Maria Monica Barzago

The Aldehyde Oxidase Gene Cluster in Mice and Rats ALDEHYDE OXIDASE HOMOLOGUE 3, A NOVEL MEMBER OF THE MOLYBDO-FLAVOENZYME FAMILY WITH SELECTIVE EXPRESSION IN THE OLFACTORY MUCOSA

Mammalian molybdo-flavoenzymes are oxidases requiring FAD and molybdopterin (molybdenum cofactor) for their catalytic activity. This family of proteins was thought to consist of four members, xanthine oxidoreductase, aldehyde oxidase 1 (AOX1), and the aldehyde oxidase homologues 1 and 2 (AOH1 and AOH2, respectively). Whereas the first two enzymes are present in humans and various other mammalian species, the last two proteins have been described only in mice. Here, we report on the identification, in both mice and rats, of a novel molybdo-flavoenzyme, AOH3. In addition, we have cloned the cDNAs coding for rat AOH1 and AOH2, demonstrating that this animal species has the same complement of molybdo-flavoproteins as the mouse. The AOH3 cDNA is characterized by remarkable similarity to AOX1, AOH1, AOH2, and xanthine oxidoreductase cDNAs. Mouse AOH3 is selectively expressed in Bowmans glands of the olfactory mucosa, although small amounts of the corresponding mRNA are present also in the skin. In the former location, two alternatively spliced forms of the AOH3 transcript with different 3′-untranslated regions were identified. The general properties of AOH3 were determined by purification of mouse AOH3 from the olfactory mucosa. The enzyme possesses aldehyde oxidase activity and oxidizes, albeit with low efficiency, exogenous substrates that are recognized by AOH1 and AOX1. The Aoh3 gene maps to mouse chromosome 1 band c1 and rat chromosome 7 in close proximity to the Aox1, Aoh1, and Aoh2 loci and has an exon/intron structure almost identical to that of the other molybdo-flavoenzyme genes in the two species.

Role of the Molybdoflavoenzyme Aldehyde Oxidase Homolog 2 in the Biosynthesis of Retinoic Acid: Generation and Characterization of a Knockout Mouse†

ABSTRACT The mouse aldehyde oxidase AOH2 (aldehyde oxidase homolog 2) is a molybdoflavoenzyme. Harderian glands are the richest source of AOH2, although the protein is detectable also in sebaceous glands, epidermis, and other keratinized epithelia. The levels of AOH2 in the Harderian gland and skin are controlled by genetic background, being maximal in CD1 and C57BL/6 and minimal in DBA/2, CBA, and 129/Sv strains. Testosterone is a negative regulator of AOH2 in Harderian glands. Purified AOH2 oxidizes retinaldehyde into retinoic acid, while it is devoid of pyridoxal-oxidizing activity. Aoh2−/− mice, the first aldehyde oxidase knockout animals ever generated, are viable and fertile. The data obtained for this knockout model indicate a significant role of AOH2 in the local synthesis and biodisposition of endogenous retinoids in the Harderian gland and skin. The Harderian glands transcriptome of knockout mice demonstrates overall downregulation of direct retinoid-dependent genes as well as perturbations in pathways controlling lipid homeostasis and cellular secretion, particularly in sexually immature animals. The skin of knockout mice is characterized by thickening of the epidermis in basal conditions and after UV light exposure. This has correlates in the corresponding transcriptome, which shows enrichment and overall upregulation of genes involved in hypertrophic responses.

Avian and Canine Aldehyde Oxidases NOVEL INSIGHTS INTO THE BIOLOGY AND EVOLUTION OF MOLYBDO-FLAVOENZYMES

Aldehyde oxidases are molybdo-flavoenzymes structurally related to xanthine oxidoreductase. They catalyze the oxidation of aldehydes or N-heterocycles of physiological, pharmacological, and toxicological relevance. Rodents are characterized by four aldehyde oxidases as follows: AOX1 and aldehyde oxidase homologs 1-3 (AOH1, AOH2, and AOH3). Humans synthesize a single functional aldehyde oxidase, AOX1. Here we define the structure and the characteristics of the aldehyde oxidase genes and proteins in chicken and dog. The avian genome contains two aldehyde oxidase genes, AOX1 and AOH, mapping to chromosome 7. AOX1 and AOH are structurally very similar and code for proteins whose sequence was deduced from the corresponding cDNAs. AOX1 is the ortholog of the same gene in mammals, whereas AOH represents the likely ancestor of rodent AOH1, AOH2, and AOH3. The dog genome is endowed with two structurally conserved and active aldehyde oxidases clustering on chromosome 37. Cloning of the corresponding cDNAs and tissue distribution studies demonstrate that they are the orthologs of rodent AOH2 and AOH3. The vestiges of dog AOX1 and AOH1 are recognizable upstream of AOH2 and AOH3 on the same chromosome. Comparison of the complement and the structure of the aldehyde oxidase and xanthine oxidoreductase genes in vertebrates and other animal species indicates that they evolved through a series of duplication and inactivation events. Purification of the chicken AOX1 protein to homogeneity from kidney demonstrates that the enzyme possesses retinaldehyde oxidase activity. Unlike humans and most other mammals, dog and chicken are devoid of liver aldehyde oxidase activity.

Regulation and biochemistry of mouse molybdo-flavoenzymes: The DBA/2 mouse is selectively deficient in the expression of aldehyde oxidase homologues 1 and 2 and represents a unique source for the purification and characterization of aldehyde oxidase

Mouse molybdo-flavoenzymes consist of xanthine oxidoreductase, aldehyde oxidase (AOX1), and two recently identified proteins, AOH1 and AOH2 (aldehyde oxidase homologues 1 and 2). Here we demonstrate that CD-1, C57BL/6, 129/Sv, and other mouse strains synthesize high levels of AOH1 in the liver and AOH2 in the skin. By contrast, the DBA/2 and CBA strains are unique, having a selective deficit in the expression of the AOH1 and AOH2 genes. DBA/2 animals synthesize trace amounts of a catalytically active AOH1 protein. However, relative to CD-1 animals, an over 2 log reduction in the steady-state levels of liver AOH1 mRNA, protein, and enzymatic activity is observed in basal conditions and following administration of testosterone. The DBA/2 mouse represents a unique opportunity to purify AOX1 and compare its enzymatic characteristics to those of the AOH1 protein. The spectroscopy and biochemistry of AOX1 are very similar to those of AOH1 except for a differential sensitivity to the non-competitive inhibitory effect of norharmane. AOX1 and AOH1 oxidize an overlapping set of aldehydes and heterocycles. For most compounds, the substrate efficiency (Vmax/Km) of AOX1 is superior to that of AOH1. Alkylic alcohols and acetaldehyde, the toxic metabolite of ethanol, are poor substrates of both enzymes. Consistent with this, the levels of acetaldehyde in the livers of ethanol administered CD-1 and DBA/2 mice are similar, indicating that neither enzyme is involved in the in vivo biotransformation of acetaldehyde.

Antitumor activity of the retinoid-related molecules (E)-3-(4'-hydroxy-3'-adamantylbiphenyl-4-yl)acrylic acid (ST1926) and 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437) in F9 teratocarcinoma : Role of retinoic acid receptor gamma and retinoid-independent pathways

The retinoid-related molecules (RRMs) ST1926 [(E)-3-(4′-hydroxy-3′-adamantylbiphenyl-4-yl)acrylic acid] and CD437 (6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid) are promising anticancer agents. We compared the retinoic acid receptor (RAR) trans-activating properties of the two RRMs and all-trans-retinoic acid (ATRA). ST1926 and CD437 are better RARγ agonists than ATRA. We used three teratocarcinoma cell lines to evaluate the significance of RARγ in the activity of RRMs: F9-wild type (WT); F9γ-/-, lacking the RARγ gene; F9γ51, aF9γ-/-derivative, complemented for the RARγ deficit. Similar to ATRA, ST1926 and CD437 activate cytodifferentiation only in F9-WT cells. Unlike ATRA, ST1926 and CD437 arrest cells in the G2/M phase of the cell cycle and induce apoptosis in all F9 cell lines. Our data indicate that RARγ and the classic retinoid pathway are not relevant for the antiproliferative and apoptotic activities of RRMs in vitro. Increases in cytosolic calcium are fundamental for apoptosis, in that intracellular calcium chelators abrogate the process. Comparison of the gene expression profiles associated with ST1926 and ATRA in F9-WT and F9γ-/-indicates that the RRM activates a conspicuous nonretinoid response in addition to the classic and RAR-dependent pathway. The pattern of genes regulated by ST1926 selectively, in a RARγ-independent manner, provides novel insights into the possible molecular determinants underlying the activity of RRMs in vitro. Furthermore, it suggests that RARγ-dependent responses are relevant to the activity of RRMs in vivo. Indeed, the receptor hinders the antitumor activity in vivo, in that both syngeneic and immunosuppressed SCID mice bearing F9γ-/- tumors have increased life spans after treatment with ST1926 and CD437 relative to their F9-WT counterparts.

Structure and evolution of vertebrate aldehyde oxidases: from gene duplication to gene suppression

Aldehyde oxidases (AOXs) and xanthine dehydrogenases (XDHs) belong to the family of molybdo-flavoenzymes. Although AOXs are not identifiable in fungi, these enzymes are represented in certain protists and the majority of plants and vertebrates. The physiological functions and substrates of AOXs are unknown. Nevertheless, AOXs are major drug metabolizing enzymes, oxidizing a wide range of aromatic aldehydes and heterocyclic compounds of medical/toxicological importance. Using genome sequencing data, we predict the structures of AOX genes and pseudogenes, reconstructing their evolution. Fishes are the most primitive organisms with an AOX gene (AOXα), originating from the duplication of an ancestral XDH. Further evolution of fishes resulted in the duplication of AOXα into AOXβ and successive pseudogenization of AOXα. AOXβ is maintained in amphibians and it is the likely precursors of reptilian, avian, and mammalian AOX1. Amphibian AOXγ is a duplication of AOXβ and the likely ancestor of reptilian and avian AOX2, which, in turn, gave rise to mammalian AOX3L1. Subsequent gene duplications generated the two mammalian genes, AOX3 and AOX4. The evolution of mammalian AOX genes is dominated by pseudogenization and deletion events. Our analysis is relevant from a structural point of view, as it provides information on the residues characterizing the three domains of each mammalian AOX isoenzyme. We cloned the cDNAs encoding the AOX proteins of guinea pig and cynomolgus monkeys, two unique species as to the evolution of this enzyme family. We identify chimeric RNAs from the human AOX3 and AOX3L1 pseudogenes with potential to encode a novel microRNA.

Human Axonal Survival of Motor Neuron (a-SMN) Protein Stimulates Axon Growth, Cell Motility, C-C Motif Ligand 2 (CCL2), and Insulin-like Growth Factor-1 (IGF1) Production

Background: Axonal SMN is a truncated product of the spinal muscular atrophy (SMA) disease gene SMN1. Results: Forced expression of axonal SMN in motoneuron-like NSC34 cells modulates growth, axonogenesis, and motility. Conclusion: Axonal SMN induces CCL2/CCL7 chemokines and the IGF-1 growth factor. CCL2 contributes to axonal SMN-induced motility and axonogenesis. Significance: Insights into the function and underlying mechanisms with relevance for axonal SMN in SMA are provided. Spinal muscular atrophy is a fatal genetic disease of motoneurons due to loss of full-length survival of motor neuron protein, the main product of the disease gene SMN1. Axonal SMN (a-SMN) is an alternatively spliced isoform of SMN1, generated by retention of intron 3. To study a-SMN function, we generated cellular clones for the expression of the protein in mouse motoneuron-like NSC34 cells. The model was instrumental in providing evidence that a-SMN decreases cell growth and plays an important role in the processes of axon growth and cellular motility. In our conditions, low levels of a-SMN expression were sufficient to trigger the observed biological effects, which were not modified by further increasing the amounts of the expressed protein. Differential transcriptome analysis led to the identification of novel a-SMN-regulated factors, i.e. the transcripts coding for the two chemokines, C-C motif ligands 2 and 7 (CCL2 and CCL7), as well as the neuronal and myotrophic factor, insulin-like growth factor-1 (IGF1). a-SMN-dependent induction of CCL2 and IGF1 mRNAs resulted in increased intracellular levels and secretion of the respective protein products. Induction of CCL2 contributes to the a-SMN effects, mediating part of the action on axon growth and random cell motility, as indicated by chemokine knockdown and re-addition studies. Our results shed new light on a-SMN function and the underlying molecular mechanisms. The data provide a rational framework to understand the role of a-SMN deficiency in the etiopathogenesis of spinal muscular atrophy.

Cellular and molecular determinants of all-trans retinoic acid sensitivity in breast cancer: Luminal phenotype and RARα expression

Forty‐two cell lines recapitulating mammary carcinoma heterogeneity were profiled for all‐trans retinoic acid (ATRA) sensitivity. Luminal and ER+ (estrogen‐receptor‐positive) cell lines are generally sensitive to ATRA, while refractoriness/low sensitivity is associated with a Basal phenotype and HER2 positivity. Indeed, only 2 Basal cell lines (MDA‐MB157 and HCC‐1599) are highly sensitive to the retinoid. Sensitivity of HCC‐1599 cells is confirmed in xenotransplanted mice. Short‐term tissue‐slice cultures of surgical samples validate the cell‐line results and support the concept that a high proportion of Luminal/ER+ carcinomas are ATRA sensitive, while triple‐negative (Basal) and HER2‐positive tumors tend to be retinoid resistant. Pathway‐oriented analysis of the constitutive gene‐expression profiles in the cell lines identifies RARα as the member of the retinoid pathway directly associated with a Luminal phenotype, estrogen positivity and ATRA sensitivity. RARα3 is the major transcript in ATRA‐sensitive cells and tumors. Studies in selected cell lines with agonists/antagonists confirm that RARα is the principal mediator of ATRA responsiveness. RARα over‐expression sensitizes retinoid‐resistant MDA‐MB453 cells to ATRA anti‐proliferative action. Conversely, silencing of RARα in retinoid‐sensitive SKBR3 cells abrogates ATRA responsiveness. All this is paralleled by similar effects on ATRA‐dependent inhibition of cell motility, indicating that RARα may mediate also ATRA anti‐metastatic effects. We define gene sets of predictive potential which are associated with ATRA sensitivity in breast cancer cell lines and validate them in short‐term tissue cultures of Luminal/ER+ and triple‐negative tumors. In these last models, we determine the perturbations in the transcriptomic profiles afforded by ATRA. The study provides fundamental information for the development of retinoid‐based therapeutic strategies aimed at the stratified treatment of breast cancer subtypes.

All-trans-retinoic Acid Modulates the Plasticity and Inhibits the Motility of Breast Cancer Cells: ROLE OF NOTCH1 AND TRANSFORMING GROWTH FACTOR (TGFβ).

Background: All-trans-retinoic acid is a promising therapeutic agent in breast cancer. Results: All-trans-retinoic acid modulates mammary tumor cell epithelial-to-mesenchymal-transition via the TGFβ and NOTCH pathways. Conclusion: The present study unveils a new aspect of all-trans-retinoic acid activity (i.e. regulation of phenotypic cell plasticity). Significance: Our results indicate that all-trans-retinoic acid is endowed with anti-metastatic properties that could be exploited at the therapeutic level. All-trans-retinoic acid (ATRA) is a natural compound proposed for the treatment/chemoprevention of breast cancer. Increasing evidence indicates that aberrant regulation of epithelial-to-mesenchymal transition (EMT) is a determinant of the cancer cell invasive and metastatic behavior. The effects of ATRA on EMT are largely unknown. In HER2-positive SKBR3 and UACC812 cells, showing co-amplification of the ERBB2 and RARA genes, ATRA activates a RARα-dependent epithelial differentiation program. In SKBR3 cells, this causes the formation/reorganization of adherens and tight junctions. Epithelial differentiation and augmented cell-cell contacts underlie the anti-migratory action exerted by the retinoid in cells exposed to the EMT-inducing factors EGF and heregulin-β1. Down-regulation of NOTCH1, an emerging EMT modulator, is involved in the inhibition of motility by ATRA. Indeed, the retinoid blocks NOTCH1 up-regulation by EGF and/or heregulin-β1. Pharmacological inhibition of γ-secretase and NOTCH1 processing also abrogates SKBR3 cell migration. Stimulation of TGFβ contributes to the anti-migratory effect of ATRA. The retinoid switches TGFβ from an EMT-inducing and pro-migratory determinant to an anti-migratory mediator. Inhibition of the NOTCH1 pathway not only plays a role in the anti-migratory action of ATRA; it is relevant also for the anti-proliferative activity of the retinoid in HCC1599 breast cancer cells, which are addicted to NOTCH1 for growth/viability. This effect is enhanced by the combination of ATRA and the γ-secretase inhibitor N-(N-(3,5-difluorophenacetyl)-l-alanyl)-S-phenylglycine t-butyl ester, supporting the concept that the two compounds act at the transcriptional and post-translational levels along the NOTCH1 pathway.

Mouse aldehyde-oxidase-4 controls diurnal rhythms, fat deposition and locomotor activity

Aldehyde-oxidase-4 (AOX4) is one of the mouse aldehyde oxidase isoenzymes and its physiological function is unknown. The major source of AOX4 is the Harderian-gland, where the enzyme is characterized by daily rhythmic fluctuations. Deletion of the Aox4 gene causes perturbations in the expression of the circadian-rhythms gene pathway, as indicated by transcriptomic analysis. AOX4 inactivation alters the diurnal oscillations in the expression of master clock-genes. Similar effects are observed in other organs devoid of AOX4, such as white adipose tissue, liver and hypothalamus indicating a systemic action. While perturbations of clock-genes is sex-independent in the Harderian-gland and hypothalamus, sex influences this trait in liver and white-adipose-tissue which are characterized by the presence of AOX isoforms other than AOX4. In knock-out animals, perturbations in clock-gene expression are accompanied by reduced locomotor activity, resistance to diet induced obesity and to hepatic steatosis. All these effects are observed in female and male animals. Resistance to obesity is due to diminished fat accumulation resulting from increased energy dissipation, as white-adipocytes undergo trans-differentiation towards brown-adipocytes. Metabolomics and enzymatic data indicate that 5-hydroxyindolacetic acid and tryptophan are novel endogenous AOX4 substrates, potentially involved in AOX4 systemic actions.