Deregulated expression and function of CFTR and Cl- secretion after activation of the Ras and Src/PyMT pathways in Caco-2 cells.
We evaluated the role of the activated Ras and Src/PyMT (Polyoma Middle T) signaling pathways on the expression of the cystic fibrosis transmembrane conductance regulator (CFTR) in human colonic Caco-2 cell lines. Control vector-transfected Caco-2 cell monolayer preparations (Caco-2-H) responded to forskolin with an increase in short circuit current (Isc) mediated by CFTR. Furthermore, Caco-2-H cells responded to ATP, a reported stimulator of intracellular Ca2+ (Cai2+), and a potential source of adenosine-mediated elevation of cAMP. In contrast, Caco-2 cells transfected with PyMT (Caco-2-MT), expressing high levels of PKC, showed no sustained Isc response to forskolin or ATP. Pretreatment of Caco-2-MT cells with 2.5 microM phorbol 12-myristate 13-acetate (PMA) for 24 hr. effectively down-regulated PKC activity and restored expression of CFTR mRNA but failed to re-establish functional CFTR. These data suggest that, stable up-regulation of PKC alpha, consequent to activation of the Ras or Src/PyMT pathways, leads to an absence of CFTR expression and Cl- secretion mediated by either cAMP or Cai2+. Moreover, Cl- secretion in the colonic Caco-2 epithelial cell line is mediated primarily by CFTR and an alternate Cai(2+)-activated Cl- channel is not functional in these cells.
Davenport
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Cystic Fibrosis/Pulmonary Research and Treatment Center, Department of Medicine, University of North Carolina at Chapel Hill 27599-7248, USA.
Mergey
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Cherqui
G
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Boucher
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RC
Gespach
C
C
Gabriel
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SE
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics*; D018938:Caco-2 Cells; D002118:Calcium / Q000378:metabolism; D018118:Chloride Channels / Q000187:drug effects; D002712:Chlorides / Q000378:metabolism*; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D011905:Genes, ras; D016391:Genes, src; D006801:Humans; D011493:Protein Kinase C / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D015398:Signal Transduction; D014158:Transcription, Genetic
D000952:Antigens, Polyomavirus Transforming; C505032:CFTR protein, human; D018118:Chloride Channels; D002712:Chlorides; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D011493:Protein Kinase C; D002118:Calcium