ABSTRACT
Synthesis of aminomethyl and bis-aminomethylfluorescein derived energy transfer terminators is described.
Subject(s)
Fluorescein/chemistry , Fluoresceins/chemical synthesis , Sequence Analysis, DNA , Energy Transfer , Fluoresceins/chemistryABSTRACT
The Ets-1 oncoprotein and the heme-catabolizing enzyme heme oxygenase (HO)-1 have been implicated in the pathogenesis of renal disease. We investigated the role of the putative Ets-binding sites (EBSs) in the transactivation of the proximal promoter of rat heme oxygenase 1 (hmox1) gene by the Ets oncoproteins Fli-1, Erg-2, and Ets-1 in mesangial cells. We examined several rat hmox1-chloramphenicol acetytransferase (CAT) constructs and EBS mutant constructs in an effort to assess the effect of ETS oncoproteins on transactivation of the rat hmox1 proximal promoter in renal glomerular mesangial cells. CAT assays demonstrated that the proximal promoter region (-1387 to -40) contains positive and negative regulatory regions and that the EBS-2, 3, and 4 play a role in basal promoter activity. Overexpression of Fli-1 and Erg-2 proteins showed a significant increase in promoter activity, whereas Ets-1 showed no effect on promoter activity. The Fli-1-induced transcriptional activation was not altered by mutation of EBSs, either independently or in combination. However, mutation of EBS-4 independently or a combined mutation of sites 3 and 4 led to a 50% reduction in Erg-2-induced transcriptional activation. Furthermore, mutation of EBS-2 and 4 completely abolished Erg-2-mediated promoter activation. Our results support a role for Ets transcription factors in the regulation of rat hmox-1 gene expression in mesangial cells.
Subject(s)
Gene Expression Regulation, Enzymologic , Glomerular Mesangium/enzymology , Heme Oxygenase (Decyclizing)/genetics , Proto-Oncogene Protein c-ets-1/genetics , Animals , Cells, Cultured , Ether-A-Go-Go Potassium Channels/genetics , Ether-A-Go-Go Potassium Channels/metabolism , Glomerular Mesangium/cytology , Heme Oxygenase (Decyclizing)/metabolism , Mice , Point Mutation , Proto-Oncogene Protein c-ets-1/metabolism , Proto-Oncogene Protein c-fli-1/genetics , Proto-Oncogene Protein c-fli-1/metabolism , Rats , Transcriptional ActivationABSTRACT
BACKGROUND/AIMS: Renal tubules undergo oxidative injury in various nephropathies. It is unknown whether tubular cells possess mechanisms to attenuate this form of injury. Heme oxygenase-1 (HO-1), the rate-limiting enzyme in heme catabolism, may provide such a mechanism by reducing levels of free heme, a prooxidant molecule, and by limiting activity of heme-containing prooxidant enzymes. Determination of the distribution of HO-1 in the nephron may identify those segments where HO-1 can afford protection against oxidative injury. METHODS: Rats were injected subcutaneously with two different inducers of HO-1: Stannous chloride and cobalt protoporphyrin. At completion of injections, frozen sections of kidneys were stained for HO-1 using a biotin-conjugated monoclonal anti-HO-1 antibody. To identify the origin of tubules staining positive for HO-1, Tetragonolobus purpureas (TP)-derived lectin and Arachnis hypogaea (AH)-derived lectin were applied to sequential sections of the kidney cortex. RESULTS: In rats injected with either HO-1 inducer, HO-1 was immunolocalized in tubules but not in glomeruli. Staining of sequential sections with TP-derived lectin, which binds mainly to proximal tubular cells, was negative in the tubules that stained positive for HO-1. Staining of sequential sections with AH-derived lectin, which binds mainly to distal and collecting tubular cells, was positive in those tubules that were also positive for HO-1. CONCLUSIONS: In kidneys of rats injected with inducers of HO-1, distal and collecting tubular cells were identified as the main segments of the nephron that express HO-1. We suggest that the distal nephron, by expressing HO-1, may be less vulnerable to oxidative injury.
