An Autoregulatory Loop Controlling CYP1A1 Gene Expression: Role of H₂O₂and NFI

Abstract

Cytochrome P450 1A1 (CYP1A1), like many monooxygenases, can produce reactive oxygen species during its catalytic cycle. Apart from the well-characterized xenobiotic-elicited induction, the regulatory mechanisms involved in the control of the steady-state activity of CYP1A1 have not been elucidated. We show here that reactive oxygen species generated from the activity of CYP1A1 limit the levels of induced CYP1A1 mRNAs. The mechanism involves the repression of the CYP1A1 gene promoter activity in a negative-feedback autoregulatory loop. Indeed, increasing the CYP1A1 activity by transfecting CYP1A1 expression vectors into hepatoma cells elicited an oxidative stress and led to the repression of a reporter gene driven by the CYP1A1 gene promoter. This negative autoregulation is abolished by ellipticine (an inhibitor of CYP1A1) and by catalase (which catalyzes H₂O₂ catabolism), thus implying that H₂O₂ is an intermediate. Down-regulation is also abolished by the mutation of the proximal nuclear factor I (NFI) site in the promoter. The transactivating domain of NFI/CTF was found to act in synergy with the arylhydrocarbon receptor pathway during the induction of CYP1A1 by 2,3,7,8-tetrachloro-p-dibenzodioxin. Using an NFI/CTF-Gal4 fusion, we show that NFI/CTF transactivating function is decreased by a high activity of CYP1A1. This regulation is also abolished by catalase or ellipticine. Consistently, the transactivating function of NFI/CTF is repressed in cells treated with H₂O₂, a novel finding indicating that the transactivating domain of a transcription factor can be targeted by oxidative stress. In conclusion, an autoregulatory loop leads to the fine tuning of the CYP1A1 gene expression through the down-regulation of NFI activity by CYP1A1-based H₂O₂ production. This mechanism allows a limitation of the potentially toxic CYP1A1 activity within the cell.

ACKNOWLEDGMENTS

This work was supported by INSERM, Université Paris V-René Descartes, Fondation pour la Recherche Médicale (grant 1000031401), and Région Ile-de-France. N.M. is supported by grants from the Swiss NSF and the Etat de Vaud.

We especially thank M. Kobr for his kind and useful help with Gal fusions experiments. We thank P. Beaune and M. Aggerbeck for critical reading of the manuscript and I. de Waziers and P. Maurel for the generous gifts of CYP1A1 cDNA and gene promoter, respectively.