Transfected β3- but not β2-adrenergic receptors regulate cystic fibrosis transmembrane conductance regulator activity via a new pathway involving the mitogen-activated protein kinases extracellular signal-regulated kinases

Amal Robay, Gilles Toumaniantz, Véronique Leblais, Chantal Gauthier

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16 Citations (Scopus)

Abstract

We have shown previously that in a heterologous mammalian expression system A549 cells, β3-adrenoceptor (β3-AR) stimulation regulates the activity of cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. The present investigation was carried out to determine the signaling pathway involved in this regulation. A549 cells were intranuclearly injected with plasmids encoding human CFTR and β3-AR. CFTR activity was functionally assessed by microcytofluorimetry. The application of 1 μM 4-[3-t-butylamino-2- hydroxypropoxy]benzimidazol-2-1 hydrochloride (CGP-12177), a β3-AR agonist, produced a CFTR activation that was not abolished by protein kinase A inhibitors. In pertussis toxin-pretreated cells, the CFTR activation induced by CGP-12177 was abolished. The overexpression of β-adrenoceptor receptor kinase, an inhibitor of βγ subunits, abolished the CGP-12177-induced CFTR activation, suggesting the involvement of βγ subunits of Gi/o proteins. The pretreatment of A549 cells with selective inhibitors of either phosphoinositide 3-kinase (PI3K), wortmannin, and 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002), or extracellular signal-regulated kinases 1 and 2 (ERK1/2) mitogen-activated protein kinase (MAPK), 2′-amino-3′-methoxyflavone (PD98059), and 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophynyltio)butadiene (U0126), abolished the effects of CGP-12177 on the CFTR activity. Immunohistochemical assays showed that only the cells expressing β3-AR exhibited MAPK activation in response to CGP-12177. Furthermore, CFTR activity increased in cells pretreated with 10% fetal bovine serum both in A549 cells injected only with CFTR and in T84 cells, which endogenously express CFTR, indicating that CFTR activity can be regulated by the MAPK independently of the β3-AR stimulation. In conclusion, we have demonstrated that CFTR is regulated through a Gi/o/PI3K/ERK1/2 MAPK signaling cascade dependently or not on an activation of β3-ARs. This pathway represents a new regulation for CFTR.

Original languageEnglish
Pages (from-to)648-654
Number of pages7
JournalMolecular Pharmacology
Volume67
Issue number3
DOIs
Publication statusPublished - Mar 2005
Externally publishedYes

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Cystic Fibrosis Transmembrane Conductance Regulator
Extracellular Signal-Regulated MAP Kinases
Mitogen-Activated Protein Kinases
Adrenergic Receptors
Mitogen-Activated Protein Kinase 3
Mitogen-Activated Protein Kinase 1
2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one
1-Phosphatidylinositol 4-Kinase
Chloride Channels
Pertussis Toxin
Protein Kinase Inhibitors
Cyclic AMP-Dependent Protein Kinases

ASJC Scopus subject areas

  • Medicine(all)
  • Molecular Medicine
  • Pharmacology

Cite this

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title = "Transfected β3- but not β2-adrenergic receptors regulate cystic fibrosis transmembrane conductance regulator activity via a new pathway involving the mitogen-activated protein kinases extracellular signal-regulated kinases",
abstract = "We have shown previously that in a heterologous mammalian expression system A549 cells, β3-adrenoceptor (β3-AR) stimulation regulates the activity of cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. The present investigation was carried out to determine the signaling pathway involved in this regulation. A549 cells were intranuclearly injected with plasmids encoding human CFTR and β3-AR. CFTR activity was functionally assessed by microcytofluorimetry. The application of 1 μM 4-[3-t-butylamino-2- hydroxypropoxy]benzimidazol-2-1 hydrochloride (CGP-12177), a β3-AR agonist, produced a CFTR activation that was not abolished by protein kinase A inhibitors. In pertussis toxin-pretreated cells, the CFTR activation induced by CGP-12177 was abolished. The overexpression of β-adrenoceptor receptor kinase, an inhibitor of βγ subunits, abolished the CGP-12177-induced CFTR activation, suggesting the involvement of βγ subunits of Gi/o proteins. The pretreatment of A549 cells with selective inhibitors of either phosphoinositide 3-kinase (PI3K), wortmannin, and 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002), or extracellular signal-regulated kinases 1 and 2 (ERK1/2) mitogen-activated protein kinase (MAPK), 2′-amino-3′-methoxyflavone (PD98059), and 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophynyltio)butadiene (U0126), abolished the effects of CGP-12177 on the CFTR activity. Immunohistochemical assays showed that only the cells expressing β3-AR exhibited MAPK activation in response to CGP-12177. Furthermore, CFTR activity increased in cells pretreated with 10{\%} fetal bovine serum both in A549 cells injected only with CFTR and in T84 cells, which endogenously express CFTR, indicating that CFTR activity can be regulated by the MAPK independently of the β3-AR stimulation. In conclusion, we have demonstrated that CFTR is regulated through a Gi/o/PI3K/ERK1/2 MAPK signaling cascade dependently or not on an activation of β3-ARs. This pathway represents a new regulation for CFTR.",
author = "Amal Robay and Gilles Toumaniantz and V{\'e}ronique Leblais and Chantal Gauthier",
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T1 - Transfected β3- but not β2-adrenergic receptors regulate cystic fibrosis transmembrane conductance regulator activity via a new pathway involving the mitogen-activated protein kinases extracellular signal-regulated kinases

AU - Robay, Amal

AU - Toumaniantz, Gilles

AU - Leblais, Véronique

AU - Gauthier, Chantal

PY - 2005/3

Y1 - 2005/3

N2 - We have shown previously that in a heterologous mammalian expression system A549 cells, β3-adrenoceptor (β3-AR) stimulation regulates the activity of cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. The present investigation was carried out to determine the signaling pathway involved in this regulation. A549 cells were intranuclearly injected with plasmids encoding human CFTR and β3-AR. CFTR activity was functionally assessed by microcytofluorimetry. The application of 1 μM 4-[3-t-butylamino-2- hydroxypropoxy]benzimidazol-2-1 hydrochloride (CGP-12177), a β3-AR agonist, produced a CFTR activation that was not abolished by protein kinase A inhibitors. In pertussis toxin-pretreated cells, the CFTR activation induced by CGP-12177 was abolished. The overexpression of β-adrenoceptor receptor kinase, an inhibitor of βγ subunits, abolished the CGP-12177-induced CFTR activation, suggesting the involvement of βγ subunits of Gi/o proteins. The pretreatment of A549 cells with selective inhibitors of either phosphoinositide 3-kinase (PI3K), wortmannin, and 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002), or extracellular signal-regulated kinases 1 and 2 (ERK1/2) mitogen-activated protein kinase (MAPK), 2′-amino-3′-methoxyflavone (PD98059), and 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophynyltio)butadiene (U0126), abolished the effects of CGP-12177 on the CFTR activity. Immunohistochemical assays showed that only the cells expressing β3-AR exhibited MAPK activation in response to CGP-12177. Furthermore, CFTR activity increased in cells pretreated with 10% fetal bovine serum both in A549 cells injected only with CFTR and in T84 cells, which endogenously express CFTR, indicating that CFTR activity can be regulated by the MAPK independently of the β3-AR stimulation. In conclusion, we have demonstrated that CFTR is regulated through a Gi/o/PI3K/ERK1/2 MAPK signaling cascade dependently or not on an activation of β3-ARs. This pathway represents a new regulation for CFTR.

AB - We have shown previously that in a heterologous mammalian expression system A549 cells, β3-adrenoceptor (β3-AR) stimulation regulates the activity of cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. The present investigation was carried out to determine the signaling pathway involved in this regulation. A549 cells were intranuclearly injected with plasmids encoding human CFTR and β3-AR. CFTR activity was functionally assessed by microcytofluorimetry. The application of 1 μM 4-[3-t-butylamino-2- hydroxypropoxy]benzimidazol-2-1 hydrochloride (CGP-12177), a β3-AR agonist, produced a CFTR activation that was not abolished by protein kinase A inhibitors. In pertussis toxin-pretreated cells, the CFTR activation induced by CGP-12177 was abolished. The overexpression of β-adrenoceptor receptor kinase, an inhibitor of βγ subunits, abolished the CGP-12177-induced CFTR activation, suggesting the involvement of βγ subunits of Gi/o proteins. The pretreatment of A549 cells with selective inhibitors of either phosphoinositide 3-kinase (PI3K), wortmannin, and 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002), or extracellular signal-regulated kinases 1 and 2 (ERK1/2) mitogen-activated protein kinase (MAPK), 2′-amino-3′-methoxyflavone (PD98059), and 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophynyltio)butadiene (U0126), abolished the effects of CGP-12177 on the CFTR activity. Immunohistochemical assays showed that only the cells expressing β3-AR exhibited MAPK activation in response to CGP-12177. Furthermore, CFTR activity increased in cells pretreated with 10% fetal bovine serum both in A549 cells injected only with CFTR and in T84 cells, which endogenously express CFTR, indicating that CFTR activity can be regulated by the MAPK independently of the β3-AR stimulation. In conclusion, we have demonstrated that CFTR is regulated through a Gi/o/PI3K/ERK1/2 MAPK signaling cascade dependently or not on an activation of β3-ARs. This pathway represents a new regulation for CFTR.

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