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Cloning, genomic organization, and osmotic response of the aldose reductase gene.

ABSTRACT: Diverse organisms accumulate organic osmolytes to adapt to hyperosmotic stress. The molecular basis of eukaryotic gene osmoregulation remains obscure. Aldose reductase [AR; alditol:NAD(P)+ 1-oxidoreductase, EC 1.1.1.21], which catalyzes the conversion of glucose to sorbitol (an organic osmolyte), is induced in renal medullary cells under hyperosmotic conditions. Elevated extracellular NaCl increases AR mRNA transcription in PAP-HT25 cells, a cell line derived from the rabbit renal papilla. We have cloned and characterized the rabbit AR gene to determine how it is regulated by hyperosmolality. The length of the gene, not including 5' or 3' flanking regions, is approximately 14.7 kilobases (kb) organized into 10 exons and 9 introns. The transcription start site is 36 base pairs upstream of the initiator methionine codon. A 5-kb fragment containing approximately 3.5 kb of 5' flanking region was isolated. The 3.5-kb sequence was examined for basal promoter activity and hyperosmotic response in luciferase reporter gene constructs. A 235-base-pair fragment (base pairs -208 to +27) was able to drive the downstream reporter gene in transfected PAP-HT25 cells under isoosmotic conditions (300 mosmol/kg of H2O). When this fragment plus the remaining upstream sequence (from approximately base pair -3429 to base pair +27) was used, cells in hyperosmotic medium (500 mosmol/kg of H2O) showed about 40-fold induction of luciferase expression compared with cells in isoosmotic medium. The upstream fragment (from approximately base pair -3429 to base pair -192) also conferred osmotic response to a heterologous promoter (B19). This finding evidences putative osmotic response element(s) (OREs) within a specific DNA fragment in a eukaryotic genome. Identification and characterization of OREs within this fragment and their associated trans-acting factors should reveal the molecular mechanisms of gene regulation in osmotic stress.

SUBMITTER: Ferraris JD

PROVIDER: S-EPMC45098 | biostudies-other | 1994 Oct

REPOSITORIES: biostudies-other

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Cloning, genomic organization, and osmotic response of the aldose reductase gene.

Ferraris J D JD Williams C K CK Martin B M BM Burg M B MB García-Pérez A A

Proceedings of the National Academy of Sciences of the United States of America 19941001 22

Diverse organisms accumulate organic osmolytes to adapt to hyperosmotic stress. The molecular basis of eukaryotic gene osmoregulation remains obscure. Aldose reductase [AR; alditol:NAD(P)+ 1-oxidoreductase, EC 1.1.1.21], which catalyzes the conversion of glucose to sorbitol (an organic osmolyte), is induced in renal medullary cells under hyperosmotic conditions. Elevated extracellular NaCl increases AR mRNA transcription in PAP-HT25 cells, a cell line derived from the rabbit renal papilla. We ha ...[more]

PMID: 7938022

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Project description:Aldose reductase (EC 1.1.1.21) catalyzes the NADPH-mediated conversion of glucose to sorbitol. The hyperglycemia of diabetes increases sorbitol production primarily through substrate availability and is thought to contribute to the pathogenesis of many diabetic complications. Increased sorbitol production can also occur at normoglycemic levels via rapid increases in aldose reductase transcription and expression, which have been shown to occur upon exposure of many cell types to hyperosmotic conditions. The induction of aldose reductase transcription and the accumulation of sorbitol, an organic osmolyte, have been shown to be part of the physiological osmoregulatory mechanism whereby renal tubular cells adjust to the intraluminal hyperosmolality during urinary concentration. Previously, to explore the mechanism regulating aldose reductase levels, we partially characterized the human aldose reductase gene promoter present in a 4.2-kb fragment upstream of the transcription initiation start site. A fragment (-192 to +31 bp) was shown to contain several elements that control the basal expression of the enzyme. In this study, we examined the entire 4.2-kb human AR gene promoter fragment by deletion mutagenesis and transfection studies for the presence of osmotic response enhancer elements. An 11-bp nucleotide sequence (TGGAAAATTAC) was located 3.7 kb upstream of the transcription initiation site that mediates hypertonicity-responsive enhancer activity. This osmotic response element (ORE) increased the expression of the chloramphenicol acetyltransferase reporter gene product 2-fold in transfected HepG2 cells exposed to hypertonic NaCl media as compared with isoosmotic media. A more distal homologous sequence is also described; however, this sequence has no osmotic enhancer activity in transfected cells. Specific ORE mutant constructs, gel shift, and DNA fragment competition studies confirm the nature of the element and identify specific nucleotides essential for enhancer activity. A plasmid construct containing three repeat OREs and a heterologous promoter increased expression 8-fold in isoosmotic media and an additional 4-fold when the transfected cells are subjected to hyperosmotic stress (total approximately 30-fold). These findings will permit future studies to identify the transcription factors involved in the normal regulatory response mechanism to hypertonicity and to identify whether and how this response is altered in a variety of pathologic states, including diabetes.

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Cloning, genomic organization, and osmotic response of the aldose reductase gene.

Publications

Cloning, genomic organization, and osmotic response of the aldose reductase gene.

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