Transforming growth factor β (TGF-β) 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-β expression. Unexpectedly, we discovered that diphenylene iodine (DIP), an inhibitor of NADP(H) oxidase, induces a robust increase in TGF-β 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-β1 mRNA was paralleled by increasing protein expression. Transient transfection of MCT cells with a reporter construct in which murine TGF-β1 enhancer/promoter elements were cloned in front of the luciferase gene, revealed that DIP, EUK-8, and Tiron all stimulated transcription of the TGF-β1 gene whereas exogenous H2O2 suppressed transcription. Antisense oligonucleotides against p22phox, but not sense oligonucleotides, also increased transcriptional activity of TGF-β1. Mutagenesis of Sp1 binding sites in the mouse TGF-β1 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-β1 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-β1 in the tubulointerstitium.
