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-Induced Phosphorylation of STAT1 on Serine 727 to Reduce Macrophage Activation1Department of Internal Medicine, Division of Endocrinology, Clinical Nutrition, and Vascular Medicine, University of California, Davis, CA 95616
Macrophages are activated by IFN-
, a proinflammatory and proatherogenic cytokine that mediates its downstream effects primarily through STAT1. IFN- signaling induces phosphorylation of two STAT1 residues: Tyr701 (Y701), which facilitates dimerization, nuclear translocation, and DNA binding; and Ser727 (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- signaling in activated macrophages. This investigation demonstrates that adding adenosine to IFN--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--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 A3 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 A3 receptor stimulation with Cl-IB-MECA reduces IFN--induced STAT1 transcriptional activity by 45% and STAT1-dependent gene expression by up to 80%. These data suggest that A3 receptor signaling is key to adenosine-mediated STAT1 modulation and anti-inflammatory action in IFN--activated mouse and human macrophages. Because STAT1 plays a key role in IFN--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.
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1 This work was supported by a Howard Hughes Medical Institute Integrating Medicine into Basic Science Fellowship, an Achievement Rewards for College Scientists Scholarship, and National Institutes of Health Grant HL55667.
2 Address correspondence and reprint requests to Dr. Kimberly E. Barnholt, 451 East Health Sciences Drive, STE 5404, University of California, Davis, CA 95616. E-mail address: kebarnholt{at}ucdavis.edu
3 Abbreviations used in this paper: GAS, -activated sequence; IRF, IFN regulatory factor; iNOS, inducible nitric oxide synthase; CaMKII, calcium/calmodulin-dependent protein kinase II; EHNA, erythro-9-(2-hydroxy-3-nonyl)adenine; CCPA, 2-chloro-N6-cyclopentyladenosine; CGS21680, 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine; NECA, N-ethylcarboxamidoadenosine; Cl-IB-MECA, 2-chloro-N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide; CPX, 8-cyclopentyl-1,3-dipropylxanthine; SCH 58261, 5-amino-7-(β-phenylethyl)-2-(8-furyl)pyrazolo(4,3-3)-1,2,4-triazolo(1,5-c)pyrimidine; MRS 1191, 3-ethyl-5-benzyl-2-meth yl-4-phenylethynyl-6-phenyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate; MRS 1220, 9-chloro-2-(2-furanyl)-5-((phenylacetyl)amino)-[1,2,4]triazolo[1,5-c]quinazoline; qRT-PCR, quantitative real-time PCR.
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