DOCSLIB.ORG

Metalloproteinase-3)-Deficient Mice Infection in Stromelysin-1 (Matrix Impaired Immunity to Intestinal Bacterial

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Metalloproteinase-3)-Deficient Mice Infection in Stromelysin-1 (Matrix Impaired Immunity to Intestinal Bacterial The Journal of Immunology Impaired Immunity to Intestinal Bacterial Infection in Stromelysin-1 (Matrix Metalloproteinase-3)-Deficient Mice1 Chris K. F. Li,* Sylvia L. F. Pender,* Karen M. Pickard,* Victoria Chance,* Judith A. Holloway,* Alan Huett,‡ Nathalie S. Gonc¸alves,* John S. Mudgett,† Gordon Dougan,‡ Gad Frankel,‡ and Thomas T. MacDonald2* Infection of mice with the intestinal bacterial pathogen Citrobacter rodentium results in colonic mucosal hyperplasia and a local Th1 inflammatory response similar to that seen in mouse models of inflammatory bowel disease. Matrix metalloproteinases (MMPs) have been shown to mediate matrix remodeling and cell migration during tissue injury and repair in the intestine. We have previously shown enhanced pathology in infected TNFRp55؊/؊, IL-12p40؊/؊, and IFN-␥؊/؊ mice, and here we show that this is associated with an increase in stromelysin-1 (MMP3) transcripts in colonic tissues. We have therefore investigated the role of MMP3 in colonic mucosal hyperplasia and the local Th1 responses using MMP3؊/؊ mice. In MMP3؊/؊ mice, similar mucosal thickening was observed after infection as in wild-type (WT) mice. Colonic tissues from MMP3؊/؊ mice showed a compensatory increase in the expression of other MMP transcripts, such as MMP7 and MMP12. However, MMP3؊/؊ mice showed delayed clearance of bacteria and delayed appearance of CD4؉ T lymphocytes into intestinal lamina propria. CSFE-labeled mesenteric lymph node CD4؉ T lymphocytes from infected WT mice migrated in fewer numbers into the mesenteric lymph nodes and colon ,of MMP3؊/؊ mice than into those of WT mice. These studies show that mucosal remodeling can occur in the absence of MMP3 ,but that MMP3 plays a role in the migration of CD4؉ T lymphocytes to the intestinal mucosa. The Journal of Immunology, 2004 173: 5171–5179. nfection of mice with the intestinal bacterial pathogen (reviewed in Ref. 7). MMPs belong to the matrixin subfamily of Citrobacter rodentium results in colonic mucosal hyperplasia the metzincin superfamily of metalloproteinases. To date, Ͼ23 dif- and a local Th1 inflammatory response similar to that seen in ferent MMPs have been cloned, and additional members continue I 3 mouse models of inflammatory bowel disease (IBD). In suscep- to be identified. Structurally, all MMPs share a similar prodomain tible mouse strains, C. rodentium infection is associated with co- and catalytic domain, which act on a broad spectrum of the extra- lonic crypt hyperplasia, goblet cell depletion, and mucosal erosion cellular matrix components. Most MMPs are secreted as proen- (1, 2). Infection of mice with live C. rodentium or intracolonic zymes and require proteolytic cleavage for activation. Inhibition of inoculation of dead bacteria also induces a large infiltrate of CD4ϩ MMPs is conducted by endogenous MMP inhibitors, tissue inhib- cells into the colonic lamina propria and a highly polarized local itor of metalloproteinases (TIMP), which contain four members Th1 response with increased transcripts for the type I cytokines (TIMP1–4). MMPs have been implicated in tissue injury and ma- IL-12, TNF-␣, and IFN-␥ (3, 4). trix remodeling in various chronic inflammatory diseases such as To elucidate the role of proinflammatory cytokines in host de- IBD, asthma, and rheumatoid arthritis. In the intestine, MMPs are fense and mucosal pathology in vivo, mice deficient in TNF-␣, highly expressed at ulcer edges in patients with chronic IBD and IFN-␥, and IL-12 were orally infected with live C. rodentium (5, play a crucial role in the tissue injury that follows T cell activation 6). These animals showed various degrees of impairment in their in explants of human fetal small bowel (8–10). In celiac disease, ability to clear infection, but remarkably, mucosal pathology was where a mucosal Th1 response to gluten drives mucosal remodel- greater in these mice than in wild-type (WT) controls. ing and growth, MMP3 is overexpressed in fibroblasts immedi- Matrix metalloproteinases (MMPs) are a family of proteases ately under the epithelium (11). important in the turnover of extracellular matrix and cell migration Taken together, these results suggest that MMPs, especially MMP3, is involved in immune-mediated tissue injury in the intes- tine. MMPs probably play a role in degrading the matrix to pro- *Division of Infection, Inflammation and Repair, University of Southampton School duce mucosal ulceration, but they may also be important in con- of Medicine, Southampton, United Kingdom; †Merck Research Laboratories, Rah- way, NJ; and ‡Department of Biochemistry, Centre for Molecular Microbiology and trolling matrix turnover and in the mucosal thickening seen in Infection, Imperial College, South Kensington, United Kingdom experimental and clinical intestinal inflammation. Received for publication February 5, 2004. Accepted for publication July 21, 2004. In this work, therefore, we first examined MMP3 in the colon of The costs of publication of this article were defrayed in part by the payment of page WT and TNF-␣, IFN-␥, and IL-12 knockout (KO) mice infected charges. This article must therefore be hereby marked advertisement in accordance with C. rodentium and subsequently investigated mucosal hyper- with 18 U.S.C. Section 1734 solely to indicate this fact. plasia and the local immune responses in MMP3null mice. 1 This work was supported by the Biotechnology and Biological Sciences Research Council. 2 Address correspondence and reprint requests to Prof. Thomas MacDonald, Mail- Materials and Methods point 813, Division of Infection, Inflammation and Repair, Southampton General Mice Hospital, Southampton, SO16 6YD, U.K. E-mail address: [email protected] 3 Abbreviations used in this paper: IBD, inflammatory bowel disease; MMP, matrix Female 6- to 8-wk-old C57BL/6J and B10R111 mice were purchased from metalloproteinase; TIMP, tissue inhibitor of metalloproteinase; DC, dendritic cell; Harlan Olac (Bichester, U.K.) or Bantin and Kingman Universal (Hull, Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ MLN, mesenteric lymph node; MLNC, mesenteric lymph node cell; KO, knockout. U.K.). TNFRp55 / , IL-12p40 / , and IFN-␥ / mice (backcrossed to Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 5172 MMP-3 AND T CELL RESPONSES IN C. rodentium INFECTION C57BL/6 background at least 10 times) were originally purchased from determination of isotype-specific Ab titers, wells were washed with PBS- The Jackson Laboratory (Bar Harbor, ME) and were maintained by ho- Tween 20 before addition of 100 ␮l of an Ig-specific HRP conjugate mozygous matings under contract at Bantin and Kingman Universal. (DAKO, High Wycombe, U.K.) diluted 1/1000 in PBS-Tween 20 contain- MMP3Ϫ/Ϫ mice were obtained from John Mudgett, Merck Research Lab- ing 0.2% (w/v) BSA for2hat37°C. Finally, after a washing with PBS- oratories (Rahway, NJ) and were maintained by homozygous matings. All Tween 20, bound Ab was detected by addition of o-phenylenediamine mice came from specific pathogen-free colonies. They were kept in specific substrate (Sigma-Aldrich) and the OD490 was measured. Titers were de- pathogen-free environment with free access to sterilized food and water. termined arbitrarily as the reciprocal of the serum dilution corresponding to All experiments used five to six mice per group and were repeated at least an OD of 0.3. twice. Protein extraction Bacterial strains and oral infection of mice Colonic samples (2 cm) were snap-frozen at Ϫ70°C and homogenized in A nalidixic acid-resistant isolate of C. rodentium (formerly Citrobacter ice-cold extraction buffer (50 mM Tris-HCl (pH 7.4), 10 mM CaCl2, 0.05% freundii biotype 4280) was used in this study. DBS255 (pCVD438) is a C. Brij 35, 0.25% Triton X-100) at maximum speed for 30 s using an IKA rodentium eae (intimin ␣) strain, which expresses biologically active in- tissue homogenizer (Fisher Scientific, Loughborough, U.K.). The homog- timin and is virulent in mice (12). Bacterial inocula were prepared by enates were centrifuged at 13,000 rpm for 10 min at 4°C, and the super- culturing bacteria overnight at 37°C in 10 ml of Luria broth containing natants were removed and assayed for protein concentration (Bio-Rad, nalidixic acid (100 ␮g/ml) plus chloramphenicol (50 ␮g/ml). Cultures were Hemel Hempstead, Hertfordshire, U.K.). Homogenates were stored at harvested by centrifugation and resuspended in 0.1 volume of PBS. Mice Ϫ70°C until required. were orally inoculated with 200 ␮l of the bacterial suspension with a ga- vage needle. The viable count of the inoculum was determined by plating Western blotting on Luria-Bertani agar containing appropriate antibiotics. In all experi- Samples were denatured in reducing treatment buffer and loaded (200 ␮g/ ϫ 9 ments, mice received 1–4 10 CFU. lane) onto 10% (v/v) Novex Bis-Tris gels (Invitrogen, Paisley, U.K.). After electrophoresis, the proteins were transferred onto nitrocellulose membrane Measurement of pathogen burden (Bio-Rad) using a Novex blotting apparatus (Invitrogen, Paisley, U.K.) and At selected times postinfection, mice were killed by cervical dislocation. detected using goat anti-mouse MMP3 (1/250) (R&D, Abingdon, U.K.), The terminal 4 cm of the colon were removed, and the colon was weighed HRP-conjugated rabbit anti-goat secondary Ab (DAKO, Cambridge, after removal of fecal pellets. In some experiments, 1-cm samples of distal U.K.), and an ECL Plus kit (Amersham Biosciences, Little Chalfont, U.K.) colon were snap-frozen in liquid nitrogen for subsequent immunohistolog- according to the manufacturer’s instructions. The Ab binding was imaged ical analysis, cytokine RT-PCR, and MMP Western blotting. Spleens and on ECL film (Amersham Biosciences). For control of protein loading, the colons were homogenized mechanically using a Seward 80 stomacher blot was stripped with Tris-HCl buffer (62.5 mM, pH 6.7) containing 10 (Seward Medica, London, U.K.). The number of viable bacteria in organ mM 2-ME for 30 min at 60°C with gentle shaking.
Load more

Recommended publications
  • Intestinal Tumorigenesis Is Suppressed in Mice Lacking the Metalloproteinase Matrilysin (Knockout͞min͞colon Cancer͞extracellular Matrix͞apc)
    Proc. Natl. Acad. Sci. USA Vol. 94, pp. 1402–1407, February 1997 Medical Sciences Intestinal tumorigenesis is suppressed in mice lacking the metalloproteinase matrilysin (knockoutyMinycolon canceryextracellular matrixyAPC) CAROLE L. WILSON*†,KATHLEEN J. HEPPNER*†,PATRICIA A. LABOSKY*‡,BRIGID L. M. HOGAN*‡, AND LYNN M. MATRISIAN*§ *Department of Cell Biology and ‡Howard Hughes Medical Institute, Vanderbilt University, Nashville, TN 37232 Communicated by Ruth Sager, Dana–Farber Cancer Institute, Boston, MA, December 12, 1996 (received for review June 27, 1996) ABSTRACT Matrix metalloproteinases (MMPs) classi- The Min (multiple intestinal neoplasia) mouse has proven to cally have been implicated in basement membrane destruction be a powerful model system to study molecules involved in the associated with late-stage tumor cell invasion and metastasis. progression of intestinal adenomas. It has been determined However, recent studies have demonstrated that one MMP that a nonsense autosomal dominant germline mutation in the family member, matrilysin, is expressed in a high percentage adenomatous polyposis coli (Apc) gene (the ApcMin allele) of early-stage human colorectal tumors. We analyzed matri- induces spontaneous intestinal tumors in these mice, providing lysin expression in benign intestinal tumors from mice het- a model that closely mimics the human hereditary colon cancer erozygous for the ApcMin allele (Miny1) and found that the syndrome, familial adenomatous polyposis (13, 14). Congenic mRNA was induced in the majority (88%) of these adenomas. C57BLy6-Min mice heterozygous for this mutant allele Protein was detected in the tumor cells, where, surprisingly, (Miny1) develop numerous benign tumors throughout the it was predominantly immunolocalized to the lumenal surface intestinal tract, although with less frequency in the large bowel, of dysplastic glands rather than the basement membrane or and have an average life span of 119 days (13, 15).
    [Show full text]
  • Discovery and Optimization of Selective Inhibitors of Meprin Α (Part II)
    pharmaceuticals Article Discovery and Optimization of Selective Inhibitors of Meprin α (Part II) Chao Wang 1,2, Juan Diez 3, Hajeung Park 1, Christoph Becker-Pauly 4 , Gregg B. Fields 5 , Timothy P. Spicer 1,6, Louis D. Scampavia 1,6, Dmitriy Minond 2,7 and Thomas D. Bannister 1,2,* 1 Department of Molecular Medicine, Scripps Research, Jupiter, FL 33458, USA; [email protected] (C.W.); [email protected] (H.P.); [email protected] (T.P.S.); [email protected] (L.D.S.) 2 Department of Chemistry, Scripps Research, Jupiter, FL 33458, USA; [email protected] 3 Rumbaugh-Goodwin Institute for Cancer Research, Nova Southeastern University, 3321 College Avenue, CCR r.605, Fort Lauderdale, FL 33314, USA; [email protected] 4 The Scripps Research Molecular Screening Center, Scripps Research, Jupiter, FL 33458, USA; [email protected] 5 Unit for Degradomics of the Protease Web, Institute of Biochemistry, University of Kiel, Rudolf-Höber-Str.1, 24118 Kiel, Germany; fi[email protected] 6 Department of Chemistry & Biochemistry and I-HEALTH, Florida Atlantic University, 5353 Parkside Drive, Jupiter, FL 33458, USA 7 Dr. Kiran C. Patel College of Allopathic Medicine, Nova Southeastern University, 3301 College Avenue, Fort Lauderdale, FL 33314, USA * Correspondence: [email protected] Abstract: Meprin α is a zinc metalloproteinase (metzincin) that has been implicated in multiple diseases, including fibrosis and cancers. It has proven difficult to find small molecules that are capable Citation: Wang, C.; Diez, J.; Park, H.; of selectively inhibiting meprin α, or its close relative meprin β, over numerous other metzincins Becker-Pauly, C.; Fields, G.B.; Spicer, which, if inhibited, would elicit unwanted effects.
    [Show full text]
  • 2335 Roles of Molecules Involved in Epithelial/Mesenchymal Transition
    [Frontiers in Bioscience 13, 2335-2355, January 1, 2008] Roles of molecules involved in epithelial/mesenchymal transition during angiogenesis Giulio Ghersi Dipartimento di Biologia Cellulare e dello Sviluppo, Universita di Palermo, Italy TABLE OF CONTENTS 1. Abstract 2. Introduction 3. Extracellular matrix 3.1. ECM and integrins 3.2. Basal lamina components 4. Cadherins. 4.1. Cadherins in angiogenesis 5. Integrins. 5.1. Integrins in angiogenesis 6. Focal adhesion molecules 7. Proteolytic enzymes 7.1. Proteolytic enzymes inhibitors 7.2. Proteolytic enzymes in angiogenesis 8. Perspective 9. Acknowledgements 10. References 1.ABSTRACT 2. INTRODUCTION Formation of vessels requires “epithelial- Growth of new blood vessels (angiogenesis) mesenchymal” transition of endothelial cells, with several plays a key role in several physiological processes, such modifications at the level of endothelial cell plasma as vascular remodeling during embryogenesis and membranes. These processes are associated with wound healing tissue repair in the adult; as well as redistribution of cell-cell and cell-substrate adhesion pathological processes, including rheumatoid arthritis, molecules, cross talk between external ECM and internal diabetic retinopathy, psoriasis, hemangiomas, and cytoskeleton through focal adhesion molecules and the cancer (1). Vessel formation entails the “epithelial- expression of several proteolytic enzymes, including matrix mesenchymal” transition of endothelial cells (ECs) “in metalloproteases and serine proteases. These enzymes with vivo”; a similar phenotypic exchange can be induced “in their degradative action on ECM components, generate vitro” by growing ECs to low cell density, or in “wound molecules acting as activators and/or inhibitors of healing” experiments or perturbing cell adhesion and angiogenesis. The purpose of this review is to provide an associated molecule functions.
    [Show full text]
  • On and Polymorphisms in Q Fever
    Matrix metalloproteinase expression, produc3on and polymorphisms in Q fever Anne F.M. Jansen1,2, Teske Schoffelen1,2, Julien Textoris3, Jean Louis Mege3, Chantal P. Bleeker-Rovers1,2, Esther van de Vosse4, Hendrik Jan Roest5, Marcel van Deuren1,2 1. Department of Internal Medicine, Division of Experimental Medicine, Radboud university medical center, Nijmegen, The Netherlands 2. Radboud Expert Centre for Q fever, Radboud university medical center, Nijmegen, the Netherlands, 3. URMITE, CNRS UMR 7278, IRD 198, INSERM 1095 Aix-Marseille University, Marseille, France 4. Department of Infec3ous Diseases, Leiden University Medical Center, Leiden, The Netherlands 5. Department of Bacteriology and TSEs, Central Veterinary Instute, part of Wageningen UR, Lelystad, the Netherlands Background C. burnei induces MMP-1 and MMP-9 produc3on in PBMCs Chronic Q fever is a life threatening condi3on caused by the Gram-negave bacterium Coxiella burnei, manifes3ng as an infec3on of aneurysms, aor3c prosthesis or heart valves. Matrix metalloproteinases (MMPs) are proteoly3c enzymes that cleave extracellular matrix and are implicated in the pathology of aneurysms and endocardi3s. Currently, the contribu3on of MMPs to the pathogenesis of chronic Q fever is unknown. Methods We inves3gated the C. burnei specific gene expression of MMPs in PBMCs and protein produc3on by ELISA in chronic Q fever paents (n=6, n=10, respec3vely), cardiovascular paents with a history of Q fever (n=10) and healthy controls (n=4, n=10, respec3vely), in some experiments, the controls had vascular disease (n=10). Circulang MMP levels were assessed with Luminex technology and these groups were also genotyped for 20 SNPs in MMP and Tissue Inhibitor of MMP (TIMP) genes.
    [Show full text]
  • Investigation of the Underlying Hub Genes and Molexular Pathogensis in Gastric Cancer by Integrated Bioinformatic Analyses
    bioRxiv preprint doi: https://doi.org/10.1101/2020.12.20.423656; this version posted December 22, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Investigation of the underlying hub genes and molexular pathogensis in gastric cancer by integrated bioinformatic analyses Basavaraj Vastrad1, Chanabasayya Vastrad*2 1. Department of Biochemistry, Basaveshwar College of Pharmacy, Gadag, Karnataka 582103, India. 2. Biostatistics and Bioinformatics, Chanabasava Nilaya, Bharthinagar, Dharwad 580001, Karanataka, India. * Chanabasayya Vastrad [email protected] Ph: +919480073398 Chanabasava Nilaya, Bharthinagar, Dharwad 580001 , Karanataka, India bioRxiv preprint doi: https://doi.org/10.1101/2020.12.20.423656; this version posted December 22, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Abstract The high mortality rate of gastric cancer (GC) is in part due to the absence of initial disclosure of its biomarkers. The recognition of important genes associated in GC is therefore recommended to advance clinical prognosis, diagnosis and and treatment outcomes. The current investigation used the microarray dataset GSE113255 RNA seq data from the Gene Expression Omnibus database to diagnose differentially expressed genes (DEGs). Pathway and gene ontology enrichment analyses were performed, and a proteinprotein interaction network, modules, target genes - miRNA regulatory network and target genes - TF regulatory network were constructed and analyzed. Finally, validation of hub genes was performed. The 1008 DEGs identified consisted of 505 up regulated genes and 503 down regulated genes.
    [Show full text]
  • Gent Forms of Metalloproteinases in Hydra
    Cell Research (2002); 12(3-4):163-176 http://www.cell-research.com REVIEW Structure, expression, and developmental function of early diver- gent forms of metalloproteinases in Hydra 1 2 3 4 MICHAEL P SARRAS JR , LI YAN , ALEXEY LEONTOVICH , JIN SONG ZHANG 1 Department of Anatomy and Cell Biology University of Kansas Medical Center Kansas City, Kansas 66160- 7400, USA 2 Centocor, Malvern, PA 19355, USA 3 Department of Experimental Pathology, Mayo Clinic, Rochester, MN 55904, USA 4 Pharmaceutical Chemistry, University of Kansas, Lawrence, KS 66047, USA ABSTRACT Metalloproteinases have a critical role in a broad spectrum of cellular processes ranging from the breakdown of extracellular matrix to the processing of signal transduction-related proteins. These hydro- lytic functions underlie a variety of mechanisms related to developmental processes as well as disease states. Structural analysis of metalloproteinases from both invertebrate and vertebrate species indicates that these enzymes are highly conserved and arose early during metazoan evolution. In this regard, studies from various laboratories have reported that a number of classes of metalloproteinases are found in hydra, a member of Cnidaria, the second oldest of existing animal phyla. These studies demonstrate that the hydra genome contains at least three classes of metalloproteinases to include members of the 1) astacin class, 2) matrix metalloproteinase class, and 3) neprilysin class. Functional studies indicate that these metalloproteinases play diverse and important roles in hydra morphogenesis and cell differentiation as well as specialized functions in adult polyps. This article will review the structure, expression, and function of these metalloproteinases in hydra. Key words: Hydra, metalloproteinases, development, astacin, matrix metalloproteinases, endothelin.
    [Show full text]
  • An Adverse Role for Matrix Metalloproteinase 12 After Spinal Cord Injury in Mice
    The Journal of Neuroscience, November 5, 2003 • 23(31):10107–10115 • 10107 Development/Plasticity/Repair An Adverse Role for Matrix Metalloproteinase 12 after Spinal Cord Injury in Mice Jennifer E. A. Wells,1 Tiffany K. Rice,1 Robert K. Nuttall,3 Dylan R. Edwards,3 Hakima Zekki,4 Serge Rivest,4 V. Wee Yong1,2 Departments of 1Clinical Neurosciences and 2Oncology, Faculty of Medicine, University of Calgary, Calgary, Alberta T2N 4N1, Canada, 3School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom, and 4Laboratory of Molecular Endocrinology and Department of Anatomy and Physiology, Laval University, Quebec, Quebec G1V 4G2, Canada We investigated the role of matrix metalloproteinases (MMPs) in acute spinal cord injury (SCI). Transcripts encoding 22 of the 23 known mammalian MMPs were measured in the mouse spinal cord at various time points after injury. Although there were significant changes in the expression levels of multiple MMPs, MMP-12 was increased 189-fold over normal levels, the highest of all MMPs examined. To evaluate the role of MMP-12 in SCI, spinal cord compression was performed in wild-type (WT) and MMP-12 null mice. Behavioral analyses were conducted for 4 weeks using the Basso–Beattie–Bresnahan (BBB) locomotor rating scale as well as the inclined plane test. The results show that MMP-12 null mice exhibited significantly improved functional recovery compared with WT controls. Twenty-eight days after injury, the BBB score in the MMP-12 group was 7, representing extensive movement of all three hindlimb joints, compared with 4 in the WT group, representing only slight movement of these joints.
    [Show full text]
  • Effects of Collagen-Derived Bioactive Peptides and Natural Antioxidant
    www.nature.com/scientificreports OPEN Efects of collagen-derived bioactive peptides and natural antioxidant compounds on Received: 29 December 2017 Accepted: 19 June 2018 proliferation and matrix protein Published: xx xx xxxx synthesis by cultured normal human dermal fbroblasts Suzanne Edgar1, Blake Hopley1, Licia Genovese2, Sara Sibilla2, David Laight1 & Janis Shute1 Nutraceuticals containing collagen peptides, vitamins, minerals and antioxidants are innovative functional food supplements that have been clinically shown to have positive efects on skin hydration and elasticity in vivo. In this study, we investigated the interactions between collagen peptides (0.3–8 kDa) and other constituents present in liquid collagen-based nutraceuticals on normal primary dermal fbroblast function in a novel, physiologically relevant, cell culture model crowded with macromolecular dextran sulphate. Collagen peptides signifcantly increased fbroblast elastin synthesis, while signifcantly inhibiting release of MMP-1 and MMP-3 and elastin degradation. The positive efects of the collagen peptides on these responses and on fbroblast proliferation were enhanced in the presence of the antioxidant constituents of the products. These data provide a scientifc, cell-based, rationale for the positive efects of these collagen-based nutraceutical supplements on skin properties, suggesting that enhanced formation of stable dermal fbroblast-derived extracellular matrices may follow their oral consumption. Te biophysical properties of the skin are determined by the interactions between cells, cytokines and growth fac- tors within a network of extracellular matrix (ECM) proteins1. Te fbril-forming collagen type I is the predomi- nant collagen in the skin where it accounts for 90% of the total and plays a major role in structural organisation, integrity and strength2.
    [Show full text]
  • ADAM10 (A-3): Sc-48400
    SAN TA C RUZ BI OTEC HNOL OG Y, INC . ADAM10 (A-3): sc-48400 BACKGROUND APPLICATIONS ADAM (a disintegrin and metalloprotease) proteins are a family of over 30 ADAM10 (A-3) is recommended for detection of ADAM10 of mouse, rat and membrane-anchored, glycosylated, Zn 2+ -dependent proteases that are involved human origin by Western Blotting (starting dilution 1:200, dilution range in cell-cell, cell-matrix interface-related processes including fertilization, mus - 1:100-1:1000), immunoprecipitation [1-2 µg per 100-500 µg of total protein cl e fusion, secretion of TNF (tumor necrosis factor α) and modulation of the (1 ml of cell lysate)], immunofluorescence (starting dilution 1:50, dilution neurogenic function of Notch and Delta. ADAM proteins possess a signal- range 1:50-1:500), immunohistochemistry (including paraffin-embedded domain, a pro-domain, a metalloprotease domain, a disintegrin domain (inte - sections) (starting dilution 1:50, dilution range 1:50-1:500) and solid phase grin ligand), a cysteine-rich region, an epidermal growth factor-like domain, ELISA (starting dilution 1:30, dilution range 1:30-1:3000). a transmembrane domain and a cytoplasmic tail. ADAMs are expressed in Suitable for use as control antibody for ADAM10 siRNA (h): sc-41410, brain, testis, epididymis, ovary, breast, placenta, liver, heart, lung, bone and ADAM10 siRNA (m): sc-41411, ADAM10 siRNA (r): sc-270165, ADAM10 muscle, and catalyze proteolysis, adhesion, fusion and intracellular signaling. shRNA Plasmid (h): sc-41410-SH, ADAM10 shRNA Plasmid (m): sc-41411- SH, ADAM10 is a TNF-processing enzyme that cleaves pro-TNF, a membrane- ADAM10 shRNA Plasmid (r): sc-270165-SH, ADAM10 shRNA (h) Lentiviral bound precusor protein, at Ala 76-Val 77, which causes membrane shedding Particles: sc-41410-V, ADAM10 shRNA (m) Lentiviral Particles: sc-41411-V of soluble TNF.
    [Show full text]
  • Neprilysin Activity Assay Kit (MAK350)
    Neprilysin Activity Assay Kit Catalog Number MAK350 Storage Temperature –20 C TECHNICAL BULLETIN Product Description Components Neprilysin, also known as neutral endopeptidase, The kit is sufficient for 100 fluorometric assays in enkephalinase, CD10, and common acute 96 well plates. lymphoblastic leukemia antigen, is a zinc-containing transmembrane metalloproteinase. It is able to NEP Assay Buffer 40 mL hydrolyze very important endogenous peptides, such as Catalog Number MAK350A natriuretic atrial factor, enkephalins, substance P, bradykinin and amyloid (A ) peptide. Thus, NEP is a Neprilysin (Lyophilized) 1 vial potentially therapeutic target in important pathological Catalog Number MAK350B conditions such as cardiovascular disease, prostate cancer, and Alzheimer’s disease. NEP has also been NEP Substrate (in DMSO) 15 L used as a biological marker in a type of childhood Catalog Number MAK350C leukemia. The detection of NEP in endometrial stromal cells has been proposed as a helpful tool in the Abz-Standard (1 mM) 100 L diagnosis of endometriosis. NEP is currently a focus of Catalog Number MAK350D major interest in cardiovascular and neurological research. Reagents and Equipment Required but Not Provided. The Neprilysin Activity Assay Kit utilizes the ability of an Pipetting devices and accessories active NEP to cleave a synthetic substrate, o-amino- (e.g., multichannel pipettor) benzoic acid (Abz)-based peptide, to release a free White opaque flatbottom 96 well plates fluorophore. The released Abz can be easily quantified Fluorescence multiwell plate reader, capable of using a fluorescence microplate reader. The substrate 37 C temperature setting is specific to NEP and can differentiate the NEP activity Refrigerated microcentrifuge capable of from trypsin and other structurally similar zinc RCF 12,000 g metalloproteinase in biological samples such as Protease Inhibitor Cocktail (contains 80 M angiotensin converting enzymes (ACE1 and ACE2) and Aprotinin) (Catalog Number P8340) endothelin converting enzymes (ECE1 and ECE2).
    [Show full text]
  • Prognostic Significance of MMP-1 and MMP-3 Functional Promoter Polymorphisms in Colorectal Cancer
    594 Vol. 11, 594–599, January 15, 2005 Clinical Cancer Research Prognostic Significance of MMP-1 and MMP-3 Functional Promoter Polymorphisms in Colorectal Cancer Franck Zinzindohoue´,1 Thierry Lecomte,2 INTRODUCTION Jean-Marc Ferraz,2 Anne-Marie Houllier,1 Matrix metalloproteinase (MMP) belongs to a large group Paul-Henri Cugnenc,2 Anne Berger,2 of proteases, which includes over 22 known human zinc- He´le`ne Blons,1 and Pierre Laurent-Puig1 dependent proteolytic enzymes. These are capable of breaking essentially all components of the extracellular matrix (1, 2). 1INSERM U490 Laboratoire de toxicologie Mole´culaire and 2 MMPs take part in high tissue turnover and remodeling, both Poˆle de cance´rologie Hoˆpital europe´en Georges Pompidou, Paris, France physiologic and pathologic conditions such as cancer. MMPs are also implicated in all steps of tumorogenesis, cancer invasion, and metastasis (3). Tumor invasion and metastasis formation ABSTRACT always begin with blood and lymphatic vessel infiltration. As Purpose: Matrix metalloproteinase (MMP) belongs to a these processes involve proteolysis of the extracellular matrix, large group of proteases capable of breaking essentially all MMPs are suggested to play a major role in tumor progression. components of the extracellular matrix. They are implicated Colorectal cancer is the most common malignancy of the in all steps of tumorogenesis, cancer invasion, and gastrointestinal tract. It is the third cause of cancer overall and metastasis. Among them, metalloproteinase type 1 (MMP- the second leading cause of cancer-related death in the Europe 1) is implicated in tumor invasion and metastasis in and the United States with an incidence of 300,000 new cases different types of cancers including colorectal cancer in (4).
    [Show full text]
  • (MMP10) Moderates Inflammation by Controlling Macrophage Activation
    Stromelysin-2 (MMP10) Moderates Inflammation by Controlling Macrophage Activation This information is current as Ryan S. McMahan, Timothy P. Birkland, Kate S. Smigiel, of September 26, 2021. Tyler C. Vandivort, Maryam G. Rohani, Anne M. Manicone, John K. McGuire, Sina A. Gharib and William C. Parks J Immunol 2016; 197:899-909; Prepublished online 17 June 2016; doi: 10.4049/jimmunol.1600502 Downloaded from http://www.jimmunol.org/content/197/3/899 Supplementary http://www.jimmunol.org/content/suppl/2016/06/16/jimmunol.160050 Material 2.DCSupplemental http://www.jimmunol.org/ References This article cites 72 articles, 15 of which you can access for free at: http://www.jimmunol.org/content/197/3/899.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 26, 2021 • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Stromelysin-2 (MMP10) Moderates Inflammation by Controlling Macrophage Activation Ryan S.
    [Show full text]