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

Adenosine A2AR 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-1 alpha by LPS (TLR4 agonist), and A2AR-dependent post-transcriptional regulation of HIF-1 alpha stability. Murine HIF-1 alpha is expressed as two mRNA isoforms: HIF-1 alpha I.1 and -I.2, which contain alternative first exons and promoters. HIF-1 alpha I.2 is expressed ubiquitously, and HIF-1 alpha I.1 is tissue-specific. We investigated the regulation of these isoforms in macrophages by TLR4 and A2AR agonists. HIF-1 alpha I.1 is induced strongly compared with HIF-1 alpha I.2 upon costimulation with LPS and A2AR agonists (NECA or CGS21680). In unstimulated cells, the I.1 isoform constituted similar to 4% of HIF-1 alpha transcripts; in LPS and NECA-or CGS21680-treated macrophages, this level was similar to 15%, indicating a substantial contribution of HIF-1 alpha I.1 to total HIF-1 alpha expression. The promoters of both isoforms were induced by LPS but not enhanced further by NECA, suggesting A2AR-mediated post-transcriptional regulation. LPS/NECA-induced expression of HIF-1 alpha I.1 was down-regulated by Bay 11-7085 (NF-kappa B inhibitor) and ZM241385 (A2AR antagonist). Although VEGF and IL-10 expression by HIF-1 alpha I.1(-/-) macrophages was equivalent to that of wild-type macrophages, TNF-alpha, MIP-1 alpha, IL-6, IL-12p40, and IL-1 alpha expression was significantly greater, suggesting a role for HIF-1 alpha I.1 in modulating expression of these cytokines. A2AR expression in unstimulated macrophages was low but was induced rapidly by LPS in a NF-kappa B-dependent manner. LPS-induced expression of A2ARs and HIF-1 beta and A2AR-dependent HIF-1 alpha mRNA and protein stabilization provide mechanisms for the synergistic effects of LPS and A2AR agonists on macrophage VEGF expression. J. Leukoc. Biol. 86: 681-689; 2009.