Transfected beta3- but not beta2-adrenergic receptors regulate cystic fibrosis transmembrane conductance regulator activity via a new pathway involving the mitogen-activated protein kinases extracellular signal-regulated kinases.
We have shown previously that in a heterologous mammalian expression system A549 cells, beta3-adrenoceptor (beta3-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 beta3-AR. CFTR activity was functionally assessed by microcytofluorimetry. The application of 1 microM 4-[3-t-butylamino-2-hydroxypropoxy]benzimidazol-2-1 hydrochloride (CGP-12177), a beta3-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 beta-adrenoceptor receptor kinase, an inhibitor of betagamma subunits, abolished the CGP-12177-induced CFTR activation, suggesting the involvement of betagamma 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 beta3-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 beta3-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 beta3-ARs. This pathway represents a new regulation for CFTR.
Robay
Amal
A
Institut du Thorax, Institut National de la Sante et de la Recherche Medicale U533, Faculté de Médecine, 1 rue Gaston Veil, BP 53508, F-44035 Nantes, France.
Toumaniantz
Gilles
G
Leblais
Véronique
V
Gauthier
Chantal
C
D002070:Butadienes / Q000494:pharmacology; D002460:Cell Line; D045744:Cell Line, Tumor; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D006801:Humans; D008168:Lung; D020935:MAP Kinase Signaling System / Q000502:physiology*; D020929:Mitogen-Activated Protein Kinase Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009570:Nitriles / Q000494:pharmacology; D018343:Receptors, Adrenergic, beta-2 / Q000235:genetics / Q000502:physiology*; D022702:Receptors, Adrenergic, beta-3 / Q000235:genetics / Q000502:physiology*; D020545:Respiratory Mucosa; D014162:Transfection
D002070:Butadienes; C505032:CFTR protein, human; D004791:Enzyme Inhibitors; D005419:Flavonoids; D009570:Nitriles; D018343:Receptors, Adrenergic, beta-2; D022702:Receptors, Adrenergic, beta-3; C113580:U 0126; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D020929:Mitogen-Activated Protein Kinase Kinases; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one