21 results on '"Stephanie, Cambier"'
Search Results
2. Tight nuclear tethering of cGAS is essential for preventing autoreactivity
- Author
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Hannah E Volkman, Stephanie Cambier, Elizabeth E Gray, and Daniel B Stetson
- Subjects
nucleic acid detection ,innate Immunity ,antiviral immunity ,cGAS ,Medicine ,Science ,Biology (General) ,QH301-705.5 - Abstract
cGAS is an intracellular innate immune sensor that detects double-stranded DNA. The presence of billions of base pairs of genomic DNA in all nucleated cells raises the question of how cGAS is not constitutively activated. A widely accepted explanation for this is the sequestration of cGAS in the cytosol, which is thought to prevent cGAS from accessing nuclear DNA. Here, we demonstrate that endogenous cGAS is predominantly a nuclear protein, regardless of cell cycle phase or cGAS activation status. We show that nuclear cGAS is tethered tightly by a salt-resistant interaction. This tight tethering is independent of the domains required for cGAS activation, and it requires intact nuclear chromatin. We identify the evolutionarily conserved tethering surface on cGAS and we show that mutation of single amino acids within this surface renders cGAS massively and constitutively active against self-DNA. Thus, tight nuclear tethering maintains the resting state of cGAS and prevents autoreactivity.
- Published
- 2019
- Full Text
- View/download PDF
3. ABCC1 transporter exports the immunostimulatory cyclic dinucleotide cGAMP
- Author
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Joanna H. Maltbaek, Stephanie Cambier, Jessica M. Snyder, and Daniel B. Stetson
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Mice ,Infectious Diseases ,Adenosine Triphosphate ,Immunology ,Immunology and Allergy ,Animals ,Humans ,Membrane Transport Proteins ,DNA ,Interferons ,Multidrug Resistance-Associated Proteins ,Nucleotides, Cyclic ,Nucleotidyltransferases - Abstract
The DNA sensor cyclic GMP-AMP synthase (cGAS) is important for antiviral and anti-tumor immunity. cGAS generates cyclic GMP-AMP (cGAMP), a diffusible cyclic dinucleotide that activates the antiviral response through the adaptor protein stimulator of interferon genes (STING). cGAMP cannot passively cross cell membranes, but recent advances have established a role for extracellular cGAMP as an "immunotransmitter" that can be imported into cells. However, the mechanism by which cGAMP exits cells remains unknown. Here, we identifed ABCC1 as a direct, ATP-dependent cGAMP exporter in mouse and human cells. We show that ABCC1 overexpression enhanced cGAMP export and limited STING signaling and that loss of ABCC1 reduced cGAMP export and potentiated STING signaling. We demonstrate that ABCC1 deficiency exacerbated cGAS-dependent autoimmunity in the Trex1
- Published
- 2022
4. Computational design of constitutively active cGAS
- Author
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Quinton M, Dowling, Hannah E, Volkman, Elizabeth E, Gray, Sergey, Ovchinnikov, Stephanie, Cambier, Asim K, Bera, Banumathi, Sankaran, Max R, Johnson, Matthew J, Bick, Alex, Kang, Daniel B, Stetson, and Neil P, King
- Abstract
Cyclic GMP-AMP synthase (cGAS) is a pattern recognition receptor critical for the innate immune response to intracellular pathogens, DNA damage, tumorigenesis and senescence. Binding to double-stranded DNA (dsDNA) induces conformational changes in cGAS that activate the enzyme to produce 2'-3' cyclic GMP-AMP (cGAMP), a second messenger that initiates a potent interferon (IFN) response through its receptor, STING. Here, we combined two-state computational design with informatics-guided design to create constitutively active, dsDNA ligand-independent cGAS (CA-cGAS). We identified CA-cGAS mutants with IFN-stimulating activity approaching that of dsDNA-stimulated wild-type cGAS. DNA-independent adoption of the active conformation was directly confirmed by X-ray crystallography. In vivo expression of CA-cGAS in tumor cells resulted in STING-dependent tumor regression, demonstrating that the designed proteins have therapeutically relevant biological activity. Our work provides a general framework for stabilizing active conformations of enzymes and provides CA-cGAS variants that could be useful as genetically encoded adjuvants and tools for understanding inflammatory diseases.
- Published
- 2021
5. Tight nuclear tethering of cGAS is essential for preventing autoreactivity
- Author
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Daniel B. Stetson, Elizabeth E. Gray, Stephanie Cambier, and Hannah E. Volkman
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0301 basic medicine ,Mouse ,medicine.disease_cause ,chemistry.chemical_compound ,0302 clinical medicine ,Immunology and Inflammation ,Cytosol ,Nuclear protein ,Biology (General) ,Cells, Cultured ,Mutation ,General Neuroscience ,Nuclear Proteins ,General Medicine ,Nucleotidyltransferases ,Cell biology ,Nuclear DNA ,antiviral immunity ,Medicine ,Research Article ,Human ,Protein Binding ,Base pair ,QH301-705.5 ,Science ,General Biochemistry, Genetics and Molecular Biology ,Cell cycle phase ,03 medical and health sciences ,Cell Line, Tumor ,medicine ,Animals ,Humans ,DNA Cleavage ,innate Immunity ,Cell Nucleus ,Innate immune system ,General Immunology and Microbiology ,nucleic acid detection ,DNA ,Mice, Inbred C57BL ,030104 developmental biology ,chemistry ,030217 neurology & neurosurgery ,DNA Damage ,HeLa Cells ,cGAS - Abstract
cGAS is an intracellular innate immune sensor that detects double-stranded DNA. The presence of billions of base pairs of genomic DNA in all nucleated cells raises the question of how cGAS is not constitutively activated. A widely accepted explanation for this is the sequestration of cGAS in the cytosol, which is thought to prevent cGAS from accessing nuclear DNA. Here, we demonstrate that endogenous cGAS is predominantly a nuclear protein, regardless of cell cycle phase or cGAS activation status. We show that nuclear cGAS is tethered tightly by a salt-resistant interaction. This tight tethering is independent of the domains required for cGAS activation, and it requires intact nuclear chromatin. We identify the evolutionarily conserved tethering surface on cGAS and we show that mutation of single amino acids within this surface renders cGAS massively and constitutively active against self-DNA. Thus, tight nuclear tethering maintains the resting state of cGAS and prevents autoreactivity.
- Published
- 2019
6. Author response: Tight nuclear tethering of cGAS is essential for preventing autoreactivity
- Author
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Daniel B. Stetson, Hannah E. Volkman, Stephanie Cambier, and Elizabeth E. Gray
- Subjects
Tethering ,Chemistry ,Cell biology - Published
- 2019
7. Human DNA-PK activates a STING-independent DNA sensing pathway
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Daniel B. Stetson, Richard C. James, Joanna H. Maltbaek, Richard Green, Michael Gale, Stephanie Cambier, and Katelyn Burleigh
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0301 basic medicine ,DNA damage ,Ubiquitin-Protein Ligases ,Immunology ,Viral Oncogene ,DNA-Activated Protein Kinase ,Herpesvirus 1, Human ,Biology ,medicine.disease_cause ,Virus ,Adenoviridae ,Cell Line ,Immediate-Early Proteins ,Mice ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Heat shock protein ,medicine ,Animals ,Humans ,Kinase activity ,Protein kinase A ,HSPA8 ,030304 developmental biology ,0303 health sciences ,Innate immune system ,Membrane Proteins ,Herpes Simplex ,General Medicine ,3. Good health ,Cell biology ,030104 developmental biology ,chemistry ,030220 oncology & carcinogenesis ,Phosphorylation ,Adenovirus E1A Proteins ,DNA - Abstract
Detection of intracellular DNA by the cGAS-STING pathway activates a type I interferon-mediated innate immune response that protects from virus infection. Whether there are additional DNA sensing pathways, and how such pathways might function, remains controversial. We show here that humans-but not laboratory mice-have a second, potent, STING-independent DNA sensing pathway (SIDSP). We identify human DNA-dependent protein kinase (DNA-PK) as the sensor of this pathway and demonstrate that DNA-PK activity drives a robust and broad antiviral response. We show that the E1A oncoprotein of human adenovirus 5 and the ICP0 protein of herpes simplex virus 1 block this response. We found heat shock protein HSPA8/HSC70 as a target for inducible phosphorylation in the DNA-PK antiviral pathway. Last, we demonstrate that DNA damage and detection of foreign DNA trigger distinct modalities of DNA-PK activity. These findings reveal the existence, sensor, a specific downstream target, and viral antagonists of a SIDSP in human cells.
- Published
- 2019
- Full Text
- View/download PDF
8. cGAS is predominantly a nuclear protein
- Author
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Hannah E. Volkman, Elizabeth E. Gray, Daniel B. Stetson, and Stephanie Cambier
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0303 health sciences ,Innate immune system ,Chemistry ,Base pair ,3. Good health ,Nuclear DNA ,Cell cycle phase ,Cell biology ,03 medical and health sciences ,genomic DNA ,Cytosol ,chemistry.chemical_compound ,0302 clinical medicine ,030220 oncology & carcinogenesis ,Nuclear protein ,DNA ,030304 developmental biology - Abstract
cGAS is an intracellular innate immune sensor that detects double-stranded DNA. The presence of billions of base pairs of genomic DNA in all nucleated cells raises the question of how cGAS is not constitutively activated. A widely accepted explanation for this is the sequestration of cGAS in the cytosol, which is thought to prevent cGAS from accessing nuclear DNA. Here, we demonstrate that cGAS is predominantly a nuclear protein, regardless of cell cycle phase or cGAS activation status. We show that nuclear cGAS is tethered tightly by a salt-resistant interaction. This tight tethering is independent of the domains required for cGAS activation, and it requires intact nuclear chromatin. We propose that tethering prevents activation of cGAS by genomic DNA, and that it might enable cGAS to distinguish between self DNA and foreign DNA within the nucleus.
- Published
- 2018
- Full Text
- View/download PDF
9. Interleukin-1β Induces Increased Transcriptional Activation of the Transforming Growth Factor-β-activating Integrin Subunit β8 through Altering Chromatin Architecture
- Author
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Arthur C. Hill, Stephen L. Nishimura, Jun Araya, Stephanie Cambier, Stephanie E. Gline, Paul J. Wolters, Sangeeta Somanath, Jennifer A. Markovics, and David M. Jablons
- Subjects
Integrin beta Chains ,Proto-Oncogene Proteins c-jun ,Interleukin-1beta ,Histone Deacetylase 2 ,Integrin alpha5 ,Biochemistry ,Chromatin remodeling ,Histones ,Histone H4 ,Transforming Growth Factor beta ,Humans ,Gene Regulation ,Promoter Regions, Genetic ,ITGB8 ,Molecular Biology ,Activating Transcription Factor 2 ,biology ,NF-kappa B ,Acetylation ,DNA ,Cell Biology ,Transforming growth factor beta ,Chromatin Assembly and Disassembly ,Chromatin ,Activating transcription factor 2 ,Nucleosomes ,Cancer research ,biology.protein ,Integrin, beta 6 ,HeLa Cells ,Transforming growth factor - Abstract
The integrin αvβ8 is a cell surface receptor for the latent domain (LAP) of the multifunctional cytokine TGF-β. Through its association with LAP, TGF-β is maintained in a latent form that must be activated to function. Binding to the integrin αvβ8 with subsequent metalloproteolytic cleavage of LAP represents a major mechanism of TGF-β activation in vivo. Altered expression of the integrin β8 subunit (ITGB8) is found in human chronic obstructive pulmonary disease, cancers, and brain vascular malformations. We have previously shown that the proinflammatory cytokine interleukin-1β (IL-1β) increases ITGB8 expression on lung fibroblasts, which increases αvβ8-mediated TGF-β activation in fibrosis and pathologic inflammation. Here we report the mechanism of increased ITGB8 expression by IL-1β. Our data support a model where the chromatin architecture of the ITGB8 core promoter is altered by nucleosomal repositioning that enhances the interaction of an AP1 complex (containing c-Jun and ATF2). This repositioning is caused by the dissociation of HDAC2 with the ITGB8 core promoter, leading to increased histone H4 acetylation and a loosening of nucleosomal-DNA interactions allowing "opening" of the chromatin structure and increased association of c-Jun and ATF-2. These changes are mediated through NFκB- and p38-dependent pathways. Ultimately, these events culminate in increasing ITGB8 transcription, αvβ8 surface expression, and αvβ8-mediated TGFβ activation.
- Published
- 2011
10. The side effects of phototherapy for neonatal jaundice: what do we know? What should we do?
- Author
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Yi Qu, Stephanie Cambier, Tao Xiong, and Dezhi Mu
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medicine.medical_specialty ,Pediatrics ,Side effect ,medicine.medical_treatment ,Treatment outcome ,Water-Electrolyte Imbalance ,Exchange transfusion ,Risk Assessment ,Fluid therapy ,Risk Factors ,medicine ,Humans ,Intensive care medicine ,business.industry ,Infant, Newborn ,Bilirubin ,Phototherapy ,Jaundice ,Infant newborn ,Jaundice, Neonatal ,Bilirubin concentration ,Treatment Outcome ,Immune System Diseases ,Pediatrics, Perinatology and Child Health ,Fluid Therapy ,medicine.symptom ,business - Abstract
Neonatal phototherapy (NNPT), a noninvasive, easily available therapy, has been widely used for the treatment of neonatal jaundice for more than half a century. Its efficiency in decreasing plasma bilirubin concentration is well documented, and NNPT leads to greatly reduced exchange transfusion rates for neonates with hyperbilirubinemia. It is generally accepted that the side effects of NNPT are not serious and seem to be well controlled. This review will focus on these possible side effects as well as the approaches to minimize them.
- Published
- 2011
11. Transcription of the Transforming Growth Factor β Activating Integrin β8 Subunit Is Regulated by SP3, AP-1, and the p38 Pathway
- Author
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Stephen L. Nishimura, Paul J. Wolters, Jun Araya, Arthur C. Hill, Jennifer A. Markovics, David M. Jablons, and Stephanie Cambier
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Integrin beta Chains ,Transcription, Genetic ,Molecular Sequence Data ,Integrin ,Biology ,p38 Mitogen-Activated Protein Kinases ,Biochemistry ,Sp3 transcription factor ,Transforming Growth Factor beta ,Cell Line, Tumor ,Sequence Homology, Nucleic Acid ,Humans ,ITGB8 ,Molecular Biology ,Transcription factor ,Inflammation ,Base Sequence ,Promoter ,Cell Biology ,Transforming growth factor beta ,Molecular biology ,Gene Expression Regulation, Neoplastic ,Transcription Factor AP-1 ,AP-1 transcription factor ,Sp3 Transcription Factor ,biology.protein ,CpG Islands ,HeLa Cells ,Transforming growth factor - Abstract
Integrin alphavbeta8 is a critical regulator of transforming growth factor beta activation in vasculogenesis during development, immune regulation, and endothelial/epithelial-mesenchymal homeostasis. Recent studies have suggested roles for integrin beta8 in the pathogenesis of chronic obstructive pulmonary disease, brain arteriovenous malformations, and select cancers (Araya, J., Cambier, S., Markovics, J. A., Wolters, P., Jablons, D., Hill, A., Finkbeiner, W., Jones, K., Broaddus, V. C., Sheppard, D., Barzcak, A., Xiao, Y., Erle, D. J., and Nishimura, S. L. (2007) J. Clin. Invest. 117, 3551-3562; Su, H., Kim, H., Pawlikowska, L., Kitamura, H., Shen, F., Cambier, S., Markovics, J., Lawton, M. T., Sidney, S., Bollen, A. W., Kwok, P. Y., Reichardt, L., Young, W. L., Yang, G. Y., and Nishimura, S. L. (2010) Am. J. Pathol. 176, 1018-1027; Culhane, A. C., and Quackenbush, J. (2009) Cancer Res. 69, 7480-7485; Cambier, S., Mu, D. Z., O'Connell, D., Boylen, K., Travis, W., Liu, W. H., Broaddus, V. C., and Nishimura, S. L. (2000) Cancer Res. 60, 7084-7093). Here we report the first identification and characterization of the promoter for ITGB8. We show that a SP binding site and a cyclic AMP response element (CRE) in the ITGB8 core promoter are required for its expression and that Sp1, Sp3, and several AP-1 transcription factors form a complex that binds to these sites in a p38-dependent manner. Furthermore, we demonstrate the requirement for Sp3, ATF-2, and p38 for the transcription and protein expression of integrin beta8. Additionally, reduction of SP3 or inhibition of p38 blocks alphavbeta8-mediated transforming growth factor beta activation. These results place integrin beta8 expression and activity under the control of ubiquitous transcription factors in a stress-activated and pro-inflammatory pathway.
- Published
- 2010
12. Transforming Growth Factor-β and Interleukin-1β Signaling Pathways Converge on the Chemokine CCL20 Promoter
- Author
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James D. Marks, Jody L. Baron, Stephen L. Nishimura, Oliver J. Brand, Stephanie Cambier, Jennifer A. Markovics, Andrew J. Bondesson, Paul J. Wolters, Sangeeta Somanath, Catherine Moermans, Mitsuo Hashimoto, Haruhiko Yanagisawa, Arthur C. Hill, David M. Jablons, and Jianlong Lou
- Subjects
Interleukin-1beta ,C-C chemokine receptor type 6 ,Inbred C57BL ,Biochemistry ,Medical and Health Sciences ,Mice ,Transforming Growth Factor beta ,CCL17 ,2.1 Biological and endogenous factors ,Aetiology ,Lung ,Cells, Cultured ,Cultured ,NF-kappa B ,hemic and immune systems ,Molecular Bases of Disease ,Biological Sciences ,respiratory system ,Respiratory ,transcription ,Signal Transduction ,TGF-β ,Biochemistry & Molecular Biology ,integrin ,Chronic Obstructive Pulmonary Disease ,1.1 Normal biological development and functioning ,Cells ,chemical and pharmacologic phenomena ,Biology ,Response Elements ,Clinical Research ,Underpinning research ,CXCL10 ,Animals ,Humans ,Molecular Biology ,CXCL16 ,Chemokine CCL20 ,Base Sequence ,chemokine ,Cell Biology ,Fibroblasts ,Molecular biology ,respiratory tract diseases ,CCL20 ,Mice, Inbred C57BL ,stomatognathic diseases ,Gene Expression Regulation ,inflammation ,Chemical Sciences ,XCL2 ,CCL25 ,CCL21 - Abstract
© 2015 by The American Society for Biochemistry and Molecular Biology, Inc. CCL20 is the only chemokine ligand for the chemokine receptor CCR6, which is expressed by the critical antigen presenting cells, dendritic cells. Increased expression of CCL20 is likely involved in the increased recruitment of dendritic cells observed in fibroinflammatory diseases such as chronic obstructive pulmonary disease (COPD). CCL20 expression is increased by the proinflammatory cytokine IL-1β. We have determined that IL-1β-dependent CCL20 expression is also dependent on the multifunctional cytokine TGF-β. TGF-β is expressed in a latent form that must be activated to function, and activation is achieved through binding to the integrin αvβ8 (itgb8). Here we confirm correlative increases in αvβ8 and IL-1β with CCL20 protein in lung parenchymal lysates of a large cohort of COPD patients. How IL-1β- and αvβ8-mediated TGF-β activation conspire to increase fibroblast CCL20 expression remains unknown, because these pathways have not been shown to directly interact. We evaluate the 5′-flanking region of CCL20 to determine that IL-1β-driven CCL20 expression is dependent on αvβ8-mediated activation of TGF-β. We identify a TGF-β-responsive element (i.e. SMAD) located on an upstream enhancer of the human CCL20 promoter required for efficient IL-1β-dependent CCL20 expression. By chromatin immunoprecipitation, this upstream enhancer complexes with the p50 subunit of NF-κB on a NF-κB-binding element close to the transcriptional start site of CCL20. These interactions are confirmed by electromobility shift assays in nuclear extracts from human lung fibroblasts. These data define a mechanism by which αvβ8-dependent activation of TGF-β regulates IL-1β-dependent CCL20 expression in COPD.
- Published
- 2014
13. Integrin αvβ8-Mediated Activation of Transforming Growth Factor-β by Perivascular Astrocytes
- Author
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Gregory Dolganov, Dezhi Mu, Rodney Collins, Jun Araya, Stephanie E. Gline, Nancy Boudreau, Stephen L. Nishimura, Stephanie Cambier, and Steven Einheber
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biology ,Endothelium ,Angiogenesis ,Transforming growth factor beta ,Pathology and Forensic Medicine ,Cell biology ,Endothelial stem cell ,Paracrine signalling ,medicine.anatomical_structure ,Immunology ,biology.protein ,medicine ,Neuroglia ,ITGB8 ,Astrocyte - Abstract
Brain hemorrhage is a severe complication of both neoplastic and nonneoplastic brain disease. Mice deficient in the αvβ8 integrin display defective brain vessel formation resulting in hemorrhage and perinatal death, but the mechanism of brain hemorrhage is unknown. Because the αvβ8 integrin is expressed by astrocytes and not expressed by endothelium, paracrine interactions between astrocytes and endothelial cells could contribute to the maintenance of brain vessel integrity. We have investigated the mechanisms underlying astrocytic-endothelial paracrine signaling and have found that integrin-mediated activation of transforming growth factor (TGF)-β by astrocytes influences endothelial cell function. Thus, we identified the integrin αvβ8 in human perivascular glial cell processes surrounding developing blood vessels. Human astrocytic αvβ8 was a major cell surface receptor for latent TGF-β, and αvβ8-dependent activation of TGF-β was the major mechanism of TGF-β activation in primary cultures of astrocytes or freshly dissociated fetal brain cells. This activation of TGF-β was sufficient to inhibit endothelial migration in fibrin gels and to alter expression of genes affecting proteolytic and angiogenic pathways. Taken together, our data suggest that astrocytic αvβ8 acts as a central regulator of brain vessel homeostasis through regulation of TGF-β activation and expression of TGF-β-responsive genes that promote vessel differentiation and stabilization, most notably plasminogen activator inhibitor-1 and thrombospondin-1.
- Published
- 2005
14. A 50-Å Separation of the Integrin αvβ3 Extracellular Domain C Termini Reveals an Intermediate Activation State
- Author
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Stephen L. Nishimura, Cédric Govaerts, Stephanie E. Gline, and Stephanie Cambier
- Subjects
Models, Molecular ,Cytoplasm ,Alpha-v beta-3 ,Protein Conformation ,Placenta ,Recombinant Fusion Proteins ,Integrin ,Alpha (ethology) ,Ligands ,Transfection ,Biochemistry ,chemistry.chemical_compound ,Protein structure ,Humans ,Binding site ,Receptor ,Beta (finance) ,Molecular Biology ,Immunosorbent Techniques ,Binding Sites ,Molecular Structure ,biology ,Chemistry ,Cell Membrane ,Cell Biology ,Alkaline Phosphatase ,Integrin alphaVbeta3 ,Peptide Fragments ,Molecular Weight ,Crystallography ,Transmembrane domain ,Chromatography, Gel ,Biophysics ,biology.protein ,Dimerization - Abstract
The integrin alpha(v)beta(3) has been shown to exist in low and high affinity conformations. Activation to the high affinity state is thought to depend on the "switchblade-like" opening, from a low affinity bent conformation with a closed headpiece to an extended form of the integrin with an open headpiece. Activation has been shown to depend on separation of the cytoplasmic domains. How cytoplasmic domain separation is related to separation of the transmembrane domains is unknown, and the distance of separation of the transmembrane domains required for activation has not been defined. A constrained secreted form of alpha(v)beta(3) was engineered that introduced a 50-A separation of the integrin C-terminal tails of the extracellular domains of the alpha(v) and beta(3) subunits. Receptor binding and recognition by ligand-induced binding state (LIBS) monoclonal antibodies demonstrated that the mutant receptor was locked into a low affinity state that was likely in a partially extended conformation but with a closed headpiece. In the presence of RGD peptide, the constrained receptor was able to fully extend, as determined by full exposure of LIBS epitopes. In the presence of the appropriate LIBS antibody, high affinity ligand binding of the constrained receptor was achieved. The results support the existence of transient intermediate activation states of secreted alpha(v)beta(3). Furthermore, these results with the secreted alpha(v)beta(3) receptor support a model for the full-length membrane-bound form of alpha(v)beta(3), whereby a 50-A lateral separation of the integrin alpha(v) and beta(3) transmembrane domains would be sufficient to enforce the switchblade-like opening to the extended conformation but insufficient for full receptor activation.
- Published
- 2004
15. Integrin αvβ8 Functions as a Receptor for Foot-and-Mouth Disease Virus: Role of the β-Chain Cytodomain in Integrin-Mediated Infection
- Author
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Stephen L. Nishimura, Terry Jackson, Stephen Berryman, Stuart Clark, Dezhi Mu, Alison Burman, Stephanie Cambier, and Andrew M. Q. King
- Subjects
Integrins ,Virus genetics ,Protein Conformation ,medicine.drug_class ,viruses ,Immunology ,Integrin ,Transfection ,Monoclonal antibody ,Microbiology ,Virus ,Cell Line ,Cricetinae ,Virology ,medicine ,Animals ,Humans ,RGD motif ,Aphthovirus ,biology ,respiratory system ,Flow Cytometry ,biology.organism_classification ,Molecular biology ,Virus-Cell Interactions ,Ectodomain ,Foot-and-Mouth Disease Virus ,Insect Science ,biology.protein ,Receptors, Virus - Abstract
Field isolates of foot-and-mouth disease virus (FMDV) have been shown to use three alphav integrins, alphavbeta1, alphavbeta3, and alphavbeta6, as cellular receptors. Binding to the integrin is mediated by a highly conserved RGD motif located on a surface-exposed loop of VP1. The RGD tripeptide is recognized by several other members of the integrin family, which therefore have the potential to act as receptors for FMDV. Here we show that SW480 cells are made susceptible to FMDV following transfection with human beta8 cDNA and expression of alphavbeta8 at the cell surface. The involvement of alphavbeta8 in infection was confirmed by showing that virus binding and infection of the transfected cells are inhibited by RGD-containing peptides and by function-blocking monoclonal antibodies specific for either the alphavbeta8 heterodimer or the alphav chain. Similar results were obtained with a chimeric alphavbeta8 including the beta6 cytodomain (alphavbeta8/6), showing that the beta6 cytodomain can substitute efficiently for the corresponding region of beta8. In contrast, virus binding to alphavbeta6 including the beta8 cytodomain (alphavbeta6/8) was lower than that of the wild-type integrin, and this binding did not lead to infection. Further, the alphavbeta6 chimera was recognized poorly by antibodies specific for the ectodomain of alphavbeta6 and displayed a relaxed sequence-binding specificity relative to that of wild-type integrin. These data suggest that the beta6 cytodomain is important for maintaining alphavbeta6 in a conformation required for productive infection by FMDV.
- Published
- 2004
16. The integrin αvβ8 mediates epithelial homeostasis through MT1-MMP–dependent activation of TGF-β1
- Author
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Stephanie Cambier, Lars Fjellbirkeland, Hisaaki Kawakatsu, Jody L. Baron, Dezhi Mu, Dean Sheppard, V. Courtney Broaddus, John S. Munger, and Stephen L. Nishimura
- Subjects
Integrins ,Lung Neoplasms ,Matrix Metalloproteinases, Membrane-Associated ,Integrin ,Cellular homeostasis ,Biology ,Transfection ,Article ,Cell Line ,Transforming Growth Factor beta1 ,03 medical and health sciences ,Paracrine signalling ,Mice ,0302 clinical medicine ,Transforming Growth Factor beta ,Matrix Metalloproteinase 14 ,Tumor Cells, Cultured ,Animals ,Homeostasis ,Humans ,ITGB8 ,Autocrine signalling ,030304 developmental biology ,Integrin binding ,0303 health sciences ,Mice, Inbred BALB C ,Cell growth ,Metalloendopeptidases ,Proteins ,Cell Biology ,Transforming growth factor beta ,TNF Receptor-Associated Factor 2 ,3. Good health ,Cell biology ,Protein Structure, Tertiary ,030220 oncology & carcinogenesis ,integrins ,transforming growth factor β ,metalloprotease ,cell cycle ,homeostasis ,biology.protein ,Oligopeptides ,Cell Division ,Protein Binding - Abstract
Integrins, matrix metalloproteases (MMPs), and the cytokine TGF-beta have each been implicated in homeostatic cell behaviors such as cell growth and matrix remodeling. TGF-beta exists mainly in a latent state, and a major point of homeostatic control is the activation of TGF-beta. Because the latent domain of TGF-beta1 possesses an integrin binding motif (RGD), integrins have the potential to sequester latent TGF-beta (SLC) to the cell surface where TGF-beta activation could be locally controlled. Here, we show that SLC binds to alpha(v)beta8, an integrin expressed by normal epithelial and neuronal cells in vivo. This binding results in the membrane type 1 (MT1)-MMP-dependent release of active TGF-beta, which leads to autocrine and paracrine effects on cell growth and matrix production. These data elucidate a novel mechanism of cellular homeostasis achieved through the coordination of the activities of members of three major gene families involved in cell-matrix interactions.
- Published
- 2002
17. Selective Targeting of TGF-β Activation to Treat Fibroinflammatory Airway Disease
- Author
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Robert Seed, Tyren Barker, Kate J. McKnelly, Jane Connor, Paul J. Wolters, Shunsuke Minagawa, Stephanie Cambier, Hideya Kitamura, Arthur C. Hill, Jun Araya, David J. Erle, Ronald Herbst, Louis F. Reichardt, Royce Ma, Mitsuo Hashimoto, David M. Jablons, Amanda Goodsell, Haruhiko Yanagisawa, Jody L. Baron, Weihua Wen, Nishimura Stephen, Shenping Wu, Ping Tsui, Cedric Govaerts, Lynne Murray, Oliver J. Brand, Andrea J. Barczak, Ran Cheng, Yifan Cheng, Anthony Cormier, Jianlong Lou, and James D. Marks
- Subjects
Integrin ,Mice, Transgenic ,Article ,Pathogenesis ,Mice ,Fibrosis ,Transforming Growth Factor beta ,medicine ,Animals ,Humans ,ITGB8 ,Receptor ,biology ,business.industry ,General Medicine ,Transforming growth factor beta ,medicine.disease ,respiratory tract diseases ,Mechanism of action ,Immunology ,Cancer research ,biology.protein ,Tracheitis ,medicine.symptom ,business ,Transforming growth factor - Abstract
Airway remodeling, caused by inflammation and fibrosis, is a major component of chronic obstructive pulmonary disease (COPD) and currently has no effective treatment. Transforming growth factor–β (TGF-β) has been widely implicated in the pathogenesis of airway remodeling in COPD. TGF-β is expressed in a latent form that requires activation. The integrin αvβ8 (encoded by the itgb8 gene) is a receptor for latent TGF-β and is essential for its activation. Expression of integrin αvβ8 is increased in airway fibroblasts in COPD and thus is an attractive therapeutic target for the treatment of airway remodeling in COPD. We demonstrate that an engineered optimized antibody to human αvβ8 (B5) inhibited TGF-β activation in transgenic mice expressing only human and not mouse ITGB8. The B5 engineered antibody blocked fibroinflammatory responses induced by tobacco smoke, cytokines, and allergens by inhibiting TGF-β activation. To clarify the mechanism of action of B5, we used hydrodynamic, mutational, and electron microscopic methods to demonstrate that αvβ8 predominantly adopts a constitutively active, extended-closed headpiece conformation. Epitope mapping and functional characterization of B5 revealed an allosteric mechanism of action due to locking-in of a low-affinity αvβ8 conformation. Collectively, these data demonstrate a new model for integrin function and present a strategy to selectively target the TGF-β pathway to treat fibroinflammatory airway diseases.
- Published
- 2014
18. Squamous metaplasia amplifies pathologic epithelial-mesenchymal interactions in COPD patients
- Author
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David J. Erle, Kirk D. Jones, Yuanyuan Xiao, Stephanie Cambier, David M. Jablons, Paul J. Wolters, Jun Araya, Walter E. Finkbeiner, V. Courtney Broaddus, Andrea Barzcak, Arthur C. Hill, Dean Sheppard, Stephen L. Nishimura, and Jennifer A. Markovics
- Subjects
Pathology ,medicine.medical_specialty ,Integrins ,Cell Communication ,Respiratory Mucosa ,Biology ,Epithelium ,Mesoderm ,Mice ,Pulmonary Disease, Chronic Obstructive ,Transforming Growth Factor beta ,Metaplasia ,medicine ,Matrix Metalloproteinase 14 ,Animals ,Humans ,Fibroblast ,Cells, Cultured ,COPD ,Lung ,Gene Expression Profiling ,Epithelial Cells ,General Medicine ,Transforming growth factor beta ,Airway obstruction ,respiratory system ,Fibroblasts ,medicine.disease ,Squamous metaplasia ,Coculture Techniques ,respiratory tract diseases ,medicine.anatomical_structure ,biology.protein ,medicine.symptom ,Airway ,Research Article ,Interleukin-1 - Abstract
Squamous metaplasia (SM) is common in smokers and is associated with airway obstruction in chronic obstructive pulmonary disease (COPD). A major mechanism of airway obstruction in COPD is thickening of the small airway walls. We asked whether SM actively contributes to airway wall thickening through alteration of epithelial-mesenchymal interactions in COPD. Using immunohistochemical staining, airway morphometry, and fibroblast culture of lung samples from COPD patients; genome-wide analysis of an in vitro model of SM; and in vitro modeling of human airway epithelial-mesenchymal interactions, we provide evidence that SM, through the increased secretion of IL-1beta, induces a fibrotic response in adjacent airway fibroblasts. We identify a pivotal role for integrin-mediated TGF-beta activation in amplifying SM and driving IL-1beta-dependent profibrotic mesenchymal responses. Finally, we show that SM correlates with increased severity of COPD and that fibroblast expression of the integrin alpha(v)beta(8), which is the major mediator of airway fibroblast TGF-beta activation, correlated with disease severity and small airway wall thickening in COPD. Our findings have identified TGF-beta as a potential therapeutic target for COPD.
- Published
- 2007
19. Integrin-Mediated Transforming Growth Factor-β Activation Regulates Homeostasis of the Pulmonary Epithelial-Mesenchymal Trophic Unit
- Author
-
Alanna Morris, Stephen L. Nishimura, Walter E. Finkbeiner, Stephanie Cambier, and Jun Araya
- Subjects
medicine.medical_specialty ,Integrins ,Matrix Metalloproteinases, Membrane-Associated ,Cellular differentiation ,Mesenchyme ,Models, Biological ,Pathology and Forensic Medicine ,Mesoderm ,Antigens, Neoplasm ,Pregnancy ,Transforming Growth Factor beta ,Internal medicine ,medicine ,Homeostasis ,Humans ,RNA, Messenger ,Fibroblast ,Autocrine signalling ,Lung ,Cells, Cultured ,Cell Proliferation ,biology ,Hepatocyte Growth Factor ,Cell Differentiation ,Epithelial Cells ,Transforming growth factor beta ,respiratory system ,Fibroblasts ,Matrix Metalloproteinases ,Cell biology ,Trachea ,Autocrine Communication ,Endocrinology ,medicine.anatomical_structure ,Hepatocyte Growth Factor Receptor ,biology.protein ,Hepatocyte growth factor ,Female ,medicine.drug ,Transforming growth factor ,Regular Articles - Abstract
Trophic interactions between pulmonary epithelial and mesenchymal cell types, known as the epithelial-mesenchymal trophic unit (EMTU), are crucial in lung development and lung disease. Transforming growth factor (TGF)-beta is a key factor in mediating these interactions, but it is expressed in a latent form that requires activation to be functional. Using intact fetal tracheal tissue and primary cultures of fetal tracheal epithelial cells and fibroblasts, we demonstrate that a subset of integrins, alpha(v)beta(6) and alpha(v)beta(8), are responsible for almost all of the TGF-beta activation in the EMTU. Both alpha(v)beta(8) and alpha(v)beta(6) contribute to fetal tracheal epithelial activation of TGF-beta, whereas only alpha(v)beta(8) contributes to fetal tracheal fibroblast activation of TGF-beta. Interestingly, fetal tracheal epithelial alpha(v)beta(8)-mediated TGF-beta activation can be enhanced by phorbol esters, likely because of the increased activity of MT1-MMP, an essential co-factor in alpha(v)beta(8)-mediated activation of TGF-beta. Autocrine alpha(v)beta(8)-mediated TGF-beta activation by fetal tracheal fibroblasts results in suppression of both transcription and secretion of hepatocyte growth factor, which is sufficient to affect phosphorylation of the airway epithelial hepatocyte growth factor receptor, c-Met, as well as airway epithelial proliferation in a co-culture model of the EMTU. These findings elucidate the function and complex regulation of integrin-mediated activation of TGF-beta within the EMTU.
- Published
- 2006
20. Integrin alpha(v)beta8-mediated activation of transforming growth factor-beta by perivascular astrocytes: an angiogenic control switch
- Author
-
Stephanie, Cambier, Stephanie, Gline, Dezhi, Mu, Rodney, Collins, Jun, Araya, Gregory, Dolganov, Steven, Einheber, Nancy, Boudreau, and Stephen L, Nishimura
- Subjects
Integrins ,Integrin beta Chains ,Reverse Transcriptase Polymerase Chain Reaction ,Gene Expression Profiling ,Blotting, Western ,Brain ,Endothelial Cells ,Cell Communication ,Flow Cytometry ,Immunohistochemistry ,Models, Biological ,Enzyme Activation ,Thrombospondin 1 ,Original Research Paper ,Transforming Growth Factor beta ,Astrocytes ,Plasminogen Activator Inhibitor 1 ,Cell Adhesion ,Humans ,Immunoprecipitation ,Cells, Cultured - Abstract
Brain hemorrhage is a severe complication of both neoplastic and nonneoplastic brain disease. Mice deficient in the alpha(v)beta8 integrin display defective brain vessel formation resulting in hemorrhage and perinatal death, but the mechanism of brain hemorrhage is unknown. Because the alpha(v)beta8 integrin is expressed by astrocytes and not expressed by endothelium, paracrine interactions between astrocytes and endothelial cells could contribute to the maintenance of brain vessel integrity. We have investigated the mechanisms underlying astrocytic-endothelial paracrine signaling and have found that integrin-mediated activation of transforming growth factor (TGF)-beta by astrocytes influences endothelial cell function. Thus, we identified the integrin alpha(v)beta8 in human perivascular glial cell processes surrounding developing blood vessels. Human astrocytic alpha(v)beta8 was a major cell surface receptor for latent TGF-beta, and alpha(v)beta8-dependent activation of TGF-beta was the major mechanism of TGF-beta activation in primary cultures of astrocytes or freshly dissociated fetal brain cells. This activation of TGF-beta was sufficient to inhibit endothelial migration in fibrin gels and to alter expression of genes affecting proteolytic and angiogenic pathways. Taken together, our data suggest that astrocytic alpha(v)beta8 acts as a central regulator of brain vessel homeostasis through regulation of TGF-beta activation and expression of TGF-beta-responsive genes that promote vessel differentiation and stabilization, most notably plasminogen activator inhibitor-1 and thrombospondin-1.
- Published
- 2005
21. Integrin alphavbeta8-mediated activation of transforming growth factor-beta inhibits human airway epithelial proliferation in intact bronchial tissue
- Author
-
Stephen L. Nishimura, Arthur C. Hill, Paul Brunetta, Gregory Dolganov, Stephanie Cambier, Lars Fjellbirkeland, David M. Jablons, and V. Courtney Broaddus
- Subjects
Male ,Integrins ,Integrin ,Bronchi ,Respiratory Mucosa ,Biology ,Pathology and Forensic Medicine ,In vivo ,Transforming Growth Factor beta ,Culture Techniques ,medicine ,Homeostasis ,Humans ,Aged ,Aged, 80 and over ,R-SMAD ,Cell adhesion molecule ,Gene Expression Profiling ,Transforming growth factor beta ,TGF beta receptor 2 ,Middle Aged ,Epithelium ,Cell biology ,medicine.anatomical_structure ,Ki-67 Antigen ,Immunology ,biology.protein ,Keratins ,Female ,Cell Division ,Transforming growth factor ,Regular Articles - Abstract
Transforming growth factor (TGF)-beta is a potent multifunctional cytokine that is an essential regulator of epithelial proliferation. Because TGF-beta is expressed almost entirely in a latent state in vivo, a major source of regulation of TGF-beta function is its activation. A subset of integrins, alphavbeta8 and alphavbeta6, which are expressed in the human airway, has recently been shown to activate latent TGF-beta in vitro, suggesting a regulatory role for integrins in TGF-beta function in vivo. Here we have developed a novel, biologically relevant experimental model of human airway epithelium using intact human bronchial tissue. We have used this model to determine the function of integrin-mediated activation of TGF-beta in the airway. In human bronchial fragments cultured in vitro, authentic epithelial-stromal interactions were maintained and integrin and TGF-beta expression profiles correlated with profiles found in normal lung. In addition, in this model, we found that either the integrin alphavbeta8 or TGF-beta could inhibit airway epithelial cell proliferation. Furthermore, we found that one mechanism of integrin-alphavbeta8-dependent inhibition of cell proliferation was through activation of TGF-beta because anti-beta8 antibody blocked the majority (76%) of active TGF-beta released from bronchial fragments. These data provide compelling evidence for a functional role for integrin-mediated activation of TGF-beta in control of human airway epithelial proliferation in vivo.
- Published
- 2003
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