Role of Bach-1 in regulation of heme oxygenase-1 in human liver cells: insights from studies with small interfering RNAS.
Heme oxygenase-1 is an antioxidant defense enzyme that converts heme to biliverdin, iron, and carbon monoxide. Bach-1 is a bZip protein that forms heterodimers with small Maf proteins and was reported recently to down-regulate the HO-1 gene in mice. Using small interfering RNAs targeted to human Bach-1 mRNA, we investigated whether modulation of human hepatic Bach-1 expression by small interfering (si)RNA technology influences heme oxygenase-1 gene expression. We found that Bach-1 siRNAs transfected into Huh-7 cells significantly reduced Bach-1 mRNA and protein levels approximately 80%, compared with non siRNA-treated cells. In contrast, transfection with the same amounts of nonspecific control duplexes or LaminB2-duplex did not reduce Bach-1 mRNA or protein levels, confirming the specificity of Bach-1 siRNA. Expression of the heme oxygenase-1 gene in Bach-1 siRNA-transfected cells was up-regulated 7-fold, compared with cells without Bach-1 siRNA. The effect of increasing concentrations of heme to up-regulate levels of heme oxygenase-1 was more pronounced when Bach-1 siRNA was present. Taken together, these results indicated that Bach-1 has a specific and selective ability to repress expression of human hepatic heme oxygenase-1. Silencing of Bach-1 by siRNAs is a useful method for up-regulating HO-1 gene expression. Exogenous heme produces additional up-regulation, beyond that produced by Bach-1 siRNAs, suggesting that heme does not act solely through its effects on Bach-1.
Department of Medicine and Pharmacology, the General Clinical Research Center of the University of Connecticut Health Center, Farmington, Connecticut 06030, USA. Shan@uchc.edu
Ying
Y
Shan
Richard W
RW
Lambrecht
Tahereh
T
Ghaziani
Susan E
SE
Donohue
Herbert L
HL
Bonkovsky
D000818:Animals; D001483:Base Sequence; D050976:Basic-Leucine Zipper Transcription Factors; D002460:Cell Line; D004247:DNA / Q000235:genetics; D051856:Fanconi Anemia Complementation Group Proteins; D015971:Gene Expression Regulation, Enzymologic; D020868:Gene Silencing; D006418:Heme / Q000235:genetics / Q000378:metabolism; D006419:Heme Oxygenase (Decyclizing) / Q000235:genetics / Q000378:metabolism*; D051547:Heme Oxygenase-1; D022781:Hepatocytes / Q000378:metabolism*; D006801:Humans; D016350:Leucine Zippers / Q000235:genetics; D008565:Membrane Proteins; D051379:Mice; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
C102108:BACH1 protein, human; D050976:Basic-Leucine Zipper Transcription Factors; D051856:Fanconi Anemia Complementation Group Proteins; D008565:Membrane Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D014157:Transcription Factors; D006418:Heme; D004247:DNA; C496541:HMOX1 protein, human; D006419:Heme Oxygenase (Decyclizing); D051547:Heme Oxygenase-1; C496542:Hmox1 protein, mouse