Mitochondrial reactive oxygen species regulate transforming growth factor-ß signaling.

TGF-ß signaling is required for normal tissue repair; however, excessive TGF-ß signaling can lead to robust profibrotic gene expression in fibroblasts, resulting in tissue fibrosis. TGF-ß binds to cell-surface receptors, resulting in the phosphorylation of the Smad family of transcription factors to initiate gene expression. TGF-ß also initiates Smad-independent pathways, which augment gene expression. Here, we report that mitochondrial reactive oxygen species (ROS) generated at complex III are required for TGF-ß-induced gene expression in primary normal human lung fibroblasts. TGF-ß-induced ROS could be detected in both the mitochondrial matrix and cytosol. Mitochondrially targeted antioxidants markedly attenuated TGF-ß-induced gene expression without affecting Smad phosphorylation or nuclear translocation. Genetically disrupting mitochondrial complex III-generated ROS production attenuated TGF-ß-induced profibrotic gene expression. Furthermore, inhibiting mitochondrial ROS generation attenuated NOX4 (NADPH oxidase 4) expression, which is required for TGF-ß induced myofibroblast differentiation. Lung fibroblasts from patients with pulmonary fibrosis generated more mitochondrial ROS than normal human lung fibroblasts, and mitochondrially targeted antioxidants attenuated profibrotic gene expression in both normal and fibrotic lung fibroblasts. Collectively, our results indicate that mitochondrial ROS are essential for normal TGF-ß-mediated gene expression and that targeting mitochondrial ROS might be beneficial in diseases associated with excessive fibrosis.

Proteasomal inhibition after injury prevents fibrosis by modulating TGF-ß(1) signalling.

BACKGROUND: The development of organ fibrosis after injury requires activation of transforming growth factor ß(1) which regulates the transcription of profibrotic genes. The systemic administration of a proteasomal inhibitor has been reported to prevent the development of fibrosis in the liver, kidney and bone marrow. It is hypothesised that proteasomal inhibition would prevent lung and skin fibrosis after injury by inhibiting TGF-ß(1)-mediated transcription. METHODS: Bortezomib, a small molecule proteasome inhibitor in widespread clinical use, was administered to mice beginning 7 days after the intratracheal or intradermal administration of bleomycin and lung and skin fibrosis was measured after 21 or 40 days, respectively. To examine the mechanism of this protection, bortezomib was administered to primary normal lung fibroblasts and primary lung and skin fibroblasts obtained from patients with idiopathic pulmonary fibrosis and scleroderma, respectively. RESULTS: Bortezomib promoted normal repair and prevented lung and skin fibrosis when administered beginning 7 days after the initiation of bleomycin. In primary human lung fibroblasts from normal individuals and patients with idiopathic pulmonary fibrosis and in skin fibroblasts from a patient with scleroderma, bortezomib inhibited TGF-ß(1)-mediated target gene expression by inhibiting transcription induced by activated Smads. An increase in the abundance and activity of the nuclear hormone receptor PPARγ, a repressor of Smad-mediated transcription, contributed to this response. CONCLUSIONS: Proteasomal inhibition prevents lung and skin fibrosis after injury in part by increasing the abundance and activity of PPARγ. Proteasomal inhibition may offer a novel therapeutic alternative in patients with dysregulated tissue repair and fibrosis.

Keratinocyte growth factor expression is suppressed in early acute lung injury/acute respiratory distress syndrome by smad and c-Abl pathways.

OBJECTIVE: Keratinocyte growth factor (KGF) is expressed primarily by fibroblasts, is important for alveolar epithelial proliferation/function, and protects against lung injury in multiple animal models. We wished to determine whether acute lung injury/acute respiratory distress syndrome (ALI/ARDS) alveolar fluid induces KGF and fibroblast genes important for alveolar repair. DESIGN: A single-center cohort study enrolling patients between 2004 and 2006. SETTING: A medical intensive care unit of a tertiary care medical center. PATIENTS: Adult patients meeting the American-European Consensus Conference definition of ALI/ARDS. INTERVENTIONS: Patients with ALI/ARDS were enrolled, and lavage fluid was collected within 48 hours of intubation. Lavage fluid was also collected from two control cohorts. The patients with ALI/ARDS were followed for 28 days or until death. MEASUREMENT AND MAIN RESULTS: Fifteen patients with ALI/ARDS, five patients with cardiogenic edema, and five normal lung parenchyma controls were enrolled from 2004 to 2006. Primary normal human lung fibroblasts were incubated with bronchoalveolar lavage fluid and assessed for KGF, connective tissue growth factor, alpha-smooth muscle actin, and collagen 1 expression by real-time reverse transcriptase-polymerase chain reaction. Fibroblasts incubated with ALI/ARDS lavage fluid expressed 50% less KGF messenger RNA than those incubated with lavage fluid from CE patients (p < 0.01) and 33% than normal parenchymal controls (p < 0.03). Lavage fluid from patients with ALI/ARDS induced more connective tissue growth factor (p < 0.05), collagen 1 (p < 0.03), and alpha-smooth muscle actin (p < 0.04) than from CE patients. Preincubation of normal human lung fibroblasts with the transforming growth factor (TGF)-beta1 receptor/smad phosphorylation inhibitor SB431542 increased ALI/ARDS-induced KGF expression by 40% (p < 0.04). In cultured human lung fibroblasts, TGF-beta1 suppressed KGF messenger RNA and protein expression, which were reversed by SB431542 and by the c-Abl inhibitor, imatinib mesylate, but not by the p38 map kinase inhibitor, SB203580. CONCLUSIONS: ALI/ARDS alveolar fluid suppresses KGF expression, in part, due to TGF-beta1. TGF-beta1 suppression of KGF requires both smad phosphorylation and c-Abl activation.