No abstract

Pulmonary fibrosis remains a significant public health burden with no proven therapies. The mitogen-activated protein kinase (MAPK)/MAPK kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling cascade is a major pathway controlling cellular processes associated with fibrogenesis, including growth, proliferation, and survival. Activation of the MAPK/ERK pathway is detected in the lungs of human fibrosis samples; however, the effect of modulating the pathway in vivo is unknown. Overexpression of transforming growth factor (TGF)-α in the lung epithelium of transgenic mice causes a progressive pulmonary fibrosis associated with increased MEK/ERK activation localized primarily in mesenchymal cells. To determine the role of the MEK pathway in the induction of TGF-α-induced lung fibrosis, TGF-α was overexpressed for 4 weeks while mice were simultaneously treated with the specific MEK inhibitor, ARRY-142886 (ARRY). Treatment with ARRY prevented increases in lung cell proliferation and total lung collagen, attenuated production of extracellular matrix genes, and protected mice from changes in lung function. ARRY administered as a rescue treatment after fibrosis was already established inhibited fibrosis progression, as assessed by lung histology, changes in body weights, extracellular matrix gene expression, and lung mechanics. These findings demonstrate that MEK inhibition prevents progression of established fibrosis in the TGF-α model, and provides proof of concept of targeting the MEK pathway in fibrotic lung disease.

Transforming growth factor (TGF)-alpha is a ligand for the epidermal growth factor receptor (EGFR). EGFR activation is associated with fibroproliferative processes in human lung disease and animal models of pulmonary fibrosis. Overexpression of TGF-alpha in transgenic mice causes progressive and severe pulmonary fibrosis; however, the intracellular signaling pathways downstream of EGFR mediating this response are unknown. Using a doxycycline-regulatable transgenic mouse model of lung-specific TGF-alpha expression, we observed increased PCNA protein and phosphorylation of Akt and p70S6K in whole lung homogenates in association with induction of TGF-alpha. Induction in the lung of TGF-alpha caused progressive pulmonary fibrosis over a 7-week period. Daily administration of rapamycin prevented accumulation of total lung collagen, weight loss, and changes in pulmonary mechanics. Treatment of mice with rapamycin 4 weeks after the induction of TGF-alpha prevented additional weight loss, increases in total collagen, and changes in pulmonary mechanics. Rapamycin prevented further increases in established pulmonary fibrosis induced by EGFR activation. This study demonstrates that mammalian target of rapamycin (mTOR) is a major effector of EGFR-induced pulmonary fibrosis, providing support for further studies to determine the role of mTOR in the pathogenesis and treatment of pulmonary fibrosis.

Pulmonary fibrosis is caused by excessive proliferation and accumulation of stromal cells. Fibrocytes are bone marrow (BM)-derived cells that contribute to pathologic stromal cell accumulation in human lung disease. However, the cellular source for these stromal cells and the degree of fibrocyte contribution to pulmonary fibrosis remain unclear. To determine the etiology of stromal cell excess during pulmonary fibrosis, we measured fibrocytes during the progression of fibrosis in the transforming growth factor (TGF)-α transgenic mouse model. Lung epithelial-specific overexpression of TGF-α led to progressive pulmonary fibrosis associated with increased accumulation of fibrocytes in the fibrotic lesions. Although reconstitution of BM cells into TGF-α mice demonstrated accumulation of these cells in fibrotic lesions, the majority of the cells did not express α-smooth muscle actin, suggesting that fibrocytes did not transform into myofibroblasts. To explore the mechanisms of fibrocytes in pulmonary fibrogenesis, adoptive cell-transfer experiments were performed. Purified fibrocytes were transferred intravenously into TGF-α transgenic mice, and fibrosis endpoints were compared with controls. Analysis of lung histology and hydroxyproline levels demonstrated that fibrocyte transfers augment TGF-α-induced lung fibrosis. A major subset of TGF-α-induced fibrocytes expressed CD44 and displayed excessive invasiveness, which is attenuated in the presence of anti-CD44 antibodies. Coculture experiments of resident fibroblasts with fibrocytes demonstrated that fibrocytes stimulate proliferation of resident fibroblasts. In summary, fibrocytes are increased in the progressive, fibrotic lesions of TGF-α-transgenic mice and activate resident fibroblasts to cause severe lung disease.