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.

Regulating the ARNT/TACC3 axis: multiple approaches to manipulating protein/protein interactions with small molecules.

For several well-documented reasons, it has been challenging to develop artificial small molecule inhibitors of protein/protein complexes. Such reagents are of particular interest for transcription factor complexes given links between their misregulation and disease. Here we report parallel approaches to identify regulators of a hypoxia signaling transcription factor complex, involving the ARNT subunit of the HIF (Hypoxia Inducible Factor) activator and the TACC3 (Transforming Acidic Coiled Coil Containing Protein 3) coactivator. In one route, we used in vitro NMR and biochemical screening to identify small molecules that selectively bind within the ARNT PAS (Per-ARNT-Sim) domain that recruits TACC3, identifying KG-548 as an ARNT/TACC3 disruptor. A parallel, cell-based screening approach previously implicated the small molecule KHS101 as an inhibitor of TACC3 signaling. Here, we show that KHS101 works indirectly on HIF complex formation by destabilizing both TACC3 and the HIF component HIF-1α. Overall, our data identify small molecule regulators for this important complex and highlight the utility of pursuing parallel strategies to develop protein/protein inhibitors.

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.