Integrin-β4 regulates the dynamic changes of phenotypic characteristics in association with epithelial-mesenchymal transition (EMT) and RhoA activity in airway epithelial cells during injury and repair

Background: In airway disease such as asthma a hyperactive cellular event of epithelial-mesenchymal transition (EMT) is considered as the mechanism of pathological airway tissue remodeling after injury to the airway epithelium. And the initiation of EMT in the airways depends on the epithelial disruption involving dissolution and/or destabilization of the adhesive structures between the cells and ECM. Previously, we have shown that integrin-β4, an epithelial adhesion molecule in bronchial epithelium is an important regulator of cell proliferation and wound repair in human airway epithelial cells. Therefore, in this study we aimed to investigate whether integrin-β4 also regulates EMT phenotypes during injury and repair in airway epithelial cells of both wild type/integrin-β4-/- mice in vivo and cultured cells treated with integrin-β4/nonsense siRNA in vitro. Methods: We induced injury to the airway epithelial cells by either repeated exposure to ozone and mechanical scratch wound, and subsequently examined the EMT-related phenotypic features in the airway epithelial cells including biomarkers expression, adhesion and cytoskeleton reorganization and cell stiffness. Results: The results show that in response to injury (ozone exposure/scratch wound) and subsequent spontaneous repair (ozone withdrawal/wound healing) both in vivo and in vitro, the airway epithelial cells underwent dynamic changes in the epithelial and mesenchymal biomarkers expression, adhesion and cytoskeleton structures as well as cell stiffness, all together exhibiting enhanced EMT phenotypic features after injury and reversal of the injury-induced effects during repair. Importantly, these injury/repair-associated EMT phenotypic changes in airway epithelial cells appeared to be dependent on integrin-β4 expression. More specifically, when integrin-β4 was deficient in mice (integrin-β4-/-) the repair of ozone-injured airway epithelium was impaired and the recovery of ozone-enhanced EMT biomarkers expression in the airway epithelium was delayed. Similarly, in the scratch wounded airway epithelial cells with integrin-β4 knockdown, the cells were impaired in all aspects related to EMT during wound and repair including cell proliferation, wound closure rate, adhesion and cytoskeleton protein expression (vinculin and vimentin), mesenchymal-like F-actin reorganization, cell stiffness and RhoA activation. Conclusion: Taken together, these results suggested that integrin-β4 may be essential in regulating the effects of injury and repair on EMT in airway epithelial cells via influencing both the cell adhesion to ECM and cells' physical phenotypes through RhoA signaling pathway.

[1]  J. Sznajder,et al.  Epithelial cell–specific loss of function of Miz1 causes a spontaneous COPD-like phenotype and up-regulates Ace2 expression in mice , 2020, Science Advances.

[2]  B. Ryffel,et al.  Ozone-Induced Aryl Hydrocarbon Receptor Activation Controls Lung Inflammation via Interleukin-22 Modulation , 2020, Frontiers in Immunology.

[3]  P. Janmey,et al.  Loss of Vimentin Enhances Cell Motility through Small Confining Spaces. , 2019, Small.

[4]  Haiyang Yu,et al.  Cell Biology International , 2019 .

[5]  J. Jones,et al.  Complexes of α6β4 integrin and vimentin act as signaling hubs to regulate epithelial cell migration , 2018, Journal of Cell Science.

[6]  Caixia Liu,et al.  CTNNAL1 inhibits ozone‐induced epithelial–mesenchymal transition in human bronchial epithelial cells , 2018, Experimental physiology.

[7]  Juan Li,et al.  Nucleoskeletal stiffness regulates stem cell migration and differentiation through lamin A/C , 2018, Journal of cellular physiology.

[8]  Jianxin Jiang,et al.  ITGB4 is essential for containing HDM‐induced airway inflammation and airway hyperresponsiveness , 2018, Journal of leukocyte biology.

[9]  M. Eisenstein,et al.  Conformational states during vinculin unlocking differentially regulate focal adhesion properties , 2017, bioRxiv.

[10]  Qing Luo,et al.  Cell stiffness determined by atomic force microscopy and its correlation with cell motility. , 2016, Biochimica et biophysica acta.

[11]  Christopher S. Chen,et al.  The mechanical regulation of integrin–cadherin crosstalk organizes cells, signaling and forces , 2016, Journal of Cell Science.

[12]  D. Costea,et al.  Vimentin-mediated regulation of cell motility through modulation of beta4 integrin protein levels in oral tumor derived cells. , 2016, The international journal of biochemistry & cell biology.

[13]  J. Garcia,et al.  Role of Integrin β4 in Lung Endothelial Cell Inflammatory Responses to Mechanical Stress , 2015, Scientific Reports.

[14]  T. Tran,et al.  Integrins as Therapeutic Targets for Respiratory Diseases , 2015, Current molecular medicine.

[15]  Steve Pawlizak,et al.  Testing the differential adhesion hypothesis across the epithelial−mesenchymal transition , 2015 .

[16]  David Grundy,et al.  Principles and standards for reporting animal experiments in The Journal of Physiology and Experimental Physiology , 2015, The Journal of physiology.

[17]  C. Dermardirossian,et al.  Protein Tyrosine Phosphatase-PEST and β8 Integrin Regulate Spatiotemporal Patterns of RhoGDI1 Activation in Migrating Cells , 2015, Molecular and Cellular Biology.

[18]  H. Meurs,et al.  Integrins: therapeutic targets in airway hyperresponsiveness and remodelling? , 2014, Trends in pharmacological sciences.

[19]  C. Figdor,et al.  Actin-binding proteins differentially regulate endothelial cell stiffness, ICAM-1 function and neutrophil transmigration , 2014, Journal of Cell Science.

[20]  X. Qu,et al.  Analysis on the Relevance of Asthma Susceptibility with the Alteration of Integrin β 4 Expression , 2014, PloS one.

[21]  M. Stepp,et al.  A central role for vimentin in regulating repair function during healing of the lens epithelium , 2014, Molecular biology of the cell.

[22]  David A Weitz,et al.  The role of vimentin intermediate filaments in cortical and cytoplasmic mechanics. , 2013, Biophysical journal.

[23]  J. Tomasek,et al.  Mechanoregulation of the Myofibroblast in Wound Contraction, Scarring, and Fibrosis: Opportunities for New Therapeutic Intervention. , 2013, Advances in wound care.

[24]  Ning Wang,et al.  Mechanotransduction at focal adhesions: from physiology to cancer development , 2013, Journal of cellular and molecular medicine.

[25]  S. O'Dea,et al.  Bone morphogenetic proteins enhance an epithelial-mesenchymal transition in normal airway epithelial cells during restitution of a disrupted epithelium , 2013, Respiratory Research.

[26]  N. D’Silva,et al.  Biomarkers of Epithelial-Mesenchymal Transition in Squamous Cell Carcinoma , 2013, Journal of dental research.

[27]  W. Seeger,et al.  E-cadherin controls bronchiolar progenitor cells and onset of preneoplastic lesions in mice. , 2012, Neoplasia.

[28]  Roger D Kamm,et al.  Integrating focal adhesion dynamics, cytoskeleton remodeling, and actin motor activity for predicting cell migration on 3D curved surfaces of the extracellular matrix. , 2012, Integrative biology : quantitative biosciences from nano to macro.

[29]  Jean Paul Thiery,et al.  Early events in cell adhesion and polarity during epithelial-mesenchymal transition , 2012, Journal of Cell Science.

[30]  Caixia Liu,et al.  Identification of Transcription Factors Regulating CTNNAL1 Expression in Human Bronchial Epithelial Cells , 2012, PloS one.

[31]  J. Y. Cho.  Recent Advances in Mechanisms and Treatments of Airway Remodeling in Asthma: A Message from the Bench Side to the Clinic , 2011, The Korean journal of internal medicine.

[32]  K. Meldrum,et al.  Arterially delivered mesenchymal stem cells prevent obstruction-induced renal fibrosis. , 2011, The Journal of surgical research.

[33]  Henry C Wong,et al.  Finite element analysis of the effects of focal adhesion mechanical properties and substrate stiffness on cell migration. , 2011, Journal of biomechanics.

[34]  Michael W. Davidson,et al.  Nanoscale architecture of integrin-based cell adhesions , 2010, Nature.

[35]  E. Reed,et al.  HLA Class I Molecules Partner with Integrin β4 to Stimulate Endothelial Cell Proliferation and Migration , 2010, Science Signaling.

[36]  Martin A. Schwartz,et al.  Cell adhesion: integrating cytoskeletal dynamics and cellular tension , 2010, Nature Reviews Molecular Cell Biology.

[37]  Dan Zeng,et al.  Integrin beta4 was downregulated on the airway epithelia of asthma patients. , 2010, Acta biochimica et biophysica Sinica.

[38]  S. T. Howard,et al.  Involvement of integrin beta4 in ozone stress-induced airway hyperresponsiveness. , 2010, Biochemical and biophysical research communications.

[39]  R. Goldman,et al.  Vimentin induces changes in cell shape, motility, and adhesion during the epithelial to mesenchymal transition , 2010, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[40]  Hui-jun Liu,et al.  Wound repair and anti-oxidative capacity is regulated by ITGB4 in airway epithelial cells , 2010, Molecular and Cellular Biochemistry.

[41]  S. Fagerholm,et al.  Filamin and filamin-binding proteins in integrin-regulation and adhesion. Focus on: "FilaminA is required for vimentin-mediated cell adhesion and spreading". , 2010, American journal of physiology. Cell physiology.

[42]  Jonathan Stricker,et al.  Mechanics of the F-actin cytoskeleton. , 2010, Journal of biomechanics.

[43]  Daniel A. Fletcher,et al.  Cell mechanics and the cytoskeleton , 2010, Nature.

[44]  P. Paré,et al.  Stress and strain in the contractile and cytoskeletal filaments of airway smooth muscle. , 2009, Pulmonary pharmacology & therapeutics.

[45]  M. Nebuloni,et al.  Direct contribution of epithelium to organ fibrosis: epithelial-mesenchymal transition. , 2009, Human pathology.

[46]  E. Neilson,et al.  Biomarkers for epithelial-mesenchymal transitions. , 2009, The Journal of clinical investigation.

[47]  Chang-Hao Yang,et al.  Overexpression of integrin alpha6 and beta4 enhances adhesion and proliferation of human retinal pigment epithelial cells on layers of porcine Bruch's membrane. , 2009, Experimental eye research.

[48]  Fei Wang,et al.  Material Properties of the Cell Dictate Stress-induced Spreading and Differentiation in Embryonic Stem Cells Growing Evidence Suggests That Physical Microenvironments and Mechanical Stresses, in Addition to Soluble Factors, Help Direct Mesenchymal-stem-cell Fate. However, Biological Responses to a L , 2022 .

[49]  J. Gern,et al.  Serial culture of murine primary airway epithelial cells and ex vivo replication of human rhinoviruses. , 2008, Journal of immunological methods.

[50]  A. Sonnenberg,et al.  Regulation of hemidesmosome disassembly by growth factor receptors. , 2008, Current opinion in cell biology.

[51]  Tae Joo Park,et al.  Dishevelled controls apical docking and planar polarization of basal bodies in ciliated epithelial cells , 2008, Nature Genetics.

[52]  Petra Beli,et al.  WAVE and Arp2/3 jointly inhibit filopodium formation by entering into a complex with mDia2 , 2008, Nature Cell Biology.

[53]  J. Miyoshi,et al.  Structural and functional associations of apical junctions with cytoskeleton. , 2008, Biochimica et biophysica acta.

[54]  Biao Hu,et al.  Wound repair and proliferation of bronchial epithelial cells regulated by CTNNAL1 , 2008, Journal of cellular biochemistry.

[55]  P. Paré,et al.  Airway smooth muscle dynamics: a common pathway of airway obstruction in asthma. , 2007, The European respiratory journal.

[56]  S. Tsukita,et al.  Requirement of ZO-1 for the formation of belt-like adherens junctions during epithelial cell polarization , 2007, The Journal of cell biology.

[57]  Anthony G. Evans,et al.  A bio-chemo-mechanical model for cell contractility , 2006, Proceedings of the National Academy of Sciences.

[58]  K. Hahn,et al.  Spatiotemporal dynamics of RhoA activity in migrating cells , 2006, Nature.

[59]  Donald E Ingber,et al.  Cell tension, matrix mechanics, and cancer development. , 2005, Cancer cell.

[60]  D. Grigoryev,et al.  Simvastatin attenuates vascular leak and inflammation in murine inflammatory lung injury. , 2005, American journal of physiology. Lung cellular and molecular physiology.

[61]  A. Sonnenberg,et al.  Keratinocytes display normal proliferation, survival and differentiation in conditional β4-integrin knockout mice , 2005, Journal of Cell Science.

[62]  G. Kirfel,et al.  Membrane ruffles in cell migration: indicators of inefficient lamellipodia adhesion and compartments of actin filament reorganization. , 2005, Experimental cell research.

[63]  D. A. Hanson,et al.  Focal adhesion kinase: in command and control of cell motility , 2005, Nature Reviews Molecular Cell Biology.

[64]  Linhong Deng,et al.  Localized mechanical stress induces time-dependent actin cytoskeletal remodeling and stiffening in cultured airway smooth muscle cells. , 2004, American journal of physiology. Cell physiology.

[65]  D. Grigoryev,et al.  Cytoskeletal activation and altered gene expression in endothelial barrier regulation by simvastatin. , 2004, American journal of respiratory cell and molecular biology.

[66]  Jeffrey A. Whitsett,et al.  Conditional gene expression in the respiratory epithelium of the mouse , 2002, Transgenic Research.

[67]  N. Balaban,et al.  Adhesion-dependent cell mechanosensitivity. , 2003, Annual review of cell and developmental biology.

[68]  D. Sheppard.  Functions of pulmonary epithelial integrins: from development to disease. , 2003, Physiological reviews.

[69]  A. Hall,et al.  Rho GTPases in cell biology , 2002, Nature.

[70]  W. Kiosses,et al.  Regulation of the small GTP‐binding protein Rho by cell adhesion and the cytoskeleton , 1999, The EMBO journal.

[71]  I. Rabinovitz,et al.  The Integrin α6β4 Functions in Carcinoma Cell Migration on Laminin-1 by Mediating the Formation and Stabilization of Actin-containing Motility Structures , 1997, The Journal of cell biology.

[72]  F. Strutz,et al.  Identification and characterization of a fibroblast marker: FSP1 , 1995, The Journal of cell biology.

[73]  J. Widdicombe,et al.  Culture and transformation of human airway epithelial cells. , 1995, The American journal of physiology.

[74]  E. Hay.  An overview of epithelio-mesenchymal transformation. , 1995, Acta anatomica.