Specificity and Mechanisms for Induction of PDE4 Up-Regulation by PGE2 and Related Agents in Lung Fibroblasts
Introduction Prostaglandin E2 (PGE2) is an important modulator of the fibrotic changes that occur in pulmonary fibrosis and COPD. It is important to understand the mechanisms that regulate the initiation and desensitization of lung fibroblast responses to PGE2. Our previous studies (Am J Respir Crit Care Med 191:A4945, 2015) showed that pretreating lung fibroblasts with PGE2 induced a desensitization of PGE2 stimulation of cyclic AMP (cAMP) accumulation that could be reversed by the phosphodiesterase 4 (PDE4) inhibitor roflumilast. PGE2 did not reduce the cAMP response to the beta-adrenergic receptor agonist isoproterenol (Iso), and Iso pretreatment reduced the response to Iso but not to PGE2. The current study more directly examined the specificity and mechanisms of PGE2 desensitization with direct assays of PDE enzyme activity.
Methods Human fetal lung (HFL-1) fibroblasts were incubated with or without 100 nM PGE2, 10 uM Iso, or 30 uM forskolin (Fsk) for 24 hr. Cells were washed to remove pretreatment drug and then lysed for PDE4 enzyme activity assays with cell lysates.
Results Pretreatment of cells with PGE2 induced a 2- to 3-fold increase in PDE enzyme activity in cell lysates, and this increased cAMP breakdown was largely reduced by including the PDE4 inhibitor roflumilast. With 24-hr pretreatments, half-maximal increases in PDE activity occurred between 1 and 10 nM PGE2. With 100 nM PGE2 pretreatments, half-maximal increases occurred between 3 and 6 hrs. In contrast to PGE2 pretreatment, pretreatment with Iso induced little or no increase in PDE activity. Although Iso did not mimic the PGE2-induced increase in PDE activity, pretreatment with the direct adenylyl cyclase activator Fsk did induce an increase in PDE activity. The mRNA synthesis inhibitor actinomycin D (2 ug/mL) and the protein synthesis inhibitor cycloheximide (5 uM) nearly completely eliminated the PGE2-induced increase in PDE activity.
Conclusions Pretreatment of HFL-1 fibroblasts with PGE2, but not with Iso, induces an increase in PDE enzyme activity that is inhibited by roflumilast, implicating PDE4 as the up-regulated isozyme. The ability of receptor-independent elevation of cAMP with Fsk to up-regulate PDE activity implicates cAMP as the likely mediator of the PDE up-regulation; the failure of cAMP elevation by Iso to induce the same response is likely related to differential localization and signaling by PGE receptors vs. beta-2 adrenergic receptors (Am J Physiol - Cell Physiol 298:L819, 2010). The inhibition by actinomycin D and cycloheximide suggests that transcriptional and translational processes mediate an up-regulation of PDE enzyme expression. These adaptive changes in PDE activity due to cAMP elevation will be important considerations for future studies to explore either PGE- or PDE-targeted therapies for fibrotic diseases of the lung.
ML Toews, NA Schulte, RS Ostrom, SI Rennard. Specificity and Mechanisms for Induction of PDE4 Up-Regulation by PGE2 and Related Agents in Lung Fibroblasts. FASEB J, 30(1):710.9, 2016.
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