A prominent feature of fibrotic tissue in general and of lungs in particular is fibroblast proliferation and accumulation. In patients overcoming fibrosis, apoptosis limits this excessive cell growth. We have previously shown resistance to Fas-induced apoptosis of primary lung fibroblasts from mice with bleomycin-induced lung fibrosis, their escape from immune surveillance, and continued accumulation in spite of overexpression of the Fas death receptor. Cellular FLICE-like inhibitory protein (c-FLIP) is a regulator of cell death receptor-induced apoptosis in many cell types. We aimed to determine c-FLIP levels in myofibroblasts from fibrotic lungs and to directly assess c-FLIP's role in apoptosis and proliferation of primary lung myofibroblasts. c-FLIP levels were determined by apoptosis gene array, flow cytometry, Western blot, and immunofluorescence before and after down-regulation with a specific small interfering RNA. Apoptosis was assessed by caspase cleavage in Western blot and by Annexin V affinity labeling after FACS and tissue immunofluorescence. Proliferation was assessed by BrdU uptake, also using FACS and immunofluorescence. We show that myofibroblasts from lungs of humans with idiopathic pulmonary fibrosis and from bleomycin-treated versus normal saline-treated mice up-regulate c-FLIP levels. Using the animal model, we show that fibrotic lung myofibroblasts divert Fas signaling from apoptosis to proliferation and that this requires signaling by TNF receptor-associated factor (TRAF) and NF-kB. c-FLIP down-regulation reverses the effect of Fas activation, causing increased apoptosis, decreased proliferation, and diminished recruitment of TRAF to the DISC complex. This indicates that c-FLIP is essential for myofibroblast accumulation and may serve as a potential target to manipulate tissue fibrosis.

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Lung fibrosis is characterized by abnormal accumulation of fibroblasts in the interstitium of the alveolar space. Two populations of myofibroblasts, distinguished by Thy1 expression, are detected in human and murine lungs. Accumulation of Thy1-negative (Thy1−) myofibroblasts was shown in the lungs of humans with idiopathic pulmonary fibrosis (IPF) and of bleomycin-treated mice. We aimed to identify genetic changes in lung myofibroblasts following Thy1 crosslinking and assess the impact of specific lung myofibroblast Thy1-deficiency, in vivo, in bleomycin-injured mouse lungs. Thy1 increased in mouse lung lymphocytes following bleomycin injury but decreased in myofibroblasts when fibrosis was at the highest point (14 days), as assessed by immunohistochemistry. Using gene chip analysis, we detected that myofibroblast Thy1 crosslinking mediates downregulation of genes promoting cell proliferation, survival, and differentiation, and reduces production of extracellular matrix (ECM) components, while concurrently mediating the upregulation of genes known to foster inflammation and immunological functions. Chimeric Thy1-deficient mice with Thy1+ lymphocytes and Thy1− myofibroblasts showed fibrosis similar to wild-type mice and an increased number of CD4/CD25 regulatory T cells, with a concomitant decrease in inflammation. Lung myofibroblasts downregulate Thy1 expression to increase their proliferation but to diminish the in vivo inflammatory milieu. Inflammation is not essential for evolution of fibrosis as was previously stated.

High doses of bleomycin administered to patients with lymphomas and other tumors lead to significant lung toxicity in general, and to apoptosis of epithelial cells, in particular. Apoptosis of alveolar epithelium is an important step in the pathogenesis of bleomycin-induced pulmonary fibrosis. The Fas-FasL pathway is one of the main apoptotic pathways involved. Telomerase is a ribonucleoprotein RNA-dependent DNA polymerase complex consisting of an RNA template and a catalytic protein, telomerase reverse transcriptase (TERT). Telomerase also possess extra-telomeric roles, including modulation of transcription of anti-apoptotic genes, differentiation signals, and more. We hypothesized that telomerase overexpression affects Fas-induced epithelial cell apoptosis by an extra-telomeric role such as regulation of anti-apoptotic genes, specifically FLICE-like inhibitory protein (FLIP). Telomerase in mouse (MLE) and human (A549) lung epithelial cell lines was upregulated by transient transfection using cDNA hTERT expression vector. Telomerase activity was detected using a real-time PCR-based system. Bleomycin, and bleomycin-induced Fas-mediated apoptosis following treatment with anti-Fas activating mAb or control IgG, were assessed by Annexin V staining, FACS analysis, and confocal microscopy; caspase cleavage by Western blot; FLIP or Fas molecule detection by Western blot and flow cytometry. hTERT transfection of lung epithelial cells resulted in a 100% increase in their telomerase activity. Fas-induced lung epithelial cell apoptosis was significantly reduced in hTERT-transfected cells compared to controls in all experiments. Lung epithelial cells with increased telomerase activity had higher levels of FLIP expression but membrane Fas expression was unchanged. Upregulation of hTERT+ in human lung epithelial cells and subsequent downregulation of FLIP by shFLIP-RNA annulled hTERT-mediated resistance to apoptosis. Telomerase-mediated FLIP overexpression may be a novel mechanism to confer protection from apoptosis in bleomycin-exposed human lung epithelial cells.

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