Shan Y., Lambrecht R.W., Bonkovsky H.L.
University of Connecticut Health Science Center, Farmington, CT, USA
Heme oxygenase (HO) catalyzes the conversion of heme to biliverdin with the release of iron and carbon monoxide. HO-1, the first isoform of HO to be identified, is highly inducible by a large number of physical and chemical factors. Many of these factors cause oxidative or other stresses to cells. In this work, we studied the regulation of the chick HO-1 gene by metalloporphyrins, using selected promoter-reporter constructs of the gene transiently or stably transfected into the chicken hepatoma cells (LMH) line. We identified and characterized the key regulatory elements in the 5’-flanking of the chick HO-1 gene which confers up-regulation of luciferase reporter gene expression in the presence of heme and cobalt protoporphyrin (CoPP). LMH cells were maintained in with 10% fetal bovine serum Waymouth's medium. Transfections of the constructs were carried out using Lipofectamine. Reporter gene expression and activation were assessed by quantitation of luciferase activity, normalized to b-galactosidase activity and protein content. To establish stable transfections, the transfected cells were incubated with 200 µg/ml geneticin beginning the day after transfection, and resistant colonies were selected over a 2-3-week period. The largest reporter plasmid, called pcHO7.1Luc, was constructed by cloning 7.1 Kb of the chick HO-1 proximal promoter into the pGL3 vector. The smaller plasmids, pcHO7.1-4.6Luc and pcHO4.6Luc, were constructed by deletion from pcHO7.1Luc. The plasmids, pcHO7.1-5.6Luc and pcHO5.6-4.6, were constructed by further deletion from pcHO7.1-4.6Luc. Site-directed mutagenesis of heme responsive elements (HeREs) was carried out using pcHO7.1-4.6Luc as template. The HeREs, located -4600, and -4676 from the transcription start site, were mutated from 5'- tgcTGTGTCA to 5'- gagTGTGTCA. The mutants were confirmed by DNA sequencing. Deletional analysis of chick HO-1 promoter/enhancer induction by heme and CoPP in transiently transfected LMH cells showed that heme and CoPP significantly up-regulated activity in pcHO7.1Luc (4.2, 5-fold), pcHO7.1-4.6Luc (3.1. 3.5-fold), and pcHO5.6-4.6Luc (4.1, 4.2-fold), but not pcHO7.1-5.6Luc (1.6, 0.6-fold), (Fig 1). These results suggested that the key regulatory elements of heme- and CoPP-mediated inductions are located 5.6 to 4.6 Kb upstream from transcription starting point of the chick HO-1 gene. Within this region, we identified two key “expanded” AP-1 sites at 4600 bp and 4676 bp upstream of the transcription starting point. The maximal induction responded particularly to heme and CoPP were 10 mM concentrations and 16 hours of exposure. Single or double mutations of these sites within pcHO7.1-4.6Luc significantly abrogated the heme-dependent, and the CoPP-dependent, up-regulation of reporter gene expression in transient transfection (Fig 2). Single and double mutations of these sites within pcHO7.1Luc in stable transfection showed the same results (data not shown). In conclusion, the chick HO-1 promoter region contains “expanded” AP-1 sites that are important for up-regulation of the gene by heme or CoPP. These key regulatory elements consist of consensus AP-1 binding sites that have been extended by three bp. [Supported by RO1DK38825 from NIH].