Glutathione (GSH) is a ubiquitous
antioxidant in lung epithelial cells and lung lining fluid.
Transforming growth factor beta1 (TGF-beta1) is a pleiotropic
cytokine involved in cellular proliferation and differentiation. The level of
TGF-beta1 is elevated in many chronic inflammatory lung disorders associated with
oxidant/
antioxidant imbalance. In this study, we show that
TGF-beta1 depletes GSH by down-regulating expression of the
enzyme responsible for its formation,
gamma-glutamylcysteine synthetase (gamma-GCS) and induces
reactive oxygen species production in type II alveolar epithelial cells (A549). To investigate the molecular mechanisms of inhibition of
glutathione synthesis, we employed reporters containing fragments from the promoter region of the gamma-GCS heavy subunit (h), the gene that encodes the catalytic subunit of gamma-GCS. We found that
TGF-beta1 reduced the expression of the long gamma-GCSh construct (-3802/GCSh-5'-Luc), suggesting that an antioxidant response element (ARE) may be responsible for mediating the
TGF-beta1 effect. Interestingly, the electrophoretic mobility shift assay revealed that the
DNA binding activity of both
activator protein-1 (AP-1) and ARE was increased in TGF-beta1-treated epithelial cells. The gamma-GCSh ARE contains a perfect
AP-1 site embedded within it, and mutation of this internal
AP-1 sequence, but not the surrounding ARE, prevented
DNA binding. Further studies revealed that c-Jun and Fra-1 dimers, members of the
AP-1 family previously shown to exert a negative effect on phase II gene expression, bound to the ARE sequence. We propose a novel mechanism of gamma-GCSh down-regulation by
TGF-beta1 that involves the binding of c-Jun and Fra-1 dimers to the distal promoter. The findings of this study provide important information, which may be used for the modulation of
glutathione biosynthesis in
inflammation.