Epithelial-mesenchymal transitions (EMT) are essential for organogenesis and triggered in carcinoma progression into an invasive state1. Transforming growth factor-β (TGF-β) cooperates with signalling pathways, such as Ras and Wnt, to induce EMT2-5, but the molecular mechanisms are not clear. Here, we report that SMAD3 and SMAD4 interact and form a complex with SNAIL1, a transcriptional repressor and promoter of EMT6, 7. The SNAIL1-SMAD3/4 complex was targeted to the gene promoters of CAR, a tight junction protein, and E-cadherin during TGF-β-driven EMT in breast epithelial cells. SNAIL1 and SMAD3/4 acted as co-repressors of CAR, occludin, claudin-3 and E-cadherin promoters in transfected cells. Conversely, co-silencing of SNAIL1 and SMAD4 by siRNA inhibited the repression of CAR and occludin during EMT. Moreover, loss of CAR and E-cadherin correlated with nuclear co-expression of SNAIL1 and SMAD3/4 in a mouse model of breast carcinoma and at the invasive fronts of human breast cancer. We propose that activation of a SNAIL1-SMAD3/4 transcriptional complex represents a novel mechanism of gene repression during EMT.
Rationale: Infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease (COVID-19), a predominantly respiratory illness. The first step in SARS-CoV-2 infection is binding of the virus to ACE2 (angiotensin-converting enzyme 2) on the airway epithelium. Objectives: The objective was to gain insight into the expression of ACE2 in the human airway epithelium. Methods: Airway epithelia sampled by fiberoptic bronchoscopy of trachea, large airway epithelia (LAE), and small airway epithelia (SAE) of nonsmokers and smokers were analyzed for expression of ACE2 and other coronavirus infection–related genes using microarray, RNA sequencing, and 10x single-cell transcriptome analysis, with associated examination of ACE2 -related microRNA. Measurements and Main Results: 1 ) ACE2 is expressed similarly in the trachea and LAE, with lower expression in the SAE; 2 ) in the SAE, ACE2 is expressed in basal, intermediate, club, mucus, and ciliated cells; 3 ) ACE2 is upregulated in the SAE by smoking, significantly in men; 4 ) levels of miR-1246 expression could play a role in ACE2 upregulation in the SAE of smokers; and 5 ) ACE2 is expressed in airway epithelium differentiated in vitro on air–liquid interface cultures from primary airway basal stem/progenitor cells; this can be replicated using LAE and SAE immortalized basal cell lines derived from healthy nonsmokers. Conclusions: ACE2 , the gene encoding the receptor for SARS-CoV-2, is expressed in the human airway epithelium, with variations in expression relevant to the biology of initial steps in SARS-CoV-2 infection.
Modified viruses are used as gene transfer vectors because of their ability to transfer genetic material efficiently to the nucleus of a target cell. To better understand intracellular translocation of adenovirus serotype 5 (Ad), fluorophores were covalently conjugated to Ad capsids, and movement of fluorescent Ad within the cytoplasm was observed during the first hour of infection of a human lung epithelial carcinoma cell line (A549). Ad translocation was characterized with respect to its ability to achieve nuclear envelope localization as well as directed movement in the cytoplasm. Whereas Ad achieved efficient nuclear localization 60 min after infection of A549 cells under control conditions, depolymerization of the microtubule cytoskeleton by addition of 25 microM nocodazole reversibly inhibited development of nuclear localization. In contrast, depolymerization of microfilaments by addition of 1 microM cytochalasin D had no effect on nuclear localization. Direct video observation of Ad motility showed that nocodazole, but not cytochalasin D, caused a reversible decrease in rapid linear translocations of Ad in the cytoplasm of A549 cells. Microinjection of function-blocking antibodies against the microtubule-dependent motor protein, cytoplasmic dynein, but not kinesin, blocked nuclear localization of Ad, consistent with net minus end-directed motility indicated by accumulation of Ad at mitotic spindles. Fluorescence ratio imaging revealed a neutral pH in the environment of translocating Ad, leading to a model in which the interaction of Ad with an intact microtubule cytoskeleton and functional cytoplasmic dynein occurs after escape from endosomes and is a necessary prerequisite to nuclear localization of adenovirus serotype 5.
The pathogenic agent, adenovirus (Ad), has taken on a new role as a vector for gene transfer in both laboratory and clinical settings. To help understand the intracellular pathways and fate of Ad gene transfer vectors, we covalently conjugated fluorophores to E1-, E3- Ad vectors and used quantitative fluorescence microscopy to assess essential steps of Ad vector gene transfer to the A549 human epithelial lung cell line including binding, internalization, escape from endosomes, translocation to the nucleus, dissociation of capsids and gene expression. The data demonstrate that Ad internalizes with a t1/2 2.5 min, breaks out of endosomes early, likely prior to endosome-endosome fusion, exhibits sustained, intracellular velocities averaging 0.58 microm/sec, and translocates to the nucleus with >80% of internalized fluorophore demonstrating nuclear localization within 60 min of infection. Interestingly, 24 hr after infection, half of the initially internalized fluorescence was detected but lacked nuclear localization, suggesting that the capsid is released from the nucleus and is likely degraded. Fluorescent labeling of virions provides a novel quantitative, morphological strategy to characterize the interaction of gene transfer vectors with the intracellular environment.
BackgroundWhereas cilia damage and reduced cilia beat frequency have been implicated as causative of reduced mucociliary clearance in smokers, theoretically mucociliary clearance could also be affected by cilia length. Based on models of mucociliary clearance predicting that cilia length must exceed the 6–7 µm airway surface fluid depth to generate force in the mucus layer, we hypothesized that cilia height may be decreased in airway epithelium of normal smokers compared to nonsmokers.Methodology/Principal FindingsCilia length in normal nonsmokers and smokers was evaluated in aldehyde-fixed, paraffin-embedded endobronchial biopsies, and air-dried and hydrated samples were brushed from human airway epithelium via fiberoptic bronchoscopy. In 28 endobronchial biopsies, healthy smoker cilia length was reduced by 15% compared to nonsmokers (p<0.05). In 39 air-dried samples of airway epithelial cells, smoker cilia length was reduced by 13% compared to nonsmokers (p<0.0001). Analysis of the length of individual, detached cilia in 27 samples showed that smoker cilia length was reduced by 9% compared to nonsmokers (p<0.05). Finally, in 16 fully hydrated, unfixed samples, smoker cilia length was reduced 7% compared to nonsmokers (p<0.05). Using genome-wide analysis of airway epithelial gene expression we identified 6 cilia-related genes whose expression levels were significantly reduced in healthy smokers compared to healthy nonsmokers.Conclusions/SignificanceModels predict that a reduction in cilia length would reduce mucociliary clearance, suggesting that smoking-associated shorter airway epithelial cilia play a significant role in the pathogenesis of smoking-induced lung disease.
ATP hydrolysis has been regarded as a general requirement for internalization processes in mammalian cells. We found, however, that treatment of ATP-depleted macrophages and fibroblasts with exogenous sphingomyelinase (SMase) rapidly induces formation of numerous vesicles that pinch off from the plasma membrane; the process is complete within 10 min after adding SMase. By electron microscopy, the SMase-induced vesicles are ∼400 nm in diameter and lack discernible coats. 15–30% of plasma membrane is internalized by SMase treatment, and there is no detectable enrichment of either clathrin or caveolin in these vesicles. When ATP is restored to the cells, the SMase-induced vesicles are able to deliver fluid-phase markers to late endosomes/lysosomes and return recycling receptors, such as transferrin receptors, back to the plasma membrane. We speculate that hydrolysis of sphingomyelin on the plasma membrane causes inward curvature and subsequent fusion to form sealed vesicles. Many cell types express a SMase that can be secreted or delivered to endosomes and lysosomes. The hydrolysis of sphingomyelin by these enzymes is activated by several signaling pathways, and this may lead to formation of vesicles by the process described here.
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