The IBMX-Insensitive PDE8A is Expressed in Human Airway Smooth Muscle Cells and Selectively Regulates Signaling Through AC6
The second messenger cAMP is produced by several different adenylyl cyclase (AC) isoforms in human airway smooth muscle (HASM). We previously defined two distinct cAMP signaling compartments in these cells, one containing AC6 and another containing AC2 (Naunyn-Schmiedebergs Arch Pharmacol, 387(4):329–39, 2014). We hypothesized that phosphodiesterase (PDE) isozymes may also be compartmentized in HASM. We detected mRNA and protein expression of the IBMX-insensitive PDE8A in cultured HASM cells. Using lentiviral delivery of shRNA we knocked down PDE8A expression and measured cAMP levels stimulated by forskolin. The broad-spectrum PDE inhibitor, IBMX, was included in these assays to eliminate contributions by most other PDE isoforms. 1 μM forskolin stimulated 2-fold higher cAMP in HASM with PDE8A knockdown than in cells treated with control lentivirus. Overexpression of AC2 did not alter this response, but overexpression of AC6 increased this response an additional 80%. We used TaqMan PCR to quantify the expression of AREG (a gene whose expression is regulated by cAMP from both AC2 and AC6) and NR4A2 (a gene regulated only by cAMP from AC6). PDE8A knockdown increased forskolin-stimulated AREG and NR4A2 expression in AC6 overexpressing HASM but had no effect on either of these genes when AC2 was overexpressed. Quantitative phosphoproteomic analysis of whole cell lysates show that forskolin treatment causes PDE8A phosphorylation in HASM but that only AC6 overexpression increased this phosphorylation. To determine receptor-mediated signaling regulated by PDE8A, we examined cAMP production in response to isoproterenol (β2AR, which couple primarily to AC6) and butaprost (EP2R, which couple to only AC2). PDE8A knockdown did not alter butaprost-stimulated cAMP in HASM overexpressing AC2, consistent with no role of this PDE isoform in AC2-mediated cAMP signaling. Isoproterenol-stimulated cAMP was reduced by 70% in HASM overexpressing AC6, as effect opposite of what was predicted based on forskolin-mediated responses. We conclude that PDE8A selectively hydrolyzes cAMP produced by AC6 with no effect on cAMP emanating from AC2. However, knockdown of PDE8A expression causes an uncoupling of β2AR to cAMP production through an undetermined mechanism.
KM Hill, F Li, AS Bogard and RS Ostrom. The IBMX-insensitive PDE8A is expressed in human airway smooth muscle cells and selectively regulates signaling through AC6. FASEB J, 30(1):1190.8, 2016.
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