Antisense oligonucleotide to PKC-ε alters cAMP-dependent stimulation of CFTR in Calu-3 cells

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Abstract
Protein kinase C (PKC) regulates cystic fibrosis transmembrane conductance regulator (CFTR) channel activity but the PKC signaling mechanism is not yet known. The goal of these studies was to identify PKC isotype(s) required for control of CFTR function. CFTR activity was measured as36Cl efflux in a Chinese hamster ovary cell line stably expressing wild-type CFTR (CHO-wtCFTR) and in a Calu-3 cell line. Chelerythrine, a PKC inhibitor, delayed increased CFTR activity induced with phorbol 12-myristate 13-acetate or with the cAMP-generating agents (−)-epinephrine or forskolin plus 8-(4-chlorophenylthio)adenosine 3′,5′- cyclic monophosphate. Immunoblot analysis of Calu-3 cells revealed that PKC-α, -βII, -δ, -ε, and -ζ were expressed in confluent cell cultures. Pretreatment of cell monolayers with Lipofectin plus antisense oligonucleotide to PKC-ε for 48 h prevented stimulation of CFTR with (−)-epinephrine, reduced PKC-ε activity in unstimulated cells by 52.1%, and decreased PKC-ε mass by 76.1% but did not affect hormone-activated protein kinase A activity. Sense oligonucleotide to PKC-ε and antisense oligonucleotide to PKC-δ and -ζ did not alter (−)-epinephrine-stimulated CFTR activity. These results demonstrate the selective regulation of CFTR function by constitutively active PKC-ε.