The neuronal oxygen-sensing pathway controls postnatal vascularization of the murine brain.

The contribution of neurons to growth and refinement of the microvasculature during postnatal brain development is only partially understood. Tissue hypoxia is the physiologic stimulus for angiogenesis by enhancing angiogenic mediators partly through activation of hypoxia-inducible factors (HIFs). Hence, we investigated the HIF oxygen-sensing pathway in postmitotic neurons for physiologic angiogenesis in the murine forebrain during postnatal development by using mice lacking the HIF suppressing enzyme prolyl-4-hydroxylase domain (PHD)2 and/or HIF-1/2α in postmitotic neurons. Perinatal activation or inactivation of the HIF pathway in neurons inversely modulated brain vascularization, including endothelial cell number and proliferation, density of total and perfused microvessels, and vascular branching. Accordingly, several angiogenesis-related genes were up-regulated in vivo and in primary neurons derived from PHD2-deficient mice. Among them, only VEGF and adrenomedullin (Adm) promoted angiogenic sprouting of brain endothelial cells. VEGF and Adm additively enhanced endothelial sprouting through activation of multiple pathways. PHD2 deficiency in neurons caused HIF-α stabilization and increased VEGF mRNA levels not only in neurons but unexpectedly also in astrocytes, suggesting a new mechanism of neuron-to-astrocyte signaling. Collectively, our results identify the PHD-HIF pathway in neurons as an important determinant for vascularization of the brain during postnatal development.-Nasyrov, E., Nolan, K. A., Wenger, R. H., Marti, H. H., Kunze, R. The neuronal oxygen-sensing pathway controls postnatal vascularization of the murine brain.

Negative Cooperativity in NAD(P)H Quinone Oxidoreductase 1 (NQO1).

NAD(P)H quinone oxidoreductase-1 (NQO1) is a homodimeric protein that acts as a detoxifying enzyme or as a chaperone protein. Dicourmarol interacts with NQO1 at the NAD(P)H binding site and can both inhibit enzyme activity and modulate the interaction of NQO1 with other proteins. We show that the binding of dicoumarol and related compounds to NQO1 generates negative cooperativity between the monomers. This does not occur in the presence of the reducing cofactor, NAD(P)H, alone. Alteration of Gly150 (but not Gly149 or Gly174) abolished the dicoumarol-induced negative cooperativity. Analysis of the dynamics of NQO1 with the Gaussian network model indicates a high degree of collective motion by monomers and domains within NQO1. Ligand binding is predicted to alter NQO1 dynamics both proximal to the ligand binding site and remotely, close to the second binding site. Thus, drug-induced modulation of protein motion might contribute to the biological effects of putative inhibitors of NQO1.

Generation of renal Epo-producing cell lines by conditional gene tagging reveals rapid HIF-2 driven Epo kinetics, cell autonomous feedback regulation, and a telocyte phenotype.

Erythropoietin (Epo) is essential for erythropoiesis and is mainly produced by the fetal liver and the adult kidney following hypoxic stimulation. Epo regulation is commonly studied in hepatoma cell lines, but differences in Epo regulation between kidney and liver limit the understanding of Epo dysregulation in polycythaemia and anaemia. To overcome this limitation, we have generated a novel transgenic mouse model expressing Cre recombinase specifically in the active fraction of renal Epo-producing (REP) cells. Crossing with reporter mice confirmed the inducible and highly specific tagging of REP cells, located in the corticomedullary border region where there is a steep drop in oxygen bioavailability. A novel method was developed to selectively grow primary REP cells in culture and to generate immortalized clonal cell lines, called fibroblastoid atypical interstitial kidney (FAIK) cells. FAIK cells show very early hypoxia-inducible factor (HIF)-2α induction, which precedes Epo transcription. Epo induction in FAIK cells reverses rapidly despite ongoing hypoxia, suggesting a cell autonomous feedback mechanism. In contrast, HIF stabilizing drugs resulted in chronic Epo induction in FAIK cells. RNA sequencing of three FAIK cell lines derived from independent kidneys revealed a high degree of overlap and suggests that REP cells represent a unique cell type with properties of pericytes, fibroblasts, and neurons, known as telocytes. These novel cell lines may be helpful to investigate myofibroblast differentiation in chronic kidney disease and to elucidate the molecular mechanisms of HIF stabilizing drugs currently in phase III studies to treat anemia in end-stage kidney disease.

Erythropoietin production by PDGFR-ß(+) cells.

PDGFR-ß-expressing cells of the kidneys are considered as a relevant site of erythropoietin (EPO) production. The origin of these cells, their contribution to renal EPO production, and if PDGFR-ß-positive cells in other organs are also capable to express EPO are less clear. We addressed these questions in mice, in which hypoxia-inducible transcription factors were stabilized in PDGFR-ß(+) cells by inducible deletion of the von Hippel-Lindau (Vhl) protein. Vhl deletion led to a 600-fold increase of plasma EPO concentration, 170-fold increase of renal EPO messenger RNA (mRNA) levels, and an increase of hematocrit values up to 70 %. Intrarenal localization of EPO-expressing cells coincided with the zonal heterogeneity and distribution of cells expressing PDGFR-ß. Amongst a variety of extrarenal organs only adrenal glands showed significant EPO mRNA expression after Vhl deletion in PDGFR-ß(+) cells. EPO mRNA, plasma EPO, and hematocrit fell to subnormal values if HIF-2α, but not HIF-1α, was deleted either alone or in combination with Vhl in PDGFR-ß(+) cells. Treatment of mice with a prolyl-hydroxylase inhibitor caused an increase of EPO mRNA abundance and plasma EPO concentrations in wild-type mice and in mice lacking HIF-1α in PDGFR-ß(+) cells but exerted no effect in mice lacking HIF-2α in PDGFR-ß(+) cells. These findings suggest that PDGFR-ß(+) cells are the only relevant site of EPO expression in the kidney and that HIF-2 is the essential transcription factor triggering EPO expression therein. Moreover, our findings suggest that PDGFR-ß(+) cells elaborating EPO might arise from the metanephric mesenchyme, rather than from the neural crest.

FIH Regulates Cellular Metabolism through Hydroxylation of the Deubiquitinase OTUB1.

The asparagine hydroxylase, factor inhibiting HIF (FIH), confers oxygen-dependence upon the hypoxia-inducible factor (HIF), a master regulator of the cellular adaptive response to hypoxia. Studies investigating whether asparagine hydroxylation is a general regulatory oxygen-dependent modification have identified multiple non-HIF targets for FIH. However, the functional consequences of this outside of the HIF pathway remain unclear. Here, we demonstrate that the deubiquitinase ovarian tumor domain containing ubiquitin aldehyde binding protein 1 (OTUB1) is a substrate for hydroxylation by FIH on N22. Mutation of N22 leads to a profound change in the interaction of OTUB1 with proteins important in cellular metabolism. Furthermore, in cultured cells, overexpression of N22A mutant OTUB1 impairs cellular metabolic processes when compared to wild type. Based on these data, we hypothesize that OTUB1 is a target for functional hydroxylation by FIH. Additionally, we propose that our results provide new insight into the regulation of cellular energy metabolism during hypoxic stress and the potential for targeting hydroxylases for therapeutic benefit.

Induction of long noncoding RNA MALAT1 in hypoxic mice.

Long thought to be "junk DNA", in recent years it has become clear that a substantial fraction of intergenic genomic DNA is actually transcribed, forming long noncoding RNA (lncRNA). Like mRNA, lncRNA can also be spliced, capped, and polyadenylated, affecting a multitude of biological processes. While the molecular mechanisms underlying the function of lncRNAs have just begun to be elucidated, the conditional regulation of lncRNAs remains largely unexplored. In genome-wide studies our group and others recently found hypoxic transcriptional induction of a subset of lncRNAs, whereof nuclear-enriched abundant/autosomal transcript 1 (NEAT1) and metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) appear to be the lncRNAs most ubiquitously and most strongly induced by hypoxia in cultured cells. Hypoxia-inducible factor (HIF)-2 rather than HIF-1 seems to be the preferred transcriptional activator of these lncRNAs. For the first time, we also found strong induction primarily of MALAT1 in organs of mice exposed to inspiratory hypoxia. Most abundant hypoxic levels of MALAT1 lncRNA were found in kidney and testis. In situ hybridization revealed that the hypoxic induction in the kidney was confined to proximal rather than distal tubular epithelial cells. Direct oxygen-dependent regulation of MALAT1 lncRNA was confirmed using isolated primary kidney epithelial cells. In summary, high expression levels and acute, profound hypoxic induction of MALAT1 suggest a hitherto unrecognized role of this lncRNA in renal proximal tubular function.

Paricalcitol protects against TGF-ß1-induced fibrotic responses in hypoxia and stabilises HIF-α in renal epithelia.

Epithelial injury and tubulointerstitial fibrosis (TIF) within a hypoxic microenvironment are associated with progressive loss of renal function in chronic kidney disease [CKD]. Transforming growth factor beta-1 (TGF-ß1) is an important mediator of renal fibrosis. Growing evidence suggests that Vitamin D [1,25-(OH)2D] and its analogues may have a renoprotective effect in CKD. Here we examined the protective effect of the vitamin D analogue paricalcitol [PC; 19-nor-1α,3ß,25-trihydroxy-9,10-secoergosta-5(Z),7(E) 22(E)-triene] on the responses of human renal epithelial cells to TGF-ß1. PC attenuated TGF-ß1-induced Smad 2 phosphorylation and upregulation of the Notch ligand Jagged-1, α-smooth muscle actin and thrombospondin-1 and prevented the TGF-ß1-mediated loss of E-Cadherin. To mimic the hypoxic milieu of CKD we cultured renal epithelial cells in hypoxia [1% O2] and observed similar attenuation by PC of TGF-ß1-induced fibrotic responses. Furthermore, in cells cultured in normoxia [21% O2], PC induced an accumulation of hypoxia-inducible transcription factors (HIF) 1α and HIF-2α in a time and concentration [1 µM-2 µM] dependent manner. Here, PC-induced HIF stabilisation was dependent on activation of the PI-3Kinase pathway. This is the first study to demonstrate regulation of the HIF pathway by PC which may have importance in the mechanism underlying renoprotection by PC.

In silico screening reveals structurally diverse, nanomolar inhibitors of NQO2 that are functionally active in cells and can modulate NF-κB signaling.

The National Cancer Institute chemical database has been screened using in silico docking to identify novel nanomolar inhibitors of NRH:quinone oxidoreductase 2 (NQO2). The inhibitors identified from the screen exhibit a diverse range of scaffolds and the structure of one of the inhibitors, NSC13000 cocrystalized with NQO2, has been solved. This has been used to aid the generation of a structure-activity relationship between the computationally derived binding affinity and experimentally measured enzyme inhibitory potency. Many of the compounds are functionally active as inhibitors of NQO2 in cells at nontoxic concentrations. To show this, advantage was taken of the NQO2-mediated toxicity of the chemotherapeutic drug CB1954. The toxicity of this drug is substantially reduced when the function of NQO2 is inhibited, and many of the compounds achieve this in cells at nanomolar concentrations. The NQO2 inhibitors also attenuated TNFα-mediated, NF-кB-driven transcriptional activity. The link between NQO2 and the regulation of NF-кB was confirmed by using short interfering RNA to NQO2 and by the observation that NRH, the cofactor for NQO2 enzyme activity, could regulate NF-кB activity in an NQO2-dependent manner. NF-кB is a potential therapeutic target and this study reveals an underlying mechanism that may be usable for developing new anticancer drugs.

Novel inhibitors of NRH:quinone oxidoreductase 2 (NQO2): crystal structures, biochemical activity, and intracellular effects of imidazoacridin-6-ones.

Imidazoacridin-6-ones are shown to be potent nanomolar inhibitors of the enzyme NQO2. By use of computational molecular modeling, a reliable QSAR was established, relating inhibitory potency with calculated binding affinity. Further, crystal structures of NQO2 containing two of the imidazoacridin-6-ones have been solved. To generate compounds with reduced off-target (DNA binding) effects, an N-oxide moiety was introduced into the tertiary aminoalkyl side chain of the imidazoacridin-6-ones. This resulted in substantially less toxicity in a panel of eight cancer cell lines, decreased protein binding, and reduced DNA binding and nuclear accumulation. Finally, one of the N-oxides showed potent ability to inhibit the enzymatic function of NQO2 in cells, and therefore, it may be useful as a pharmacological probe to study the properties of the enzyme in vitro and in vivo.

In silico identification and biochemical evaluation of novel inhibitors of NRH:quinone oxidoreductase 2 (NQO2).

The NCI chemical database has been screened using in silico docking to identify novel inhibitors of NRH:quinone oxidoreductase 2 (NQO2). Compounds identified from the screen exhibit a diverse range of scaffolds and inhibitory potencies are generally in the micromolar range. Some of the compounds also have the ability to inhibit NQO1. The modes of binding of the different compounds to the two enzymes are illustrated and discussed.

Triazoloacridin-6-ones as novel inhibitors of the quinone oxidoreductases NQO1 and NQO2.

A range of triazoloacridin-6-ones functionalized at C5 and C8 have been synthesized and evaluated for ability to inhibit NQO1 and NQO2. The compounds were computationally docked into the active site of NQO1 and NQO2, and calculated binding affinities were compared with IC(50) values for enzyme inhibition. Excellent correlation coefficients were demonstrated suggesting a predictive QSAR model for this series of structurally similar analogues. From this we have identified some of these triazoloacridin-6-ones to be the most potent NQO2 inhibitors so far reported.

Synthesis and biological evaluation of coumarin-based inhibitors of NAD(P)H: quinone oxidoreductase-1 (NQO1).

The synthesis is reported here of two novel series of inhibitors of human NAD(P)H quinone oxidoreductase-1 (NQO1), an enzyme overexpressed in several types of tumor cell. The first series comprises substituted symmetric dicoumarol analogues; the second series contains hybrid compounds where one 4-hydroxycoumarin system is replaced by a different aromatic moiety. Several compounds show equivalent or improved NQO1 inhibition over dicoumarol, both in the presence and in the absence of added protein. Further, correlation is demonstrated between the ability of these agents to inhibit NQO1 and computed binding affinity. We have solved the crystal structure of NQO1 complexed to a hybrid compound and find good agreement with the in silico model. For both MIA PaCa-2 pancreatic tumor cells and HCT116 colon cancer cells, dicoumarol shows the greatest toxicity of all compounds. Thus, we provide a computational, synthetic, and biological platform to generate competitive NQO1 inhibitors with superior pharmacological properties to dicoumarol. This will allow a more definitive study of NQO1 activity in cells, in particular, its drug activating/detoxifying properties and ability to modulate oncoprotein stability.

Analysis of a promoter polymorphism in the SMDF neuregulin 1 isoform in Schizophrenia.

BACKGROUND/AIMS: Neuregulin 1 (NRG1) is a positional candidate gene in schizophrenia (SZ). Two major susceptibility loci in the NRG1 gene approximately one million nucleotides apart have been identified in genetic studies. Several candidate functional allelic variants have been described that might be involved in disease susceptibility. However, the findings are still preliminary. We recently mapped active promoters and other regulatory domains in several SZ and bipolar disorder (BD) candidate genes using ChIP-chip (chromatin immunoprecipitation hybridized to microarrays). One was the promoter for the NRG1 isoform, SMDF, which maps to the 3' end of the gene complex. Analysis of the SNP database revealed several polymorphisms within the approximate borders of the region immunoprecipitated in our ChIP-chip experiments, one of which is rs7825588. METHODS: This SNP was analyzed in patients with SZ and BD and its effect on promoter function was assessed by electromobility gel shift assays and luciferase reporter constructs. RESULTS: A significant increase in homozygosity for the minor allele was found in patients with SZ (genotype distribution chi(2) = 7.32, p = 0.03) but not in BD (genotype distribution chi(2) = 0.52, p = 0.77). Molecular studies demonstrated modest, but statistically significant allele-specific differences in protein binding and promoter function. CONCLUSION: The findings suggest that homozygosity for rs725588 could be a risk genotype for SZ.

Coumarin-based inhibitors of human NAD(P)H:quinone oxidoreductase-1. Identification, structure-activity, off-target effects and in vitro human pancreatic cancer toxicity.

The enzyme human NAD(P)H quinone oxidoreductase-1 (NQO1), which is overexpressed in several types of tumor cell, is considered a design target for cancer therapeutics. We identify new coumarin-based competitive inhibitors of NQO1, one of which is nanomolar. Using computational docking and molecular dynamics, we obtain insights into the structural basis of inhibition. Selected inhibitors were then assessed for off-target effects associated with dicoumarol and were found to have differing effects on superoxide formation and mitochondrial respiration. A comparison of NQO1 inhibition and off-target effects for dicoumarol and its derivatives suggests that the ability of dicoumarol to kill cancer cells is independent of NQO1 inhibition, that cellular superoxide production by dicoumarol does not seem linked to NQO1 inhibition but may be related to mitochondrial decoupling, and that superoxide does not appear to be a major determinant of cytotoxicity. Implications are discussed for NQO1 inhibition as an anticancer drug design target and superoxide generation as the dicoumarol-mediated mechanism of cytotoxicity.

In silico identification and biochemical characterization of novel inhibitors of NQO1.

From in silico docking and COMPARE analysis, novel inhibitors of human NAD(P)H quinone oxidoreductase (NQO1) have been identified from the NCI compound database, the most potent of which has an observed IC(50) of 0.7muM. The inhibitors exhibit a diverse range of scaffolds. The ability of docking calculations to predict experimentally determined binding affinities for NQO1 is discussed, considering the influence of target flexibility and scoring function.