CFTR dysfunction in smooth muscle drives TGFß dependent airway hyperreactivity.

BACKGROUND: The primary underlying defect in cystic fibrosis (CF) is disrupted ion transport in epithelia throughout the body. It is unclear if symptoms such as airway hyperreactivity (AHR) and increased airway smooth muscle (ASM) volume in people with CF are due to inherent abnormalities in smooth muscle or are secondary to epithelial dysfunction. Transforming Growth Factor beta 1 (TGFß) is an established genetic modifier of CF lung disease and a known driver of abnormal ASM function. Prior studies have demonstrated that CF mice develop greater AHR, goblet cell hyperplasia, and ASM hypertrophy after pulmonary TGFß exposure. However, the mechanism driving these abnormalities in CF lung disease, specifically the contribution of CFTR loss in ASM, was unknown. METHODS: In this study, mice with smooth muscle-specific loss of CFTR function (Cftrfl/fl; SM-Cre mice) were exposed to pulmonary TGFß. The impact on lung pathology and physiology was investigated through examination of lung mechanics, Western blot analysis, and pulmonary histology. RESULTS: Cftrfl/fl; SM-Cre mice treated with TGFß demonstrated greater methacholine-induced AHR than control mice. However, Cftrfl/fl; SM-Cre mice did not develop increased inflammation, ASM area, or goblet cell hyperplasia relative to controls following TGFß exposure. CONCLUSIONS: These results demonstrate a direct smooth muscle contribution to CF airway obstruction mediated by TGFß. Dysfunction in non-epithelial tissues should be considered in the development of CF therapeutics, including potential genetic therapies.

Neutrophil extracellular traps activate IL-8 and IL-1 expression in human bronchial epithelia.

Neutrophil extracellular traps (NETs) provide host defense but can contribute to the pathobiology of diverse human diseases. We sought to determine the extent and mechanism by which NETs contribute to human airway cell inflammation. Primary normal human bronchial epithelial cells (HBEs) grown at air-liquid interface and wild-type (wt)CFBE41o- cells (expressing wtCFTR) were exposed to cell-free NETs from unrelated healthy volunteers for 18 h in vitro. Cytokines were measured in the apical supernatant by Luminex, and the effect on the HBE transcriptome was assessed by RNA sequencing. NETs consistently stimulated IL-8, TNF-α, and IL-1α secretion by HBEs from multiple donors, with variable effects on other cytokines (IL-6, G-CSF, and GM-CSF). Expression of HBE RNAs encoding IL-1 family cytokines, particularly IL-36 subfamily members, was increased in response to NETs. NET exposure in the presence of anakinra [recombinant human IL-1 receptor antagonist (rhIL-1RA)] dampened NET-induced changes in IL-8 and TNF-α proteins as well as IL-36α RNA. rhIL-36RA limited the increase in expression of proinflammatory cytokine RNAs in HBEs exposed to NETs. NETs selectively upregulate an IL-1 family cytokine response in HBEs, which enhances IL-8 production and is limited by rhIL-1RA. The present findings describe a unique mechanism by which NETs may contribute to inflammation in human lung disease in vivo. NET-driven IL-1 signaling may represent a novel target for modulating inflammation in diseases characterized by a substantial NET burden.

Subacute TGFß Exposure Drives Airway Hyperresponsiveness in Cystic Fibrosis Mice through the PI3K Pathway.

Cystic fibrosis (CF) is a lethal genetic disease characterized by progressive lung damage and airway obstruction. The majority of patients demonstrate airway hyperresponsiveness (AHR), which is associated with more rapid lung function decline. Recent studies in the neonatal CF pig demonstrated airway smooth muscle (ASM) dysfunction. These findings, combined with observed CF transmembrane conductance regulator (CFTR) expression in ASM, suggest that a fundamental defect in ASM function contributes to lung function decline in CF. One established driver of AHR and ASM dysfunction is transforming growth factor (TGF) ß1, a genetic modifier of CF lung disease. Prior studies demonstrated that TGFß exposure in CF mice drives features of CF lung disease, including goblet cell hyperplasia and abnormal lung mechanics. CF mice displayed aberrant responses to pulmonary TGFß, with elevated PI3K signaling and greater increases in lung resistance compared with controls. Here, we show that TGFß drives abnormalities in CF ASM structure and function through PI3K signaling that is enhanced in CFTR-deficient lungs. CF and non-CF mice were exposed intratracheally to an adenoviral vector containing the TGFß1 cDNA, empty vector, or PBS only. We assessed methacholine-induced AHR, bronchodilator response, and ASM area in control and CF mice. Notably, CF mice demonstrated enhanced AHR and bronchodilator response with greater ASM area increases compared with non-CF mice. Furthermore, therapeutic inhibition of PI3K signaling mitigated the TGFß-induced AHR and goblet cell hyperplasia in CF mice. These results highlight a latent AHR phenotype in CFTR deficiency that is enhanced through TGFß-induced PI3K signaling.

Longitudinal free-breathing MRI measurement of murine lung physiology in a progressive model of lung fibrosis.

To longitudinally monitor progressive fibrosis in the transforming growth factor-α (TGF-α) transgenic mouse model of lung fibrosis, we used retrospective self-gating ultrashort echo time (UTE) magnetic resonance imaging (MRI) to image mouse lung at baseline and after 4 and 8 wk of fibrosis initiation via doxycycline administration. Only bitransgenic mice were used in this study and divided into two cohorts: six mice were fed doxycycline food to induce lung fibrosis (referred to as Dox cohort), and five other mice were fed normal food (referred to as control cohort). Lung mechanics, histology, and hydroxyproline were assessed after the final MRI. A linear mixed-effects model was used to analyze MRI-derived longitudinal lung-function parameters. Tidal volume decreased at a rate of -0.016 ± 0.002 ml/week [χ2(1) = 16.48, P < 0.001] for Dox cohort and increased at a rate of 0.010 ± 0.003 ml/week [χ2(1) = 6.37, P = 0.01] for control cohort. Minute ventilation decreased at a rate of -1.71 ± 0.26 ml·min-1·wk-1 [χ2(1) = 14.04, P < 0.001] for Dox cohort but did not change significantly over time for control cohort. High-density lung volume percentage increased at a rate of 3.9 ± 0.7%/wk for Dox cohort [χ2(1) = 11.47, P < 0.001] but did not change significantly over time for control cohort. MRI-derived lung structure and function parameters were strongly correlated with pleural thickness, hydroxyproline content, lung compliance, airway resistance, and airway elastance. We conclude that self-gating UTE MRI could be used to longitudinally monitor lung fibrosis in the TGF-α transgenic mouse model. NEW & NOTEWORTHY Self-gating UTE MRI was used to monitor morphology and physiology in lung fibrosis in a transforming growth factor-α transgenic mouse model. Tidal volume was shown for the first time to correlate strongly with conventional metrics of fibrosis such as hydroxyproline and pleural thickness.

Subacute TGFß expression drives inflammation, goblet cell hyperplasia, and pulmonary function abnormalities in mice with effects dependent on CFTR function.

Cystic fibrosis (CF) produces variable lung disease phenotypes that are, in part, independent of the CF transmembrane conductance regulator ( CFTR) genotype. Transforming growth factor-ß (TGFß) is the best described genetic modifier of the CF phenotype, but its mechanism of action is unknown. We hypothesized that TGFß is sufficient to drive pathognomonic features of CF lung disease in vivo and that CFTR deficiency enhances susceptibility to pathological TGFß effects. A CF mouse model and littermate controls were exposed intratracheally to an adenoviral vector containing the TGFß1 cDNA (Ad-TGFß), empty vector, or PBS only. Studies were performed 1 wk after treatment, including lung mechanics, collection of bronchoalveolar lavage fluid, and analysis of lung histology, RNA, and protein. CF and non-CF mice showed similar weight loss, inflammation, goblet cell hyperplasia, and Smad pathway activation after Ad-TGFß treatment. Ad-TGFß produced greater abnormalities in lung mechanics in CF versus control mice, which was uniquely associated with induction of phosphoinositide 3-kinase and mitogen-activated protein kinase signaling. CFTR transcripts were reduced, and epithelial sodium channel transcripts were increased in CF and non-CF mice, whereas the goblet cell transcription factors, forkhead ortholog A3 and SAM-pointed domain-containing ETS-like factor, were increased in non-CF but not CF mice following Ad-TGFß treatment. Pulmonary TGFß1 expression was sufficient to produce pulmonary remodeling and abnormalities in lung mechanics that were associated with both shared and unique cell signaling pathway activation in CF and non-CF mice. These results highlight the multifunctional impact of TGFß on pulmonary pathology in vivo and identify cellular-response differences that may impact CF lung pathology.

Magnetic resonance imaging of disease progression and resolution in a transgenic mouse model of pulmonary fibrosis.

Pulmonary fibrosis contributes to morbidity and mortality in a range of diseases, and there are no approved therapies for reversing its progression. To understand the mechanisms underlying pulmonary fibrosis and assess potential therapies, mouse models are central to basic and translational research. Unfortunately, metrics commonly used to assess murine pulmonary fibrosis require animals to be grouped and euthanized, increasing experimental difficulty and cost. We examined the ability of magnetic resonance imaging (MRI) to noninvasively assess lung fibrosis progression and resolution in a doxycycline (Dox) regulatable, transgenic mouse model that overexpresses transforming growth factor-α (TGF-α) under control of a lung-epithelial-specific promoter. During 7 wk of Dox treatment, fibrotic lesions were readily observed as high-signal tissue. Mean weighted signal and percent signal volume were found to be the most robust MRI-derived measures of fibrosis, and these metrics correlated significantly with pleural thickness, histology scores, and hydroxyproline content (R = 0.75-0.89). When applied longitudinally, percent high signal volume increased by 1.5% wk-1 (P < 0.001) and mean weighted signal increased at a rate of 0.0065 wk-1 (P = 0.0062). Following Dox treatment, lesions partially resolved, with percent high signal volume decreasing by -3.2% wk-1 (P = 0.0034) and weighted mean signal decreasing at -0.015 wk-1 (P = 0.0028). Additionally, longitudinal MRI revealed dynamic remodeling in a subset of lesions, a previously unobserved behavior in this model. These results demonstrate MRI can noninvasively assess experimental lung fibrosis progression and resolution and provide unique insights into its pathobiology.

Unique and Redundant Functions of p70 Ribosomal S6 Kinase Isoforms Regulate Mesenchymal Cell Proliferation and Migration in Pulmonary Fibrosis.

The p70 ribosomal S6 kinase (p70S6K) is a downstream substrate that is phosphorylated and activated by the mammalian target of rapamycin complex and regulates multiple cellular processes associated with pulmonary fibrogenesis. Two isoforms of the p70S6K have been identified (S6K1 and S6K2), but their relative contributions in mediating pulmonary fibrosis are unknown. To interrogate the roles of the p70S6K isoforms, we overexpressed transforming growth factor (TGF)-α in mice deficient for the S6K1 or S6K2 genes and measured changes in lung histology, morphometry, total lung collagen, lung function, and proliferation between wild-type and isoform-deficient mice. Deficiency of S6K1, but not S6K2, had a significant effect on reducing proliferation in subpleural fibrotic lesions during TGF-α-induced fibrosis. Migration was significantly decreased in mesenchymal cells isolated from the lungs of S6K1 knockout mice compared with wild-type or S6K2 knockout mice. Conversely, increases in subpleural thickening were significantly decreased in S6K2-deficient mice compared with wild type. Deficiency of S6K2 significantly reduced phosphorylation of the downstream S6 ribosomal protein in lung homogenates and isolated mesenchymal cells after TGF-α expression. However, deficiency of neither isoform alone significantly altered TGF-α-induced collagen accumulation or lung function decline in vivo. Furthermore, deficiency in neither isoform prevented changes in collagen accumulation or lung compliance decline after administration of intradermal bleomycin. Together, these findings demonstrate that the p70S6K isoforms have unique and redundant functions in mediating fibrogenic processes, including proliferation, migration, and S6 phosphorylation, signifying that both isoforms must be targeted to modulate p70S6K-mediated pulmonary fibrosis.

p70 ribosomal S6 kinase regulates subpleural fibrosis following transforming growth factor-α expression in the lung.

The p70 ribosomal S6 kinase (S6K) is a downstream substrate that is phosphorylated and activated by the mammalian target of rapamycin complex and regulates multiple cellular processes associated with fibrogenesis. Recent studies demonstrate that aberrant mTORC1-S6K signaling contributes to various pathological conditions, but a direct role in pulmonary fibroproliferation has not been established. Increased phosphorylation of the S6K pathway is detected immediately following transforming growth factor-α (TGF-α) expression in a transgenic model of progressive lung fibrosis. To test the hypothesis that the S6K directly regulates pulmonary fibroproliferative disease we determined the cellular sites of S6K phosphorylation during the induction of fibrosis in the TGF-α model and tested the efficacy of specific pharmacological inhibition of the S6K pathway to prevent and reverse fibrotic disease. Following TGF-α expression increased phosphorylation of the S6K was detected in the airway and alveolar epithelium and the mesenchyme of advanced subpleural fibrotic regions. Specific inhibition of the S6K with the small molecule inhibitor LY-2584702 decreased TGF-α and platelet-derived growth factor-ß-induced proliferation of lung fibroblasts in vitro. Administration of S6K inhibitors to TGF-α mice prevented the development of extensive subpleural fibrosis and alterations in lung mechanics, and attenuated the increase in total lung hydroxyproline. S6K inhibition after fibrosis was established attenuated the progression of subpleural fibrosis. Together these studies demonstrate targeting the S6K pathway selectively modifies the progression of pulmonary fibrosis in the subpleural compartment of the lung.

Inhibition of the αvß6 integrin leads to limited alteration of TGF-α-induced pulmonary fibrosis.

A number of growth factors and signaling pathways regulate matrix deposition and fibroblast proliferation in the lung. The epidermal growth factor receptor (EGFR) family of receptors and the transforming growth factor-ß (TGF-ß) family are active in diverse biological processes and are central mediators in the initiation and maintenance of fibrosis in many diseases. Transforming growth factor-α (TGF-α) is a ligand for the EGFR, and doxycycline (Dox)-inducible transgenic mice conditionally expressing TGF-α specifically in the lung epithelium develop progressive fibrosis accompanied with cachexia, changes in lung mechanics, and marked pleural thickening. Although recent studies demonstrate that EGFR activation modulates the fibroproliferative effects involved in the pathogenesis of TGF-ß induced pulmonary fibrosis, in converse, the direct role of EGFR induction of the TGF-ß pathway in the lung is unknown. The αvß6 integrin is an important in vivo activator of TGF-ß activation in the lung. Immunohistochemical analysis of αvß6 protein expression and bronchoalveolar analysis of TGF-ß pathway signaling indicates activation of the αvß6/TGF-ß pathway only at later time points after lung fibrosis was already established in the TGF-α model. To determine the contribution of the αvß6/TGF-ß pathway on the progression of established fibrotic disease, TGF-α transgenic mice were administered Dox for 4 wk, which leads to extensive fibrosis; these mice were then treated with a function-blocking anti-αvß6 antibody with continued administration of Dox for an additional 4 wk. Compared with TGF-α transgenic mice treated with control antibody, αvß6 inhibition significantly attenuated pleural thickening and altered the decline in lung mechanics. To test the effects of genetic loss of the ß6 integrin, TGF-α transgenic mice were mated with ß6-null mice and the degree of fibrosis was compared in adult mice following 8 wk of Dox administration. Genetic ablation of the ß6 integrin attenuated histological and physiological changes in the lungs of TGF-α transgenic mice although a significant degree of fibrosis still developed. In summary, inhibition of the ß6 integrin led to a modest, albeit significant, effect on pleural thickening and lung function decline observed with TGF-α-induced pulmonary fibrosis. These data support activation of the αvß6/TGF-ß pathway as a secondary effect contributing to TGF-α-induced pleural fibrosis and suggest a complex contribution of multiple mediators to the maintenance of progressive fibrosis in the lung.

Dual targeting of MEK and PI3K pathways attenuates established and progressive pulmonary fibrosis.

Pulmonary fibrosis is often triggered by an epithelial injury resulting in the formation of fibrotic lesions in the lung, which progress to impair gas exchange and ultimately cause death. Recent clinical trials using drugs that target either inflammation or a specific molecule have failed, suggesting that multiple pathways and cellular processes need to be attenuated for effective reversal of established and progressive fibrosis. Although activation of MAPK and PI3K pathways have been detected in human fibrotic lung samples, the therapeutic benefits of in vivo modulation of the MAPK and PI3K pathways in combination are unknown. Overexpression of TGFα in the lung epithelium of transgenic mice results in the formation of fibrotic lesions similar to those found in human pulmonary fibrosis, and previous work from our group shows that inhibitors of either the MAPK or PI3K pathway can alter the progression of fibrosis. In this study, we sought to determine whether simultaneous inhibition of the MAPK and PI3K signaling pathways is a more effective therapeutic strategy for established and progressive pulmonary fibrosis. Our results showed that inhibiting both pathways had additive effects compared to inhibiting either pathway alone in reducing fibrotic burden, including reducing lung weight, pleural thickness, and total collagen in the lungs of TGFα mice. This study demonstrates that inhibiting MEK and PI3K in combination abolishes proliferative changes associated with fibrosis and myfibroblast accumulation and thus may serve as a therapeutic option in the treatment of human fibrotic lung disease where these pathways play a role.

Bone marrow-derived stromal cells are invasive and hyperproliferative and alter transforming growth factor-α-induced 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.

Resistin-like molecule α1 (Fizz1) recruits lung dendritic cells without causing pulmonary fibrosis.

BACKGROUND: Resistin-like molecule alpha or found in inflammatory zone protein (Fizz1) is increased in pulmonary epithelial cells and also in limited amounts by other lung cells during various lung injuries and fibrosis. However, the direct role of Fizz1 produced in the pulmonary epithelium has not been determined. METHODS: Fizz1 Transgenic mice (CCSP/Fizz1) were generated that overexpress Fizz1 in the lung epithelium under the control of a doxycycline (Dox) inducible lung epithelial cell specific promoter Scgb1a1 (Clara cell secretory protein, CCSP). Histology and FACS analysis of lung cells were used to identify the direct effects of Fizz1 in the transgenic mice (Dox treated) when compared with control (CCSP/-) mice. Intratracheal bleomycin sulfate or silica in saline and saline alone were used to study the role of Fizz1 during bleomycin- and silica-induced pulmonary fibrosis in CCSP/Fizz1 and CCSP/- mice. Weight change, pulmonary inflammation, and fibrosis were assessed 10 days post bleomycin or 28 days post silica challenge. RESULTS: When CCSP/Fizz1 mice were fed Dox food, elevated Fizz1 protein was detected in lung homogenates by western blot. Lungs of mice in which Fizz1 was induced in the epithelium contained increased lung cells staining for CD11c and F4/80 by FACS analysis consistent with increased dendritic cells however, no changes were observed in the percentage of interstitial macrophages compared to CCSP/- controls. No significant changes were found in the lung histology of CCSP/Fizz1 mice after up to 8 weeks of overexpression compared to CCSP/- controls. Overexpression of Fizz1 prior to challenge or following challenge with bleomycin or silica did not significantly alter airway inflammation or fibrosis compared to control mice. CONCLUSIONS: The current study demonstrates that epithelial cell derived Fizz1 is sufficient to increase the bone-marrow derived dendritic cells in the lungs, but it is not sufficient to cause lung fibrosis or alter chemical or particle-induced fibrosis.

MEK-ERK pathway modulation ameliorates pulmonary fibrosis associated with epidermal growth factor receptor activation.

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.

Rapamycin Regulates Bleomycin-Induced Lung Damage in SP-C-Deficient Mice.

Injury to the distal respiratory epithelium has been implicated as an underlying cause of idiopathic lung diseases. Mutations that result in SP-C deficiencies are linked to a small subset of spontaneous and familial cases of interstitial lung disease (ILD) and interstitial pulmonary fibrosis (IPF). Gene-targeted mice that lack SP-C (Sftpc(-/-)) develop an irregular ILD-like disease with age and are a model of the human SP-C related disease. In the current study, we investigated whether rapamycin could ameliorate bleomycin-induced fibrosis in the lungs of Sftpc(-/-) mice. Sftpc(+/+) and -/- mice were exposed to bleomycin with either preventative administration of rapamycin or therapeutic administration beginning eight days after the bleomycin injury. Rapamycin-treatment increased weight loss and decreased survival of bleomycin-treated Sftpc(+/+) and Sftpc(-/-) mice. Rapamycin did not reduce the fibrotic disease in the prophylactic or rescue experiments of either genotype of mice. Further, rapamycin treatment augmented airway resistance and reduced lung compliance of bleomycin-treated Sftpc(-/-) mice. Rapamycin treatment was associated with an increased expression of profibrotic Th2 cytokines and reduced expression of INF-γ. These findings indicate that novel therapeutics will be required to treat individuals with SP-C deficient ILD/IPF.

Inhibition of PI3K by PX-866 prevents transforming growth factor-alpha-induced pulmonary fibrosis.

Transforming growth factor-alpha (TGFalpha) 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. EGFR signaling activates several intracellular signaling pathways including phosphatidylinositol 3'-kinase (PI3K). We previously showed that induction of lung-specific TGFalpha expression in transgenic mice caused progressive pulmonary fibrosis over a 4-week period. The increase in levels of phosphorylated Akt, detected after 1 day of doxycycline-induced TGFalpha expression, was blocked by treatment with the PI3K inhibitor, PX-866. Daily administration of PX-866 during TGFalpha induction prevented increases in lung collagen and airway resistance as well as decreases in lung compliance. Treatment of mice with oral PX-866 4 weeks after the induction of TGFalpha prevented additional weight loss and further increases in total collagen, and attenuated changes in pulmonary mechanics. These data show that PI3K is activated in TGFalpha/EGFR-mediated pulmonary fibrosis and support further studies to determine the role of PI3K activation in human lung fibrotic disease, which could be amenable to targeted therapy.

Rapamycin prevents transforming growth factor-alpha-induced pulmonary fibrosis.

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.

EGF receptor tyrosine kinase inhibitors diminish transforming growth factor-alpha-induced pulmonary fibrosis.

Transforming growth factor-alpha (TGF-alpha) is a ligand for the EGF receptor (EGFR). EGFR activation is associated with fibroproliferative processes in human lung disease and animal models of pulmonary fibrosis. We determined the effects of EGFR tyrosine kinase inhibitors gefitinib (Iressa) and erlotinib (Tarceva) on the development and progression of TGF-alpha-induced pulmonary fibrosis. Using a doxycycline-regulatable transgenic mouse model of lung-specific TGF-alpha expression, we determined effects of treatment with gefitinib and erlotinib on changes in lung histology, total lung collagen, pulmonary mechanics, pulmonary hypertension, and expression of genes associated with synthesis of ECM and vascular remodeling. Induction in the lung of TGF-alpha caused progressive pulmonary fibrosis over an 8-wk period. Daily administration of gefitinib or erlotinib prevented development of fibrosis, reduced accumulation of total lung collagen, prevented weight loss, and prevented changes in pulmonary mechanics. Treatment of mice with gefitinib 4 wk after the induction of TGF-alpha prevented further increases in and partially reversed total collagen levels and changes in pulmonary mechanics and pulmonary hypertension. Increases in expression of genes associated with synthesis of ECM as well as decreases of genes associated with vascular remodeling were also prevented or partially reversed. Administration of gefitinib or erlotinib did not cause interstitial fibrosis or increases in lavage cell counts. Administration of small molecule EGFR tyrosine kinase inhibitors prevented further increases in and partially reversed pulmonary fibrosis induced directly by EGFR activation without inducing inflammatory cell influx or additional lung injury.

Genomic profile of matrix and vasculature remodeling in TGF-alpha induced pulmonary fibrosis.

Expression of transforming growth factor alpha (TGF-alpha) in the respiratory epithelium of transgenic mice caused pulmonary fibrosis, cachexia, pulmonary hypertension, and altered lung function. To identify genes and molecular pathways mediating lung remodeling, mRNA microarray analysis was performed at multiple times after TGF-alpha expression and revealed changes consistent with a role for TGF-alpha in the regulation of extracellular matrix and vasculogenesis. Transcripts for extracellular matrix proteins were augmented along with transcripts for genes previously identified to have roles in pulmonary fibrosis, including tenascin C, osteopontin, and serine (or cysteine) peptidase inhibitor, clade F, member 1. Transcripts regulating vascular processes including endothelin receptor type B, endothelial-specific receptor tyrosine kinase, and caveolin, caveolae protein 1 were decreased. When TGF-alpha expression was no longer induced, lung remodeling partially reversed and lung function and pulmonary hypertension normalized. Transcripts increased during resolution included midkine, matrix metalloproteinase 2, and hemolytic complement. Hierarchical clustering revealed that genes regulated by TGF-alpha were similar to those altered in the lungs of patients with idiopathic pulmonary fibrosis. These studies support a role for epithelial cell-derived TGF-alpha in the regulation of processes that alter the airway and vascular architecture and function.

The genetics of ray pattern variation in Caenorhabditis briggsae.

BACKGROUND: How does intraspecific variation relate to macroevolutionary change in morphology? This question can be addressed in species in which derived characters are present but not fixed. In rhabditid nematodes, the arrangement of the nine bilateral pairs of peripheral sense organs (rays) in tails of males is often the most highly divergent character between species. The development of ray pattern involves inputs from hometic gene expression patterns, TGFbeta signalling, Wnt signalling, and other genetic pathways. In Caenorhabditis briggsae, strain-specific variation in ray pattern has provided an entree into the evolution of ray pattern. Some strains were fixed for a derived pattern. Other strains were more plastic and exhibited derived and ancestral patterns at equal frequencies. RESULTS: Recombinant inbred lines (RILs) constructed from crosses between the variant C. briggsae AF16 and HK104 strains exhibited a wide range of phenotypes including some that were more extreme than either parental strain. Transgressive segregation was significantly associated with allelic variation in the C. briggsae homolog of abdominal B, Cb-egl-5. At least two genes that affected different elements of ray pattern, ray position and ray fusion, were linked to a second gene, mip-1. Consistent with this, the segregation of ray position and ray fusion phenotypes were only partially correlated in the RILs. CONCLUSIONS: The evolution of ray pattern has involved allelic variation at multiple loci. Some of these loci impact the specification of ray identities and simultaneously affect multiple ray pattern elements. Others impact individual characters and are not constrained by covariance with other ray pattern elements. Among the genetic pathways that may be involved in ray pattern evolution is specification of anteroposterior positional information by homeotic genes.