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

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 (PKI␣) and have compared it to H-89, a commonly used small molecule PKA inhibitor. Initial studies established efficient gene transfer and confirmed functionality of PKI␣ 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-1␤ and monocyte chemotactic protein (MCP)-1, respectively. These effects were abolished by PKI␣. 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 PKI␣ 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.Through highly coordinated changes in the rate of synthesis and degradation, cAMP mediates the effect of a large number of hormones, autacoids, and neurotransmitters. Current dogma holds that agonism of Gs-coupled receptors augments the basal activity of one or more isoforms of adenylyl cyclase. The cAMP signal then is propagated and amplified through the activation of PKA, ultimately to effect a change in cell function (see Beavo and Brunton, 2002). In the inactive state, PKA is a tetramer composed of two catalytic and two regulatory subunits (Francis and Corbin, 1999). cAMP, when elevated, binds to the regulatory subunits, resulting in the dissociation of the inactive holoenzyme and the release of

Prostanoid receptor expression by human airway smooth muscle cells and regulation of the secretion of granulocyte colony-stimulating factor

“…that many historical results obtained with this and other structurally similar compounds are erroneous. Indeed, we and others have recently shown that while H-89 blocks PKA it is completely unselective for this protein kinase (17,49).…”

Selective Prostacyclin Receptor Agonism Augments Glucocorticoid-Induced Gene Expression in Human Bronchial Epithelial Cells

“…Ϫ /E3 Ϫ replication-deficient adenovirus vector (Ad5.CMV.PKI␣) containing a 251-base pair DNA fragment encoding the complete amino acid sequence of the ␣-isoform of cAMP-dependent protein kinase (PKA) inhibitor ␣ (PKI␣) downstream of the constitutively active CMV immediate early promoter (28). After 48 h, cells were processed for the assessment of GRE-dependent transcription as described above.…”

“…To control for biological effects of the virus per se, the vector Ad5.CMV.Null, expressing no transgene, was used in parallel. Using this experimental protocol, we have previously reported that Ͼ90% of BEAS-2B cells are infected with Ad5.CMV.PKI␣, resulting in the expression of a completely functional transgene with no adverse effect on cell viability (28).…”

“…7a, taprostene increased cAMP in a concentration-dependent manner with a p[A] 50 of 8.01 Ϯ 0.01. In subsequent experiments, BEAS-2B GRE reporter cells were infected with an adenovirus vector, Ad5.CMV.PKI␣, which directs overexpression of PKI␣, a highly selective inhibitor of PKA (28). In uninfected cells, PKI␣ was not detected by Western blotting in any experiment.…”

PKCθ and PKA are antagonistic partners in the NF-AT transactivation pathway of primary mouse CD3+ T lymphocytes

“…This was consistent with the function of the Rp cAMP isomer as potent PKA inhibitor. H89, an extensively used enzymatic PKA inhibitor, was not able to rescue PDE4 LMWI effects, indicating lack of this PKA inhibition in intact cells (data not shown, Meja et al 28 ). Therefore Rp cAMP but not H89 was used in this study.…”