Hypoxia-inducible factor 1 (HIF-1) is a master regulator of oxygen homeostasis that controls angiogenesis, erythropoiesis, and glycolysis via transcriptional activation of target genes under hypoxic conditions. O₂-dependent binding of the von Hippel-Lindau (VHL) tumor suppressor protein targets the HIF-1α subunit for ubiquitination and proteasomal degradation. The activity of the HIF-1α transactivation domains is also O₂regulated by a previously undefined mechanism. Here, we report the identification of factor inhibiting HIF-1 (FIH-1), a protein that binds to HIF-1α and inhibits its transactivation function. In addition, we demonstrate that FIH-1 binds to VHL and that VHL also functions as a transcriptional corepressor that inhibits HIF-1α transactivation function by recruiting histone deacetylases. Involvement of VHL in association with FIH-1 provides a unifying mechanism for the modulation of HIF-1α protein stabilization and transcriptional activation in response to changes in cellular O₂concentration.

Thioredoxin (TRX) is a pleiotropic cellular factor that has thiol-mediated redox activity and is important in regulation of cellular processes, including proliferation, apoptosis, and gene expression. The activity of several transcription factors is posttranslationally altered by redox modification(s) of specific cysteine residue(s). One such factor is nuclear factor (NF)-κB, whose DNA-binding activity is markedly augmented by TRX treatment in vitro . Similarly, the DNA-binding activity of activator protein 1 (AP-1) is modified by a DNA repair enzyme, redox factor 1 (Ref-1), which is identical to a DNA repair enzyme, AP endonuclease. Ref-1 activity is in turn modulated by various redox-active compounds, including TRX. We here report the molecular cascade of redox regulation of AP-1 mediated by TRX and Ref-1. Phorbol 12-myristate 13 acetate efficiently translocated TRX into the HeLa cell nucleus where Ref-1 preexists. This process seems to be essential for AP-1 activation by redox modification because co-overexpression of TRX and Ref-1 in COS-7 cells potentiated AP-1 activity only after TRX was transported into the nucleus by phorbol 12-myristate 13 acetate treatment. To prove the direct active site-mediated association between TRX and Ref-1, we generated a series of substitution-mutant cysteine residues of TRX. In both an in vitro diamide-induced cross-linking study and an in vivo mammalian two-hybrid assay we proved that TRX can associate directly with Ref-1 in the nucleus; also, we demonstrated the requirement of cysteine residues in the TRX catalytic center for the potentiation of AP-1 activity. This report presents an example of a cascade in cellular redox regulation.

Stimulation of human colon cancer cells with insulin-like growth factor 1 (IGF-1) induces expression of theVEGF gene, encoding vascular endothelial growth factor. In this article we demonstrate that exposure of HCT116 human colon carcinoma cells to IGF-1 induces the expression of HIF-1α, the regulated subunit of hypoxia-inducible factor 1, a known transactivator of the VEGF gene. In contrast to hypoxia, which induces HIF-1α expression by inhibiting its ubiquitination and degradation, IGF-1 did not inhibit these processes, indicating an effect on HIF-1α protein synthesis. IGF-1 stimulation of HIF-1α protein and VEGF mRNA expression was inhibited by treating cells with inhibitors of phosphatidylinositol 3-kinase and MAP kinase signaling pathways. These inhibitors also blocked the IGF-1- induced phosphorylation of the translational regulatory proteins 4E-BP1, p70 S6 kinase, and eIF-4E, thus providing a mechanism for the modulation of HIF-1α protein synthesis. Forced expression of a constitutively active form of the MAP kinase kinase, MEK2, was sufficient to induce HIF-1α protein and VEGF mRNA expression. Involvement of the MAP kinase pathway represents a novel mechanism for the induction of HIF-1α protein expression in human cancer cells.

Recent works have shown the importance of reduction/oxidation (redox) regulation in various biological phenomena. Thioredoxin (TRX) is one of the major components of the thiol reducing system and plays multiple roles in cellular processes such as proliferation, apoptosis, and gene expression. To investigate the molecular mechanism of TRX action, we used a yeast two-hybrid system to identify TRX-binding proteins. One of the candidates, designated as thioredoxin-binding protein-2 (TBP-2), was identical to vitamin D₃ up-regulated protein 1 (VDUP1). The association of TRX with TBP-2/VDUP1 was observed in vitro and in vivo. TBP-2/VDUP1 bound to reduced TRX but not to oxidized TRX nor to mutant TRX, in which two redox active cysteine residues are substituted by serine. Thus, the catalytic center of TRX seems to be important for the interaction. Insulin reducing activity of TRX was inhibited by the addition of recombinant TBP-2/VDUP1 protein in vitro. In COS-7 and HEK293 cells transiently transfected with TBP-2/VDUP1 expression vector, decrease of insulin reducing activity of TRX and diminishment of TRX expression was observed. These results suggested that TBP-2/VDUP1 serves as a negative regulator of the biological function and expression of TRX. Treatment of HL-60 cells with 1α,25-dihydroxyvitamin D₃ caused an increase of TBP-2/VDUP1 expression and down-regulation of the expression and the reducing activity of TRX. Therefore, the TRX-TBP-2/VDUP1 interaction may be an important redox regulatory mechanism in cellular processes, including differentiation of myeloid and macrophage lineages.

Understanding molecular mechanisms regulating angiogenesis may lead to novel therapies for ischemic disorders. Hypoxia-inducible factor 1 (HIF-1) activates vascular endothelial growth factor (VEGF) gene expression in hypoxic/ischemic tissue. In this study we demonstrate that exposure of primary cultures of cardiac and vascular cells to hypoxia or AdCA5, an adenovirus encoding a constitutively active form of HIF-1α, modulates the expression of genes encoding the angiogenic factors angiopoietin-1 (ANGPT1), ANGPT2, placental growth factor, and platelet-derived growth factor-B. Loss-of-function effects were also observed in HIF-1α–null embryonic stem cells. Depending on the cell type, expression of ANGPT1 and ANGPT2 was either activated or repressed in response to hypoxia or AdCA5. In all cases, there was complete concordance between the effects of hypoxia and AdCA5. Injection of AdCA5 into mouse eyes induced neovascularization in multiple capillary beds, including those not responsive to VEGF alone. Analysis of gene expression revealed increased expression of ANGPT1, ANGPT2, platelet-derived growth factor-B, placental growth factor, and VEGF mRNA in AdCA5-injected eyes. These results indicate that HIF-1 functions as a master regulator of angiogenesis by controlling the expression of multiple angiogenic growth factors and that adenovirus-mediated expression of a constitutively active form of HIF-1α is sufficient to induce angiogenesis in nonischemic tissue of an adult animal.

Oxidative stresses such as UV irradiation to mammalian cells triggers a variety of oxistress responses including activation of transcription factors. Recently, activation of nuclear factor-κB (NF-κB) has been shown to be under oxidoreduction (redox) regulation controlled by thioredoxin (TRX), which is one of major endogenous redox-regulating molecules with thiol reducing activity. In order to elucidate where in the cellular compartment TRX participates in NF-κB regulation, we investigated the intracellular localization of TRX. UVB irradiation induced translocation of TRX from the cytoplasm into the nucleus. In our in vitro diamide-induced cross-linking study, we showed that TRX can associate directly with NF-κB p50. Overexpression of wild-type TRX suppressed induction of luciferase activity under NF-κB-binding sites in response to UV irradiation compared with the mock transfectant. In contrast, overexpression of nuclear-targeted TRX enhanced the luciferase activity. Thus, TRX seems to play dual and opposing roles in the regulation of NF-κB. In the cytoplasm, it interferes with the signals to IκB kinases and blocks the degradation of IκB. In the nucleus, however, TRX enhances NF-κB transcriptional activities by enhancing its ability to bind DNA. This two-step TRX-dependent regulation of the NF-κB complex may be a novel activation mechanism of redox-sensitive transcription factors.