Published online before print May 28, 2009

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(Journal of Leukocyte Biology. 2009;86:681-689.)
© 2009 Society for Leukocyte Biology

Differential regulation of HIF-1 isoforms in murine macrophages by TLR4 and adenosine A_2A receptor agonists

Madhuri Ramanathan^*, Wenting Luo^*, Balázs Csóka, György Haskó, Dmitry Lukashev, Michail V. Sitkovsky and Samuel Joseph Leibovich^*,1

^* Department of Cell Biology and Molecular Medicine and The Cardiovascular Research Institute and
Department of Surgery, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, New Jersey, USA; and
New England Inflammation and Tissue Protection Institute, Northeastern University, Boston, Massachusetts, USA

1. Correspondence: Department of Cell Biology and Molecular Medicine, New Jersey Medical School, UMDNJ, 185 S. Orange Avenue, Newark, NJ 07103, USA. E-mail: leibovic{at}umdnj.edu

ABSTRACT

Adenosine A_2AR and TLR agonists synergize to induce an "angiogenic switch" in macrophages, down-regulating TNF- and up-regulating VEGF expression. This switch involves transcriptional regulation of VEGF by HIF-1, transcriptional induction of HIF-1 by LPS (TLR4 agonist), and A_2AR-dependent post-transcriptional regulation of HIF-1 stability. Murine HIF-1 is expressed as two mRNA isoforms: HIF-1I.1 and -I.2, which contain alternative first exons and promoters. HIF-1I.2 is expressed ubiquitously, and HIF-1I.1 is tissue-specific. We investigated the regulation of these isoforms in macrophages by TLR4 and A_2AR agonists. HIF-1I.1 is induced strongly compared with HIF-1I.2 upon costimulation with LPS and A_2AR agonists (NECA or CGS21680). In unstimulated cells, the I.1 isoform constituted 4% of HIF-1 transcripts; in LPS and NECA- or CGS21680-treated macrophages, this level was 15%, indicating a substantial contribution of HIF-1I.1 to total HIF-1 expression. The promoters of both isoforms were induced by LPS but not enhanced further by NECA, suggesting A_2AR-mediated post-transcriptional regulation. LPS/NECA-induced expression of HIF-1I.1 was down-regulated by Bay 11-7085 (NF-B inhibitor) and ZM241385 (A_2AR antagonist). Although VEGF and IL-10 expression by HIF-1I.1–/– macrophages was equivalent to that of wild-type macrophages, TNF-, MIP-1, IL-6, IL-12p40, and IL-1β expression was significantly greater, suggesting a role for HIF-1I.1 in modulating expression of these cytokines. A_2AR expression in unstimulated macrophages was low but was induced rapidly by LPS in a NF-B-dependent manner. LPS-induced expression of A_2ARs and HIF-1 and A_2AR-dependent HIF-1 mRNA and protein stabilization provide mechanisms for the synergistic effects of LPS and A_2AR agonists on macrophage VEGF expression.

Key Words: endotoxin • VEGF • TNF- • inflammation • transcription factors • alternative activation

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