are expressed in SMCs, and ADAM-15 and -17 are expressed in endothelial cells.49 Asakura et al32 implicated ADAM-12 in PE-induced HB-EGF shedding and cardiac hypertrophy, using yeast-2 hybrid assays. Furthermore, KB-R7785, which specifically inactivates ADAM-12,32 abolished HB-EGF-mediated EGFR transactivation that was induced by PE, angiotensin, and endothelin-1, and attenuated PE-induced cardiac hypertrophy.32 In our experiments, BiPS partially, and KB-R7785 completely, inhibited PE-induced protein synthesis in SMCs. The IC50 for KB-R7785 against HB-EGF shedding is 0.23 µmol/L, versus 4.5 µmol/L for TNF{alpha}-TACE–mediated shedding.32 Our observation that 0.5 and 1 µmol/L KB-R7785 abolished PE growth (Figure 4) suggests that ADAM-12 may mediate HB-EGF shedding induced by {alpha}1-AR stimulation of SMCs, in agreement with cardiomyocytes.32

Additional studies are needed to determine how the metalloproteinase responsible for pro-HB-EGF cleavage is activated by {alpha}1-ARs. Our previous data suggest that H2O2 is the key ROS.12 However, it is not known if H2O2 acts from an intracellular or extracellular location, because H2O2 diffuses through cell membranes at a rate (113 µm/s) similar to water (110 µm/s).50 It is also unclear how H2O2 activates metalloproteinases. Low levels of H2O2 (4 µmol/L) can activate MMPs in cultured SMCs.51 Hydrogen peroxide can also activate MT1-MMP in endothelial cell lysates, leading to activation of a proteinase cascade consisting of MMP2 followed by activation of secreted latent MMPs.52 In addition, exposure of endothelial cells to sublethal levels of H2O2 (1.5 to 32 µmol/L) led to appearance of MMP activity in the culture media.52

Limitations
Binding of HB-EGF to cell surface heparan sulfate proteoglycans (HSPG), which restricts diffusion, provides a matrix depot, and increases the affinity of HB-EGF for EGFR, is important for EGFR activation.23 However, binding of released HB-EGF interferes with measurement of its shedding into matrix and culture media.39 Because no assays exist for direct measurement of HB-EGF shedding in intact tissues, our supportive evidence necessarily derives from indirect approaches: competitive antagonism of HB-EGF (P22), internalization of pro-HB-EGF (CRM197), selective (putatively) inhibition of pro-HB-EGF cleavage (KB-R7785), HB-EGF neutralizing antibody, and use of HB-EGF–/– SMCs.

In conclusion, our results identify important new steps in the signal transduction pathway mediating catecholamine induction of growth of smooth muscle cells and adventitial fibroblasts. Furthermore, they provide a potential mechanism that may underlie why injury causes prevailing levels of catecholamines in the vascular wall to become strongly trophic.6,7,9,10 That is, arterial injury activates or increases all of the elements identified in the adrenergic trophic pathway, ie, ROS,53 metalloproteinases,47 HB-EGF, EGFR,26,27 and MAPKs.54,55 Thus, injury produces conditions that favor synergistic amplification of adrenergic growth.


*    Acknowledgments
 
Support was provided by the following National Institutes of Health grants: NIH-HL62584 (to J.E.F.) and NIH-CA43973 (to D.C.L.). The authors thank Kirk McNaughton for histology.


*    Footnotes
 
Original received June 1, 2004; revision received September 23, 2004; accepted October 6, 2004.


*    References
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