Adenosine blocks IFN-gamma-induced phosphorylation of STAT1 on serine 727 to reduce macrophage activation.
Macrophages are activated by IFN-gamma, a proinflammatory and proatherogenic cytokine that mediates its downstream effects primarily through STAT1. IFN-gamma signaling induces phosphorylation of two STAT1 residues: Tyr(701) (Y701), which facilitates dimerization, nuclear translocation, and DNA binding; and Ser(727) (S727), which enables maximal STAT1 transcription activity. Immunosuppressive molecules such as adenosine in the cellular microenvironment can reduce macrophage inflammatory and atherogenic functions through receptor-mediated signaling pathways. We hypothesized that adenosine achieves these protective effects by interrupting IFN-gamma signaling in activated macrophages. This investigation demonstrates that adding adenosine to IFN-gamma-stimulated murine RAW 264.7 and human THP-1 macrophages results in unique modulation of STAT1 serine and tyrosine phosphorylation events. We show that adenosine inhibits IFN-gamma-induced STAT1 S727 phosphorylation by >30% and phosphoserine-mediated transcriptional activity by 58% but has no effect on phosphorylation of Y701 or receptor-associated JAK tyrosine kinases. Inhibition of the adenosine A(3) receptor with a subtype-specific antagonist (MRS 1191 in RAW 264.7 cells and MRS 1220 in THP-1 cells) reverses this adenosine suppressive effect on STAT1 phosphoserine status by 25-50%. Further, RAW 264.7 A(3) receptor stimulation with Cl-IB-MECA reduces IFN-gamma-induced STAT1 transcriptional activity by 45% and STAT1-dependent gene expression by up to 80%. These data suggest that A(3) receptor signaling is key to adenosine-mediated STAT1 modulation and anti-inflammatory action in IFN-gamma-activated mouse and human macrophages. Because STAT1 plays a key role in IFN-gamma-induced inflammation and foam cell transformation, a better understanding of the mechanisms underlying STAT1 deactivation by adenosine may improve preventative and therapeutic approaches to vascular disease.
Department of Internal Medicine, Division of Endocrinology, Clinical Nutrition, and Vascular Medicine, University of California, Davis, CA 95616, USA. kebarnholt@ucdavis.edu
Kimberly E
KE
Barnholt
Rama S
RS
Kota
Hnin Hnin
HH
Aung
John C
JC
Rutledge
D000241:Adenosine / Q000494:pharmacology*; D058918:Adenosine A3 Receptor Antagonists; D000700:Analgesics / Q000494:pharmacology*; D000818:Animals; D002460:Cell Line; D004095:Dihydropyridines / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects / Q000276:immunology; D006801:Humans; D007371:Interferon-gamma / Q000276:immunology / Q000494:pharmacology; D053612:Janus Kinases / Q000276:immunology / Q000378:metabolism; D008262:Macrophage Activation / Q000187:drug effects / Q000276:immunology; D008264:Macrophages / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism; D051379:Mice; D046228:Microarray Analysis; D010766:Phosphorylation / Q000187:drug effects* / Q000276:immunology; D011799:Quinazolines / Q000494:pharmacology; D043684:Receptor, Adenosine A3 / Q000276:immunology / Q000378:metabolism; D050794:STAT1 Transcription Factor / Q000037:antagonists & inhibitors / Q000276:immunology* / Q000378:metabolism; D012694:Serine / Q000276:immunology / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000276:immunology; D015533:Transcriptional Activation / Q000187:drug effects / Q000276:immunology; D014230:Triazoles / Q000494:pharmacology; D014443:Tyrosine / Q000276:immunology / Q000378:metabolism
C110041:9-chloro-2-(2-furyl)-5-phenylacetylamino(1,2,4)triazolo(1,5-c)quinazoline; D058918:Adenosine A3 Receptor Antagonists; D000700:Analgesics; D004095:Dihydropyridines; C107728:MRS 1191; D011799:Quinazolines; D043684:Receptor, Adenosine A3; D050794:STAT1 Transcription Factor; D014230:Triazoles; D014443:Tyrosine; D012694:Serine; D007371:Interferon-gamma; D053612:Janus Kinases; D000241:Adenosine