Transforming growth factor-β1 (TGF-β1) is a multifunctional cytokine involved in differentiation, growth, and survival of mesenchymal cells while inhibiting growth/survival of most other cell types. The mechanism(s) of pro-survival signaling by TGF-β1 in mesenchymal cells is unclear. In this report, we demonstrate that TGF-β1 protects against serum deprivation-induced apoptosis of mesenchymal cells isolated from patients with acute lung injury and of normal human fetal lung fibroblasts (IMR-90). TGF-β receptor(s)-activated signaling in these cells involves rapid activation of the Smad and p38 MAPK pathways within minutes of TGF-β1 treatment followed by a more delayed activation of the pro-survival phosphatidylinositol 3-kinase-protein kinase B (PKB)/Akt pathway. Pharmacological inhibition of p38 MAPK with SB203580 or expression of a p38 kinase-deficient mutant protein inhibits TGF-β1-induced PKB/Akt phosphorylation. Conditioned medium from TGF-β1-treated cells rapidly induces PKB/Akt activation in an SB203580- and suramin-sensitive manner, suggesting p38 MAPK-dependent production of a secreted growth factor that activates this pro-survival pathway by an autocrine/paracrine mechanism. Inhibition of the phosphatidylinositol 3-kinase-PKB/Akt pathway blocks TGF-β1-induced resistance to apoptosis. These results demonstrate the activation of a novel TGF-β1-activated pro-survival/anti-apoptotic signaling pathway in mesenchymal cells/fibroblasts that may explain cell-specific actions of TGF-β1 and provide mechanistic insights into its pro-fibrotic and tumor-promoting effects.

Pulmonary fibrosis is characterized by alterations in fibroblast phenotypes resulting in excessive extracellular matrix accumulation and anatomic remodeling. Current therapies for this condition are largely ineffective. Peroxisome proliferator-activated receptor-γ (PPAR-γ) is a member of the nuclear hormone receptor superfamily, the activation of which produces a number of biological effects, including alterations in metabolic and inflammatory responses. The role of PPAR-γ as a potential therapeutic target for fibrotic lung diseases remains undefined. In the present study, we show expression of PPAR-γ in fibroblasts obtained from normal human lungs and lungs of patients with idiopathic interstitial pneumonias. Treatment of lung fibroblasts and myofibroblasts with PPAR-γ agonists results in inhibition of proliferative responses and induces cell cycle arrest. In addition, PPAR-γ agonists, including a constitutively active PPAR-γ construct (VP16-PPAR-γ), inhibit the ability of transforming growth factor-β1 to induce myofibroblast differentiation and collagen secretion. PPAR-γ agonists also inhibit fibrosis in a murine model, even when administration is delayed until after the initial inflammation has largely resolved. These observations indicate that PPAR-γ is an important regulator of fibroblast/myofibroblast activation and suggest a role for PPAR-γ ligands as novel therapeutic agents for fibrotic lung diseases.

Myofibroblasts are crucial to the pathogenesis of tissue fibrosis. Their formation of stress fibers results in the release of myocardin-related transcription factor (MRTF), a transcriptional coactivator of serum response factor (SRF). MRTF-A (Mkl1)-deficient mice are protected from lung fibrosis. We hypothesized that the SRF/MRTF pathway inhibitor CCG-203971 would modulate myofibroblast function in vitro and limit lung fibrosis in vivo. Normal and idiopathic pulmonary fibrosis lung fibroblasts were treated with/without CCG-203971 (N-[4-chlorophenyl]-1-[3-(2-furanyl)benzoyl]-3-piperidine carboxamide) and/or Fas-activating antibody in the presence/absence of transforming growth factor (TGF)-β1, and apoptosis was assessed. In vivo studies examined the effect of therapeutically administered CCG-203971 on lung fibrosis in two distinct murine models of fibrosis induced by bleomycin or targeted type II alveolar epithelial injury. In vitro, CCG-203971 prevented nuclear localization of MRTF-A; increased the apoptotic susceptibility of normal and idiopathic pulmonary fibrosis fibroblasts; blocked TGF-β1-induced myofibroblast differentiation; and inhibited TGF-β1-induced expression of fibronectin, X-linked inhibitor of apoptosis, and plasminogen activator inhibitor-1. TGF-β1 did not protect fibroblasts or myofibroblasts from apoptosis in the presence of CCG-203971. In vivo, CCG-203971 significantly reduced lung collagen content in both murine models while decreasing alveolar plasminogen activator inhibitor-1 and promoting myofibroblast apoptosis. These data support a central role of the SRF/MRTF pathway in the pathobiology of lung fibrosis and suggest that its inhibition can help resolve lung fibrosis by promoting fibroblast apoptosis.