Interferon-gamma down-regulates adenosine 2b receptor-mediated signaling and short circuit current in the intestinal epithelia by inhibiting the expression of adenylate cyclase.
Adenosine is an endogenous signaling molecule that is highly up-regulated in inflammatory states. Adenosine acts through the A2b receptor, a G protein-coupled receptor that couples positively to Galpha(s) and activates adenylate cyclase. This leads to cAMP-mediated electrogenic chloride secretion in intestinal epithelia. To better understand the regulation of the A2b receptor in intestinal epithelia, we studied the effects of interferon-gamma (IFN-gamma), a potent immunomodulatory cytokine, in the T84 cell line. Pretreatment of cells with 500 units/ml IFN-gamma for 12 h inhibited an adenosine-induced short circuit current (Isc) without affecting the transepithelial resistance. Under these conditions, IFN-gamma did not inhibit the protein expression or membrane recruitment of the A2b receptor, shown to be essential for its function. Interestingly, IFN-gamma inhibited cAMP levels as well as its downstream signaling pathway as shown by the inhibition of adenosine-induced phosphorylation of cAMP response element-binding protein and protein kinase A activity. Similar studies with forskolin, a direct activator of adenylate cyclase, also demonstrated inhibition of cAMP and its downstream response by IFN-gamma. However, IFN-gamma did not affect secretory responses to the calcium-dependent secretagogue carbachol or cAMP analog 8-bromo-cAMP, indicating that normal secretory responses to adequate second messengers in IFN-gamma-treated cells are achievable. Moreover, IFN-gamma inhibited the expression of adenylate cyclase isoforms 5 and 7. In conclusion, we demonstrate that IFN-gamma down-regulates adenosine-mediated signaling possibly through the direct inhibition of adenylate cyclase expression. We propose that IFN-gamma may acutely affect global cAMP-mediated responses in the intestinal epithelia, thereby decreasing secretory responses, which may consequently aggravate inflammatory processes.
Kolachala
Vasantha
V
Division of Digestive Diseases, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
Asamoah
Vivian
V
Wang
Lixin
L
Srinivasan
Shanthi
S
Merlin
Didier
D
Sitaraman
Shanthi V
SV
D000262:Adenylyl Cyclases / Q000378:metabolism*; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D016207:Cytokines / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D004591:Electrophoresis, Polyacrylamide Gel; D004594:Electrophysiology; D004789:Enzyme Activation; D004848:Epithelium / Q000473:pathology*; D005786:Gene Expression Regulation; D006801:Humans; D007249:Inflammation; D007371:Interferon-gamma / Q000378:metabolism; D007413:Intestinal Mucosa / Q000378:metabolism*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D020033:Protein Isoforms; D043704:Receptor, Adenosine A2B / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D013997:Time Factors
D016207:Cytokines; D020033:Protein Isoforms; D043704:Receptor, Adenosine A2B; D007371:Interferon-gamma; D000242:Cyclic AMP; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases; D000262:Adenylyl Cyclases