Yasuaki Kabe

Hypoxic regulation of the cerebral microcirculation is mediated by a carbon monoxide-sensitive hydrogen sulfide pathway

Enhancement of cerebral blood flow by hypoxia is critical for brain function, but signaling systems underlying its regulation have been unclear. We report a pathway mediating hypoxia-induced cerebral vasodilation in studies monitoring vascular disposition in cerebellar slices and in intact mouse brains using two-photon intravital laser scanning microscopy. In this cascade, hypoxia elicits cerebral vasodilation via the coordinate actions of H2S formed by cystathionine β-synthase (CBS) and CO generated by heme oxygenase (HO)-2. Hypoxia diminishes CO generation by HO-2, an oxygen sensor. The constitutive CO physiologically inhibits CBS, and hypoxia leads to increased levels of H2S that mediate the vasodilation of precapillary arterioles. Mice with targeted deletion of HO-2 or CBS display impaired vascular responses to hypoxia. Thus, in intact adult brain cerebral cortex of HO-2–null mice, imaging mass spectrometry reveals an impaired ability to maintain ATP levels on hypoxia.

A new APE1/Ref-1-dependent pathway leading to reduction of NF-κB and AP-1, and activation of their DNA-binding activity

APE1/Ref-1 is thought to be a multifunctional protein involved in reduction-oxidation (redox) regulation and base excision DNA repair, and is required for early embryonic development in mice. APE1/Ref-1 has redox activity and AP endonuclease activity, and is able to enhance DNA-binding activity of several transcription factors, including NF-kappaB, AP-1 and p53, through reduction of their critical cysteine residues. However, it remains elusive exactly how APE1/Ref-1 carries out its essential functions in vivo. Here, we show that APE1/Ref-1 not only reduces target transcription factors directly but also facilitates their reduction by other reducing molecules such as glutathione or thioredoxin. The new activity of APE1/Ref-1, termed redox chaperone activity, is exerted at concentration significantly lower than that required for its redox activity and is neither dependent on its redox activity nor on its AP endonuclease activity. We also show evidence that redox chaperone activity of APE1/Ref-1 is critical to NF-kappaB-mediated gene expression in human cells and is mediated through its physical association with target transcription factors. Thus, APE1/Ref-1 may play multiple roles in an antioxidative stress response pathway through its different biochemical activities. These findings also provide new insight into the mechanism of intracellular redox regulation.

NF-Y Is Essential for the Recruitment of RNA Polymerase II and Inducible Transcription of Several CCAAT Box-Containing Genes

ABSTRACT Osteoclast differentiation factor (ODF)/receptor activator of NF-κB ligand is essential for inducing the differentiation of mature osteoclasts. We find that nuclear factor Y (NF-Y) binds to the CCAAT box on the ODF promoter and regulates its basal transcriptional activity. The CCAAT box on the ODF gene is required for its transcriptional induction by vitamin D3, suggesting that NF-Y coregulates this promoter along with VDR. Chromatin immunoprecipitation analysis reveals that NF-Y is required for the recruitment of RNA polymerase II (RNAPII) and TATA box binding protein on the ODF promoter. Stimulation with vitamin D3 facilitates the recruitment of VDR and p300 onto the ODF promoter, resulting in acetylation of histone H4 in an NF-Y-independent manner. ODF gene induction by parathyroid hormone or prostaglandin E is also dependent on NF-Y. Furthermore, NF-Y is essential for the recruitment of RNAPII onto other CCAAT box-containing promoters, such as those of osteopontin, CYP24, and E2F1. These results suggest that NF-Y recruits RNAPII and general transcription factors onto various CCAAT box-containing promoters in response to various inductions to permit strong transcriptional activation independently of histone modifications.

The Ape-1/Ref-1 Redox Antagonist E3330 Inhibits the Growth of Tumor Endothelium and Endothelial Progenitor Cells : Therapeutic Implications in Tumor Angiogenesis

The apurinic/apyrimidinic endonuclease 1/redox factor‐1 (Ape‐1/Ref‐1) is a multi‐functional protein, involved in DNA repair and the activation of redox‐sensitive transcription factors. The Ape‐1/Ref‐1 redox domain acts as a cytoprotective element in normal endothelial cells, mitigating the deleterious effects of apoptotic stimuli through induction of survival signals. We explored the role of the Ape‐1/Ref‐1 redox domain in the maintenance of tumor‐associated endothelium, and of endothelial progenitor cells (EPCs), which contribute to tumor angiogenesis. We demonstrate that E3330, a small molecule inhibitor of the Ape‐1/Ref‐1 redox domain, blocks the in vitro growth of pancreatic cancer‐associated endothelial cells (PCECs) and EPCs, which is recapitulated by stable expression of a dominant‐negative redox domain mutant. Further, E3330 blocks the differentiation of bone marrow‐derived mesenchymal stem cells (BMSCs) into CD31+ endothelial progeny. Exposure of PCECs to E3330 results in a reduction of H‐ras expression and intracellular nitric oxide (NO) levels, as well as decreased DNA‐binding activity of the hypoxia‐inducible transcription factor, HIF‐1α. E3330 also reduces secreted and intracellular vascular endothelial growth factor expression by pancreatic cancer cells, while concomitantly downregulating the cognate receptor Flk‐1/KDR on PCECs. Inhibition of the Ape‐1/Ref‐1 redox domain with E3330 or comparable angiogenesis inhibitors might be a potent therapeutic strategy in solid tumors. J. Cell. Physiol. 219: 209–218, 2009.

HIF-1α induction suppresses excessive lipid accumulation in alcoholic fatty liver in mice

BACKGROUND & AIMS Chronic alcohol intake stimulates hepatic oxygen consumption and subsequently causes liver hypoxia, leading to activation of hypoxia inducible factor-1 (HIF-1). Although HIF-1 plays a crucial role in the metabolic switch from aerobic to anaerobic metabolism in response to hypoxia, its roles in the regulation of lipid metabolism in alcoholic fatty liver remain unknown. METHODS Wild-type and hepatocyte-specific HIF-1α-null mice were subjected to a 6% ethanol-containing liquid diet for 4 weeks, and functional effects of loss of the HIF-1α gene on lipid metabolism were examined in the liver. RESULTS Hepatocyte-specific HIF-1α-null mice developed severe hypertriglyceridemia with enhanced accumulation of lipids in the liver of mice exposed to a 6% ethanol-containing liquid diet for 4 weeks. Sterol regulatory element-binding protein 1c (SREBP-1c) and its downstream target acetyl-CoA carboxylase were greatly activated as the hepatic steatosis progressed, and these alterations were inversely correlated with the expression of the HIF-1-regulated gene DEC1. Overexpression of DEC1 in the mutant liver abrogated the detrimental effects of loss of HIF-1α gene on ethanol-induced fatty liver with reduced SREBP-1c expression. Conversely, co-administration of the HIF hydroxylase inhibitor dimethyloxalylglycine for the last 2 weeks improved markedly the ethanol-induced fatty liver in mice. CONCLUSIONS The current results provide direct evidence for protective roles of HIF-1 induction in the development of ethanol-induced fatty liver via activation of the HIF-1-regulated transcriptional repressor DEC1.

Development and application of high-performance affinity beads: Toward chemical biology and drug discovery

In drug development research, the elucidation and understanding of the interactions between physiologically active substances and proteins that numerous genes produce is important. Currently, most commercially available drugs and physiologically active substances have been brought to market without knowledge of factors interacting with the drugs and the substances. Affinity purification is a useful and powerful technique employed to understand factors that are targeted by drugs and physiologically active substances. However, use of conventional matrices for affinity chromatography often causes a decrease in efficiency of affinity purification and, as a result, more practical matrices for affinity purification have been developed for application in drug discovery research. In this paper, we describe the development of high-performance affinity beads (SG beads and FG beads) that enable one-step affinity purification of drug targets and the elucidation of the mechanism of the action of the drugs. We also describe a chemical screening system using our affinity beads. We hope that utilization of the affinity beads will contribute to the progress of research in chemical biology.

Porphyrin Accumulation in Mitochondria Is Mediated by 2-Oxoglutarate Carrier

Heme (Fe-protoporphyrin IX), an endogenous porphyrin derivative, is an essential molecule in living aerobic organisms and plays a role in a variety of physiological processes such as oxygen transport, respiration, and signal transduction. For the biosynthesis of heme or the mitochondrial heme proteins, heme or its biosynthetic precursor porphyrin must be transported into mitochondria from cytosol. The mechanism of porphyrin accumulation in the mitochondrial inner membrane is unclear. In the present study, we analyzed the mechanism of mitochondrial translocation of porphyrin derivatives. We showed that palladium meso-tetra(4-carboxyphenyl)porphyrin (PdTCPP), a phosphorescent porphyrin derivative, accumulated in the mitochondria of several cell lines. Using affinity latex beads, we showed that 2-oxoglutarate carrier (OGC), the mitochondrial transporter of 2-oxoglutarate, bound to PdTCPP, and in vitro PdTCPP inhibited 2-oxoglutarate uptake into mitochondria in a competitive manner (Ki = 15 μm). Interestingly, all types of porphyrin derivatives examined in this study competitively inhibited 2-oxoglutarate uptake into mitochondria, including protoporphyrin IX, coproporphyrin III, and hemin. Furthermore, mitochondrial accumulation of porphyrins was inhibited by 2-oxoglutarate or OGC inhibitor. These results suggested that porphyrin accumulation in mitochondria is mediated by OGC and that porphyrins are able to competitively inhibit 2-oxoglutarate uptake into mitochondria. This is the first report of a putative mechanism for accumulation of porphyrins in the mitochondrial inner membrane.

Haem-dependent dimerization of PGRMC1/Sigma-2 receptor facilitates cancer proliferation and chemoresistance

Progesterone-receptor membrane component 1 (PGRMC1/Sigma-2 receptor) is a haem-containing protein that interacts with epidermal growth factor receptor (EGFR) and cytochromes P450 to regulate cancer proliferation and chemoresistance; its structural basis remains unknown. Here crystallographic analyses of the PGRMC1 cytosolic domain at 1.95 Å resolution reveal that it forms a stable dimer through stacking interactions of two protruding haem molecules. The haem iron is five-coordinated by Tyr113, and the open surface of the haem mediates dimerization. Carbon monoxide (CO) interferes with PGRMC1 dimerization by binding to the sixth coordination site of the haem. Haem-mediated PGRMC1 dimerization is required for interactions with EGFR and cytochromes P450, cancer proliferation and chemoresistance against anti-cancer drugs; these events are attenuated by either CO or haem deprivation in cancer cells. This study demonstrates protein dimerization via haem–haem stacking, which has not been seen in eukaryotes, and provides insights into its functional significance in cancer.

GABP, HCF-1 and YY1 are involved in Rb gene expression during myogenesis

Muscle cell differentiation, or myogenesis, is a well‐characterized process and involves the expression of specific sets of genes in an orderly manner. A prerequisite for myogenesis is the exit from the cell cycle, which is associated with the up‐regulation of the tumor suppressor Rb. In this study, we set to investigate the regulatory mechanism of the Rb promoter that allows adequate up‐regulation in differentiating myoblasts. We report that Rb expression is regulated by the transcription factors GABP, HCF‐1 and YY1. Before induction of differentiation, Rb is expressed at a low level and GABP and YY1 are both present on the promoter. YY1, which exerts an inhibitory effect on Rb expression, is removed from the promoter as cells advance through myogenesis and translocates from the nucleus to the cytoplasm. On the other hand, upon induction of differentiation, the GABP cofactor HCF‐1 is recruited to and coactivates the promoter with GABP. RNAi‐mediated knock‐down of HCF‐1 results in inhibition of Rb up‐regulation as well as myotube formation. These results indicate that the Rb promoter is subject to regulation by positive and negative factors and that this intricate activation mechanism is critical to allow the accurate Rb gene up‐regulation observed during myogenesis.

hnRNP-U enhances the expression of specific genes by stabilizing mRNA.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are thought to be involved in pre‐mRNA processing. hnRNP‐U, also termed scaffold attachment factor A (SAF‐A), binds to pre‐mRNA and nuclear matrix/scaffold attachment region DNA elements. However, its role in the regulation of gene expression is as yet poorly understood. In the present study, we show that hnRNP‐U specifically enhances the expression of tumor necrosis factor α mRNA by increasing its stability, possibly through binding to the 3′ untranslated region. We also show that hnRNP‐U enhances the expression of several other genes as well, including GADD45A, HEXIM1, HOXA2, IER3, NHLH2, and ZFY, by binding to and stabilizing these mRNAs. These results suggest that hnRNP‐U enhances the expression of specific genes by regulating mRNA stability.