Characterization of the osmotic response element of the human aldose reductase gene promoter.

Abstract

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 ofthe 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 "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.Aldose reductase (EC 1.1.1.21) catalyzes the NADPHmediated reduction of a variety of carbonyl compounds to their respective alcohols with a wide range of catalytic efficiencies (1). The conversion of D-glucose to sorbitol constitutes the first step of the polyol pathway, an accessory pathway of glucose metabolism that converts glucose to fructose. The catalytic efficiency of aldose reductase for D-glucose (1) is relatively low and is reflected by a Km(Dglucose) of 100 mM, a substrate concentration that is 20-fold higher than prevailing normal glycemic...

Identification and Characterization of Multiple Osmotic Response Sequences in the Human Aldose Reductase Gene

“…The sequence from positions Ϫ650 to ϩ617 is identical to the available published sequence with the exception of 8 bp (30), indicating that the 2.8-kb fragment contains the region upstream of the AR gene, and it is unlikely that it is part of an AR pseudogene. A discrepancy became apparent between this region and a corresponding region of a putative aldose reductase gene promoter containing a functional ORE (clone 6A3a) located ϳ3.7 kb upstream of the transcription start site (21). The sequence of this 5Ј-flanking upstream region was found to be entirely different from that of AR5 upstream of position Ϫ1066 (an MboI site), whereas the sequences downstream of that position are identical in the two clones.…”

“…In addition, while we were preparing this report, the 11-bp osmotic response element (ORE) essential for osmoregulation of the rabbit AR gene was reported and found to be located 1105 bp upstream of the transcription start site (20). Recently, we identified a putative functional ORE found to be located 3.7 kb upstream of the transcription start site of the aldose reductase gene (21). We hereby show that this putative AR ORE resulted from a cloning artifact occurring at 1066 bp (MboI restriction site) upstream of the initiation transcription site in a human aldose reductase gene isolated from a commercially obtained genomic library constructed by partial digestion with MboI restriction enzyme.…”

Fat-specific protein 27 modulates nuclear factor of activated T cells 5 and the cellular response to stress

“…In addition, the DNA binding activity of NFAT5 is increased in diabetics with microvascular complications ( 41 ). Moreover, aldose reductase, a key enzyme in the polyol pathway that has been mechanistically linked with hyperglycemiainduced complications ( 42 ), is a target gene of NFAT5 ( 43 ). In addition to TNF-␣ and MCP1, which are direct NFAT5 target genes ( 16,44 ), IL-1, IL-6, and IL-18 contain putative NFAT5 consensus sites in their promoter regions and can be regulated by NFAT5 under hypertonic conditions ( 20 ).…”

Aldose reductase functions as a detoxification system for lipid peroxidation products in vasculitis

“…Its pattern of tissue expression with preferential production in the renal medulla has suggested a contribution in the regulation of tissue osmolarity (24), and an osmotic response element has been identified in the promoter region of AR (25,26). The enzyme has also been detected in the Schwann cell sheath of peripheral nerves, the lens, the retina, and in arterial endothelium (14,17,19,20,27,28).…”