Antioxidant treatment induces transcription and expression of transforming growth factor beta in cultured renal proximal tubular cells.
Transforming growth factor beta (TGF-beta) plays an important role in the development of tubulointerstitial fibrosis in chronic renal disease. We were interested whether interference with oxygen radicals may modulate TGF-beta expression. Unexpectedly, we discovered that diphenylene iodine (DIP), an inhibitor of NADP(H) oxidase, induces a robust increase in TGF-beta transcript expression in cultured mouse proximal tubular cells (MCT cells). A similar increase was seen with EUK-8, a synthetic salen-manganese complex with high oxyradical scavenger activities. This induction of TGF-beta1 mRNA was paralleled by increasing protein expression. Transient transfection of MCT cells with a reporter construct in which murine TGF-beta1 enhancer/promoter elements were cloned in front of the luciferase gene, revealed that DIP, EUK-8, and Tiron all stimulated transcription of the TGF-beta1 gene whereas exogenous H2O2 suppressed transcription. Antisense oligonucleotides against p22phox, but not sense oligonucleotides, also increased transcriptional activity of TGF-beta1. Mutagenesis of Sp1 binding sites in the mouse TGF-beta1 enhancer/promoter abolished the stimulatory effect of the antioxidants. Gel shift experiments revealed that DIP as well as EUK-8 activated binding of nuclear proteins to Sp1 consensus sequence. Our data provide evidence that TGF-beta1 transcription is negatively regulated in MCT cells under basal conditions by NADP(H) oxidase-mediated oxygen radicals. Thus, antioxidant therapy may increase local synthesis of TGF-beta1 in the tubulointerstitium.
Wolf
G
G
Department of Medicine, University Hospital Eppendorf, University of Hamburg, Germany. wolf@uke.uni-hamburg.de
Hannken
T
T
Schroeder
R
R
Zahner
G
G
Ziyadeh
F N
FN
Stahl
R A
RA
D014013:1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium Salt / Q000494:pharmacology; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology*; D015153:Blotting, Western; D002460:Cell Line; D016384:Consensus Sequence / Q000235:genetics; D004247:DNA / Q000235:genetics / Q000378:metabolism; D005029:Ethylenediamines / Q000494:pharmacology; D017930:Genes, Reporter / Q000235:genetics; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007455:Iodine / Q000737:chemistry; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D026901:Membrane Transport Proteins; D051379:Mice; D009154:Mutation / Q000235:genetics; D009252:NADPH Dehydrogenase / Q000235:genetics; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000235:genetics; D009942:Organometallic Compounds / Q000494:pharmacology; D010750:Phosphoproteins / Q000235:genetics; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020218:Response Elements / Q000235:genetics; D016329:Sp1 Transcription Factor / Q000378:metabolism; D015533:Transcriptional Activation / Q000187:drug effects*; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000235:genetics* / Q000378:metabolism; D053773:Transforming Growth Factor beta1
D000975:Antioxidants; D005029:Ethylenediamines; D026901:Membrane Transport Proteins; D016376:Oligonucleotides, Antisense; D009942:Organometallic Compounds; D010750:Phosphoproteins; D012333:RNA, Messenger; D016329:Sp1 Transcription Factor; C508960:TGFB1 protein, human; C508961:Tgfb1 protein, mouse; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D014013:1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium Salt; C090577:N,N'-bis(salicylideneamino)ethane-manganese(II); D004247:DNA; D007455:Iodine; D006861:Hydrogen Peroxide; D019255:NADPH Oxidases; C086986:CYBA protein, human; D009252:NADPH Dehydrogenase