Differential regulation of HIF-1alpha isoforms in murine macrophages by TLR4 and adenosine A(2A) receptor agonists.
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.
Ramanathan
Madhuri
M
Department of Cell Biology and Molecular Medicine and The Cardiovascular Research Institute, University of Medicine and Dentistry of New Jersey, Newark, New Jersey 07103, USA.
Luo
Wenting
W
Csóka
Balázs
B
Haskó
György
G
Lukashev
Dmitry
D
Sitkovsky
Michail V
MV
Leibovich
Samuel Joseph
SJ
D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D058908:Adenosine A2 Receptor Agonists; D058917:Adenosine A2 Receptor Antagonists; D019830:Adenosine-5'-(N-ethylcarboxamide) / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D004338:Drug Combinations; D005260:Female; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology; D049410:Luciferases, Renilla / Q000378:metabolism; D017737:Macrophages, Peritoneal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D020297:Mice, Congenic; D008810:Mice, Inbred C57BL; D016328:NF-kappa B / Q000037:antagonists & inhibitors; D009570:Nitriles / Q000494:pharmacology; D010627:Phenethylamines / Q000494:pharmacology; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D058906:Purinergic P1 Receptor Agonists; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013450:Sulfones / Q000494:pharmacology; D051197:Toll-Like Receptor 4 / Q000819:agonists* / Q000378:metabolism; D014162:Transfection; D014227:Triazines / Q000494:pharmacology; D014230:Triazoles / Q000494:pharmacology; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism
D058908:Adenosine A2 Receptor Agonists; D058917:Adenosine A2 Receptor Antagonists; C416282:BAY 11-7085; D004338:Drug Combinations; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008070:Lipopolysaccharides; D016328:NF-kappa B; D009570:Nitriles; D010627:Phenethylamines; D020033:Protein Isoforms; D058906:Purinergic P1 Receptor Agonists; D012333:RNA, Messenger; D013450:Sulfones; D051197:Toll-Like Receptor 4; D014227:Triazines; D014230:Triazoles; D042461:Vascular Endothelial Growth Factor A; C097270:ZM 241385; C061282:2-(4-(2-carboxyethyl)phenethylamino)-5'-N-ethylcarboxamidoadenosine; D019830:Adenosine-5'-(N-ethylcarboxamide); D049410:Luciferases, Renilla; D000241:Adenosine