Hypoxia-inducible regulation of placental BOK expression.

BOK (BCL-2-related ovarian killer) is a member of the pro-apoptotic BCL-2 family that is highly expressed in the human placenta. BOK excess causes increased trophoblast autophagy and apoptosis in pre-eclampsia, a pathological condition of hypoxia and oxidative stress. In the present study, we identified an HRE (hypoxia-response element) at the junction of exon-1 and intron-1 (+229 to +279) in the human BOK gene, as well as an antisense transcript driven by a promoter located in intron-2. The isolated BOK-HRE bound hypoxia-inducible HIF (hypoxia-inducible factor) proteins in vitro as well as in trophoblastic JEG3 cells and was functional in its natural position as well as in front of a heterologous promoter. Being a reverted repeat, the BOK-HRE functioned in both orientations. This directionless feature of the BOK-HRE facilitates hypoxia regulation via HIF of both BOK and its antisense transcript as demonstrated by RNAi knockdown of the HIF system. Although the antisense transcript was expressed in several human carcinoma cell lines, including choriocarcinoma-derived JEG3 cells, no antisense-regulated mechanism for BOK expression was noted. Taken together, these findings indicate that hypoxia-induced expression of BOK in placental cells is regulated via HIF and is not affected by its antisense transcript.

A novel function of novobiocin: disrupting the interaction of HIF 1α and p300/CBP through direct binding to the HIF1α C-terminal activation domain.

Hypoxia-inducible factor 1α (HIF1α) is an important cellular survival protein under hypoxic conditions, regulating the cellular response to low oxygen tension via recruitment of a transcriptional co-activator, p300/CBP. p300/CBP induces expression of multiple genes involved in cell survival, proliferation, angiogenesis, and tumor development. Thus, a strategy to inhibit hypoxic responses in tumors may be to target the protein-protein interaction between HIF1α and p300/CBP. Here, we document, for the first time, that the aminocoumarin antibiotic, novobiocin, directly blocks the protein-protein interaction between the HIF1α C-terminal activation domain (CTAD) and the cysteine-histidine rich (CH1) region of p300/CBP. Also, novobiocin down-regulated HIF1α-controlled gene expression, specifically CA9, which is related to tumorigenesis. In a monolayer cell culture, novobiocin inhibited cell proliferation and colony formation in the MCF-7 human breast adenocarcinoma cell line and the A549 human lung cancer cell line. Rescue experiments revealed that the recombinant CTAD fragment of HIF1α partially reversed novobiocin's inhibitory effects on cell proliferation and colony formation in MCF-7 cells. These findings suggest a novel mechanism of action for novobiocin which has the potential for innovative therapeutic use in tumor treatment.

Gene transcripts encoding hypoxia-inducible factor (HIF) exhibit tissue- and muscle fiber type-dependent responses to hypoxia and hypercapnic hypoxia in the Atlantic blue crab, Callinectes sapidus.

Hypoxia inducible factor (HIF) is a transcription factor that under low environmental oxygen regulates the expression of suites of genes involved in metabolism, angiogenesis, erythropoiesis, immune function, and growth. Here, we isolated and sequenced partial cDNAs encoding hif-α and arnt/hif-ß from the Atlantic blue crab, Callinectes sapidus, an estuarine species that frequently encounters concurrent hypoxia (low O(2)) and hypercapnia (elevated CO(2)). We then examined the effects of acute exposure (1h) to hypoxia (H) and hypercapnic hypoxia (HH) on relative transcript abundance for hif-α and arnt/hif-ß in different tissues (glycolytic muscle, oxidative muscle, hepatopancreas, gill, and gonads) using quantitative real-time RT-PCR. Our results indicate that hif-α and arnt/hif-ß mRNAs were constitutively present under well-aerated normoxia (N) conditions in all tissues examined. Further, H and HH exposure resulted in both tissue-specific and muscle fiber type-specific effects on relative hif-α transcript abundance. In the gill and glycolytic muscle, relative hif-α mRNA levels were significantly lower under H and HH, compared to N, while no change (or a slight increase) was detected in oxidative muscle, hepatopancreas and gonadal tissues. H and HH did not affect relative transcript abundance for arnt/hif-ß in any tissue or muscle fiber type. Thus, in crustaceans the HIF response to H and HH appears to involve changes in hif transcript abundance, with variation in hif-α and arnt/hif-ß transcriptional dynamics occurring in both a tissue- and muscle fiber type-dependent manner.

Homodimerization of the PAS-B domains of hypoxia-inducible factors.

The Per-Arnt-Sim (PAS) domains of hypoxia-inducible transcription factors (HIF) mediate heterodimer formation between the HIF-α forms that are induced in the event of cellular hypoxia and the constitutive HIF-ß variants. Previous efforts toward structural characterization of the HIF-1α PAS domains were limited by protein stability. Using homology modeling based on the published crystal structure of the PAS-B domain of the homologous protein HIF-2α in complex with the partner HIF-ß (also known as ARNT), we have identified a variant of HIF-1α with improved solubility, monodispersity, and stability. Purified solutions of the PAS-B domains of HIF-1α and HIF-2α differ in their propensity for homodimer formation. In an attempt to understand the structural basis for this difference, and to document the structural changes that accompany homodimer formation, we have undertaken a comparative NMR study of the PAS-B domains of HIF-1α and HIF-2α and mutants of HIF-1α that mimic the behavior of HIF-2α. The NMR spectra of all of these domains are very similar, consistent with the similarity of their amino acid sequences. However, the greater propensity of the HIF-1α PAS-B domain to form dimers as the concentration was increased allowed us to determine the site of homodimerization and pointed toward possible sequence changes in HIF-1α that might discourage the formation of homodimers.

cDNA cloning, gene organization and variant specific expression of HIF-1 alpha in high altitude yak (Bos grunniens).

Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric basic-helix-loop-helix-PER-ARNT-SIM (bHLH-PAS) transcription factor consisting of HIF-1alpha and HIF-1beta subunits. HIF-1alpha is the oxygen-regulated subunit of HIF-1, which regulates the transcription of genes involved in oxygen homeostasis in response to hypoxia. Yak (Bos grunniens), a mammal native to high altitude (HA) region ( approximately 3500-5500 m), has successfully adapted over many generations to the chronic hypoxia of HA. In the present work, cDNA encoding HIF-1alpha has been cloned from the blood of yak. Tissue specific expression of the mRNA was analyzed in blood, heart, lung, liver and kidney by RT-PCR with primers from three different regions of cDNA. The HIF-1alpha expression was liver and blood specific. The HIF-1alpha mRNA contains 823 bp long 3'UTR that is AU-rich and contains ten AUUUA pentamers and two overlapping copies of the nonamer UUAUUUAUUUAUU. Three potential microRNAs, hsa-miR-107/mmu-miR-107/rno-miR-107, hsa-miR-18b and hsa-miR-135a/mmu-miR-135a/rno-miR-135a, targeting 3'UTR of yak HIF-1alpha, were identified by using target prediction software. The CDS encodes for 823 residues of amino acids and showed 99%, 95%, 92%, 90% and 90% similarity to domestic cattle, human, plateau pika, mouse and rat HIF-1alpha, respectively. HIF-1alpha cDNA, cloned and sequenced in the present work has revealed the evolutionary conservation through multiple sequence alignment. Liver and blood specific stability of HIF-1alpha mRNA appears miR-107 regulated.

Characterization of hARD2, a processed hARD1 gene duplicate, encoding a human protein N-alpha-acetyltransferase.

BACKGROUND: Protein acetylation is increasingly recognized as an important mechanism regulating a variety of cellular functions. Several human protein acetyltransferases have been characterized, most of them catalyzing epsilon-acetylation of histones and transcription factors. We recently described the human protein acetyltransferase hARD1 (human Arrest Defective 1). hARD1 interacts with NATH (N-Acetyl Transferase Human) forming a complex expressing protein N-terminal alpha-acetylation activity. RESULTS: We here describe a human protein, hARD2, with 81 % sequence identity to hARD1. The gene encoding hARD2 most likely originates from a eutherian mammal specific retrotransposition event. hARD2 mRNA and protein are expressed in several human cell lines. Immunoprecipitation experiments show that hARD2 protein potentially interacts with NATH, suggesting that hARD2-NATH complexes may be responsible for protein N-alpha-acetylation in human cells. In NB4 cells undergoing retinoic acid mediated differentiation, the level of endogenous hARD1 and NATH protein decreases while the level of hARD2 protein is stable. CONCLUSION: A human protein N-alpha-acetyltransferase is herein described. ARD2 potentially complements the functions of ARD1, adding more flexibility and complexity to protein N-alpha-acetylation in human cells as compared to lower organisms which only have one ARD.