TRANSFORMING GROWTH FACTOR-BETA(1) IS A POTENT INHIBITOR OF GLUTATHIONE SYNTHESIS IN THE LUNG EPITHELIAL-CELL LINE A549 - TRANSCRIPTIONAL EFFECT ON THE GSH RATE-LIMITING ENZYME GAMMA-GLUTAMYLCYSTEINE SYNTHETASE

Glutathione (GSH) is an essential antioxidant tripeptide that protects mammalian cells against oxidants and xenobiotics. Patients with fibro tic lung disorders have very low levels of GSH in their alveolar epith elial lining fluid (ELF), whereas transforming growth factor (TGF)-bet a is overexpressed in their alveolar epithelial cells. We observed tha t TGF-beta(1) increased susceptibility of the human alveolar epithelia l cell line A549 to H2O2-mediated cytotoxicity (P < 0.05), decreased t he activities of the antioxidant enzymes glutathione reductase and cat alase by 31%, and markedly decreased GSH content in A549 cells (P < 0. 01). GSH depletion was associated with an equivalent decrease in the a ctivity of the rate-limiting enzyme in GSH synthesis, gamma-glutamylcy steine synthetase (gamma-GCS) (P < 0.01). Western blot analysis confir med that the loss of gamma-GCS activity was associated with a marked d ecrease in gamma-GCS heavy subunit (gamma-GCShs) protein. TGF-beta(1) suppressed the steady-state level of messenger RNA (mRNA) for the gamm a-GCShs gene, with a maximal effect at 24 h. The half-life of gamma-GC Shs mRNA was not affected by TGF-beta(1), but transcription of the gen e was downregulated as determined with nuclear run-on assays. Our find ings indicate for the first time that TGF-beta(1) is a potent inhibito r of GSH synthesis in human lung epithelial cells, and that the inhibi tion is mediated, at least in part, by a transcriptional effect on the gene encoding gamma-GCShs. Regulation of gamma-GCShs gene expression by TGF-beta(1) is likely to play an important role in lower respirator y tract GSH homeostasis, and may represent a novel target for therapeu tic efforts in lung fibrosis.