Two cAMP signaling compartments centering around adenylyl cyclase (AC) exist in human airway smooth muscle (HASM) cells, one containing ß2AR-AC6 and another containing E prostanoid receptors (EPR)-AC2. We hypothesized that different phosphodiesterase (PDE) isozymes selectively regulate cAMP signaling in each compartment. According to RNA-seq data, 18 of 24 PDE genes were expressed in primary HASM cells derived from age- and gender-matched donors with and without asthma. PDE8A was the third most abundant of the cAMP-degrading PDE genes, after PDE4A and PDE1A. Knockdown of PDE8A using shRNA evoked 2-fold greater cAMP responses to 1 DM forskolin in the presence of IBMX. Overexpression of AC2 did not alter this response, but overexpression of AC6 increased cAMP responses an additional 80%. We examined cAMP dynamics in live HASM cells using a fluorescent sensor. PF-04957325, a PDE8-selective inhibitor, increased basal cAMP levels by itself, indicating a significant basal level of cAMP synthesis. In the presence of an AC inhibitor to reduce basal signaling, PF-04957325 accelerated cAMP production and increased the inhibition of cell proliferation induced by isoproterenol, but had no effect on cAMP levels or cell proliferation regulated by PGE2. Lipid raft fractionation of HASM cells revealed PDE8A immunoreactivity in buoyant fractions containing caveolin-1 and AC5/6 immunoreactivity. Thus, PDE8 is expressed in lipid rafts of HASM cells where it specifically regulates ß2AR-AC6 signaling without effects on signaling by the EP2/4R-AC2 complex. In airway diseases such as asthma and COPD, PDE8 may represent a novel therapeutic target to modulate HASM responsiveness and airway remodeling.
Johnstone TB, Smith KH, Koziol-White CJ, et al. PDE8 is expressed in human airway smooth muscle and selectively regulates cAMP signaling by ß2AR-AC6. Am J Respir Cell Mol Biol. 2017. doi: 10.1165/rcmb.2017-0294OC
American Thoracic Society