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This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.05-5328fjev1 20/11/1936    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Chansel, D. Articles by Tharaux, P.-L. Search for Related Content PubMed PubMed Citation Articles by Chansel, D. Articles by Tharaux, P.-L.

Heparin binding EGF is necessary for vasospastic response to endothelin

Dominique Chansel^*,1, Magali Ciroldi^*,1, Sophie Vandermeersch^*, Leslie F Jackson, Ana-Maria Gomez, Daniel Henrion, David C. Lee, Thomas M. Coffman^||, Sylvain Richard, Jean-Claude Dussaule^*,¶ and Pierre-Louis Tharaux^*,2

^* INSERM U702; Hôpital Tenon; Université Pierre et Marie Curie, Paris, France;

Department of Biochemistry and Biophysics, UNC Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA;

INSERM U637; Université Montpellier 1; CHU Arnaud de Villeneuve, France;

CNRS UMR 6188; Faculté de Médecine, Université d’Angers, France;

^|| Division of Nephrology, Department of Medicine, Duke University and Durham Veterans Affairs Medical Center, Durham, North Carolina, USA; and

^¶ Université Pierre et Marie Curie, AP-HP; School of Medicine Saint-Antoine, Paris, France

²Correspondence: INSERM U702, Hôpital Tenon, Université Pierre et Marie Curie, 4 rue de la Chine, Paris 75020, France. E-mail: pierre-louis.tharaux{at}tnn.aphp.fr

SPECIFIC AIMS

We addressed the hypothesis that transactivation of the EGF (EGF) receptor pathway by heparin binding epidermal growth factor EGF (HB-EGF) is rapid enough to play an important role in the contractile actions of endothelin (ET-1). Using two strains of mice and pharmacological blockers of the EGF receptor phosphoinositide3-kinase (PI3K), metalloproteinases, and HB-EGF, we showed that endothelin-induced vasocontraction and its acute pressor effects require HB-EGF, EGFR, and PI3K. These three steps are necessary to full calcium mobilization by ET-1 in mouse and human vascular smooth muscle cells.

PRINCIPAL FINDINGS

1. Alteration of vasocontractile response to ET-1 of HB-EGF-deficient arteries
In carotid artery segments from wild-type (WT) mice, ET-1 induced robust contraction. However, the dose-response curve to ET-1 was markedly shifted to the right in HB-EGF–/– vessels (Fig. 1 ). EC₅₀ were 42 ± 5 and 265 ± 56 nmol/l in HB-EGF +/+ and HB-EGF–/– carotids, respectively (P<0.01). Even at considerably high ET-1 concentrations (0.1–10 µmol/l), HB-EGF–/– carotids could not achieve full contraction. The maximal tone induced by ET-1 was 3-fold higher in HB-EGF+/+ than in HB-EGF–/– carotids (3.20±0.48 vs. 1.05±0.21 mN, respectively, P<0.01). By contrast, KCl produced robust contraction of similar magnitude in segments arteries from HB-EGF+/+ and –/– mice. HB-EGF binds to EGFR, which transduces numerous cellular signaling cascades. Therefore, we tested whether acute EGFR pharmacological blockade would impair vasocontraction to ET-1 and confirmed previous data obtained in mouse aorta (Fig. 1) . This result ruled out the involvement of a developmental action of congenital HB-EGF deficiency or EGFR loss of activity in ET-1 response in the HB-EGF–/– strain. Consistently, HB-EGF-deficient carotids and aortas were of normal size and structure (not shown).

View larger version (16K): [in this window] [in a new window]   Figure 1. Effects of endothelial denudation or of EGFR inhibition with AG1478 (500 nmol/l) or of PI3K inhibition with LY294002 (10 µmol/l) on ET-1-induced isovolumetric tone in HB-EGF+/+ control carotids (upper panel) and HB-EGF-deficient carotids (lower panel). (#P<0.05 vs. ET-1-only treated controls with endothelium. *P <0.05 vs. ET-1-only treated controls with endothelium).

Next, preincubation of carotids with PI3K inhibitor LY294002 for 2 min prior to ET-1 addition prevented an ET-1-induced rise in isovolumetric tone in normal but not in HB-EGF-deficient arteries within a wide range of ET-1 concentrations (P<0.01 vs. controls) (Fig. 1) . This result indicates that PI3K is critical for the full normal response to ET-1.

2. Involvement of EGFR transactivation in the ET-1-induced increase in [Ca²⁺]_i in mouse VSMCs. Comparison of ET-1 [Ca²⁺]_i response in VSMC from wild-type, EGFR kinase-deficient wa2 mice, and HB-EGF –/– mice
Basal [Ca²⁺]_i in suspended VSMCs from waved-2 (wa2) mice, which display a point mutation that alter EGFR tyrosine kinase activity by >80%, and from wild-type mice was the same: 278 ± 15 nmol/l (n=37) vs. 275 ± 8 (n=81). In both cases [Ca²⁺]_i release was immediate (time to peak<3 s) after stimulation by 100 nmol/l ET-1. The [Ca²⁺]_i response was biphasic, with a peak followed by a subsequent sustained phase. In VSMCs from wa2 mice, the peak value was significantly reduced: 373 ± 27 (n=37) vs. 487 ± 28 nmol/l (n=45) in wild-type cells (P<0.01) (Fig. 2 ).

View larger version (16K): [in this window] [in a new window]   Figure 2. Effect of genetically determined loss of function of EGFR tyrosine kinase on calcium signaling in suspended VSMCs. Mean acute maximum change in [Ca2+]i (peak) upon stimulation of wa2 VSMCs and normal VSMCs from wild-type littermates with 100 nmol/l ET-1. ([Ca2+]i: difference in [Ca2+]i between peak and baseline). (*P<0.01 vs. controls).

To ascertain the role of EGFR transactivation in ET-1 [Ca²⁺]_i increase, we used AG1478, a specific EGFR inhibitor. Wild-type VSMCs were incubated for 10 min, with or without 250 nmol/l AG1478, before stimulation with 100 nmol/l ET-1. We verified that AG1478 had no effect by itself on baseline [Ca²⁺]_i. Inhibition of ET-1-induced peak above baseline value with AG1478 was marked (47%) (182±20 vs. 341±32 nmol/l for AG1478 incubated and control cells, respectively; P<0.01, n=6).

To verify that endogenous HB-EGF mediates the ET-1-induced [Ca²⁺]_i response in VSMCs, we assessed the response of HB-EGF–/– and control VSMCs derived from mouse aortas. Whereas basal [Ca²⁺]_i in VSMCs from HB-EGF–/– and from wild-type mice was the same (290±15 vs. 270±12 nmol/l, respectively, P>0.05, NS), HB-EGF deficiency was associated with significant 32% lower ET-1-stimulated increase in [Ca²⁺]_i (peak minus baseline values: 322±39 vs. 475±22 nmol/l in HB-EGF–/– and HB-EGF+/+ VSMCs, respectively, P<0.05, n=13 and 7 measures per group).

3. Role of PI3-kinase on the ET-1-induced increase in [Ca²⁺]_i in VSMC
To study the role of PI3K in ET-1-stimulated [Ca²⁺] increase, we used wortmannin and LY 294002, two unrelated specific inhibitors of PI3K. The cells were incubated with various concentrations of both drugs for 10 min before stimulation with 100 nmol/l ET-1. Both inhibitors had no action on basal [Ca²⁺]_i. By contrast, addition of each of these two inhibitors prior to ET-1 blunted [Ca²⁺]_i peak value in a concentration-dependent manner. The maximal inhibition (50% of control, P<0.05), was obtained with 1 µmol/l wortmannin (314±54 vs. 626±40 nmol/l with and without wortmannin, respectively, n=8); nearly the same inhibition (44% of control, P<0.01) was obtained with 50 µmol/l LY 294002 (382±36 with LY 294002 vs. 673±92 nmol/l for controls, respectively).

4. Neutralization of HB-EGF impairs ET-1-mediated [Ca²⁺]_i in human VSMCs
Human proHB-EGF has been shown to be the endogenous receptor for Corynebacterium diphtheriae toxin (DT). Mutated DT toxin (CRM 197) mutated to be devoid of toxicity, is able to neutralize HB-EGF binding to EGFR. VSMCs from human internal mammary arteries were cultured and incubated with CRM 197 for 1 h before the experiments. In this condition, human VSMCs had a significantly lower Ca²⁺ response to ET-1 than untreated controls (38 nmol/l ET-1 increased the specific fluorescence by 1.28±0.03 fold in 189 human mammary VSMC in control conditions and 1.15±0.01 fold in 157 human mammary VSMC treated with 10 mg/ml CRM197, P<0.001), suggesting a role of HB-EGF in mediating ET-1-induced Ca²⁺ signaling.

CONCLUSIONS AND SIGNIFICANCE

We observed that HB-EGF did not influence the control of blood pressure in physiological conditions but was required for ET-1-induced vasoconstriction and increase in blood pressure in vivo. We previously demonstrated that pharmacologic blockade of the EGFR prevented or reversed ET-1-induced vasoconstriction of mouse aorta. However, we were not able to identify the mechanism involving EGFR tyrosine kinase.

We now demonstrate that HB-EGF is the major ligand for EGFR, which mediates ET-1-induced a pressive response in vivo and vasoconstriction in freshly isolated mouse arteries (Fig. 1) . This action is caused at least in part by the participation of EGFR activity in the ET-1-stimulated rise in [Ca²⁺]_i in mouse and human VSMC (Fig. 2) .

We further report that functional transactivation of EGFR was extremely rapid, since the [Ca²⁺]_i transient peak was blunted by 30–40% within 3 s after the addition of ET-1 in either wa2 cells (Fig. 2) or AG1478-treated cells. This indicates rapid GPCR-EGFR functional crosstalk. This confers on the HB-EGF-EGFR pathway an acute physiological role that is distinct from that described in previous studies, in which the alpha1 receptor agonists phenylephrine and thrombin were both shown to induce subacute EGFR autophosphorylation in vitro and to display similar requirements for HB-EGF in order to promote protein synthesis in rat and mouse VSMCs after several hours, an in vitro trophic effect that was mimicked by HB-EGF itself.

Little is yet known about how EGFR can modulate calcium signaling on transactivation by GPCRs. A significant finding of our study is that PI3K activity is required for full ET-1-mediated vasoconstrictive action and [Ca²⁺]_i signaling. When in the present study both PI3K and EGFR were pharmacologically inhibited, no additive inhibition of the ET-1 stimulated [Ca²⁺]_i peak was observed. In addition, LY294002 and wortmaninn had no effect in EGFR tyrosine kinase-deficient wa2 VSMCs. Although these data and most of the literature suggest that PI3K is activated downstream of EGFR, we cannot exclude the possibility that ET receptors may also activate PI3K independently and in parallel.

HB-EGF alone, even at concentrations that induced a strong phosphorylation of EGFR, failed to induce any rise in [Ca²⁺]_i or in arterial tone of normal mouse aorta. We suggest that concomitant signaling cascades from ET receptors and EGFR are both required for [Ca²⁺]_i peak in VSMCs and vasoconstriction. In this regard, our findings may explain interesting data by Watts and colleagues indicating that EGF failed to increase tone in aorta isolated from normal rats but enhanced aortic tone from DOCA/salt-treated hypertensive rats, a condition where accumulation of ET-1 in the vascular wall has been demonstrated.

In conclusion, our results identify important new steps in the signal transduction pathway mediating ET-1 induction of Ca²⁺ signaling and vasoconstriction. Moreover, the strong requirement for HB-EGF and EGFR for early Ca²⁺ signaling in mouse and human VSMCs may constitute an amplifying loop with subsequent release and synthesis of HB-EGF, since [Ca²⁺]_i increase is known to stimulate metalloproteinase cleavage and secretion of HB-EGF. Furthermore, our results may suggest a mechanism whereby injury causes prevailing levels of ET-1 in the vascular wall to become strongly vasoconstrictive and trophic (Fig. 3 ). That is, hypertension, nephrosclerosis, or atherosclerosis increase levels of key intermediates of the endothelin-HB-EGF pathway (i.e., metalloproteinases, HB-EGF, and PI3K).

View larger version (24K): [in this window] [in a new window]   Figure 3. Schematic diagram of a pathophysiological model: ET-1 requires EGFR tyrosine kinase activity to induce full [Ca2+]i response and vasocontraction. EGFR activation and subsequent calcium transients occur within seconds after release of active HB-EGF through a batimastat-sensitive metalloproteinase activity. PI3K, a known downstream effector of EGFR and of ET receptors, is also required with the same fast kinetic. ET-1 may also induce synthesis of MMPs or ADAMs and of HB-EGF (yellow arrows), thereby amplifying its pathophysiological actions.

FOOTNOTES

¹ These authors contributed equally to this work.

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5328fje

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This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.05-5328fjev1 20/11/1936    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Chansel, D. Articles by Tharaux, P.-L. Search for Related Content PubMed PubMed Citation Articles by Chansel, D. Articles by Tharaux, P.-L.