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 E_2:A Comparison with H-89

摘要

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 [~3H]arachidonic acid (AA)release in response to interleukin-1 betaand monocyte chemotactic protein (MCP)-1,respectively.These effects were abolished by PKIalpha.In contrast,H-89 behaved unpre-dictably 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 PKIlpha 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.