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
- Koremu K. Meja,
- Matthew C. Catley,
- Lisa M. Cambridge,
- Peter J. Barnes,
- Hazel Lum,
- Robert Newton and
- Mark A. Giembycz
- Thoracic Medicine, National Heart and Lung Institute, Imperial College London, London, United Kingdom (K.K.M., M.C.C., L.M.C., P.J.B.); Department of Pharmacology, Rush University Medical Center, Chicago, Illinois (H.L.); Department of Biological Sciences, Biomedical Research Institute, University of Warwick, Coventry, United Kingdom (R.N.); and Department of Pharmacology and Therapeutics, Respiratory Research Group, University of Calgary, Calgary, Alberta, Canada (M.A.G.)
- Address correspondence to:
Dr. Mark A. Giembycz, Department of Pharmacology and Therapeutics, Respiratory Research Group, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, Canada T2N 4N1. E-mail: giembycz{at}ucalgary.ca
Abstract
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 (PKIα) and have compared it to H-89, a commonly used small molecule PKA inhibitor. Initial studies established efficient gene transfer and confirmed functionality of PKIα 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 [3H]arachidonic acid (AA) release in response to interleukin-1β and monocyte chemotactic protein (MCP)-1, respectively. These effects were abolished by PKIα. 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 [3H]AA release and enhanced the inhibitory effect of submaximal concentrations of rolipram and 8-bromo-cAMP. We suggest that expression of PKIα 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.
Footnotes
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This work was supported by GlaxoSmithKline (Stevenage, UK) and by Novartis Horsham Research Centre (Horsham, UK). M.C.C. was supported by a Medical Research Council/Aventis collaborative studentship. M.A.G. is an Alberta Heritage Senior Medical Scholar and is funded by the Canadian Institutes of Health Research. Org 9935 was kindly donated by Dr. Mohammad Shahid (Organon Laboratories).
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DOI: 10.1124/jpet.103.060020.
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ABBREVIATIONS. PKA, cAMP-dependent protein kinase; PDE, phosphodiesterase; PKG, cGMP-dependent protein kinase; GEF, guanine nucleotide exchange factor; PKI, heat-stable inhibitor of PKA; CMV, cytomegalovirus; HEK, human embryonic kidney; GFP, green fluorescent protein; moi, multiplicity of infection; DAPI, 4′,6-diamidino-2-phenylindole; 8-Br-cAMP, 8-bromo-cAMP; PGE2, prostaglandin E2; MOPS, 4-mor-pholinepropanesulfonic acid; CREB, cAMP response element-binding protein; BSA, bovine serum albumin; PBS, phosphate-buffered saline; GM-CSF, granulocyte/macrophage colony-stimulating factor; IL-1β, interleukin-1β; ELISA, enzyme-linked immunosorbent assay; AA, arachidonic acid; MCP, monocyte chemotactic peptide; CRE, cAMP response element; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; Cα, α-isoform of the catalytic subunit of PKA.
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- Received September 15, 2003.
- Accepted January 26, 2004.
- The American Society for Pharmacology and Experimental Therapeutics
