Deregulated Expression and Function of CFTR and Cl−Secretion after Activation of theRasandSrc/PyMTPathways in Caco-2 Cells

We evaluated the role of the activatedRasandSrc/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+(Ca2+i), 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 ISCresponse to forskolin or ATP. Pretreatment of Caco-2-MT cells with 2.5 μM 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 PKCα, consequent to activation of theRasorSrc/PyMTpathways, leads to an absence of CFTR expression and Cl−secretion mediated by either cAMP or Ca2+i. Moreover, Cl−secretion in the colonic Caco-2 epithelial cell line is mediated primarily by CFTR and an alternate Ca2+i-activated Cl−channel is not functional in these cells.