Transforming Growth Factor- β₁ Is a Potent Inhibitor of Glutathione Synthesis in the Lung Epithelial Cell Line A549: Transcriptional Effect on the GSH Rate-limiting Enzyme γ -Glutamylcysteine Synthetase

Abstract

Glutathione (GSH) is an essential antioxidant tripeptide that protects mammalian cells against oxidants and xenobiotics. Patients with fibrotic lung disorders have very low levels of GSH in their alveolar epithelial lining fluid (ELF), whereas transforming growth factor (TGF)-beta is overexpressed in their alveolar epithelial cells. We observed that TGF-beta1 increased susceptibility of the human alveolar epithelial cell line A549 to H2O2-mediated cytotoxicity (P < 0.05), decreased the activities of the antioxidant enzymes glutathione reductase and catalase by 31%, and markedly decreased GSH content in A549 cells (P < 0.01). GSH depletion was associated with an equivalent decrease in the activity of the rate-limiting enzyme in GSH synthesis, gamma-glutamylcysteine synthetase (gamma-GCS) (P < 0.01). Western blot analysis confirmed that the loss of gamma-GCS activity was associated with a marked decrease in gamma-GCS heavy subunit (gamma-GCShs) protein. TGF-beta1 suppressed the steady-state level of messenger RNA (mRNA) for the gamma-GCShs gene, with a maximal effect at 24 h. The half-life of gamma-GCShs mRNA was not affected by TGF-beta1, but transcription of the gene was downregulated as determined with nuclear run-on assays. Our findings indicate for the first time that TGF-beta1 is a potent inhibitor of GSH synthesis in human lung epithelial cells, and that the inhibition is mediated, at least in part, by a transcriptional effect on the gene encoding gamma-GCShs. Regulation of gamma-GCShs gene expression by TGF-beta1 is likely to play an important role in lower respiratory tract GSH homeostasis, and may represent a novel target for therapeutic efforts in lung fibrosis.

Endocrine disruptors that deplete glutathione levels in APC promote Th2 polarization in mice leading to the exacerbation of airway inflammation

“…While it has been reported that reduced levels of GSH in the epithelial lining fluid were found in asthmatic lungs compared with those in normal subjects [37], results on GSH levels in APC in asthmatic patients are not available to date. However, it is possible that the GSH levels in APC in the patients with asthma are decreased, since TGF-b1, which acts as a potent inhibitor of GSH synthesis [38], has been shown to be systemically elevated in asthmatic patients [39].…”

Transforming growth factor-β1 is a potent inhibitor of secretory leukoprotease inhibitor expression in a bronchial epithelial cell line

“…TGF-b 1 , however, may not only interfere with the defence mechanisms of the epithelium of the lung by reducing SLPI levels. In a recent study ARSALANE et al [38] demonstrated that TGF-b 1 is able to inhibit glutathione synthesis in the human alveolar epithelial cell line A549. Glutathione is the most important extracellular antioxidant of the human lung [39,40].…”

Oxidative stress and regulation of glutathione in lung inflammation

“…Recent studies have shown increased expression of TGF-b1 in bronchiolar and alveolar epithelium in IPF and COPD patients, and higher levels in the BALF of atopic asthmatics as compared to healthy subjects [154,155]. TGF-b1 also downregulates c-GCS-HS mRNA and glutathione synthesis in human alveolar epithelial cells and pulmonary artery endothelial cells in vitro [109,110]. Interestingly, recent studies by FACTOR et al [156] showed decreased glutathione synthesis in a TGF transgenic (overexpression) mouse model and increased susceptibility to oxidant-mediated injury.…”