ABSTRACT
Idiopathic pulmonary fibrosis (IPF) is a chronic progressive interstitial lung disease characterized by patchy scarring of the distal lung with limited therapeutic options and poor prognosis. Here, we show that conditional deletion of the ubiquitin ligase Nedd4-2 (Nedd4l) in lung epithelial cells in adult mice produces chronic lung disease sharing key features with IPF including progressive fibrosis and bronchiolization with increased expression of Muc5b in peripheral airways, honeycombing and characteristic alterations in the lung proteome. NEDD4-2 is implicated in the regulation of the epithelial Na+ channel critical for proper airway surface hydration and mucus clearance and the regulation of TGFß signaling, which promotes fibrotic remodeling. Our data support a role of mucociliary dysfunction and aberrant epithelial pro-fibrotic response in the multifactorial disease pathogenesis. Further, treatment with the anti-fibrotic drug pirfenidone reduced pulmonary fibrosis in this model. This model may therefore aid studies of the pathogenesis and therapy of IPF.
Subject(s)
Epithelial Cells/pathology , Idiopathic Pulmonary Fibrosis/genetics , Lung/pathology , Nedd4 Ubiquitin Protein Ligases/genetics , Nedd4 Ubiquitin Protein Ligases/metabolism , Adult , Aged , Animals , Biopsy , Disease Models, Animal , Epithelial Sodium Channels/metabolism , Humans , Idiopathic Pulmonary Fibrosis/drug therapy , Idiopathic Pulmonary Fibrosis/pathology , Lung/cytology , Mice , Mice, Knockout , Middle Aged , Mucin-5B/metabolism , Proteomics , Pyridones/administration & dosage , UbiquitinationABSTRACT
Activation of protein kinase Akt via its direct phosphorylation by mammalian target of rapamycin (mTOR) complex 2 (mTORC2) couples extracellular growth and survival cues with pathways controlling cell growth and proliferation, yet how growth factors target the activity of mTORC2 toward Akt is unknown. In this study, we examine the localization of the obligate mTORC2 component, mSin1, inside cells and report the development of a reporter to examine intracellular localization and regulation by growth factors of the endogenous mTORC2 activity. Using a combination of imaging and biochemical approaches, we demonstrate that inside cells, mTORC2 activity localizes to the plasma membrane, mitochondria, and a subpopulation of endosomal vesicles. We show that unlike the endosomal pool, the activity and localization of mTORC2 via the Sin1 pleckstrin homology domain at the plasma membrane is PI3K and growth factor independent. Furthermore, we show that membrane recruitment is sufficient for Akt phosphorylation in response to growth factors. Our results indicate the existence of spatially separated mTORC2 populations with distinct sensitivity to PI3K inside cells and suggest that intracellular localization could contribute to regulation of mTORC2 activity toward Akt.
Subject(s)
Carrier Proteins/metabolism , Multiprotein Complexes/metabolism , TOR Serine-Threonine Kinases/metabolism , Carrier Proteins/genetics , Cell Membrane/enzymology , Endosomes/enzymology , HEK293 Cells , HeLa Cells , Humans , MCF-7 Cells , Mechanistic Target of Rapamycin Complex 2 , Microscopy, Confocal , Mitochondria/enzymology , Monomeric GTP-Binding Proteins , Multiprotein Complexes/genetics , Phosphorylation , Protein Interaction Domains and Motifs , Proto-Oncogene Proteins c-akt/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Signal Transduction , TOR Serine-Threonine Kinases/genetics , Time Factors , Time-Lapse Imaging , TransfectionABSTRACT
Non-small cell lung cancer (NSCLC) is characterized by early metastasis and has the highest mortality rate among all solid tumors, with the majority of patients diagnosed at an advanced stage where curative therapeutic options are lacking. In this study, we identify a targetable mechanism involving TGFß elevation that orchestrates tumor progression in this disease. Substantial activation of this pathway was detected in human lung cancer tissues with concomitant downregulation of BAMBI, a negative regulator of the TGFß signaling pathway. Alterations of epithelial-to-mesenchymal transition (EMT) marker expression were observed in lung cancer samples compared with tumor-free tissues. Distinct alterations in the DNA methylation of the gene regions encoding TGFß pathway components were detected in NSCLC samples compared with tumor-free lung tissues. In particular, epigenetic silencing of BAMBI was identified as a hallmark of NSCLC. Reconstitution of BAMBI expression in NSCLC cells resulted in a marked reduction of TGFß-induced EMT, migration, and invasion in vitro, along with reduced tumor burden and tumor growth in vivo In conclusion, our results demonstrate how BAMBI downregulation drives the invasiveness of NSCLC, highlighting TGFß signaling as a candidate therapeutic target in this setting. Cancer Res; 76(13); 3785-801. ©2016 AACR.
Subject(s)
Adenocarcinoma/pathology , Biomarkers, Tumor/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Carcinoma, Squamous Cell/pathology , Lung Neoplasms/pathology , Membrane Proteins/metabolism , Transforming Growth Factor beta/metabolism , Adenocarcinoma/genetics , Adenocarcinoma/metabolism , Aged , Animals , Apoptosis , Biomarkers, Tumor/genetics , Blotting, Western , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/metabolism , Case-Control Studies , Cell Movement , Cell Proliferation , DNA Methylation , Down-Regulation , Epigenesis, Genetic , Epithelial-Mesenchymal Transition , Female , Fluorescent Antibody Technique , Follow-Up Studies , Humans , Immunoenzyme Techniques , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Male , Membrane Proteins/genetics , Mice , Mice, Nude , Neoplasm Invasiveness , Neoplasm Staging , Prognosis , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , Transforming Growth Factor beta/genetics , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
In the following work the influence of air-ions and ozone on the selected microorganisms, first of all on Staphylococcus epidermidis is discussed. Carried out investigations proved that the levels of air-ions concentrations, causing specific changes in the development of particular microorganisms, are very differentiated and depend on individual parameters of those organisms. These levels are generally characterized by high values of concentrations, range n +/- =4 x 10(6) ions/cm3 and are connected with the type of the air-ions used. However investigations did not confirm any negative influence of the positive air-ions seen as a destructive factor upon any of the investigated microorganisms.
