Takeshi Iwata

Osmotic Response Element Is Required for the Induction of Aldose Reductase by Tumor Necrosis Factor-α

Induction of aldose reductase (AR) was observed in human cells treated with tumor necrosis factor-α (TNF-α). AR protein expression increased severalfold in human liver cells after 1 day of exposure to 100 units/ml TNF-α. An increase in AR transcripts was also observed in human liver cells after 3 h of TNF-α treatment, reaching a maximum level of 11-fold at 48 h. Among the three inflammatory cytokines: TNF-α, interleukin-1, and interferon-γ, TNF-α (100 units/ml) gave the most induction of AR. Differences in the pattern of AR induction were observed in human liver, lens, and retinal pigment epithelial cells with increasing concentrations of TNF-α. A similar pattern of AR promoter response was observed between TNF-α and osmotically stressed human liver cells. The deletion of the osmotic response element (ORE) abolished the induction by TNF-α and osmotic stress. A point mutation that converts ORE to a nuclear factor-κB (NF-κB) sequence abolished the osmotic response but maintained the TNF-α response. Electrophoretic gel mobility shift assays showed two NF-κB proteins, p50 and p52, capable of binding ORE sequence, and gel shift Western assay detected NF-κB proteins p50 and p65 in the ORE complex. Inhibitors of NF-κB signaling, lactacystin, and MG132 abolished the AR promoter response to TNF-α.

Structural organization of the human sorbitol dehydrogenase gene (SORD).

The primary structure of human sorbitol dehydrogenase (SORD) was determined by cDNA and genomic cloning. The nucleotide sequence of the mRNA covers 2471 bp including an open reading frame that yields a protein of 356 amino acid residues. The gene structure of SORD spans approximately 30 kb divided into 9 exons and 8 introns. The gene was localized to chromosome 15q21.1 by in situ hybridization. Two transcription initiation sites were detected. Three Sp1 sites and a repetitive sequence (CAAA)5 were observed in the 5 noncoding region; no classical TATAA or CCAAT elements were found. The related alcohol dehydrogenases and zeta-crystallin have the same gene organization split by 8 introns, but no splice points coincide between SORD and these gene types. The deduced amino acid sequence of the SORD structure differs at a few positions from the directly determined protein sequence, suggesting allelic forms of the enzyme. High levels of SORD transcripts were observed in lens and kidney, as judged from Northern blot analysis.

Structural organization and chromosomal localization of the human ribosomal protein L9 gene

The intron-containing gene for the human ribosomal protein L9 has been cloned, sequenced and localized. The gene is approximately 5.5 kb in length and contains 8 exons. Splice sites follow the AG/GT consensus rule. The message for human rpL9 is 712 nt in length and is detected in all tissues examined. In the adult, expression is highest in retina and liver while brain shows highest expression among the fetal tissues tested. The transcription start site contains an oligopyrimidine tract, TTCTTTCTT, similar to those found in other ribosomal protein genes. As in other previously characterized ribosomal protein genes, a TATA box is absent from the 5 flanking region but a number of elements recognized by common transcription factors are present including Sp1 sites, CACCC boxes, inverted CCAAT boxes, and GATA elements. Another possible element of interest in the rpL9 5 flanking region is RFX1 also found in the well characterized rat rpL30 promoter. The gene was mapped by fluorescent in situ hybridization to band 13p of chromosome 4. At least 8 possible pseudogenes are present in the human genome, one of which is on Xp. As assessed by Southern Zoo-blot analysis and direct cDNA sequence comparison, the human ribosomal protein L9 gene, like other ribosomal protein genes, is highly conserved among mammals.

Identification of a Novel cis-Element Required for the Constitutive Activity and Osmotic Response of the Rat Aldose Reductase Promoter

A new and essential cis-element AEE (aldose reductase enhancerelement), necessary for the constitutive activity and the osmotic stress response of rat aldose reductase transcription in a rat liver cell line, has been identified. In transient transfection assays, an increase in promoter activity, up to 3.8-fold, was observed with osmotic stress (600 mosm/kg H2O) using a luciferase reporter gene construct containing aldose reductase promoter sequence from −1,094 base pair (bp) to +23 bp. A deletion between −1,071 and −895 bp reduced the constitutive activity and abolished the osmotic response of the promoter. Exonuclease III mediated in vivoDNA footprinting and dimethyl sulfate in vivo footprinting revealed DNA protection of a 32-bp region and two guanosines (G) within this region protected from methylation, respectively. Electrophoretic gel mobility shift assays using whole liver cell extracts showed protein binding, under both normal and stressed conditions. Deletion of the sequence between the two guanosines protected by in vivo dimethyl sulfate DNA footprinting (GAAGAGTG) in a luciferase construct (−1,094 bp to +23 bp) abolished the constitutive promoter activity. One copy of AEE fused to the thymidine kinase promoter gave a maximum constitutive activity of 7.7-fold and a maximum osmotic response activity of 6.7-fold.

Expression, purification and preliminary crystallographic analysis of human sorbitol dehydrogenase.

Human sorbitol dehydrogenase (SDH) was expressed in Escherichia coli BL21 cells and purified using ammonium sulfate precipitation and anion-exchange and dye-affinity chromatography. Purified SDH was crystallized from polyethylene glycol solutions using the hanging-drop vapour-diffusion method. X-ray data were collected to 2.75 A resolution. The crystals belong to the monoclinic C2 space group, with unit-cell parameters a = 145.9, b = 52.3, c = 169.0 A, beta = 101.8 degrees. This is the first crystallization report of human sorbitol dehydrogenase.

Discovery of Potential Sorbitol Dehydrogenase Inhibitors from Virtual Screening

Sorbitol dehydrogenase (SDH) is the second enzyme in the polyol pathway of glucose metabolism and is a possible target for the treatment of the complications of diabetes. In this study the molecular modelling program DOCK was used to analyse 249,071 compounds from the National Cancer Institute Database and predict those with high affinity for SDH. From a total of 21 tested the 7 compounds including flavin adenine dinucleotide disodium hydrate, (+)-Amethopterin, 3-hydroxy-2-napthoic(2-hydroxybenzylidene) hydrazide, folic acid, N-2,4-dinitrophenyl-L-cysteic acid, Vanillin azine and 1H-indole-2,3-dione,5-bromo-6-nitro-1-(2,3,4-tri-O-acetyl-alpha-L-arabinopyranosyl)-(9Cl), were shown to inhibit SDH and displayed IC50 values of 0.192 microM, 1.1 microM, 1.2 microM, 4.5 microM, 5.3 microM, 7 microM and 28 microM, respectively. These compounds may aid the design of pharmaceutical agents for the treatment of diabetes complications.

GENE REGULATION OF ALDOSE REDUCTASE UNDER OSMOTIC STRESS

Aldose reductase (AR) catalyzes the reduction of various sugars to their respective sugar alcohols, including glucose to sorbitol. The accumulation of sorbitol from excess glucose in diabetic patients is believed to be responsible for initiating diabetic complications, such as retinopathy and cataract (van Heyningen, 1959; Kinoshita, 1974). Inhibition of AR by AR inhibitors in diabetic animal models has been shown to reduce the accumulation of sorbitol and prevent or retard these diabetic complications.