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We have recently shown that vasoactive peptides such as angiotensin II (Ang II) and endothelin-1 (ET-1) increase the expression of Gi proteins and the proliferation of A10 vascular smooth muscle cells (VSMC) through mitogen-activated protein (MAP) kinase--phosphoinositide (PI) 3-kinase pathways. This study was intended to examine the implication of epidermal growth factor receptor (EGFR) activation in ET-1-induced enhanced expression of Gi proteins and proliferation of A10 VSMC, and to further investigate the underlying mechanisms responsible for these increases. Cell proliferation was determined by [³thymidine incorporation and the expression of Gi proteins; extracellular signal-regulated kinases 1 and 2 (ERK1/2) and EGFR phosphorylation was determined by Western blotting. Treatment of A10 VSMC with ET-1 enhanced the expression of Gi proteins, which was attenuated by BQ123 and BQ788, antagonists of [ET.sub.A] and [ET.sub.B] receptor respectively. In addition, ET-1 enhanced the phosphorylation of EGFR in A10 VSMC, which was restored to the control levels by EGFR inhibitor and [ET.sub.A] and [ET.sub.B] receptor antagonists. Furthermore, ET-1 also augmented the proliferation and ERK1/2 phosphorylation of A10 VSMC, which were restored to the control levels by inhibition of EGFR. These data suggest that ET-1 transactivates EGFR, which, through MAP kinase signaling, may contribute to the enhanced expression of Gi proteins and thus increased proliferation of A10 VSMC. Key words: ET-1, EGFR, ERK1/2, Gi proteins, VSMC proliferation. Nous avons recemment montre que les peptides vasoactifs comme l'angiotensine II (Ang II) et l'endotheline-1 (ET-1) augmentent l'expression des proteines Gi et la proliferation des cellules musculaires lisses vasculaires (CMLV) par l'intermediaire des voies MAP kinase--PI 3-kinase. La presente etude a ete realisee afin d'examiner l'implication de l'activation du recepteur de l'EGF (EGFR) dans l'augmentation de l'expression des proteines Gi et de la proliferation des CMLV A10 induites par l'ET-1, et d'examiner plus en profondeur les mecanismes sous-jacents responsables de ces augmentations. La proliferation cellulaire a ete determinee par incorporation de [³H]thymidine, et l'expression des proteines Gi ainsi que la phosphorylation de ERK1/2 et du EGFR ont ete determinees par buvardage Western. Le traitement de cellules A10 avec l'ET-1 augmentait l'expression des proteines Gi, laquelle etait attenuee par le BQ123 et le BQ788, des antagonistes des recepteurs [ET.sub.A] et [E.sub.TB] respectivement. En outre, l'ET-1 augmentait la phosphorylation du EGFR chez les cellules A10, laquelle etait ramenee aux niveaux controles par un inhibiteur du EGFR et par les antagonistes des recepteurs [ET.sub.A] et [ET.sub.B]. De plus, l'ET-1 augmentait aussi la proliferation et la phosphorylation de ERK1/2 chez les cellules A10, lesquelles etaient ramenees aux niveaux controles par un inhibiteur du EGFR. Ces donnees suggerent que l'ET-1 trans-active le EGFR qui, par l'intermediaire de la signalisation par les MAP kinases, peut contribuer a l'augmentation de l'expression des proteines Gi et consequemment, stimuler la proliferation des cellules A10. [Traduit par la Redaction] Mots-cles: ET-1, EGFR, ERK1/2, proteines Gi, proliferation des CMLV., Introduction Guanine-nucleotide-binding regulatory proteins (G proteins), a family of GTP-binding proteins play an important role in the regulation of a variety of physiological functions, including blood pressure, through the activation [...]

We showed previously that vascular smooth muscle cells (VMSC) from spontaneously hypertensive rats (SHR) exhibit increased proliferation. The present study was undertaken to examine whether the enhanced levels of endogenous angiotensin (ANG) II and endothelin (ET)-1 contribute to the enhanced proliferation of VSMC from SHR and to further investigate the underlying mechanisms responsible for this response. The enhanced proliferation of VSMC from SHR compared with Wistar-Kyoto (WKY) rats was attenuated by losartan, BQ-123, BQ-788, and AG-1478, inhibitors of [AT.sub.1], [ET.sub.A], [ET.sub.B] and epidermal growth factor (EGF-R) receptors, respectively. In addition, BQ-123 and BQ-788 also attenuated the enhanced production of superoxide anion ([O.sup.-.sub.2]) and NADPH oxidase activity. Furthermore, diphenyleneiodonium (DPI, inhibitor of NADPH oxidase), N-acetyl-L-cysteine (NAC, [O.sup.-.sub.2] scavenger), and PP2 (inhibitor of c-Src) also inhibited the augmented proliferation of VSMC from SHR to WKY levels. In addition, the enhanced phosphorylation of EGF-R in VSMC from SHR compared with WKY was also attenuated by inhibitors of [AT.sub.1], [ET.sub.A], [ET.sub.B], and EGF-R but not by inhibitors of platelet-derived growth factor receptor or insulin-like growth factor receptor. Furthermore, the enhanced phosphorylation of ERK1/2 in VSMC from SHR was also attenuated by [AT.sub.1], [ET.sub.A], [ET.sub.B], c-Src, and EGF-R inhibitors. The phosphorylation of c-Src was significantly augmented in VSMC from SHR compared with VSMC from WKY and was attenuated by DPI and NAC. These data suggest that endogenous vasoactive peptides, through increased oxidative stress and resultant activation of c-Src, transactivate EGF-R, which through mitogen-activated protein (MAP) kinase signaling may contribute to the hyperproliferation of VSMC from SHR. oxidative stress; extracellular signal-regulated kinase 1/2; vascular smooth muscle cell proliferation; spontaneously hypertensive rat doi: 10.1152/ajpheart.00526.2010.

We have recently shown that vasoactive peptides such as angiotensin II (Ang II) and endothelin-1 (ET-1) increased the expression of [G.sub.i] proteins and proliferation of A10 vascular smooth muscle cells (VSMC) through MAP kinase/PI3 kinase pathways. The present study was undertaken to examine the implication of growth factor receptor activation in Ang II-induced enhanced expression of [G.sub.i] proteins and proliferation of A10 VSMC and to further investigate the underlying mechanisms responsible for these increases. Cell proliferation was determined by [³H]thymidine incorporation, and the expression of [G.sub.i] proteins and the phosphorylation of ERK1/2 and epidermal growth factor receptor (EGFR) was determined by Western blotting. Treatment of A10 VSMC with Ang II enhanced the expression of [G.sub.i] proteins, which was attenuated by Ang II [AT.sub.1] receptor antagonist but not by [AT.sub.2] receptor antagonist. The inhibitor of EGFR also attenuated the enhanced expression of [G.sub.i] proteins induced by Ang II to control levels. In addition, Ang II enhanced the phosphorylation of EGFR in A10 VSMC, and this was restored to control levels by the EGFR inhibitor and antioxidants. Furthermore, Ang II also augmented the proliferation and ERK1/2 phosphorylation of A10 VSMC, which were restored to control levels by the EGFR inhibitor. These data suggest that the Ang II-induced increase in oxidative stress transactivates EGFR, which through MAP kinase signaling may contribute to the enhanced expression of [G.sub.i] proteins and thereby to the increased proliferation of A10 VSMC. Key words: Ang II, EGFR, ERK1/2, [G.sub.i] proteins, oxidative stress, VSMC proliferation. Nous avons montre recemment que les peptides vasoactifs tels que l'angiotensine II (Ang II) et l'endotheline-1 (ET-1) augmentent l'expression des proteines [G.sub.i] et la proliferation des cellules du muscle lisse vasculaire (CMLV) A10, et ce, par les voies des MAP kinases/PI3 kinases. La presente etude a eu pour but d'examiner le role de l'activation du recepteur du facteur des croissance dans l'augmentation par Ang II de l'expression des proteines [G.sub.i] et de la proliferation des CMLV A10, ainsi que d'etudier plus en detail les meecanismes a l'origine de ces augmentations. Nous avons mesure la proliferation des cellules par incorporation de [³H]thymidine et determine par immunobuvardage de type western l'expression des proteines [G.sub.i] ainsi que la phosphorylation des recepteurs de la proteine ERK1/2 et du facteur de croissance epidermique (R-EGF). Le traitement des CMLV A10 par Ang II a stimule l'expression des proteines [G.sub.i], qui a ete attenuee par l'antagoniste du recepteur [AT.sub.1] de l'Ang II, mais pas par l'antagoniste du recepteur [AT.sub.2]. De plus, l'inhibiteur du REGF a aussi diminuee au niveau des valeurs teemoins l'augmentation par Ang II de l'expression des proteines [G.sub.i].Dememe, l'Ang II a stimule; la phosphorylation du R-EGF dans les CMLV A10, laquelle a ete ramenee aux taux teemoins par l'inhibiteur du R-EGF ainsi que par des antioxydants. L'Ang II a aussi augmented la proliferation et la phosphorylation de la proteine ERK1/2 des CMLV A10, lesquelles ont ete rameneees aux valeurs temoins par l'inhibiteur du R-EGF. Ces resultants donnent a penser que l'augmentation du stress oxydatif par l'Ang II transactive le R-EGF qui, par la voie de signalisation des MAP kinases, pourrait jouer un role dans l'augmentation de l'expression des proteines [G.sub.i] et, par consequent, dans l'augmentation de la proliferation des CMLV A10. Mots-cles: Ang II, EGFR, ERK1/2, proteines [G.sub.i], stress oxydatif, proliferation des CMLV. [Traduit par la Redaction] doi: 10.1139/Y09-089, Introduction Guanine nucleotide regulatory proteins (G proteins) are a family of GTP-binding proteins that play an important role in the regulation of a variety of signal transduction systems, including the [...]