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Induction of murine adenosine A2A receptor expression by LPS: analysis of the 5′ upstream promoter

Genes & Immunity volume 14, pages 147–153 (2013)Cite this article

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Abstract

Non-activated macrophages express low levels of A2ARs and lipopolysaccharides (LPS) upregulates A2AR expression in an NF-κB-dependent manner. The murine A2AR gene is encoded by three exons, m1, m2 and m3. Exons m2 and m3 are conserved, while m1 encodes the 5′ untranslated UTR. Three m1 variants have been defined, m1A, m1B and m1C, with m1C being farthest from the transcriptional start site. LPS upregulates A2ARs in primary murine peritoneal and bone-marrow-derived macrophages and RAW264.7 cells by selectively splicing m1C to m2, through a promoter located upstream of m1C. We have cloned ∼1.6 kb upstream of m1C into pGL4.16(luc2CP/Hygro) promoterless vector. This construct in RAW 264.7 cells responds to LPS, and adenosine receptor agonists augmented LPS responsiveness. The NF-κB inhibitors BAY-11 and triptolide inhibited LPS-dependent induction. Deletion of a key proximal NF-κB site (402–417) abrogated LPS responsiveness, while deletion of distal NF-κB and C/EBPβ sites did not. Site-directed mutagenesis of CREB (309–320), STAT1 (526–531) and AP2 (566–569) sites had little effect on LPS and adenosine receptor agonist responsiveness; however, mutation of a second STAT1 site (582–588) abrogated this responsiveness. Further analysis of this promoter should provide valuable insights into regulation of A2AR expression in macrophages in response to inflammatory stimuli.

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Acknowledgements

This work was supported by a grant from the US Public Health Service National Institutes of Health RO1-GM068636 (SJL) and RO1-GM66189 (GH). Shalini Outram was supported by a training grant from the NIH (T32-HL069752). We are grateful to Li Hao in the New Jersey Medical School Bioinformatics Center for assistance with the bio-informatic analyses.

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Authors and Affiliations

  1. Department of Cell Biology and Molecular Medicine and The Cardiovascular Research Institute, Newark, NJ, USA

    G Elson, M Eisenberg, C Garg, S Outram, C J Ferrante & S J Leibovich

  2. Department of Surgery, New Jersey Medical School, UMDNJ, Newark, NJ, USA

    G Hasko

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  1. G Elson
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  2. M Eisenberg
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Correspondence to S J Leibovich.

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Elson, G., Eisenberg, M., Garg, C. et al. Induction of murine adenosine A2A receptor expression by LPS: analysis of the 5′ upstream promoter. Genes Immun 14, 147–153 (2013). https://doi.org/10.1038/gene.2012.60

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