Adenovirus-mediated delivery and expression of a cAMP-dependent protein kinase inhibitor gene to BEAS-2B epithelial cells abolishes the anti-inflammatory effects of rolipram, salbutamol, and prostaglandin E2: a comparison with H-89.
cAMP-elevating drugs are thought to mediate their biological effects by activating the cAMP/cAMP-dependent protein kinase (PKA) cascade. However, this hypothesis is difficult to confirm due to a lack of selective inhibitors. Here, we have probed the role of PKA in mediating inhibitory effects of several cAMP-elevating drugs in BEAS-2B epithelial cells using an adenovirus vector encoding a PKA inhibitor protein (PKIalpha) and have compared it to H-89, a commonly used small molecule PKA inhibitor. Initial studies established efficient gene transfer and confirmed functionality of PKIalpha 48 h after virus infection. All cAMP-elevating drugs tested promoted the phosphorylation of cAMP response element-binding protein (CREB), activated a cAMP response element (CRE)-driven luciferase reporter gene, and suppressed both granulocyte/macrophage colony-stimulating factor (GM-CSF) generation and [(3)H]arachidonic acid (AA) release in response to interleukin-1beta and monocyte chemotactic protein (MCP)-1, respectively. These effects were abolished by PKIalpha. In contrast, H-89 behaved unpredictably under the same conditions. Thus, although CREB phosphorylation evoked by a range of cAMP-elevating drugs was abolished by H-89, neither activation of the CRE-dependent luciferase reporter gene construct nor the inhibition of GM-CSF generation was inhibited. Paradoxically, H-89 antagonized MCP-1-induced [(3)H]AA release and enhanced the inhibitory effect of submaximal concentrations of rolipram and 8-bromo-cAMP. We suggest that expression of PKIalpha in susceptible cells provides a simple and unambiguous way to assess the role of PKA in cAMP signaling and to probe the mechanism of action of other drugs and cAMP-dependent responses where the participation of PKA is equivocal. Furthermore, these data suggest that H-89 is not a selective inhibitor of PKA and should be avoided.
Meja
Koremu K
KK
Thoracic Medicine, National Heart and Lung Institute, Imperial College London, London, United Kingdom.
Catley
Matthew C
MC
Cambridge
Lisa M
LM
Barnes
Peter J
PJ
Lum
Hazel
H
Newton
Robert
R
Giembycz
Mark A
MA
D051697:Activating Transcription Factor 1; D048868:Adaptor Proteins, Signal Transducing; D000249:Adenosine Monophosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D000256:Adenoviridae / Q000235:genetics; D000420:Albuterol / Q000494:pharmacology*; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000494:pharmacology*; D002478:Cells, Cultured; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D004268:DNA-Binding Proteins; D015232:Dinoprostone; D004347:Drug Interactions; D004847:Epithelial Cells / Q000187:drug effects*; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007546:Isoquinolines / Q000494:pharmacology*; D010766:Phosphorylation; D011817:Rabbits; D020889:Rolipram / Q000494:pharmacology*; D013449:Sulfonamides; D014157:Transcription Factors / Q000378:metabolism; D014316:Tritium
D051697:Activating Transcription Factor 1; D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D007546:Isoquinolines; C491526:PKIA protein, human; D013449:Sulfonamides; D014157:Transcription Factors; D014316:Tritium; C015722:8-bromo-2'-AMP; D016718:Arachidonic Acid; D000249:Adenosine Monophosphate; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D017868:Cyclic AMP-Dependent Protein Kinases; D020889:Rolipram; D015232:Dinoprostone; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide; D000420:Albuterol