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.
Acetyltransferase complexes of the MYST family with distinct substrate specificities and functions maintain a conserved association with different ING tumor suppressor proteins. ING complexes containing the HBO1 acetylase are a major source of histone H3 and H4 acetylation in vivo and play critical roles in gene regulation and DNA replication. Here, our molecular dissection of HBO1/ING complexes unravels the protein domains required for their assembly and function. Multiple PHD finger domains present in different subunits bind the histone H3 N-terminal tail with a distinct specificity toward lysine 4 methylation status. We show that natively regulated association of the ING4/5 PHD domain with HBO1-JADE determines the growth inhibitory function of the complex, linked to its tumor suppressor activity. Functional genomic analyses indicate that the p53 pathway is a main target of the complex, at least in part through direct transcription regulation at the initiation site of p21/CDKN1A. These results demonstrate the importance of ING association with MYST acetyltransferases in controlling cell proliferation, a regulated link that accounts for the reported tumor suppressor activities of these complexes. Members of the ING (inhibitor of growth) family of growth regulators are present in all eukaryotes, with the five human proteins (ING1 to ING5) and the three from Saccharomyces cerevisiae (Yng1, Yng2, and Pho23) being the most studied. Their homology is highest at the carboxyl termini within a plant homeodomain (PHD) finger-a motif common to many chromatin regulatory proteins (8, 54). Expression analyses of several tumor types show that ING genes are either mutated or downregulated in many forms of cancer (43,73), and a number of studies have implicated the ING proteins in the regulation of the cell cycle and proliferation, cellular aging and senescence, hormone signaling pathways, brain tumor growth, and angiogenesis (reviewed in reference 51). These functions stem from direct mechanistic roles in chromatin modification and remodeling, gene-specific transcription regulation, and DNA repair, recombination, and replication (2, 54, 61).The multisubunit protein complexes containing ING family members have been purified and characterized from yeast and human cells (reviewed in references 2 and 54). The human INGs can be divided into three groups-ING1/2, ING3, and ING4/5-based on their association with three distinct types of protein complexes (8). Each of these complexes regulates chromatin modification and structure via histone acetylation and deacetylation. The ING complexes that carry out histone acetylation contain members of the MYST family of histone acetyltransferases (HATs) as their catalytic subunits (Fig. 1A). Human MYST HATs include Tip60 (KAT5), HBO1 (KAT7), MOZ (KAT6A), MORF (KAT6B), and MOF (KAT8). These enzymes are also known to play crucial roles in transcription activation and in DNA repair, recombination, and replication and are implicated in development and many human diseases, most notably cancer (2,14,...
Hypoxia-inducible factor-1 (HIF-1) is a key transcription factor for responses to low oxygen. Different nonhypoxic stimuli, including hormones and growth factors, are also important HIF-1 activators in the vasculature. Angiotensin II (Ang II), the main effecter hormone in the renin-angiotensin system, is a potent HIF-1 activator in vascular smooth muscle cells (VSMCs). HIF-1 activation by Ang II involves intricate mechanisms of HIF-1␣ transcription, translation, and protein stabilization. Additionally, the generation of reactive oxygen species (ROS) is essential for HIF-1 activation during Ang II treatment. However, the role of the different VSMC ROS generators in HIF-1 activation by Ang II remains unclear. This work aims at elucidating this question. Surprisingly, repression of NADPH oxidase-generated ROS, using Vas2870, a specific inhibitor or a p22 phox siRNA had no significant effect on HIF-1 accumulation by Ang II. In contrast, repression of mitochondrial-generated ROS, by complex III inhibition, by Rieske Fe-S protein siRNA, or by the mitochondrial-targeted antioxidant SkQ1, strikingly blocked HIF-1 accumulation. Furthermore, inhibition of mitochondrial-generated ROS abolished HIF-1␣ protein stability, HIF-1-dependent transcription and VSMC migration by Ang II. A large number of studies implicate NADPH oxidase-generated ROS in Ang II-mediated signaling pathways in VSMCs. However, our work points to mitochondrial-generated ROS as essential intermediates for HIF-1 activation in nonhypoxic conditions. INTRODUCTIONHypoxia-inducible factor-1 (HIF-1) is a key hypoxia-inducible transcription factor responsible for the adaptation of cells and tissues to low oxygen by regulating such responses as cell metabolism, proliferation and survival, erythropoiesis, and angiogenesis (Semenza, 2003). HIF-1 binds to hypoxia-response elements (HRE) found in promoter or enhancer DNA regions of target hypoxia-inducible genes that include vascular endothelial growth factor (VEGF), glucose transporter-1 (Glut-1), nitric oxide synthases, and likely between 100 and 200 others (Kaelin and Ratcliffe, 2008).The active HIF-1 complex is a heterodimer consisting of an oxygen-sensitive HIF-1␣ subunit and a constitutively expressed HIF-1 subunit. HIF-1␣ possesses an oxygen-dependent degradation domain (ODDD) containing two key proline residues which are, in the presence of oxygen, hydroxylated by HIF prolyl-hydroxylases (PHDs; Kaelin and Ratcliffe, 2008). HIF-1␣ prolyl-hydroxylation allows for recognition by pVHL, the product of the von Hippel-Lindau tumor suppressor gene, the substrate recognition component of an E3 ubiquitin ligase complex that polyubiquitinates and targets HIF-1␣ for proteasomal degradation. In hypoxic conditions, low oxygen leads to HIF-1␣ stabilization due to inhibition of prolyl-hydroxylation and subsequent decreases in HIF-1␣ ubiquitination and degradation (Cockman et al., 2000;Epstein et al., 2001;Schofield and Ratcliffe, 2004).In addition to hypoxia, there are also different nonhypoxic HIF activators that include g...
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