Mark J. Stewart

The role of nuclear factor NF-Y/CP1 in the transcriptional regulation of the human aldehyde dehydrogenase 2-encoding gene.

Mitochondrial aldehyde dehydrogenase (ALDH2) activity is produced at low levels in many tissues, with highest production in liver. Transfection assays using the first 600 bp of upstream DNA provided evidence for both positive and negative regulatory elements in the proximal promoter. A region from -79 to -116 bp was protected in DNase I footprinting assays and bound in electrophoretic mobility shift assays (EMSA) by a nuclear factor found in all cell lines and tissues tested. This region, denoted FP160, contained the consensus recognition sites for Sp1 and AP2, and a CCAAT box. The CCAAT box was specifically protected by a nuclear factor in methylation interference assays. Mutagenesis of specific bp within the CCAAT box eliminated protein binding in vitro and decreased transcriptional activity from the ALDH2 promoter approximately 50% in reporter gene assays. Competition experiments showed that the nuclear factor binding to the FP160 oligodeoxyribonucleotide (oligo) was competed by oligos corresponding to an NY-Y/CP1-binding site to a greater extent than by those containing sites for CTF/NF1, C/EPB or CP2. The heat stability, resistance to proteinase K digestion, sensitivity to inhibition of DNA binding by o-phenanthroline, and immunological properties of the liver factor binding to FP160 were very similar to the corresponding properties of NF-Y/CP1. Thus, the proximal ALDH2 promoter was bound by NF-Y/CP1 and this transcription factor may be responsible for the basal expression of the gene observed in most tissues. The NFY-CP1 present in rat liver has similar properties to that previously characterized in M12 B-lymphoma cells and LMTK mouse fibroblasts.

A direct repeat (DR-1) element in the first exon modulates transcription of the preproenkephalin A gene

The preproenkephalin A gene is regulated by upstream cis-acting elements which respond to various signals, such as cAMP, calcium, and phorbol esters. An additional regulatory element was detected downstream of the transcription start site of the human preproenkephalin A gene in transfection experiments. The element was localized by DNAse I footprinting and methylation interference assays to a direct repeat (DR-1) element in the first (untranslated) exon. Deletion or mutation of this site reduced transcriptional activity of promoter-reporter constructs by over 50%. Antibodies against COUP-TF beta/ARP-1 and RXR transcription factors altered the pattern seen on electrophoretic mobility shift assays using double-stranded oligonucleotide containing the exon 1 protein binding site. This suggests that the factors that bind this site and modulate transcription of the PPE gene include members of the COUP-TF and retinoid X receptor families.

Activity of the Human Aldehyde Dehydrogenase 2 Promoter Is Influenced by the Balance between Activation by Hepatocyte Nuclear Factor 4 and Repression by Perosixome Proliferator Activated Receptor δ, Chicken Ovalbumin Upstream Promoter-Transcription Factor, and Apolipoprotein Regulatory Protein-1

We have had a long-standing interest in the regulation of expression of mitochondrial aldehyde dehydrogenase (ALDH2). This enzyme has the lowest Km for acetaldehyde among many known aldehyde dehydrogenases, and a syndrome of genetic deficiency in ALD(Crabb et al., 1989) is known to markedly impair oxidation of acetaldehyde after consumption of ethanol (Enomoto et al., 1991). This impairment of acetaldehyde disposition is the cause of the Asian alcohol flush reaction and is very strongly protective against the development of alcoholism (Goedde et al., 1983; Harada et al., 1982; Thomas- son et., 1991). We reason that factors that alter the expression of the enzyme might also influence risk of alcoholism and have therefore studied the control of the ALDH2 gene, with particular attention to its expression in liver.

Dexamethasone represses phorbol ester-, forskolin-, and calcium-stimulated expression of a preproenkephalin A promoter-chloramphenicol acetyltransferase gene via a receptor-mediated mechanism

CV-1 cells were stably transfected with a preproenkephalin A (PPE) promoter-chloramphenicol acetyltransferase (CAT) reporter plasmid containing -176 to +171 bp of the human PPE gene. Low levels of CAT were expressed constitutively. The reporter enzyme activity was induced by treatment of the cells for 6 h with drugs that increased intracellular cAMP (forskolin and 8-bromo-cAMP), intracellular calcium (A23187), or protein kinase C activity (tetradecanoyl phorbol-4-acetate, TPA) in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine. Co-administration of dexamethasone reduced the magnitude of phorbol ester-stimulated CAT activity by about 50%, while there were smaller but not significant effects on forskolin- or A23187-stimulated expression of this reporter construct. In transient transfections which included the PPE-CAT reporter gene and a glucocorticoid receptor expression plasmid, dexamethasone significantly reduced stimulated expression of the reporter by TPA, forskolin, and A23187. The effect was observed with 10(-8)-10(-6) M dexamethasone and was blocked by the presence of the glucocorticoid antagonist RU486, suggesting that the effect of dexamethasone was mediated by the glucocorticoid receptor. The promoter region contained in this construct lacks a classical glucocorticoid response element or known negative elements; thus, dexamethasone may reduce stimulated expression of the PPE promoter via indirect effects.

Tissue-specific expression and preliminary functional analysis of the 5' flanking regions of the human mitochondrial aldehyde dehydrogenase (ALDH2) gene.

Mitochondrial aldehyde dehydrogenase (ALDH2) is a member of a large group of enzymes that catalyze the irreversible oxidation of aldehydes to carboxylic acids. The ALDH2 enzyme has a very low Km (<1 μM) for short aliphatic aldehydes such as acetaldehyde and propionaldehyde. In human beings, this isozyme appears to play a major role in the removal of acetaldehyde generated from the oxidation of ethanol. This conclusion is based on the study of individuals with a genetically determined deficiency in ALDH2 activity. Liver or hair root extracts from these individuals show the absence of the ALDH2 activity band on starch gels or isoelectric focusing gels (also known in the literature as the ALDH I or E2 band) (Harada et al., 1980). The deficiency results from a point mutation that substitutes a lysine for glutamate at position 487 (Yoshida et al., 1984; Crabb et al., 1989). Individuals with the deficient phenotype experience alcohol-induced flushing that is secondary to high levels of circulating acetaldehyde (Harada et al., 1981; Enomoto et al., 1991b). This is commonly referred to as the Oriental alcohol flush reaction. Assays of liver extracts from individuals carrying a deficient allele have about half the aldehyde dehydrogenase activity of extracts from subjects without the deficiency (S.-J. Yin, personal communication). Thus, ALDH2 activity makes a major contribution to the total aldehyde oxidizing capacity of the liver.