Volume 394, Issue 4

No abstract available

Hypoxia-inducible transcription factor (HIF), an αβ dimer, is the key inducer of hypoxia-responsive genes that operate both during normal development and pathological processes in association with decreased oxygen availability. The products of HIF target genes function in, e.g., hematopoiesis, angiogenesis, iron transport, glucose utilization, resistance to oxidative stress, cell proliferation, survival and apoptosis, extracellular matrix homeostasis, and tumorigenesis and metastasis. HIF is accumulated in hypoxia, whereas it is rapidly degraded in normoxic cells. The oxygen-sensing mechanism behind this phenomenon is provided by HIF prolyl 4-hydroxylases (HIF-P4Hs, commonly known as PHDs and EglNs) that require oxygen in their reaction. In normoxia, two prolines in the oxygen-dependent degradation domain of the HIFα subunit become hydroxylated by the HIF-P4Hs. The 4-hydroxyproline residues formed serve as recognition sites for the von Hippel-Lindau E3 ubiquitin ligase complex and result in subsequent ubiquitination and instant proteasomal degradation of HIFα in normoxia. The HIF-P4H reaction is inhibited in hypoxia. HIFα evades degradation and forms a functional dimer with HIFβ, leading to activation of the HIF target genes. The central role of HIF-P4Hs in the regulation of the hypoxia response pathway has provided an attractive possibility as a drug candidate for treatment of, e.g., severe anemias and ischemic conditions, and several companies are currently carrying out clinical studies on the use of HIF-P4H inhibitors to treat anemia in patients with a kidney disease. Therefore, it is important to understand the effects of individual HIF-P4H isoenzymes on the hypoxia response and potential other pathways in vivo . The common and specific functions of the HIF-P4H isoenzymes are discussed in this review on the basis of available data from cell biological studies and gene-modified animals.

The prolyl 4-hydroxylase domain protein 3 (PHD3) belongs to 2-oxoglutarate and iron-dependent dioxygenases. Together with the two closest paralogues, PHD1 and PHD2, these enzymes have been identified as cellular oxygen sensors that can mark the hypoxia-inducible factor α (HIF-α) for von Hippel-Lindau protein-mediated proteasomal destruction. Although having overlapping functions with PHD1 and PHD2, PHD3 markedly differs from the two isoforms. PHD3 shows a different expression pattern and subcellular localization as well as activity under low oxygen tension. Moreover, it has the widest range of non-HIF targets underlying its diverse functions. The functions of PHD3 differ depending on the cell type and also partially on the microenvironmental conditions it is expressed at. Under normoxia, PHD3 has been shown to be proapoptotic, but under hypoxia, it can have cell survival or proliferation-supporting functions. Here we discuss the regulation, targets, and functions of PHD3.

By driving the primary transcriptional response, the h ypoxia i nducible f actor (HIF) is a master player of the hypoxia-signaling cascade, activation of which is essential to maintain oxygen homeostasis. HIF is formed by the interaction of a constitutive HIF-1β subunit with a HIF-α subunit tightly regulated through the concerted action of the p rolyl h ydroxylase d omain containing proteins (PHDs) and factor inhibiting HIF. In well-oxygenated cells, HIF-α prolyl-hydroxylation by PHDs is the recognition signal for the binding of the ubiquitin E3 ligase pVHL, allowing protein poly-ubiquitination and degradation by the proteasome. Factor inhibiting HIF-mediated asparaginyl hydroxylation prevents interaction with the CBP/p300 coactivator and hence reduces HIF-dependent transcriptional activity. Upon low oxygen availability, HIF-α hydroxylation is blocked, resulting in protein stabilization and HIF complex activation. Post-translational modifications other than hydroxylation appear to be important in the cellular response to hypoxia. S mall u biquitin-like mo difier (SUMO) is a 10 kDa protein readily conjugated to the lysine (K) residues of numerous cellular substrates in a sequential process termed SUMOylation. Recent data support the idea that a fine balance in SUMOylation/deSUMOylation is required for the adequate activation of the hypoxia-signaling cascade. In the present review, we will concentrate on the mechanisms of SUMOylation and its consequences in the cellular response to hypoxia.

Many inflammatory diseases are characterised by persistent and inappropriate neutrophil activation, systemic or localised hypoxia, and bacterial colonisation. Hypoxia represents an important regulator of inflammatory responses because it inhibits neutrophil apoptosis, a process central to the timely resolution of inflammation. Progress in understanding how cells respond to hypoxia has led to the identification of hypoxia-inducible transcription factors (HIFs) and their hydroxylation by the prolyl hydroxylase enzymes. There is now a significant body of data to support a critical role for this HIF pathway in regulating neutrophil function. Moreover, manipulations of specific components of this pathway have very divergent effects on myeloid cell function. In this review, we will discuss the role individual members of the HIF pathway play in regulating key neutrophil functions and the implications this has for the development of effective therapeutic strategies that selectively target inappropriate neutrophil persistence while maintaining a fully competent immune response.

Hypoxia is associated with a diverse range of physiological and pathophysiological processes, including development, wound healing, inflammation, vascular disease and cancer. The requirement that eukaryotic cells have for molecular oxygen as the terminal electron acceptor for the electron transport chain means that the maintenance of oxygen delivery is key for bioenergetic homeostasis. Metazoans have evolved an effective way to adapt to hypoxic stress at the molecular level through a transcription factor termed the hypoxia inducible factor. A family of oxygen-sensing hydroxylases utilizes molecular oxygen as a co-substrate for the hydroxylation of hypoxia inducible factor α subunits, thereby reducing its expression and transcriptional activity when oxygen is available. Recent studies have indicated that other hypoxia-responsive transcriptional pathways may also be hydroxylase-dependent. In this review, we will discuss the role of hydroxylases in the regulation of NF-κB, a key regulator of immunity and inflammation. Developing our understanding of the role of hydroxylases in hypoxic inflammation may identify novel therapeutic approaches in chronic inflammatory disease.

Dendritic cells (DCs) are an important link between innate and adaptive immunity. DCs get activated in inflamed tissues where oxygen tension is usually low, which requires the transcription factor hypoxia inducible factor (HIF)-1 for cellular adaptation. To investigate whether the HIF-1 transcriptional complex plays a pivotal role in the function of DCs, we compared the effects of exogenous inflammatory stimuli and hypoxia on HIF-1α in bone marrow-derived DCs from wild type and myeloid-specific HIF-1α knock-out mice. We showed that the Toll-like receptor ligands lipopolysaccharides and cytosine-phosphatidyl-guanines significantly induce HIF-1α mRNA and protein, leading to elevated HIF-1 target gene expression of vascular endothelial growth factor. In contrast, polyinosinic:polycytidylic acid did not show comparable effects. Furthermore the potential to up-regulate inflammatory cytokines critically influences DC function. Our data demonstrate that HIF-1α protein is needed for adequate production of interferon-α and -β. In co-cultures of DCs and cytotoxic T cells, we observed that DCs lacking active HIF-1α protein induce significantly less CD278 and granzyme B mRNA in T cells. We conclude that HIF-1α plays a crucial role in DC interferon production and T cell activation, linking the innate and adaptive immune system.

Hypoxia-inducible transcription factors (HIFs) mediate the cellular response to hypoxia. HIF-DNA binding triggers a transcriptional program that acts to both restore oxygen homeostasis and adapt cells to low oxygen availability. In this context, HIF is centrally involved in many physiologic and pathophysiological processes such as development, high altitude adaptation, ischemic disease, inflammation, and cancer. The recent development of chromatin immunoprecipitation coupled to genome-wide DNA sequence analysis allows the position and extent of HIF binding to DNA to be characterized across the entire genome and correlated with genetic, epigenetic, and transcriptional analyses. This review summarizes recent pan-genomic analyses of HIF binding and HIF-dependent transcriptional regulation.

The histone variant 2AX (H2AX) is phosphorylated at Serine 139 by the PI3K-like kinase family members ATM, ATR and DNA-PK. Genotoxic stress, such as tumor radio- and chemotherapy, is considered to be the main inducer of phosphorylated H2AX (γH2AX), which forms distinct foci at sites of DNA damage where DNA repair factors accumulate. γH2AX accumulation under severe hypoxic/anoxic (0.02% oxygen) conditions has recently been reported to follow replication fork stalling in the absence of detectable DNA damage. In this study, we found HIF-dependent accumulation of γH2AX in several cancer cell lines and mouse embryonic fibroblasts exposed to physiologically relevant chronic hypoxia (0.2% oxygen), which did not induce detectable levels of DNA strand breaks. The hypoxic accumulation of γH2AX was delayed by the RNAi-mediated knockdown of HIF-1α or HIF-2α and further decreased when both HIF-αs were absent. Conversely, basal phosphorylation of H2AX was increased in cells with constitutively stabilized HIF-2α. These results suggest that both HIF-1 and HIF-2 are involved in γH2AX accumulation by tumor hypoxia, which might increase a cancer cell’s capacity to repair DNA damage, contributing to tumor therapy resistance.

Despite multiple advances in cancer therapies, patients with glioblastoma (GBM) still have a poor prognosis. Numerous glioma models are used not only for the development of innovative therapies but also to optimize conventional ones. Given the significance of hypoxia in drug and radiation resistance and that hypoxia is widely observed among GBM, the establishment of a reliable method to map hypoxia in preclinical human models may contribute to the discovery and translation of future and more targeted therapies. The aim of this study was to compare the hypoxic status of two commonly used human orthotopic glioma models (U87 and U251) developed in rats and studied by noninvasive hypoxia imaging with 3-[ 18 F]fluoro-1-(2-nitro-1-imidazolyl)-2-propanol-micro-positron emission tomography ([ 18 F]-FMISO-μPET). In parallel, because of the relationships between angiogenesis and hypoxia, we used magnetic resonance imaging (MRI), histology, and immunohistochemistry to characterize the tumoral vasculature. Although all tumors were detectable in T2-weighted MRI and 2-deoxy-2-[ 18 F]fluoro- d -glucose-μPET, only the U251 model exhibited [ 18 F]-FMISO uptake. Additionally, the U251 tumors were less densely vascularized than U87 tumors. Our study demonstrates the benefits of noninvasive imaging of hypoxia in preclinical models to define the most reliable one for translation of future therapies to clinic based on the importance of intratumoral oxygen tension for the efficacy of chemotherapy and radiotherapy.

Rhizobium leguminosarum produces large amounts of exopolysaccharide (EPS) that has been shown to be an important determinant of successful nitrogen-fixing symbiosis with legume plants. EPS is assembled in a Wzx/Wzy-dependent manner, and proteins involved in the process are proposed to form a complex that enables coupling the synthesis of EPS subunits with their polymerization and transport. Pss proteins, which are encoded within the chromosomal polysaccharide synthesis cluster of Rhizobium leguminosarum bv. trifolii TA1, were subjected to interaction analysis. PssN was shown to form multimeric complexes in the outer membrane and interact with the extracellular PssO protein and the inner membrane oligomeric PssP co-polymerase. PssO was demonstrated to form oligomers in the presence of the cross-linker. Bacterial two-hybrid analysis showed that PssP interacts with PssL and PssT, counterparts of Gram-negative bacteria Wzx and Wzy proteins. Membrane topology of PssT is discussed in the context of its plausible Wzy-like polymerase activity, interactions with PssP and a possible impact of these interactions on EPS polymerization and chain length determination. The importance of protein-protein and putative protein-polysaccharide interactions in EPS transport is discussed. A topology model for the EPS transport system, with highlights on localization, functions and interactions between the Pss proteins, is proposed.

To look for oral proteasome inhibitors, daily injested food is the best source for cancer chemoprevention. A combination of active components from vegetables, coffee, tea, and fruit could be more efficient to inhibit 26S proteasome activities for preventing cancer diseases. Tannic acid and quercetin have been shown to strongly inhibit 26S proteasome activity, but the molecular target involved remains unknown. Overlay assay, peptide assay, Western blot, and 2-D gels were used to assess the combination of quercetin and tannic acid as a potential inhibitor. Here, we demonstrated that the combination of quercetin and tannic acid (1) synergistically suppresses chymotrypsin-, caspase-, and trypsin-like proteolytic activities, (2) are tightly binding substrates, (3) do not perturb the proteasome structure, (4) inhibit the 26S proteasome affected by ubiquitin, ATP, or β-casein, and (5) inhibit β-casein degradation by the 26S proteasome in vitro . Finally, the inhibition of the proteasome by a combination of quercetin plus tannic acid in Hep-2 cells resulted in the induction of S5a at low dose, accumulation of ubiquitin, and the cleavage of pro-caspase-3, followed by the induction of apoptotic cell death. Evaluating the combination of quercetin and tannic acid as an oral drug to prevent cancer may provide a pharmacological rationale to pursue preclinical trials of this combination.