Antioxidant treatment induces transcription and expression of transforming growth factor β in cultured renal proximal tubular cells

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Abstract
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.

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