Hypoxia-inducible factor 1 (HIF-1) regulates many genes induced by low oxygen conditions. The expression of important hypoxic genes such as glucose transporter 1 and vascular endothelial growth factor are increased in macrophages during wound healing and in the presence of the endotoxin, lipopolysaccharide (LPS). Recent studies have demonstrated that nonhypoxic stimuli can also activate HIF-1 in a cell-specific manner. Here, we demonstrate that in macrophages, LPS can control the activation of hypoxia-regulated genes through the HIF-1 pathway. We show that in these cells, protein expression levels of HIF-1␣ are strongly increased to levels comparable to hypoxic induction. HIF-1␣ mRNA levels are markedly increased following LPS stimulation, suggesting a transcriptional induction. In functional studies, the LPS-induced HIF-1 complex could specifically bind to the HIF-1 DNA-binding motif. Additionally, when cells were transfected with an HIF-1-specific reporter construct, LPS could strongly activate the expression of the reporter to levels that surpassed those observed after hypoxic induction. This induction was blocked by the cotransfection of a dominant-negative form of HIF-1␣. These results indicate that the HIF-1 complex is involved in macrophage gene activation following LPS exposure and identify a novel pathway that could play a determinant role during inflammation and wound healing. IntroductionHypoxia activates a number of genes that are important in the cellular and tissue adaptation to low oxygen conditions. 1 These genes include erythropoietin, glucose transporters, glycolytic enzymes, and vascular endothelial growth factor (VEGF). The hypoxic expression of these different genes is controlled at the transcriptional level by the ubiquitously expressed transcription factor, hypoxia-inducible factor 1 (HIF-1). HIF-1 is a member of the basic helix-loop-helix superfamily of transcription factors. Only active as a heterodimer, HIF-1 is composed of 2 subunits: HIF-1␣ and HIF-1␤. 2 Whereas the HIF-1␤ protein is readily found in all cells, HIF-1␣ is virtually undetectable in normal oxygen conditions. When cells are subjected to hypoxic conditions (1% oxygen), levels of the HIF-1␣ subunit are rapidly increased. Instead of acting on HIF-1␣ transcription or translation, hypoxia increases HIF-1␣ protein levels by inhibiting the rapid ubiquitination and degradation of HIF-1␣ by the proteasome. An elegant series of studies has shown that when oxygen is present, HIF-1␣ is modified by prolyl hydroxylation, which permits the binding of the von Hippel-Lindau protein (pVHL), a recognition component of the E3 ligase complex. This promotes the ubiquitin degradation of HIF-1␣. [3][4][5][6][7][8] In a very recent study, acetylation of HIF-1␣ in normoxic conditions also targets HIF-1␣ for proteasomal degradation. 9 Under hypoxic conditions, prolyl hydroxylation of HIF-1␣ is blocked and acetylation is down-regulated, which permits HIF-1␣ protein stabilization. HIF-1␣ is then free to bind with HIF-1␤ to form the HIF-1 transcripti...

Hypoxia-inducible factor-1 (HIF-1) regulates the transcription of many genes induced by low oxygen conditions. Recent studies have demonstrated that non-hypoxic stimuli can also activate HIF-1 in a cell-specific manner. Here, we define two key mechanisms that are implicated in increasing the active subunit of the HIF-1 complex, HIF-1␣, following the stimulation of vascular smooth muscle cells (VSMC) with angiotensin II (Ang II). We show that, in contrast to hypoxia, the induction of HIF-1␣ by Ang II in VSMC is dependent on active transcription and ongoing translation. We demonstrate that stimulation of VSMC by Ang II strongly increases HIF-1␣ gene expression. The activation of diacylglycerol-sensitive protein kinase C (PKC) plays a major role in the increase of HIF-1␣ gene transcription. We also demonstrate that Ang II relies on ongoing translation to maintain elevated HIF-1␣ protein levels. Ang II increases HIF-1␣ translation by a reactive oxygen species (ROS)-dependent activation of the phosphatidylinositol 3-kinase pathway, which acts on the 5-untranslated region of HIF-1␣ mRNA. These results establish that the non-hypoxic induction of the HIF-1 transcription factor via vasoactive hormones (Ang II and thrombin) is triggered by a dual mechanism, i.e. a PKC-mediated transcriptional action and a ROS-dependent increase in HIF-1␣ protein expression. Elucidation of these signaling pathways that up-regulate the vascular endothelial growth factor (VEGF) could have a strong impact on different aspects of vascular biology.

Hypoxia-inducible factor-1 (HIF-1) is a decisive element for the transcriptional regulation of many genes induced under low oxygen conditions. Under normal oxygen conditions, HIF-1␣, the active subunit of HIF-1, is hydroxylated on proline residues by specific HIF prolyl-hydroxylases, leading to ubiquitination and degradation by the proteasome. In hypoxia, hydroxylation and ubiquitination are blocked and HIF-1␣ accumulates in cells. Recent studies have shown that in normal oxygen conditions G-protein-coupled receptor agonists, including angiotensin (Ang) II and thrombin, potently induce and activate HIF-1 in vascular smooth muscle cells. The current study identifies HIF-1␣ protein stabilization as a key mechanism for HIF-1 induction by Ang II. We show that hydroxylation on proline 402 is altered by Ang II, decreasing pVHL binding to HIF-1␣ and allowing HIF-1␣ protein to escape subsequent ubiquitination and degradation mechanisms. We show that HIF-1␣ stability is mediated through the Ang II-mediated generation of hydrogen peroxide and a subsequent decrease in ascorbate levels, leading to decreased HIF prolyl-hydroxylase activity and HIF-1␣ stabilization. These findings identify novel and intricate signaling mechanisms involved in HIF-1 complex activation and will lead to the elucidation of the importance of HIF-1 in different Ang II-related cell responses. INTRODUCTIONOxygen is an essential element in the biology of every aerobic organism. Tissue and cellular regulation of oxygen supply is essential to mediate adaptation mechanisms during low oxygen conditions. At the cellular level, the hypoxiainducible transcription factor, HIF-1, is a key regulator of responses in low oxygen conditions. HIF-1 specifically binds hypoxic response element (HRE)-driven promoters on a number of genes that include vascular endothelial growth factor (VEGF), heme oxygenase, glucose transporter-1, and erythropoietin (Semenza et al., 1991;Semenza and Wang, 1992;Lee et al., 1997). HIF-1 is a heterodimeric complex composed of HIF-1␣ and HIF-1␤. HIF-1␤ is found in all cells, whereas HIF-1␣ is the oxygen-regulated subunit (Wang et al., 1995).HIF-1␣ is highly unstable and the mechanisms controlling HIF-1␣ degradation in normoxic conditions have been well described (Schofield and Ratcliffe, 2005). Human HIF-1␣ is hydroxylated on two proline residues; Pro 402 and Pro 564 . Both residues are situated in the oxygen-dependent degradation domain (ODDD) of HIF-1␣. Three different HIF prolyl-hydroxylases have been described, termed PHD1, PHD2, and PHD3 for their prolyl hydroxylase domain (Bruick and McKnight, 2001;Epstein et al., 2001). PHD enzymes hydroxylate HIF-1␣ using oxygen and 2-oxoglutarate as substrates and iron and ascorbate as essential cofactors. Although all three PHDs have been shown to regulate HIF-1␣, the key isoform responsible for HIF-1␣ regulation in many cell types is PHD2 (Berra et al., 2003;Appelhoff et al., 2004). It has been shown that PHD1 and PHD3 also hydroxylate HIF-1␣ in vivo and in vitro (Epstein et al., 2001;Appelhoff e...