CHARACTERIZATION OF THE OSMOTIC RESPONSE ELEMENT OF THE HUMAN ALDOSE REDUCTASE GENE PROMOTER

Aldose reductase (EC 1.1.1.21) catalyzes the NADPH-mediated conversion of glucose to sorbitol. The hyperglycemia of diabetes increases sorbi tol production primarily through substrate availability and is thought to contribute to the pathogenesis of many diabetic complications. Inc reased sorbitol production can also occur at normoglycemic levels via rapid increases in aldose reductase transcription and expression, whic h have been shown to occur upon exposure of many cell types to hyperos motic 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 tu bular cells adjust to the intraluminal hyperosmolality during urinary concentration. Previously, to explore the mechanism regulating aldose reductase levels, we partially characterized the human aldose reductas e gene promoter present in a 4.2-kb fragment upstream of the transcrip tion initiation start site. A fragment (-192 to +31 bp) mas shown to c ontain several elements that control the basal expression of the enzym e. In this study, we examined the entire 9.2-kb human AR gene promoter fragment by deletion mutagenesis and transfection studies for the pre sence of osmotic response enhancer elements. An 11-bp nucleotide seque nce (TGGAAAATTAC) was located 3.7 kb upstream of the transcription ini tiation site that mediates hypertonicity-responsive enhancer activity. This osmotic response element (ORE) increased the expression of the c hloramphenicol acetyltransferase reporter gene product 2-fold in trans fected HepG2 cells exposed to hypertonic NaCl media as compared with i soosmotic 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 co mpetition studies confirm the nature of the element and identify speci fic nucleotides essential for enhancer activity. A plasmid construct c ontaining three repeat OREs and a heterologous promoter increased expr ession 8-fold in isoosmotic media and an additional 4-fold when the tr ansfected cells are subjected to hyperosmotic stress (total approximat e to 30-fold). These findings will permit future studies to identify t he transcription factors involved in the normal regulatory response me chanism to hypertonicity and to identify whether and how this response is altered in a variety of pathologic states, including diabetes.