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Molecular Endocrinology, doi:10.1210/me.2003-0309
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Molecular Endocrinology 18 (9): 2344-2354
Copyright © 2004 by The Endocrine Society

Urotensin II Promotes Hypertrophy of Cardiac Myocytes via Mitogen-Activated Protein Kinases

Döne Onan, Luisa Pipolo, Eunice Yang, Ross D. Hannan and Walter G. Thomas

Molecular Endocrinology (D.O., L.P., W.G.T.) and Gene Transcription (E.Y., R.D.H.) Laboratories, Baker Heart Research Institute, Melbourne 8008, Victoria, Australia; and Department of Biochemistry and Molecular Biology (D.O.), Monash University, Melbourne 3800, Victoria, Australia

Address all correspondence and requests for reprints to: Dr Walter Thomas, Baker Heart Research Institute, P.O. Box 6492, St. Kilda Road Central, Melbourne 8008, Victoria, Australia. E-mail: walter.thomas{at}baker.edu.au.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Urotensin II and its receptor are coexpressed in the heart and up-regulated during cardiac dysfunction. In cultured neonatal cardiomyocytes, we mimicked this up-regulation using an adenovirus to increase expression of the urotensin receptor. In this model system, urotensin II promoted strong hypertrophic growth and phenotypic changes, including cell enlargement and sarcomere reorganization. Urotensin II potently activated the MAPKs, ERK1/2 and p38, and blocking these kinases with PD098059 and SB230580, respectively, significantly inhibited urotensin II-mediated hypertrophy. In contrast, urotensin II did not activate JNK. The activation of ERK1/2 and p38 as well as cellular hypertrophy was independent of protein kinase C, and calcium and phosphoinositide 3-kinase, yet dependent on the capacity of the urotensin receptor to trans-activate the epidermal growth factor receptor. Urotensin II promoted the tyrosine phosphorylation of epidermal growth factor receptors, which was inhibited by the selective epidermal growth factor receptor kinase inhibitor, AG1478. These data indicate that perturbations in cardiac homeostasis, which lead to up-regulation of urotensin II receptors, promote urotensin II-mediated cardiomyocyte hypertrophy via ERK1/2 and p38 signaling pathways in an epidermal growth factor receptor-dependent manner.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE VASOACTIVE FISH peptide hormone urotensin II (U-II) has been cloned recently from humans (1, 2) and identified as the endogenous ligand of a seven-transmembrane spanning, G protein-coupled receptor (GPCR) referred to as the U-II receptor (UT-IIR) (1, 3, 4). U-II stimulation of UT-IIRs activates G{alpha}q-phospholipase Cß to generate diacylglycerol and inositol trisphosphate (1, 4, 5), which stimulates protein kinase C (PKC) and releases calcium from intracellular stores. In nonhuman primates, U-II is the most potent vasoconstrictor known, exerting complex hemodynamic effects that culminate in cardiovascular collapse and death (1, 6). In humans, the vasoactive role of U-II remains somewhat elusive due to inconsistent in vitro and in vivo data (reviewed in Ref. 7); however, a putative role for U-II and its receptor in disease is emerging. U-II can inhibit the release of insulin from pancreatic cells in vitro (8), and elevated levels of U-II have been reported in the plasma and/or urine of diabetic (9) and renal failure patients (10, 11). U-II has mitogenic growth properties in vascular smooth muscle cells (12, 13, 14) and tumor cells (15, 16, 17), implicating a role for U-II in atherosclerosis and cancer, respectively.

Although the expression of UT-IIRs are low to undetectable in the healthy myocardium, U-II and its receptor are up-regulated in patients with end-stage heart failure (18). In addition, expression of UT-IIRs are up-regulated in the ischemic (19), chronic hypoxic (20), and postmyocardial infarct (21) rat myocardium. Interestingly, UT-IIR up-regulation in the myocardium of hypoxic rats was accompanied by ventricular hypertrophy (20). In vivo, short-term hypertrophic growth of cardiomyocytes is an adaptive response that increases cardiac output in cardiac dysfunction (heart failure, hypertension) and injury (myocardial infarction), whereas sustained hypertrophic growth is maladaptive and can cause adverse cardiac remodeling, which is characterized by inflammation, fibrosis, and cardiac ventricular hypertrophy. We recently developed an in vitro neonatal cardiomyocyte culture model expressing recombinant UT-II receptors, to mimic the up-regulation observed in the diseased myocardium (21). In this in vitro setting, U-II promotes hypertrophic growth (increase in cell size and protein content in the absence of proliferation) of cardiomyocytes (21), mimicking the hypertrophy observed in vivo (20). Alterations in the expression of fetal genes indicative of hypertrophy (atrial natriuretic peptide, myosin light chain-2 and {alpha}-skeletal actin genes) and an increased expression of fibronectin {alpha}1(I) and {alpha}1(III) and procollagen mRNA transcripts suggestive of fibrosis were also observed (21). Thus, U-II may be beneficial to cardiac output in the healthy myocardium (22) and may promote cardiac fibrosis and hypertrophy in the failing myocardium (where UT-IIR expression levels are increased). At present, the signaling mechanisms that couple U-II to hypertrophy are unknown.

In the current study, the signaling pathways that control U-II-mediated cardiomyocyte hypertrophy were assessed in our in vitro model. This has allowed us to identify a key role for the epidermal growth factor receptor (EGFR) trans-activation and the activation of ERK1/2 and p38 MAPK in U-II-mediated hypertrophy.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Characterization of Adenoviral Vectors Expressing the UT-IIR (AdUT-IIR)
Cardiomyocyte cultures were infected with an adenovirus, AdUT-IIR, that coexpresses the UT-IIR and the marker protein, green fluorescent protein (GFP), under separate cytomegalovirus promoters (23); viral infection was monitored by following GFP expression. AdUT-IIR was titrated up to 20 x 103 pfu per well, and the level of UT-IIR expressed was determined by radioligand binding of [125I]U-II. In the absence of adenoviral infection, cultured neonatal cardiomyocytes expressed very low levels of UT-IIR, whereas the addition of increasing amounts of AdUT-IIR led to enhanced receptor expression, up to 1000 fmol receptor/mg protein at 20 x 103 pfu (Fig. 1AGo). At this maximal level of adenovirus infection, more than 95% of cardiomyocyte cells displayed GFP expression (Fig. 1BGo). Adenoviral-directed UT-II receptors displayed high affinity for U-II [dissociation constant (kd) = 3 nM; Fig. 1CGo].



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  		Fig. 1. Characterization of AdUT-IIR

A, Cardiomyocytes were infected with increasing amounts [0–20 x 103 pfu (PFU)] of AdUT-IIR and UT-IIR expression was determined by radioligand binding assay using [125I]U-II. Receptor expression (femtomoles receptor/mg protein) was determined as described elsewhere (59 ) using a kd of 3 nM. B, When infected with 20 x 103 pfu of AdUT-IIR (which coexpresses GFP), greater than 90% of cardiomyocytes displayed fluorescence. Left panel, Phase contrast; right panel, GFP fluorescence. C, Competition binding was performed on cardiomyocytes 72 h after AdUT-IIR infection (5 x 103 pfu, 430 fmol receptor/mg protein), using increasing concentrations of unlabeled U-II. Binding is expressed as a percent of Bmax, which is the level of binding in the absence of unlabeled U-II. Data are the average of four experiments and the mean kd was determined as 3 nM.

 
UT-IIR-Directed Cardiomyocyte Hypertrophy
Hypertrophy is defined as a significant increase in cellular size and protein levels without changes in cell number and DNA content (24, 25). The hypertrophic effect of U-II (100 nM) was assessed in cardiomyocytes infected over a range of AdUT-IIR (0 to 20 x 103 pfu). In the absence of AdUT-IIR infection, U-II stimulation did not cause hypertrophy, whereas infection with AdUT-IIR promoted U-II-dependent hypertrophy (Fig. 2AGo). The lowest level of adenovirus infection that resulted in significant U-II-dependent hypertrophy was 3 x 103 pfu/per well (expressing 280 fmol of receptor/mg protein). Cells infected with 5 x 103 pfu (expressing 430 fmol receptor/mg protein) hypertrophied at a level equivalent to that produced by the {alpha}1adrenergic receptor agonist phenylephrine (U-II 133 ± 7%; P > 0.001 vs. phenylephrine 128 ± 5%; Fig. 2AGo), and subsequent experiments were undertaken at this level of UT-IIR expression. Dose-response curves confirmed the potent hypertrophic effect of U-II (EC50 value of 0.26 nM) (Fig. 2BGo).



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  		Fig. 2. UT-IIR Directs Cardiomyocyte Hypertrophy

A, Cardiomyocytes were infected with increasing amounts of AdUT-IIR (0–20 x 103 pfu) and stimulated with U-II (100 nM, gray bars). Uninfected myocytes were stimulated with phenylephrine (25 µM) in the presence of propranolol (1 µM) as a positive control for hypertrophy (black bar). Cells were harvested after 72 h stimulation and assayed for total protein content. Data are expressed as the average (±SEM) percent change in protein content compared with uninfected, unstimulated control, which had a protein content of 120 ± 6 µg/well. *, P < 0.05; and **, P < 0.001 vs. control cells (n = 4). B, AdUT-IIR-infected cardiomyocytes (5 x 103 pfu, 430 fmol receptor/mg protein) were stimulated with increasing concentrations of U-II (0.01 nM-100 nM) and total cell protein was determined. Basal protein content in the absence of stimulation was 112 ± 4 µg protein/well (mean ± SEM, n = 3).

 
U-II Induces Sarcomeric Reorganization
In addition to an increase in cellular protein (Fig. 2AGo), hypertrophic myocytes display the organization of the contractile protein actin into sarcomeres (contractile units) (reviewed in Ref. 26). U-II-mediated hypertrophy was also confirmed morphologically using phase and fluorescence microscopy (Fig. 3Go). In the absence of U-II stimulation, both uninfected and AdUT-IIRinfected cardiomyocytes appeared the same, indicating that receptor expression alone does not promote hypertrophic changes (panels 1 and 4, respectively). U-II stimulation of uninfected cells also failed to modify cell phenotype (panel 2), whereas in AdUT-IIR-infected cells, U-II stimulated an increase in cell size (panel 5) and an increase in sarcomeric fiber bundles (panel 8). These phenotypic changes were similar to those observed after the {alpha}1-adrenergic receptor stimulation of uninfected cardiomyocytes (panels 3 and 9).



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  		Fig. 3. UT-IIR-Mediated Hypertrophy Is Associated with Changes in Cell Morphology

Uninfected cardiomyocytes were not stimulated (panel 1), stimulated with U-II (panel 2), or treated with the {alpha}1-adrenergic agonist, phenylephrine, as a positive control (panel 3). Cardiomyocytes infected with AdUT-IIR were unstimulated (panel 4) or stimulated with U-II and visualized by phase (panel 5) or fluorescence microscopy (panel 6). Panels 7–9, Cardiomyocytes were fixed with 4% paraformaldehyde and stained with tetramethylrhodamine B isothiocyanate-labeled phalloidin to visualize sarcomeric reorganization. Cardiomyocytes were infected with AdUT-IIR and left unstimulated (panel 7), or stimulated with U-II (panel 8). Uninfected cells were stimulated with phenylephrine (panel 9). For all panels x20 objective magnification was used.

 
U-II Activates MAPK
Given the central role proposed for MAPKs in hypertrophy, we examined the involvement of the three major MAPK signaling pathways in U-II-mediated hypertrophy, specifically ERK1/2, JNK, and p38. U-II stimulation of AdUT-IIR-infected cardiomyocytes resulted in a robust activation of ERK1/2 at 5–10 min and remained above basal up to 60 min after stimulation (Fig. 4AGo). U-II stimulation of AdUT-IIR-infected cardiomyocytes also produced a strong activation of p38 at 5 min, which persisted up to 60 min after stimulation (Fig. 4BGo). U-II failed to activate JNK in these cells compared with transient UV stimulation, a robust activator of JNK (Fig. 4CGo).



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  		Fig. 4. U-II Activation of MAPK

Cardiomyocytes were infected with AdUT-IIR and stimulated with U-II (100 nM) for various time points (0–60 min). Western blots of cell extracts were probed with antibodies to activated ERK1/2 (A, upper blot), p38 (B, upper blot), and JNK (C, upper blot). Blots were stripped and reprobed with total antibodies for the corresponding MAPK (lower blots). U-II activates ERK1/2 and p38 but does not activate JNK over a 60-min time point. Positive control for JNK was human embryonic kidney 293 cells stimulated with UV light for 30 min. Blots are representative of three experiments. Activated ERK1/2 (A, bottom panel) and p38 (B, bottom panel) were normalized for the corresponding total MAPK blots and expressed as a fold increase in activation over basal (0 time, unstimulated).

 
Mechanisms of MAPK Activation by U-II
Trans-activation of the EGFR is a major route for GPCR-mediated MAPK activation. U-II stimulation of cells infected with AdUT-IIR resulted in the tyrosine phosphorylation of the EGFR, and this trans-activation was completely blocked by the EGFR kinase inhibitor AG1478 (Fig. 5AGo). We confirmed that EGF stimulation of cardiomyocytes also activates ERK1/2 and p38 and that AG1478 completely inhibited this activation (Fig. 5BGo). Interestingly, AG1478 inhibited U-II-activated ERK1/2 by 40 ± 7% and p38 by 83 ± 6% (Fig. 5CGo). This suggests that U-II activation of p38 is largely dependent on EGFR trans-activation, whereas U-II activation of ERK1/2 is less so.



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  		Fig. 5. EGFR trans-Activation in U-II-Dependent MAPK Activation

A, Cardiomyocytes were infected with AdUT-IIR for 72 h and left untreated or stimulated with 100 nM U-II for 3 min in the presence or absence of the EGFR inhibitor, AG1478 (5 µM). Cells were also stimulated with EGF (10 nM) as a positive control for EGFR phosphorylation. The EGFR was immunoprecipitated from cell extracts and Western blotted using antibodies for phosphotyrosine (anti-P Tyr, top panel) and total EGFR protein (bottom panel). This blot is representative of three experiments. B, Cardiomyocytes were stimulated with EGF (10 nM, 10 min), with or without AG1478 pretreatment (5 µM), and extracts were Western blotted for activated and total ERK1/2 and p38. C, Cardiomyocytes were infected with AdUT-IIR for 72 h, preincubated with AG1478 (5 µM), and left unstimulated or stimulated with U-II (100 nM, 10 min), as indicated. Extracts were Western blotted and probed for activated and total ERK1/2 or p38; fold changes in ERK1/2 and p38 activity are indicated.

 
Two major mechanisms for GPCR-dependent trans-activation of the EGFR have been proposed. The first involves the release of heparin-bound EGF (HB-EGF) from a membrane-bound precursor by the actions of matrix metalloproteases that are activated by GPCR stimulation (27). Another mechanism of EGFR trans-activation involves the GPCR second messenger PKC and/or calcium and the nonreceptor tyrosine kinases Src and Pyk2(27). Furthermore, PKC or calcium can directly activate MAPK pathway-independent EGFR trans-activation (28). Inhibition of HB-EGF with heparin reduced U-II-mediated ERK1/2 and p38 activation by approximately 60%; however, inhibition of PKC with bisindolylmaleimide I (BIM-I) and chelation of calcium with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-2 AM) had no effect, although basal p38 activation was high in the presence of BAPTA-2 AM (Fig. 6Go). Furthermore, inhibition of phosphoinositide 3-kinase (PI3K) with wortmannin did not have any effect on ERK1/2 or p38 activation (Fig. 6Go), and this was also confirmed using LY294002 and the mammalian target of rapamycin inhibitor rapamycin (data not shown). These data indicate that U-II trans-activates the EGFR (and activates MAPK) via HB-EGF release.



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  		Fig. 6. Mechanisms of MAPK Activation by U-II

Cardiomyocytes were infected with AdUT-IIR for 72 h, preincubated with heparin (100 µg/ml), BIM (50 µM), BAPTA-2 AM (10 µM), and wortmannin (0.2 µM), and left unstimulated or stimulated with U-II (100 nM, 10 min), as indicated. Extracts were Western blotted and probed for activated and total ERK1/2 or p38; fold changes in ERK1/2 and p38 activity are indicated.

 
Signaling Mechanisms Involved in U-II Hypertrophy
First, we confirmed that PD98059 and SB203580 selectively and completely inhibited ERK1/2 and p38, respectively, under the conditions used in these experiments (Fig. 7AGo). PD098059 inhibited U-II-hypertrophy by about 50%, from 133 ± 7% to 118 ± 7% (P < 0.05), and SB203580 treatment inhibited U-II-mediated hypertrophy by 60%, from 133 ± 7% to 113 ± 10% (P < 0.05) (Fig. 7BGo). PD098059 and SB203580 had no effect on basal cellular protein levels in the absence of U-II stimulation, 102 ± 2% and 98 ± 8%, respectively (Fig. 7BGo). These data indicate that ERK1/2 and p38 signaling pathways both contribute to U-II-mediated hypertrophic growth.



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  		Fig. 7. Signaling Mechanisms of U-II-Mediated Hypertrophy

A, Cardiomyocytes were infected with AdUT-IIR for 72 h, preincubated with PD098059 (20 µM) or SB203580 (20 µM), and left unstimulated or stimulated with U-II (100 nM, 10 min), as indicated. Extracts were Western blotted and probed for activated and total ERK1/2 or p38; fold changes in ERK1/2 and p38 activity are indicated. B, Cardiomyocytes were infected with AdUT-IIR, preincubated with PD098059 (20 µM), SB203580 (20 µM), or AG1478 (5 µM), and left unstimulated (white bars) or stimulated with U-II (100 nM) (black bars). Hypertrophy (± SEM, n = 4) was measured 72 h after U-II stimulation. **, P < 0.001 vs. unstimulated control; *, P < 0.05 vs. U-II stimulated. The addition of PD098059, SB203580, and AG1478 had no effect on protein levels in the absence of U-II stimulation (white bars). C (left panel), Cardiomyocytes were stimulated with EGF (10 nM) in the presence or absence of AG1478 (5 µM) pretreatment and hypertrophy was determined 72 h later (±SEM, n = 3); right panel, uninfected or AdUT-IIR-infected cardiomyocytes were stimulated with U-II (100 nM) or PMA (100 nM) in the presence or absence of BIM-I (50 µM), as indicated. Hypertrophy (±SEM, n = 3) was measured 72 h after U-II stimulation. *, P < 0.05 vs. PMA stimulated; ns, not significant.

 
AG1478 significantly reduced U-II-mediated hypertrophy, from 133 ± 7% to 117 ± 6% [P < 0.05 (Fig. 7BGo)], whereas AG1478 did not affect cellular protein levels in the absence of U-II stimulation (100 ± 6%). This demonstrates that a component of U-II-hypertrophy involves EGFR trans-activation signaling pathways, in line with its contribution to ERK and p38 activation (Fig. 5CGo). Indeed, direct stimulation of cardiomyocytes with EGF also led to hypertrophy, which was completely inhibited by AG1478 (Fig. 7CGo, left panel). Furthermore, the combined inhibition of ERK1/2 and the EGFR completely reduced U-IImediated hypertrophy from 133 ± 7% to 106 ± 5% (Fig. 7BGo), suggesting that U-II-mediated hypertrophy involves EGFR trans-activation (for ERK1/2 and p38 activation) as well as other signaling pathways that activate ERK1/2.

Phorbol 12-myristate 13-acetate (PMA)-mediated ERK1/2 activation (data not shown) and hypertrophy (Fig. 7CGo) were completely blocked by inhibition of PKC with BIM-I (Fig. 7CGo). U-II-mediated activation of ERK1/2 and p38 was not inhibited by BIM-I (Fig. 6Go) nor was U-II-mediated hypertrophy (129 ± 7% compared with 133 ± 7% for U-II stimulation alone) (Fig. 7CGo). Hence, U-II hypertrophic signaling appears to be PKC independent.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Endogenous levels of UT-IIRs are very low in the healthy myocardium of humans (18) and rats (19, 20, 21) but are increased in disease (18, 19, 20, 21). In hypoxic rats, UT-IIRs are up-regulated in the myocardium, which is hypertrophied (20). There is no information available detailing the signal transduction pathways that facilitate U-II-mediated hypertrophic growth in cardiomyocytes. In this study, we have used a previously developed in vitro model of U-II-mediated cardiac hypertrophy (21) to show that hypertrophic growth and phenotypic changes in cardiomyocytes are dependent upon the level of UT-IIRs, and the capacity of U-II to activate ERK1/2, p38, and EGFR-dependent signaling pathways.

In our hands, [125I]U-II binding studies revealed that UT-IIRs are below the level of detection in uninfected cardiomyocyte cultures, and thus U-II stimulation does not cause hypertrophy under these conditions. In contrast to our findings, Zou et al. (30) reported U-II-dependent hypertrophic changes in isolated cardiomyocyte cultures. These somewhat contradictory results most likely reflect differences in the preparation of cardiomyocyte cultures. Standard preparations of cultured neonatal cardiomyocytes contain approximately 3–5% of cardiac fibroblasts, which rapidly increase if the antimitotic agent, 5-bromo-2'-deoxyuridine, is not added to the media. KCl (50 mM) is also added to prevent spontaneous contractions that can cause hypertrophic growth of high-density cardiomyocyte cultures, which is independent of growth factors and GPCR activation (24, 31). In the experiments of Zou et al. (30), fibroblast content and contractility were not controlled for, which may account for the discrepancy between the two studies. Like U-II, we have also reported that the prototypical hypertrophic agonist, angiotensin II, does not cause hypertrophy in purified neonatal cardiomyocyte cultures (25). This was also due to low levels of AT1A receptor expression and not the lack of appropriate adaptor molecules and signaling pathways in these cells. Indeed, infection of cardiomyocytes with adenovirus expressing the AT1A receptor was necessary to produce angiotensin II-dependent hypertrophy. Therefore, it appears that receptor levels need to be above a threshold for cardiomyocyte hypertrophy to occur and, in agreement with this, the extent of hypertrophy observed after U-II stimulation was proportional to the level of UT-II receptor expressed.

GPCRs regulate cell growth by a network of interrelated signaling pathways including Gq-activated PKC and calcium, as well as MAPK and tyrosine kinase pathways, although the relative contribution of each differs between receptors and tissues. In the present study, we provide the first evidence for the involvement of ERK1/2 and p38, but not JNK, in U-II-mediated hypertrophic growth of isolated rat cardiomyocytes. In line with this, other studies have also found ERK1/2 (32, 33, 34, 35) and p38 (35, 36, 37, 38) signaling pathways to be important in the development of cardiomyocyte hypertrophy in vitro and in vivo. EGFR trans-activation is a major route for GPCRs to activate ERK1/2 (25, 39, 40) and p38 MAPK (39, 41), and we show that a significant component of U-II-mediated hypertrophy involves the trans-activation of the EGFR, presumably via its capacity to initiate ERK and p38 signaling. Indeed, we have demonstrated that U-II-activation of ERK1/2 was partially dependent, whereas activation of p38 was strongly dependent on EGFR trans-activation. However, inhibition of both the EGFR and ERK1/2 was required to completely prevent U-II-directed hypertrophy, suggesting the additional involvement of an EGFR-independent pathway for ERK1/2 activation and hypertrophy by U-II.

Despite much evidence supporting a role for specific PKC isoforms in ERK1/2 activation and cardiac hypertrophy (reviewed in Refs. 28 and 42), we observed no effect of PKC inhibition on the hypertrophy of cardiomyocytes in response to U-II stimulation. Interestingly, angiotensin II-induced hypertrophy in cardiomyocytes is also PKC independent, although in contrast to U-II, it is completely dependent on EGFR-driven ERK1/2 signaling pathways (25). To contrast further, another Gq-coupled GPCR agonist, endothelin-1, requires PKC signaling pathways and EGFR trans-activation for the activation of ERK1/2 and hypertrophy (Hannan, R.D., and J. Osborne, unpublished observations). Finally, our observations for hypertrophic growth in cardiomyocytes contrast with those observed for proliferative U-II growth in vascular cells, which is primarily PKC and ERK dependent, although it does involve a contribution of the EGFR (13, 14). Therefore, although a defined group of signal transduction pathways can contribute to cell growth, the relative involvement is contextual.

The EGFR family has important roles in normal and pathological growth of the heart (43, 44), and GPCRs can activate EGFRs in a number of ways. The EGFR can bind ligands (e.g. HB-EGF, neuregulins) that are shed from the cell surface by GPCR-induced matrix-metalloprotease activation, which activates the small GTP-binding protein Ras and downstream kinases (Raf and MAPKs) (45). Another mechanism of EGFR trans-activation involves intracellular nonreceptor tyrosine kinases, such as Src or Pyk2, which are activated by the Gq-signaling mediators, calcium and/or PKC (40, 45). The capacity of U-II to activate MAPK and trans-activate the EGFR was not dependent on PKC or calcium, but it appeared to be dependent on HB-EGF, because inhibition of HB-EGF with heparin inhibited U-II-mediated ERK and p38 activation.

We have previously reported (21) that inhibition of Gq or the small GTP-binding protein Ras does not completely inhibit (~50% reduction) the activation of the hypertrophic marker atrial natriuretic peptide by U-II, indicating that other G proteins may contribute to U-II-mediated hypertrophy. Consistent with this, activation of p38, which we have identified as important for U-II-mediated hypertrophy, usually requires the involvement of G proteins other than Ras (e.g. the Rho members, RhoA, Rac, and Cdc42). In addition, although ß{gamma}-subunits (which are released from activated G proteins) can stimulate MAPK through PI3K and tyrosine kinases, we found that pharmacological inhibition of PI3K and downstream mammalian target of rapamycin did not hinder the capacity of U-II to activate ERK1/2 or p38, indicating that the PI3K pathway is not involved.

Moderate levels of Gq signaling stimulate adaptive hypertrophy (46, 47, 48), whereas high levels of Gq activation result in maladaptive cardiomyocyte apoptosis (49, 50, 51, 52). Interestingly, and in marked contrast to the AT1A receptor (25), we observed that UT-IIR expression above 1000 fmol/mg protein (titers of AdUT-IIR greater than reported in the current study), resulted in U-II-dependent cell death (Onan, D., and W. G. Thomas, unpublished observations). This observation supports the contention that extended activation of Gq by some GPCRs promotes a transition from hypertrophy to apoptosis (53). Unlike angiotensin II, vasoconstriction to U-II is noticeably sustained (1, 54, 55), suggesting that regulatory mechanisms that commonly terminate GPCR signaling (i.e. receptor phosphorylation and internalization) may not be evoked for the UT-IIR. Indeed, we have evidence that the UT-IIR is poorly phosphorylated and internalized after U-II stimulation (Onan, D., and W. G. Thomas, manuscript in preparation). Whether these differences in AT1A and UT-IIR regulation impact on the relative contribution of various signaling pathways and contribute to the strength and duration of signaling, to facilitate the transition from hypertrophic to apoptotic signaling requires further investigation.

Finally, observations made in vitro (neonatal cardiomyocyte cultures) have been confirmed in vivo (26, 56), strengthening the validity of cell culture models. Accordingly, we propose that U-II is likely to mediate cardiomyocyte hypertrophy in vivo during cardiovascular disease states (e.g. heart failure and myocardial infarction) where cardiac UT-IIRs are increased. This hypertrophy requires activation of MAPKs, specifically ERK1/2 and p38, through a mechanism that involves EGFR trans-activation. Future in vivo studies will be undertaken to investigate the contribution of U-II, its receptor, and these specific signaling pathways in cardiac health and disease.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Generation of UT-IIR Expressing Adenovirus
The rat UT-IIR was cloned into the mammalian expression vector pRc/CMV by PCR from reverse transcribed heart mRNA using sense (5'-GAATCCCAGTGCGGCCGCCACCATGG CTCTGAGCCTGGA GTCTACAAC-3') and antisense (5'-CCTATGTCTAGATTGCACAGTGCACTCTCAGAG-3') primers corresponding to positions 799–846 and 1976–2008, respectively, of the published sequence (57). An optimized Kozak ribosome binding site (underlined) surrounding the methionine start codon (italicized) was included in the sense primer. The NotI (sense) and XbaI (antisense) restriction sites are shown in bold. The pAd-Easy1 virus and shuttle plasmid, pAdTrack/CMV, were obtained from Dr. B. Volgenstein (Johns Hopkins Oncology Center, Baltimore, MD). UT-IIR cDNA was subcloned into the NotI and XbaI sites of the shuttle vector, pAdTrack/CMV, yielding pAdTrack-UT-R. Recombinant adenovirus (AdUT-IIR) was generated by bacterial homologous recombination between PmeI linearized pAdTrack-UT-R and pAdEasy-1(23). Large-scale amplification and purification of AdUT-IIR virus were performed as described elsewhere (58). The titer of AdUT-IIR viral stock used for these studies was measured by plaque titration on human embryonic kidney 293 cells and determined as 0.02 x 109 pfu/ml.

Cell Culture
Neonatal cardiac myocytes were isolated from the ventricles of 1-d-old Sprague Dawley rat pups (Precinct Animal Centre, Alfred Medical Research and Education Precinct) and plated on 12-well culture plates (coated with 0.1% gelatin) at high density (1250 cells/mm2) or low density (330 cells/mm2) as previously described (24, 31). Myocyte cultures were incubated in DMEM with 10% FBS and 0.1 mM of bromodeoxyuridine overnight to allow attachment. Cells were transferred to defined media (DMEM containing 0.037% NaHCO3, 0.01% antibiotic/antimycotic, 0.01% vitamins, 0.02% nonessential amino acids, 0.1% sodium pyruvate, 2 mg insulin, 30 mg 5-bromo-2'-deoxyuridine, and 10 mg apotransferrin per liter) containing 50 mM KCl and infected with adenovirus. Cardiomyocyte cultures prepared in this way have been shown to contain 3–5% cardiac fibroblasts (24); hence the antimitotic agent, 5-bromo-2'-deoxyuridine, is added to culture media to prevent the otherwise rapid proliferation of fibroblasts and to ensure results were cardiomyocyte specific. KCl (50 mM) was also added to the medium to prevent spontaneous contraction characteristic of neonatal cardiac myocytes plated at high density (24).

Adenoviral Infection of Cardiac Myocytes
Cardiac myocytes were transferred to defined media 24 h after plating and infected with AdUT-IIR. Virus (20 x 103 pfu) infected at least 90% of 0.45 x 106 myocytes (1250 cells/mm2) 72–96 h after infection, as defined by GFP fluorescence (see Fig. 1BGo). For experiments, a much lower level (5 x 103 pfu of virus) was used, which resulted in a moderate level of UT-IIR expression (430 fmol receptor/mg protein) in cardiomyocyte cultures.

AdUT-IIR Binding
UT-IIR expression levels in neonatal cardiomyocyte cultures (1250 cells/mm2) were determined 72 h after infection with increasing doses of AdUT-IIR, from 0–200 x 103 pfu, using whole-cell binding of 125I-labeled rat U-II (specific activity, ~1000 Ci/mmol, Austin Biomedical Services, Melbourne, Australia). Briefly, cells were washed twice with Hank’s buffered salt solution and equilibrated with UT-IIR binding buffer (200 mM Tris-Cl, pH 7.4; 120 mM NaCl; 5 mM MgCl2; 2 mg/ml D-glucose; and 0.1% BSA) at 4 C. 125I-labeled rat U-II (30 pM) was added per well in the absence or presence of excess unlabeled U-II (10–6 M) for 5 h at 4 C. Cells were washed three times with UT-IIR binding buffer, extracted with 0.25% sodium dodecyl sulfate/0.25 M NaOH solution, and counted on a {gamma}-counter. Receptor affinities were determined using displacement by a series of U-II concentrations (10–11–10–6 M).

Cardiomyocyte Hypertrophy Assay
One day after viral infection the cardiac myocytes were treated with the appropriate pharmacological inhibitors [PD098059 (20 µM), AG1478 (5 µM), BIM (50 µM), and SB203580 (20 µM)], wortmannin (0.2 µM), heparin (100 µg/ml), BAPTA-2 AM (10 µM), rapamycin (0.02 µM), and LY294002 (20 µM), for 45 min followed by stimulation with U-II (100 nM). After 72 h of U-II stimulation, cells were trypsinized and the cells were washed and resuspended in HBSS. Half of the sample was used to determine total protein content using the Lowry protein assay and the other half was used to determine DNA content using the Burton assay. Hypertrophy was defined as a significant increase in protein content in the absence of a significant change in DNA content.

Sarcomeric Organization
Cardiomyocyte cultures (330 cells/mm2) were stimulated with phenylephrine (25 µM) and propranolol (1 µM), or U-II (100 nM) 24 h after infection with 5 x 103 pfu of virus, and incubated for an additional 72 h. Cells were then washed with PBS and fixed with 4% paraformaldehyde for 30 min at room temperature. Fixed cells were then incubated with 0.1% Triton X-100/3% BSA for 30 min, followed by a 1-h incubation with tetramethylrhodamine B isothiocyanate-labeled phalloidin (10 µg/ml) at room temperature. Cells were washed thoroughly with PBS and visualized by fluorescence microscopy.

MAPK Assays
Activation of ERK1/2, JNK, and p38 in response to U-II stimulation, in the absence or presence of specific inhibitors [PD098059 (20 µM), AG1478 (5 µM), BIM (50 µM), and SB203580 (20 µM), wortmannin (0.2 µM), heparin (100 µg/ml), BAPTA-2 AM (10 µM), rapamycin (0.02 µM), and LY294002 (20 µM)], was examined by Western blot as described elsewhere (25). Briefly, 72 h after cardiac myocytes (1250 cells/mm2) were infected with 5 pfu AdUT-IIR, cells were stimulated with U-II (100 nM) for various time points in the presence or absence of the indicated MAPK inhibitor, which was added 45 min before U-II stimulation. For active and total ERK and p38 MAPK blots, cells were lysed with 250 µl of RIPA buffer (50 mM Tris, pH 7.4, 100 mM NaCl, 2 mM EDTA, 50 mM sodium fluoride, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 10 mM sodium pyrophosphate, 1 µg/ml aprotinin, 5 µg/ml leupeptin, 1 µg/ml pepstatin); however, 100 µl of 1x sodium dodecyl sulfate sample buffer was used to lyse cells for active and total JNK blots. Cell extracts were Western blotted and probed for active MAPKs with monoclonal antibodies to phospho-p44/42 (ERK1/2) (Cell Signaling Technology, Beverley, MA; catalog no. 9106L), phospho-JNK (Cell Signaling Technology, catalog no. 9255) and phospho-p38 (Promega V-121A). Blots were stripped and reprobed for total MAPK using anti-ERK1 antibodies (C-16; Santa Cruz Biotechnology, Inc., Santa Cruz, CA; catalog no. sc-93), anti-JNK antibodies (Cell Signaling Technology; catalog no. 9252), and anti-p38 antibodies (Santa Cruz Biotechnology, Inc., catalog no. sc-728), respectively. The relative levels of active and total MAPK were quantified using Scion Image (Scion Corp., Frederick, MD). The level of active MAPK was normalized for total MAPK loading on the gel and expressed as a fold increase of the values obtained for MAPK activity in uninfected, unstimulated cells.

EGFR Phosphorylation
Cardiomyocyte cultures grown in 10-cm2 dishes (1250 cells/mm2) were infected with 5 x 103 pfu of AdUT-IIR 24 h after preparation. Cultures were stimulated 72 h after infection for 3 min with EGF (10 nM), or U-II (100 nM) in the presence or absence of the EGFR inhibitor AG1478 (5 µM). Extracts were immunoprecipitated using anti-EGFR antibodies (Santa Cruz catalog no. sc-1005). Western blots were probed sequentially with antiphosphotyrosine antibody (Clone 4G10, Upstate Biotechnology, Inc., Lake Placid, NY; catalog no. 05–321) and the anti-EGFR antibody (Santa Cruz catalog no. sc-1005).

Statistical Analysis
For multigroup comparisons, data were analyzed by one-way ANOVA followed by Newman-Keuls comparison between all groups. Data are presented as mean ± SEM, and P < 0.05 was considered to be significantly different.


   ACKNOWLEDGMENTS
 
We thank Karen Stewart and Catherine Hamilton for the synthesis and purification of the U-II peptide. We also thank Anna Jenkins for cardiomyocyte preparations and Thao Pham for assistance with adenovirus preparation.


   FOOTNOTES
 
This work was supported by the National Health and Medical Research Council of Australia Block Grant 993001 and Project Grants 166900 (to R.D.H) and 225123 (to W.G.T.).

Current Address for R.D.H.: Growth Control Laboratory, Peter MacCallum Cancer Research Centre, Locked Bag 1, A’Beckett Street, Melbourne 8006, Victoria, Australia.

Abbreviations: AdUT-IIR, Adenovirus expressing the urotensin receptor; BAPTA-2AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester); BIM-I, bisindolylmaleimide-I; EGFR, epidermal growth factor receptor; GFP, green fluorescent protein; GPCR, G protein-coupled receptor; HB-EGF, heparin-bound epidermal growth factor; JNK, c-Jun N-terminal kinase; kd, dissociation constant; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; U-II, urotensin-II; UT-IIR, urotensin II receptor.

Received for publication August 14, 2003. Accepted for publication June 1, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Ames RS, Sarau HM, Chambers JK, Willette RN, Aiyar NV, Romanic AM, Louden CS, Foley JJ, Sauermelch CF, Coatney RW, Ao Z, Disa J, Holmes SD, Stadel JM, Martin JD, Liu WS, Glover GI, Wilson S, McNulty DE, Ellis CE, Elshourbagy NA, Shabon U, Trill JJ, Hay DW, Douglas SA, et al 1999 Human urotensin-II is a potent vasoconstrictor and agonist for the orphan receptor GPR14. Nature 401:282–286[CrossRef][Medline]
  2. Coulouarn Y, Lihrmann I, Jegou S, Anovar Y, Tostivint H, Beauvillain JC, Conlon JM, Bern HA, Vaudry H 1998 Cloning of the cDNA encoding the urotensin II precursor in frog and human reveals intense expression of the urotensin II gene in motoneurons of the spinal cord. Proc Natl Acad Sci USA 95:15803–15808[Abstract/Free Full Text]
  3. Liu Q, Pong SS, Zeng Z, Zhang Q, Howard AD, Williams Jr DL, Davidoff M, Wang R, Austin CP, McDonald TP, Bai C, George SR, Evans JF, Caskey CT 1999 Identification of urotensin II as the endogenous ligand for the orphan G-protein-coupled receptor GPR14. Biochem Biophys Res Commun 266:174–178[CrossRef][Medline]
  4. Nothacker HP, Wang Z, McNeill AM, Saito Y, Mertens S, O’Dowd B, Duckles SP, Civelli O 1999 Identification of the natural ligand of an orphan G-protein-coupled receptor involved in the regulation of vasoconstriction. Nat Cell Biol 1:383–385[CrossRef][Medline]
  5. Saetrum Opgaard O, Nothacker H, Ehlert FJ, Krause DN 2000 Human urotensin II mediates vasoconstriction via an increase in inositol phosphates. Eur J Pharmacol 406:265–271[CrossRef][Medline]
  6. Zhu YZ, Wang ZJ, Zhu YC, Zhang L, Oakley RM, Chung CW, Lim KW, Lee HS, Ozoux ML, Linz W, Bohm M, Kostenis E 2004 Urotensin II causes fatal circulatory collapse in anesthesized monkeys in vivo: a "vasoconstrictor" with a unique hemodynamic profile. Am J Physiol 286:H830–H836
  7. Maguire JJ, Davenport AP 2002 Is urotensin-II the new endothelin? Br J Pharmacol 137:579–588[CrossRef][Medline]
  8. Silvestre RA, Rodriguez-Gallardo J, Egido EM, Marco J 2001 Inhibition of insulin release by urotensin II—a study on the perfused rat pancreas. Horm Metab Res 33:379–381[CrossRef][Medline]
  9. Totsune K, Takahashi K, Arihara Z, Sone M, Ito S, Murakami O 2003 Increased plasma urotensin II levels in patients with diabetes mellitus. Clin Sci (Colch) 104:1–5[Medline]
  10. Totsune K, Takahashi K, Arihara Z, Sone M, Satoh F, Ito S, Kimura Y, Sasano H, Murakami O 2001 Role of urotensin II in patients on dialysis. Lancet 358:810–811[CrossRef][Medline]
  11. Matsushita M, Shichiri M, Imai T, Iwashina M, Tanaka H, Takasu N, Hirata Y 2001 Co-expression of urotensin II and its receptor (GPR14) in human cardiovascular and renal tissues. J Hypertens 19:2185–2190[CrossRef][Medline]
  12. Sauzeau V, Le Mellionnec E, Bertoglio J, Scalbert E, Pacaud P, Loirand G 2001 Human urotensin II-induced contraction and arterial smooth muscle cell proliferation are mediated by RhoA and Rho-kinase. Circ Res 88:1102–1104[Abstract/Free Full Text]
  13. Watanabe T, Pakala R, Katagiri T, Benedict CR 2001 Synergistic effect of urotensin II with serotonin on vascular smooth muscle cell proliferation. J Hypertens 19:2191–2196[CrossRef][Medline]
  14. Watanabe T, Pakala R, Katagiri T, Benedict CR 2001 Synergistic effect of urotensin II with mildly oxidized LDL on DNA synthesis in vascular smooth muscle cells. Circulation 104:16–18[Abstract/Free Full Text]
  15. Takahashi K, Totsune K, Murakami O, Shibahara S 2001 Expression of urotensin II and urotensin II receptor mRNAs in various human tumor cell lines and secretion of urotensin II-like immunoreactivity by SW-13 adrenocortical carcinoma cells. Peptides 22:1175–1179[CrossRef][Medline]
  16. Takahashi K, Totsune K, Murakami O, Arihara Z, Noshiro T, Hayashi Y, Shibahara S 2003 Expression of urotensin II and its receptor in adrenal tumors and stimulation of proliferation of cultured tumor cells by urotensin II. Peptides 24:301–306[CrossRef][Medline]
  17. Shenouda A, Douglas SA, Ohlstein EH, Giaid A 2002 Localization of urotensin-II immunoreactivity in normal human kidneys and renal carcinoma. J Histochem Cytochem 50:885–889[Abstract/Free Full Text]
  18. Douglas SA, Tayara L, Ohlstein EH, Halawa N, Giaid A 2002 Congestive heart failure and expression of myocardial urotensin II. Lancet 359:1990–1997[CrossRef][Medline]
  19. Zhou P, Wu SY, Yu CF, Wang H, Tang CS, Lin L, Yuan WJ 2003 [Effects of urotensin II on isolated rat hearts under normal perfusion and ischemia reperfusion]. Sheng Li Xue Bao 55:442–448[Medline]
  20. Zhang Y, Li J, Cao J, et al 2002 Effect of chronic hypoxia on contents of urotensin II and its functional receptors in rat myocardium. Heart Vessels 16:64–68[CrossRef][Medline]
  21. Tzanidis A, Hannan RD, Thomas WG, Onan D, Autelitano DJ, See F, Kelly DJ, Gilbert RE, Krum H 2003 Direct actions of urotensin II on the heart. Implications for cardiac fibrosis and hypertrophy. Circ Res 93:246–253[Abstract/Free Full Text]
  22. Russell FD, Molenaar P, O’Brien DM 2001 Cardiostimulant effects of urotensin-II in human heart in vitro. Br J Pharmacol 132:5–9[CrossRef][Medline]
  23. He TC, Zhou S, da Costa LT, Yu J, Kinzler KW, Vogelstein B 1998 A simplified system for generating recombinant adenoviruses. Proc Natl Acad Sci USA 95:2509–2514[Abstract/Free Full Text]
  24. Hannan RD, Luyken J, Rothblum LI 1996 Regulation of ribosomal DNA transcription during contraction-induced hypertrophy of neonatal cardiomyocytes. J Biol Chem 271:3213–3220[Abstract/Free Full Text]
  25. Thomas WG, Brandenburger Y, Autelitano DJ, Pham T, Qian H, Hannan RD 2002 Adenoviral-directed expression of the type 1A angiotensin receptor promotes cardiomyocyte hypertrophy via transactivation of the epidermal growth factor receptor. Circ Res 90:135–142[Abstract/Free Full Text]
  26. Chien KR, Knowlton KU, Zhu H, Chien S 1991 Regulation of cardiac gene expression during myocardial growth and hypertrophy: molecular studies of an adaptive physiologic response. FASEB J 5:3037–3046[Abstract]
  27. Smith NJ, Chan H-W, Osborne JE, Thomas WG, Hannan RD, Hijacking of EGF receptors by angiotensin II: new possibilities for understanding and treating cardiac hypertrophy. Cell Mol Life Sci, in press
  28. Sugden PH, Clerk A 1998 Cellular mechanisms of cardiac hypertrophy. J Mol Med 76:725–746[CrossRef][Medline]
  29. Itoh H, McMaster D, Lederis K 1988 Functional receptors for fish neuropeptide urotensin II in major rat arteries. Eur J Pharmacol 149:61–66[CrossRef][Medline]
  30. Zou Y, Nagai R, Yamazaki T 2001 Urotensin II induces hypertrophic responses in cultured cardiomyocytes from neonatal rats. FEBS Lett 508:57–60[CrossRef][Medline]
  31. Hannan RD, Stefanovsky V, Taylor L, Moss T, Rothblum LI 1996 Overexpression of the transcription factor UBF1 is sufficient to increase ribosomal DNA transcription in neonatal cardiomyocytes: implications for cardiac hypertrophy. Proc Natl Acad Sci USA 93:8750–8755[Abstract/Free Full Text]
  32. Yue TL, Gu JL, Wang C, Reith AD, Lee JC, Mirabile RC, Kreutz R, Wang Y, Maleeff B, Parsons AA, Ohlstein EH 2000 Extracellular signal-regulated kinase plays an essential role in hypertrophic agonists, endothelin-1 and phenylephrine-induced cardiomyocyte hypertrophy. J Biol Chem 275:37895–37901[Abstract/Free Full Text]
  33. Post GR, Goldstein D, Thuerauf DJ, Glembotski CC, Brown JH 1996 Dissociation of p44 and p42 mitogen-activated protein kinase activation from receptorinduced hypertrophy in neonatal rat ventricular myocytes. J Biol Chem 271:8452–8457[Abstract/Free Full Text]
  34. Esposito G, Prasad SV, Rapacciuolo A, Mao L, Koch WJ, Rockman HA 2001 Cardiac overexpression of a G(q) inhibitor blocks induction of extracellular signal-regulated kinase and c-Jun NH(2)-terminal kinase activity in in vivo pressure overload. Circulation 103:1453–1458[Abstract/Free Full Text]
  35. Ng DC, Long CS, Bogoyevitch MA 2001 A role for the extracellular signal-regulated kinase and p38 mitogen-activated protein kinases in interleukin-1 ß-stimulated delayed signal transducer and activator of transcription 3 activation, atrial natriuretic factor expression, and cardiac myocyte morphology. J Biol Chem 276:29490–29498[Abstract/Free Full Text]
  36. Sugden PH, Clerk A 1998 "Stress-responsive" mitogen-activated protein kinases (c-Jun N-terminal kinases and p38 mitogen-activated protein kinases) in the myocardium. Circ Res 83:345–352[Free Full Text]
  37. Zechner D, Thuerauf DJ, Hanford DS, McDonough PM, Glembotski CC 1997 A role for the p38 mitogenactivated protein kinase pathway in myocardial cell growth, sarcomeric organization, and cardiac-specific gene expression. J Cell Biol 139:115–127[Abstract/Free Full Text]
  38. Bogoyevitch MA 2000 Signalling via stress-activated mitogen-activated protein kinases in the cardiovascular system. Cardiovasc Res 45:826–842[Abstract/Free Full Text]
  39. Eguchi S, Dempsey PJ, Frank GD, Motley ED, Inagami T 2001 Activation of MAPKs by angiotensin II in vascular smooth muscle cells. Metalloprotease-dependent EGF receptor activation is required for activation of ERK and p38 MAPK but not for JNK. J Biol Chem 276:7957–7962[Abstract/Free Full Text]
  40. Shah BH, Catt KJ 2003 A central role of EGF receptor transactivation in angiotensin II-induced cardiac hypertrophy. Trends Pharmacol Sci 24:239–244[Medline]
  41. Kanda Y, Mizuno K, Kuroki Y, Watanabe Y 2001 Thrombin-induced p38 mitogen-activated protein kinase activation is mediated by epidermal growth factor receptor transactivation pathway. Br J Pharmacol 132:1657–1664[CrossRef][Medline]
  42. Clerk A, Sugden PH 1999 Activation of protein kinase cascades in the heart by hypertrophic G protein-coupled receptor agonists. Am J Cardiol 83:64H–69H
  43. Asakura M, Kitakaze M, Takashima S, Liao Y, Ishikura F, Yoshinaka T, Ohmoto H, Node K, Yoshino K, Ishiguro H, Asanuma H, Sanada S, Matsumura Y, Takeda H, Beppu S, Tada M, Hori M, Higashiyama S 2002 Cardiac hypertrophy is inhibited by antagonism of ADAM12 processing of HB-EGF: metalloproteinase inhibitors as a new therapy. Nat Med 8:35–40[CrossRef][Medline]
  44. Zhao YY, Sawyer DR, Baliga RR, Opel DJ, Han X, Marchionni MA, Kelly RA 1998 Neuregulins promote survival and growth of cardiac myocytes. Persistence of ErbB2 and ErbB4 expression in neonatal and adult ventricular myocytes. J Biol Chem 273:10261–10269[Abstract/Free Full Text]
  45. Prenzel N, Fischer OM, Streit S, Hart S, Ullrich A 2001 The epidermal growth factor receptor family as a central element for cellular signal transduction and diversification. Endocr Relat Cancer 8:11–31[Abstract]
  46. Milano CA, Dolber PC, Rockman HA, Bond RA, Venable ME, Allen LF, Lefkowitz RJ 1994 Myocardial expression of a constitutively active {alpha} 1B-adrenergic receptor in transgenic mice induces cardiac hypertrophy. Proc Natl Acad Sci USA 91:10109–10113[Abstract/Free Full Text]
  47. D’Angelo DD, Sakata Y, Lorenz JN, Boivin GP, Walsh RA, Liggett SB, Dorn II GW 1997 Transgenic G{alpha}q overexpression induces cardiac contractile failure in mice. Proc Natl Acad Sci USA 94:8121–8126[Abstract/Free Full Text]
  48. LaMorte VJ, Thorburn J, Absher D, Spiegel A, Brown JH, Chien KR, Feramisco JR, Knowlton KU 1994 Gq- and ras-dependent pathways mediate hypertrophy of neonatal rat ventricular myocytes following {alpha}1-adrenergic stimulation. J Biol Chem 269:13490–13496[Abstract/Free Full Text]
  49. Sakata Y, Hoit BD, Liggett SB, Walsh RA, Dorn II GW 1998 Decompensation of pressure-overload hypertrophy in G{alpha}q-overexpressing mice. Circulation 97:1488–1495[Abstract/Free Full Text]
  50. Adams JW, Pagel AL, Means CK, Oksenberg D, Armstrong RC, Brown JH 2000 Cardiomyocyte apoptosis induced by G{alpha}q signaling is mediated by permeability transition pore formation and activation of the mitochondrial death pathway. Circ Res 87:1180–1187[Abstract/Free Full Text]
  51. Althoefer H, Eversole-Cire P, Simon MI 1997 Constitutively active G{alpha}q and G{alpha}13 trigger apoptosis through different pathways. J Biol Chem 272:24380–24386[Abstract/Free Full Text]
  52. Adams JW, Sakata Y, Davis MG, Sah VP, Wang Y, Liggett SB, Chien KR, Brown JH, Dorn II GW 1998 Enhanced G{alpha}q signaling: a common pathway mediates cardiac hypertrophy and apoptotic heart failure. Proc Natl Acad Sci USA 95:10140–10145[Abstract/Free Full Text]
  53. Adams JW, Brown JH 2001 G-proteins in growth and apoptosis: lessons from the heart. Oncogene 20:1626–1634[CrossRef][Medline]
  54. Douglas SA, Sulpizio AC, Piercy V, Sarau HM, Ames RS, Aiyar NV, Ohlstein EH, Willette RN 2000 Differential vasoconstrictor activity of human urotensin-II in vascular tissue isolated from the rat, mouse, dog, pig, marmoset and cynomolgus monkey. Br J Pharmacol 131:1262–1274[CrossRef][Medline]
  55. Camarda V, Rizzi A, Calo G, Gendron G, Perron SI, Kostenis E, Zamboni P, Mascoli F, Regoli D 2002 Effects of human urotensin II in isolated vessels of various species; comparison with other vasoactive agents. Naunyn Schmiedebergs Arch Pharmacol 365:141–149[CrossRef][Medline]
  56. Sugden PH 1999 Signaling in myocardial hypertrophy: life after calcineurin? Circ Res 84:633–646[Free Full Text]
  57. Tal M, Ammar DA, Karpuj M, Krizhanovsky V, Naim M, Thompson DA 1995 A novel putative neuropeptide receptor expressed in neural tissue, including sensory epithelia. Biochem Biophys Res Commun 209:752–759[CrossRef][Medline]
  58. Brandenburger Y, Jenkins A, Autelitano DJ, Hannan RD 2001 Increased expression of UBF is a critical determinant for rRNA synthesis and hypertrophic growth of cardiac myocytes. FASEB J 15:2051–2053[Free Full Text]
  59. Swillens S 1992 How to estimate the total receptor concentration when the specific radioactivity of the ligand is unknown. Trends Pharmacol Sci 13:430–434[Medline]



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