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Differential regulation of HIF-1alpha isoforms in murine macrophages by TLR4 and adenosine A(2A) receptor agonists.

ABSTRACT: Adenosine A(2A)R and TLR agonists synergize to induce an "angiogenic switch" in macrophages, down-regulating TNF-alpha and up-regulating VEGF expression. This switch involves transcriptional regulation of VEGF by HIF-1, transcriptional induction of HIF-1alpha by LPS (TLR4 agonist), and A(2A)R-dependent post-transcriptional regulation of HIF-1alpha stability. Murine HIF-1alpha is expressed as two mRNA isoforms: HIF-1alphaI.1 and -I.2, which contain alternative first exons and promoters. HIF-1alphaI.2 is expressed ubiquitously, and HIF-1alphaI.1 is tissue-specific. We investigated the regulation of these isoforms in macrophages by TLR4 and A(2A)R agonists. HIF-1alphaI.1 is induced strongly compared with HIF-1alphaI.2 upon costimulation with LPS and A(2A)R agonists (NECA or CGS21680). In unstimulated cells, the I.1 isoform constituted approximately 4% of HIF-1alpha transcripts; in LPS and NECA- or CGS21680-treated macrophages, this level was approximately 15%, indicating a substantial contribution of HIF-1alphaI.1 to total HIF-1alpha expression. The promoters of both isoforms were induced by LPS but not enhanced further by NECA, suggesting A(2A)R-mediated post-transcriptional regulation. LPS/NECA-induced expression of HIF-1alphaI.1 was down-regulated by Bay 11-7085 (NF-kappaB inhibitor) and ZM241385 (A(2A)R antagonist). Although VEGF and IL-10 expression by HIF-1alphaI.1-/- macrophages was equivalent to that of wild-type macrophages, TNF-alpha, MIP-1alpha, IL-6, IL-12p40, and IL-1beta expression was significantly greater, suggesting a role for HIF-1alphaI.1 in modulating expression of these cytokines. A(2A)R expression in unstimulated macrophages was low but was induced rapidly by LPS in a NF-kappaB-dependent manner. LPS-induced expression of A(2A)Rs and HIF-1alpha and A(2A)R-dependent HIF-1alpha mRNA and protein stabilization provide mechanisms for the synergistic effects of LPS and A(2A)R agonists on macrophage VEGF expression.

SUBMITTER: Ramanathan M

PROVIDER: S-EPMC2796622 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature

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Differential regulation of HIF-1alpha isoforms in murine macrophages by TLR4 and adenosine A(2A) receptor agonists.

Ramanathan Madhuri M Luo Wenting W Csóka Balázs B Haskó György G Lukashev Dmitry D Sitkovsky Michail V MV Leibovich Samuel Joseph SJ

Journal of leukocyte biology 20090528 3

Adenosine A(2A)R and TLR agonists synergize to induce an "angiogenic switch" in macrophages, down-regulating TNF-alpha and up-regulating VEGF expression. This switch involves transcriptional regulation of VEGF by HIF-1, transcriptional induction of HIF-1alpha by LPS (TLR4 agonist), and A(2A)R-dependent post-transcriptional regulation of HIF-1alpha stability. Murine HIF-1alpha is expressed as two mRNA isoforms: HIF-1alphaI.1 and -I.2, which contain alternative first exons and promoters. HIF-1alph ...[more]

PMID: 19477908

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Project description:The A(2A) adenosine receptor (A(2A)AR) mediates anti-inflammatory actions of adenosine in a variety of cell types. LPS (lipopolysaccharide) was reported to induce a small (<2-fold) increase in the expression of A(2A)AR mRNA in human monocytes and monocytic cell lines. We investigated the effects of LPS on the expression of adenosine receptor mRNAs in primary mouse IPMPhi (intraperitoneal macrophages), human macrophages and Wehi-3 cells. Treatment with 10 ng/ml LPS for 4 h produced a >100-fold increase in A(2A)AR mRNA. LPS-induced increases in mRNA for A(2A)AR and TNFalpha (tumour necrosis factor alpha) are reduced by 90% in IPMPhi pretreated with the NF-kappaB (nuclear factor kappaB) inhibitor, BAY 11-7082 {(E)3-[(4-methylphenyl)sulphonyl]-2-propenenitrile; 10 microM}. In Wehi-3 cells exposed to LPS, A(2A)AR and A(2B)AR transcripts are elevated by 290- and 10-fold respectively, the A(1)AR transcript is unchanged and the A(3)AR transcript is decreased by 67%. The induction of A(2A)AR mRNA by LPS is detectable after 1 h, reaches a peak at 6 h at 600 times control and remains elevated beyond 24 h. The ED50 (effective dose) of LPS is 2.3 ng/ml. A(2A)AR receptor number, measured by 125I-ZM241385 binding to whole cells, is undetectable in naïve cells and increases linearly at a rate of 23 receptors x cell(-1) x min(-1) to a B(max) of 348 fmol/mg (28000 receptors/cell) in 20 h. The increase in receptor number is correlated with an increase in the potency of an A(2A) agonist (4-{3-[6-amino-9-(5-ethylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl}-cyclohexanecarboxylic acid methyl ester; referred to as ATL146e) to stimulate cAMP in these cells. After LPS pretreatment, the potency of the A(2A) agonist, ATL146e, to reduce TNFalpha release from IPMPhi was increased by 200-fold. The results support the hypothesis that regulation of adenosine receptor expression, especially up-regulation of the A(2A)AR, is part of a delayed feedback mechanism initiated through NF-kappaB to terminate the activation of human and mouse macrophages.

Project description:Influences of adenosine 2A receptor (A2AR) activity on the cardiac transcriptome and genesis of endotoxemic myocarditis are unclear. We applied transcriptomic profiling (39 K Affymetrix arrays) to identify A2AR-sensitive molecules, revealed by receptor knockout (KO), in healthy and endotoxemic hearts. Baseline cardiac function was unaltered and only 37 A2AR-sensitive genes modified by A2AR KO (≥1.2-fold change, <5 % FDR); the five most induced are Mtr, Ppbp, Chac1, Ctsk and Cnpy2 and the five most repressed are Hp, Yipf4, Acta1, Cidec and Map3k2. Few canonical paths were impacted, with altered Gnb1, Prkar2b, Pde3b and Map3k2 (among others) implicating modified G protein/cAMP/PKA and cGMP/NOS signalling. Lipopolysaccharide (LPS; 20 mg/kg) challenge for 24 h modified >4100 transcripts in wild-type (WT) myocardium (≥1.5-fold change, FDR < 1 %); the most induced are Lcn2 (+590); Saa3 (+516); Serpina3n (+122); Cxcl9 (+101) and Cxcl1 (+89) and the most repressed are Car3 (-38); Adipoq (-17); Atgrl1/Aplnr (-14); H19 (-11) and Itga8 (-8). Canonical responses centred on inflammation, immunity, cell death and remodelling, with pronounced amplification of toll-like receptor (TLR) and underlying JAK-STAT, NFκB and MAPK pathways, and a 'cardio-depressant' profile encompassing suppressed ß-adrenergic, PKA and Ca2+ signalling, electromechanical and mitochondrial function (and major shifts in transcripts impacting function/injury including Lcn2, S100a8/S100a9, Icam1/Vcam and Nox2 induction, and Adipoq, Igf1 and Aplnr repression). Endotoxemic responses were selectively modified by A2AR KO, supporting inflammatory suppression via A2AR sensitive shifts in regulators of NFκB and JAK-STAT signalling (IκBζ, IκBα, STAT1, CDKN1a and RRAS2) without impacting the cardio-depressant gene profile. Data indicate A2ARs exert minor effects in un-stressed myocardium and selectively suppress NFκB and JAK-STAT signalling and cardiac injury without influencing cardiac depression in endotoxemia.

Dataset Information

Differential regulation of HIF-1alpha isoforms in murine macrophages by TLR4 and adenosine A(2A) receptor agonists.

Publications

Differential regulation of HIF-1alpha isoforms in murine macrophages by TLR4 and adenosine A(2A) receptor agonists.

Similar Datasets

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