Zea Borok

Induction of Epithelial-Mesenchymal Transition in Alveolar Epithelial Cells by Transforming Growth Factor-β1: Potential Role in Idiopathic Pulmonary Fibrosis

The hallmark of idiopathic pulmonary fibrosis (IPF) is the myofibroblast, the cellular origin of which in the lung is unknown. We hypothesized that alveolar epithelial cells (AECs) may serve as a source of myofibroblasts through epithelial-mesenchymal transition (EMT). Effects of chronic exposure to transforming growth factor (TGF)-beta1 on the phenotype of isolated rat AECs in primary culture and a rat type II cell line (RLE-6TN) were evaluated. Additionally, tissue samples from patients with IPF were evaluated for cells co-expressing epithelial (thyroid transcription factor (TTF)-1 and pro-surfactant protein-B (pro-SP-B), and mesenchymal (alpha-smooth muscle actin (alpha-SMA)) markers. RLE-6TN cells exposed to TGF-beta1 for 6 days demonstrated increased expression of mesenchymal cell markers and a fibroblast-like morphology, an effect augmented by tumor necrosis factor-alpha (TNF-alpha). Exposure of rat AECs to TGF-beta1 (100 pmol/L) resulted in increased expression of alpha-SMA, type I collagen, vimentin, and desmin, with concurrent transition to a fibroblast-like morphology and decreased expression of TTF-1, aquaporin-5 (AQP5), zonula occludens-1 (ZO-1), and cytokeratins. Cells co-expressing epithelial markers and alpha-SMA were abundant in lung tissue from IPF patients. These results suggest that AECs undergo EMT when chronically exposed to TGF-beta1, raising the possibility that epithelial cells may serve as a novel source of myofibroblasts in IPF.

Inhibition of Wnt/β-catenin/CREB binding protein (CBP) signaling reverses pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF)/usual interstitial pneumonia is a ravaging condition of progressive lung scarring and destruction. Anti-inflammatory therapies including corticosteroids have limited efficacy in this ultimately fatal disorder. An important unmet need is to identify new agents that interact with key molecular pathways involved in the pathogenesis of pulmonary fibrosis to prevent progression or reverse fibrosis in these patients. Because aberrant activation of the Wnt/β-catenin signaling cascade occurs in lungs of patients with IPF, we have targeted this pathway for intervention in pulmonary fibrosis using ICG-001, a small molecule that specifically inhibits T-cell factor/β-catenin transcription in a cyclic AMP response-element binding protein binding protein (CBP)-dependent fashion. ICG-001 selectively blocks the β-catenin/CBP interaction without interfering with the β-catenin/p300 interaction. We report here that ICG-001 (5 mg/kg per day) significantly inhibits β-catenin signaling and attenuates bleomycin-induced lung fibrosis in mice, while concurrently preserving the epithelium. Administration of ICG-001 concurrent with bleomycin prevents fibrosis, and late administration is able to reverse established fibrosis and significantly improve survival. Because no effective treatment for IPF exists, selective inhibition of Wnt/β-catenin-dependent transcription suggests a potential unique therapeutic approach for pulmonary fibrosis.

Pulmonary Langerhans cell granulomatosis (histiocytosis X). A clinicopathologic study of 48 cases.

We report the clinical and histologic findings of lung biopsies from 48 patients with pulmonary Langerhans cell granulomatosis (PLCG) and show how special techniques such as immunohistochemistry, electron microscopy (EM), and high resolution computerized tomography (HRCT) of the lungs can be useful in diagnostically challenging cases. Nineteen patients were men and 29 were women. The median age was 33 years (range 15–54 years). Two had pituitary involvement. Bone lesions were observed in four patients and biopsy proven in two. All patients were cigarette smokers. In six patients HRCT revealed a combination of thin-walled cystic and nodular lesions. The pathologic diagnosis was established on the basis of open lung biopsy specimens in 44 cases and trans-bronchial biopsy specimens in 4 of 10 cases. In two trans-bronchial biopsies, diagnostic PLCG infiltrates were seen in toluidine blue-stained thick sections in the tissue processed for EM but not on the tissue processed for histology. EM in both of these cases revealed Birbeck granules within LCs. The diagnosis was supported by a positive bone biopsy in one of these patients and characteristic HRCT findings in the other. The antibody to S100 protein stained the LC infiltrates in the five cases studied. This staining and the characteristic findings on HRCT confirmed the diagnosis in one case in which the PLCG lesions were obscured by atelectasis. The frequent finding of intraluminal fibrosis (78% of open lung biopsies) supports the recent suggestion that this alteration plays an important role in the pathogenesis of fibrotic remodeling in PLCG. The strong association of PLCG with cigarette smoking and the frequent peribronchiolar location of PLCG lesions (87% of open lung biopsies) in our cases are consistent with the concept that in adults this disorder is associated with an abnormal response to cigarette smoke.

Effect of glutathione aerosol on oxidant-antioxidant imbalance in idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is characterised by alveolar inflammation, exaggerated release of oxidants, and subnormal concentrations of the antioxidant glutathione in respiratory epithelial lining fluid (ELF). Glutathione (600 mg twice daily for 3 days) was given by aerosol to 10 patients with IPF. Total ELF glutathione rose transiently, ELF oxidised glutathione concentrations increased, and there was a decrease in spontaneous superoxide anion release by alveolar macrophages. Thus, glutathione by aerosol could be a means of reversing the oxidant-antioxidant imbalance in IPF.

Interactions Between β-Catenin and Transforming Growth Factor-β Signaling Pathways Mediate Epithelial-Mesenchymal Transition and Are Dependent on the Transcriptional Co-activator cAMP-response Element-binding Protein (CREB)-binding Protein (CBP)

Background: Direct evidence for molecular interdependence between transforming growth factor-β (TGF-β) and Wnt pathways in mesenchymal gene regulation during epithelial-mesenchymal transition (EMT) is limited. Results: TGF-β induction of α-smooth muscle actin (α-SMA) involves ternary complex formation among Smad3, β-catenin, and CBP. Conclusion: TGF-β and β-catenin/CBP-dependent pathways coordinately regulate α-SMA induction. Significance: Inhibition of β-catenin/CBP-dependent effects of TGF-β suggests a novel therapeutic approach to EMT/fibrosis. Interactions between transforming growth factor-β (TGF-β) and Wnt are crucial to many biological processes, although specific targets, rationale for divergent outcomes (differentiation versus block of epithelial proliferation versus epithelial-mesenchymal transition (EMT)) and precise mechanisms in many cases remain unknown. We investigated β-catenin-dependent and transforming growth factor-β1 (TGF-β1) interactions in pulmonary alveolar epithelial cells (AEC) in the context of EMT and pulmonary fibrosis. We previously demonstrated that ICG-001, a small molecule specific inhibitor of the β-catenin/CBP (but not β-catenin/p300) interaction, ameliorates and reverses pulmonary fibrosis and inhibits TGF-β1-mediated α-smooth muscle actin (α-SMA) and collagen induction in AEC. We now demonstrate that TGF-β1 induces LEF/TCF TOPFLASH reporter activation and nuclear β-catenin accumulation, while LiCl augments TGF-β-induced α-SMA expression, further confirming co-operation between β-catenin- and TGF-β-dependent signaling pathways. Inhibition and knockdown of Smad3, knockdown of β-catenin and overexpression of ICAT abrogated effects of TGF-β1 on α-SMA transcription/expression, indicating a requirement for β-catenin in these Smad3-dependent effects. Following TGF-β treatment, co-immunoprecipitation demonstrated direct interaction between endogenous Smad3 and β-catenin, while chromatin immunoprecipitation (ChIP)-re-ChIP identified spatial and temporal regulation of α-SMA via complex formation among Smad3, β-catenin, and CBP. ICG-001 inhibited α-SMA expression/transcription in response to TGF-β as well as α-SMA promoter occupancy by β-catenin and CBP, demonstrating a previously unknown requisite TGF-β1/β-catenin/CBP-mediated pro-EMT signaling pathway. Clinical relevance was shown by β-catenin/Smad3 co-localization and CBP expression in AEC of IPF patients. These findings suggest a new therapeutic approach to pulmonary fibrosis by specifically uncoupling CBP/catenin-dependent signaling downstream of TGF-β.

Epithelium-specific deletion of TGF-β receptor type II protects mice from bleomycin-induced pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a chronic fibroproliferative pulmonary disorder for which there are currently no treatments. Although the etiology of IPF is unknown, dysregulated TGF-β signaling has been implicated in its pathogenesis. Recent studies also suggest a central role for abnormal epithelial repair. In this study, we sought to elucidate the function of epithelial TGF-β signaling via TGF-β receptor II (TβRII) and its contribution to fibrosis by generating mice in which TβRII was specifically inactivated in mouse lung epithelium. These mice, which are referred to herein as TβRIINkx2.1-cre mice, were used to determine the impact of TβRII inactivation on (a) embryonic lung morphogenesis in vivo; and (b) the epithelial cell response to TGF-β signaling in vitro and in a bleomycin-induced, TGF-β-mediated mouse model of pulmonary fibrosis. Although postnatally viable with no discernible abnormalities in lung morphogenesis and epithelial cell differentiation, TβRIINkx2.1-cre mice developed emphysema, suggesting a requirement for epithelial TβRII in alveolar homeostasis. Absence of TβRII increased phosphorylation of Smad2 and decreased, but did not entirely block, phosphorylation of Smad3 in response to endogenous/physiologic TGF-β. However, TβRIINkx2.1-cre mice exhibited increased survival and resistance to bleomycin-induced pulmonary fibrosis. To our knowledge, these findings are the first to demonstrate a specific role for TGF-β signaling in the lung epithelium in the pathogenesis of pulmonary fibrosis.

Future Directions in Idiopathic Pulmonary Fibrosis Research. An NHLBI Workshop Report

The median survival of patients with idiopathic pulmonary fibrosis (IPF) continues to be approximately 3 years from the time of diagnosis, underscoring the lack of effective medical therapies for this disease. In the United States alone, approximately 40,000 patients die of this disease annually. In November 2012, the NHLBI held a workshop aimed at coordinating research efforts and accelerating the development of IPF therapies. Basic, translational, and clinical researchers gathered with representatives from the NHLBI, patient advocacy groups, pharmaceutical companies, and the U.S. Food and Drug Administration to review the current state of IPF research and identify priority areas, opportunities for collaborations, and directions for future research. The workshop was organized into groups that were tasked with assessing and making recommendations to promote progress in one of the following six critical areas of research: (1) biology of alveolar epithelial injury and aberrant repair; (2) role of extracellular matrix; (3) preclinical modeling; (4) role of inflammation and immunity; (5) genetic, epigenetic, and environmental determinants; (6) translation of discoveries into diagnostics and therapeutics. The workshop recommendations provide a basis for directing future research and strategic planning by scientific, professional, and patient communities and the NHLBI.

Role of Endoplasmic Reticulum Stress in Epithelial–Mesenchymal Transition of Alveolar Epithelial Cells: Effects of Misfolded Surfactant Protein

Endoplasmic reticulum (ER) stress has been implicated in alveolar epithelial type II (AT2) cell apoptosis in idiopathic pulmonary fibrosis. We hypothesized that ER stress (either chemically induced or due to accumulation of misfolded proteins) is also associated with epithelial-mesenchymal transition (EMT) in alveolar epithelial cells (AECs). ER stress inducers, thapsigargin (TG) or tunicamycin (TN), increased expression of ER chaperone, Grp78, and spliced X-box binding protein 1, decreased epithelial markers, E-cadherin and zonula occludens-1 (ZO-1), increased the myofibroblast marker, α-smooth muscle actin (α-SMA), and induced fibroblast-like morphology in both primary AECs and the AT2 cell line, RLE-6TN, consistent with EMT. Overexpression of the surfactant protein (SP)-C BRICHOS mutant SP-C(ΔExon4) in A549 cells increased Grp78 and α-SMA and disrupted ZO-1 distribution, and, in primary AECs, SP-C(ΔExon4) induced fibroblastic-like morphology, decreased ZO-1 and E-cadherin and increased α-SMA, mechanistically linking ER stress associated with mutant SP to fibrosis through EMT. Whereas EMT was evident at lower concentrations of TG or TN, higher concentrations caused apoptosis. The Src inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4]pyramidine) (PP2), abrogated EMT associated with TN or TG in primary AECs, whereas overexpression of SP-C(ΔExon4) increased Src phosphorylation, suggesting a common mechanism. Furthermore, increased Grp78 immunoreactivity was observed in AT2 cells of mice after bleomycin injury, supporting a role for ER stress in epithelial abnormalities in fibrosis in vivo. These results demonstrate that ER stress induces EMT in AECs, at least in part through Src-dependent pathways, suggesting a novel role for ER stress in fibroblast accumulation in pulmonary fibrosis.

EMT and Interstitial Lung Disease: A Mysterious Relationship

Purpose of reviewPathogenesis of interstitial lung diseases (ILD) has largely been investigated in the context of the most frequent ILD, idiopathic pulmonary fibrosis (IPF). We review studies of epithelial-to-mesenchymal transition (EMT) and discuss its potential contribution to collagen-producing (myo)fibroblasts in IPF. Recent findingsEndoplasmic reticulum (ER) stress leading to epithelial apoptosis has been reported as a potential etiologic factor in fibrosis. Recent studies further suggest EMT as a link between ER stress and fibrosis. Combinatorial interactions among Smad3, &bgr;-catenin and other transcriptional co-activators at the &agr;-smooth muscle actin (&agr;-SMA) promoter provide direct evidence for crosstalk between transforming growth factor-&bgr; (TGF&bgr;) and &bgr;-catenin pathways during EMT. Lineage tracing yielded conflicting results, with two recent studies supporting and one opposing a role for EMT in lung fibrosis. SummaryAdvances have been made in elucidating causes and mechanisms of EMT, potentially leading to new treatment options, although contributions of EMT to lung fibrosis in vivo remain controversial. In addition to EMT providing a direct source of (myo)fibroblasts, expression of mesenchymal markers may reflect epithelial injury, in which case inhibition of EMT might be deleterious. EMT-derived cells may also contribute to aberrant epithelial–mesenchymal crosstalk that promotes fibrogenesis.

Regulation of breast cancer metastasis by Runx2 and estrogen signaling: the role of SNAI2

IntroductionIn contrast to its role in breast cancer (BCa) initiation, estrogen signaling has a protective effect in later stages, where estrogen receptor (ER)α loss associates with aggressive metastatic disease. We asked whether the beneficial effect of estrogen signaling in late-stage BCa is attributable to the recently reported estrogen-mediated antagonism of the pro-metastatic transcription factor Runx2.MethodsMCF7/Rx2dox breast cancer cells were engineered with a lentivirus expressing Runx2 in response to doxycycline (dox). Cells treated with dox and/or estradiol (E2) were subjected to genome-wide expression profiling, RT-qPCR analysis of specific genes, and Matrigel™ invasion assays. Knockdown of genes of interest was performed using lentiviruses expressing appropriate shRNAs, either constitutively or in response to dox. Gene expression in BCa tumors was investigated using a cohort of 557 patients compiled from publicly available datasets. Association of gene expression with clinical metastasis was assessed by dichotomizing patients into those expressing genes of interest at either high or low levels, and comparing the respective Kaplan-Meier curves of metastasis-free survival.ResultsRunx2 induced epithelial-mesenchymal transition (EMT) evidenced by acquisition of a fibroblastic morphology, decreased expression of E-cadherin, increased expression of vimentin and invasiveness. Runx2 stimulated SNAI2 expression in a WNT- and transforming growth factor (TGF)β-dependent manner, and knockdown of SNAI2 abrogated the pro-metastatic activities of Runx2. E2 antagonized the pro-metastatic activities of Runx2, including SNAI2 upregulation. In primary BCa tumors, Runx2 activity, SNAI2 expression, and metastasis were positively correlated, and SNAI2 expression was negatively correlated with ERα. However, the negative correlation between SNAI2 and ERα in bone-seeking BCa cells was weaker than the respective negative correlation in tumors seeking lung. Furthermore, the absence of ERα in primary tumors was associated with lung- and brain- but not with bone metastasis, and tumor biopsies from bone metastatic sites displayed the unusual combination of high Runx2/SNAI2 and high ERα expression.ConclusionsE2 antagonizes Runx2-induced EMT and invasiveness of BCa cells, partly through attenuating expression of SNAI2, a Runx2 target required for mediating its pro-metastatic property. That ERα loss promotes non-osseous metastasis by unleashing Runx2/SNAI2 is supported by the negative correlation observed in corresponding tumors. Unknown mechanisms in bone-seeking BCa allow high Runx2/SNAI2 expression despite high ERα level