DOCSLIB.ORG

A Significant Soluble Keratin Fraction In

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
A Significant Soluble Keratin Fraction In Journal of Cell Science 105, 433-444 (1993) 433 Printed in Great Britain © The Company of Biologists Limited 1993 A significant soluble keratin fraction in ‘simple’ epithelial cells Lack of an apparent phosphorylation and glycosylation role in keratin solubility Chih-Fong Chou*, Carrie L. Riopel, Lusijah S. Rott and M. Bishr Omary† Palo Alto Veterans Administration Medical Center and the Digestive Disease Center at Stanford University, School of Medicine, 3801 Miranda Avenue, GI 111, Palo Alto, CA 94304, USA *Author for reprint requests †Author for correspondence SUMMARY We studied the solubility of keratin polypeptides 8 and aments in vitro as determined by electron microscopy. 18 (K8/18), which are the predominant intermediate fil- Cross-linking of soluble K8/18 followed by immunopre- aments in the human colonic epithelial cell line HT29. cipitation resulted in dimeric and tetrameric forms, We find that asynchronously growing cells (G0/G1 stage based on migration in SDS-polyacrylamide gels. In of the cell cycle) have a substantial pool of soluble ker- addition, cross-linked and native soluble K8/18 showed atin that constitutes approx. 5% of total cellular ker- similar migration on nondenaturing gels and similar atin. This soluble keratin pool was observed after sedimentation after sucrose density gradient centrifu- immunoprecipitation of K8/18 from the cytosolic frac- gation. Our results indicate that simple epithelial ker- tion of cells disrupted using three detergent-free meth- atins are appreciably more soluble than previously rec- ods. Several other cell lines showed a similar significant ognized. The soluble keratin form is assembly competent soluble cytosolic K8/18 pool. Arrest of HT29 cells in and appears to be primarily tetrameric. Although K8/18 G2/M stage of the cell cycle was associated with a con- solubility was found to increase during mitotic arrest, current increase in keratin solubility. Comparison of glycosylation and phosphorylation did not play an obvi- K8/18 obtained from the soluble cytosolic fraction and ous role in generating the soluble fraction, suggesting the insoluble high-speed pellet fraction showed similar an alternate mechanism for keratin solubility. levels of phosphorylation and glycosylation and similar tryptic radiolabeled phospho- and glycopeptide pat- Key words: keratin glycosylation, keratin phosphorylation, keratin terns. Soluble K8/18 can form characteristic 10 nm fil- solubility INTRODUCTION for a number of IF. For example, newly synthesized vimentin was detected in a precursor soluble pool with sub- Intermediate filaments (IF) represent one of the three major sequent incorporation into filaments (Blikstad and classes of cytoskeletal proteins that are present in most Lazarides, 1983). The soluble pool may also include pre- eukaryotic cells (Steinert and Roop, 1988). Of the three existing older molecules in addition to newly synthesized major cytoskeletal proteins, IF are felt to be the least sol- vimentin, as determined by pulse-chase labeling experi- uble; and within IF, keratins are considered insoluble in ments (Söellner et al., 1985). These observations suggested aqueous salt solutions (Steinert et al., 1982; Bershadsky and an exchange between the soluble and insoluble IF fractions, Vasiliev, 1988; Lazarides, 1982). In fact, a generally uti- which was supported by the observation that post-transla- lized method for keratin isolation makes use of their insol- tional generation of vimentin filaments from a soluble pool ubility in high-salt buffer systems (Achtstaetter et al., 1986). may occur with or without cotranslational filament forma- Also, complete solubilization of keratins generally requires tion, depending on the tissue source (Isaacs et al., 1989). a high concentration of urea, although different keratin pairs Similarly, cytosolic fractions from rat livers contained a exhibit different solubility in urea. For example, keratin keratin-like pool based on its filament-forming ability and polypeptides 5 and 14 (K5/14, nomenclature and numeri- partial peptide mapping analysis (Sahyoun et al., 1982). cal classification are based on that of Moll et al., 1982), Neurofilaments also appeared to be formed from soluble which are expressed in basal epidermal cells, can be solu- precursors that can be chased with time into the cytoskele- bilized in 2 M urea; whereas K1 and K10/11 (expressed in tal fraction (Black et al., 1986). In most cases, the soluble suprabasal cells) required 4-6 M urea (Eichner and Kahn, IF pool was not quantitated but was implied to be ‘small’. 1990). For example, a small soluble K8 and K18 pool was iden- The presence of a ‘small’ soluble pool has been shown tified in the human hepatocellular cell line PLC (Franke et 434 C.-F. Chou and others al., 1987). In addition, using immunoblot analysis, Xeno - or Eagle’s MEM supplemented with 10% fetal calf serum. Arrest pus laevis oocytes and eggs were estimated to contain 1 to of cells in G2/M using colcemid (0.5 mg/ml) and cell cycle analy- 10% of their total keratin in a soluble form (Gall and sis was carried out exactly as described (Chou and Omary, 1993). Karsenti, 1987). When quantitated, soluble vimentin was Monoclonal antibodies to K8/18 used were: L2A1 coupled to ® estimated to be less than 0.4% of total vimentin as deter- Sepharose beads (Chou et al., 1992), or to Affinica agarose beads mined by pulse labeling with [35S]methionine for 30 min as per manufacturer’s recommendation (Schleicher & Schuell, Keene, NH); and CK5 (Sigma). Other reagents used were UDP- then chasing for up to 15 h (Söellner et al., 1985). Fur- [4,5-3H]galactose (34.6 Ci/mmol), N-[6-3H]glucosamine-HCl 32 35 thermore, for some IF (e.g. neurofilaments), solubility (40.4 Ci/mmol), orthophosphate ( PO4), [ S]protein-labeling appears to be tissue dependent. For example, mice injected mix (1186 Ci/mmol), liquid and spray ENHANCE® (Du Pont- intravitreally with [35S]methionine showed a substantial New England Nuclear); catalase (Worthington Biochemical Corp., soluble pool of retinal but not optic axon neurofilament-L Freehold, NJ); human immunoglobulin G (IgG), galactosyltrans- (Nixon et al., 1989). ferase, bovine serum albumin (BSA), goat anti-mouse IgG- The factors involved in generating a soluble IF pool are Sepharose conjugate (Sigma); peroxidase-labeled goat anti-mouse not known. On the basis of in vitro studies of IF assembly, Ig (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD); and the polar nature of the phosphate group, phosphoryla- electron microscopy (EM) grids (Ted Pella Inc., Redding, CA); tion is an attractive potential regulator of IF solubility. For disuccinimidyl suberate (DSS) crosslinking agent (Pierce, Rock- ford, IL). example, in vitro phosphorylation of filamentous vimentin (Inagaki et al., 1987; Chou et al., 1989), rat liver K8/18 Preparation of supernatant (S), pellet (P) and high- (Yano et al., 1991), desmin (Inagaki et al., 1988) and neu- salt extract (HSE) fractions rofilaments (Gonda et al., 1990) resulted in filament disas- sembly as determined by negative staining electron Cells grown asynchronously (primarily G0/G1 cells (G cells)) or arrested in G2/M (M cells) were washed twice with phosphate microscopy. However, analysis of the soluble vimentin buffered saline (PBS), then suspended in 1.5 ml of PBS, buffer fraction (Blikstad and Lazarides, 1983; Isaacs et al., 1989; A (50 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 25 mM KCl), or Söellner et al., 1985; Lamb et al., 1989), the soluble ker- buffer B (10 mM Tris-HCl, pH 7.5, 1 mM MgCl2, 130 mM KCl, atin fraction (Gall and Karsenti, 1987) or soluble neurofil- 5 mM EDTA, 5 mM NaCl). All buffers and detergent solutions aments (Black et al., 1986; Nixon et al., 1989) indicated also contained 0.1 mM phenylmethylsulfonyl fluoride (added that phosphorylation does not appear to play a role in gen- fresh), 25 mg/ml aprotinin, 10 mM leupeptin and 10 mM pepstatin. erating the soluble or insoluble fractions. To generate the S and P fractions, cells were disrupted in buffer Recently we showed that K8/18 in HT29 cells undergo A, B or PBS (1.5 ml) using a cell disruption bomb (no. 4639, Parr 2 a dynamic O-linked glycosylation, with multiple glycosy- Instrument Company, Maline, IL) at 1000 lbf/in for 5 min, fol- lation sites consisting of single N-acetylglucosamine lowed by ultracentrifugation (300,000 g; 90 min, 4°C, SW50.1 rotor). Alternatively, cells were disrupted in buffer B using a (GlcNAc) residues (Chou et al., 1992). The dynamic nature Dounce homogenizer (100 strokes) or by freeze-thawing three of this modification was reflected by the faster rate of K8/18 times (- 80°C, 5 min, followed by rapid thawing in a 37°C water carbohydrate biosynthesis and degradation, when compared bath) followed by ultracentrifugation. with the corresponding rates for the protein backbone (Chou High-salt extraction was performed in a manner similar to that et al., 1992). Mitotic arrest of HT29 cells, using colcemid described before (Achtstaetter et al., 1986). Briefly, HT29 cells or nocodazole, resulted in a dramatic increase in both gly- (one confluent 100 mm dish) or the P fraction obtained after bomb- cosylation and phosphorylation of K8/18 (Chou and Omary, ing and then ultracentrifugation were mixed with 1 ml of 1% 1993). Threonine was a major site of glycosylation, whereas Triton X-100 (TX-100) 5 mM EDTA in PBS for 2 min (4°C) fol- serine was the primary site of phosphorylation (Chou and lowed by centrifugation (16,000 g, 10 min). The supernatant was Omary, 1993). Here we quantitate the soluble K8/18 frac- removed and used for immunoprecipitation or discarded. The pellet was homogenized in a high-salt buffer (1 ml) containing 10 tion and ask what happens to K8/18 solubility in mitotic mM Tris-HCl (pH 7.6), 140 mM NaCl, 1.5 M KCl, 5 mM EDTA, arrest, and what role does phosphorylation and glycosyla- 0.5% TX-100, followed by a 30 min incubation and then pellet- tion play in generating the soluble fraction.
Load more

Recommended publications
  • Structures of the ß-Keratin Filaments and Keratin Intermediate Filaments in the Epidermal Appendages of Birds and Reptiles (Sauropsids)
    G C A T T A C G G C A T genes Review Structures of the ß-Keratin Filaments and Keratin Intermediate Filaments in the Epidermal Appendages of Birds and Reptiles (Sauropsids) David A.D. Parry School of Fundamental Sciences, Massey University, Private Bag 11-222, Palmerston North 4442, New Zealand; [email protected]; Tel.: +64-6-9517620; Fax: +64-6-3557953 Abstract: The epidermal appendages of birds and reptiles (the sauropsids) include claws, scales, and feathers. Each has specialized physical properties that facilitate movement, thermal insulation, defence mechanisms, and/or the catching of prey. The mechanical attributes of each of these appendages originate from its fibril-matrix texture, where the two filamentous structures present, i.e., the corneous ß-proteins (CBP or ß-keratins) that form 3.4 nm diameter filaments and the α-fibrous molecules that form the 7–10 nm diameter keratin intermediate filaments (KIF), provide much of the required tensile properties. The matrix, which is composed of the terminal domains of the KIF molecules and the proteins of the epidermal differentiation complex (EDC) (and which include the terminal domains of the CBP), provides the appendages, with their ability to resist compression and torsion. Only by knowing the detailed structures of the individual components and the manner in which they interact with one another will a full understanding be gained of the physical properties of the tissues as a whole. Towards that end, newly-derived aspects of the detailed conformations of the two filamentous structures will be discussed and then placed in the context of former knowledge.
    [Show full text]
  • A Dysfunctional Desmin Mutation in a Patient with Severe Generalized Myopathy
    Proc. Natl. Acad. Sci. USA Vol. 95, pp. 11312–11317, September 1998 Genetics A dysfunctional desmin mutation in a patient with severe generalized myopathy ANA M. MUN˜OZ-MA´RMOL*†‡,GERALDINE STRASSER‡§,MARCOS ISAMAT*†,PIERRE A. COULOMBE¶,YANMIN YANG§, i XAVIER ROCA†,ELENA VELA*†,JOSE´ L. MATE†,JAUME COLL ,MARI´A TERESA FERNA´NDEZ-FIGUERAS†, JOSE´ J. NAVAS-PALACIOS†,AURELIO ARIZA†, AND ELAINE FUCHS§** *Fundacio´n Echevarne, 08037 Barcelona, Spain; Departments of †Pathology and iNeurology, Hospital Universitari Germans Trias i Pujol, Universitat Auto´noma de Barcelona, 08916 Badalona, Barcelona, Spain; ¶Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, MD, 21205; and §Howard Hughes Medical Institute and Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, IL 60637 Contributed by Elaine Fuchs, July 29, 1998 ABSTRACT Mice lacking desmin produce muscle fibers desmin functions in cellular transmission of both active and with Z disks and normal sarcomeric organization. However, passive force (14, 15). the muscles are mechanically fragile and degenerate upon Desminopathies are a heterogeneous group of human myop- repeated contractions. We report here a human patient with athies characterized by abnormal aggregations of desmin in severe generalized myopathy and aberrant intrasarcoplasmic skeletal andyor cardiac muscle fibers (for review, see ref. 16). The accumulation of desmin intermediate filaments. Muscle tissue clumping of desmin in muscle cells of these patients bears a from this patient lacks the wild-type desmin allele and has a striking resemblance to the keratin aggregates that accumulate in desmin gene mutation encoding a 7-aa deletion within the degenerating epithelial cells of various human keratin disorders coiled-coil segment of the protein.
    [Show full text]
  • Keratins Determine Network Stress Responsiveness in Reconstituted Actin-Keratin Filament Systems
    bioRxiv preprint doi: https://doi.org/10.1101/2020.12.27.424392; this version posted December 28, 2020. The copyright holder for this preprint (which was not certified by peer review)Please is the author/funder, do not adjust who margins has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. ARTICLE Keratins determine network stress responsiveness in reconstituted actin‐keratin filament systems† *af‡ ab‡ ab cd *abe Iman Elbalasy , Paul Mollenkopf , Cary Tutmarc , Harald Herrmann and Jörg Schnauß The cytoskeleton is a major determinant of cell mechanics, a property that is altered during many pathological situations. To understand these alterations, it is essential to investigate the interplay between the main filament systems of the cytoskeleton in the form of composite networks. Here, we investigate the role of keratin intermediate filaments (IFs) in network strength by studying in vitro reconstituted actin and keratin 8/18 composite networks via bulk shear rheology. We co‐polymerized these structural proteins in varying ratios and recorded how their relative content affects the overall mechanical response of the various composites. For relatively small deformations, we found that all composites exhibited an intermediate linear viscoelastic behavior compared to that of the pure networks. In stark contrast, the composites displayed increasing strain stiffening behavior as a result of increased keratin content when larger deformations were imposed. This strain stiffening behavior is fundamentally different from behavior encountered with vimentin IF as a composite network partner for actin. Our results provide new insights into the mechanical interplay between actin and keratin in which keratin provides reinforcement to actin.
    [Show full text]
  • Biological Functions of Cytokeratin 18 in Cancer
    Published OnlineFirst March 27, 2012; DOI: 10.1158/1541-7786.MCR-11-0222 Molecular Cancer Review Research Biological Functions of Cytokeratin 18 in Cancer Yu-Rong Weng1,2, Yun Cui1,2, and Jing-Yuan Fang1,2,3 Abstract The structural proteins cytokeratin 18 (CK18) and its coexpressed complementary partner CK8 are expressed in a variety of adult epithelial organs and may play a role in carcinogenesis. In this study, we focused on the biological functions of CK18, which is thought to modulate intracellular signaling and operates in conjunction with various related proteins. CK18 may affect carcinogenesis through several signaling pathways, including the phosphoinosi- tide 3-kinase (PI3K)/Akt, Wnt, and extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) signaling pathways. CK18 acts as an identical target of Akt in the PI3K/Akt pathway and of ERK1/2 in the ERK MAPK pathway, and regulation of CK18 by Wnt is involved in Akt activation. Finally, we discuss the importance of gaining a more complete understanding of the expression of CK18 during carcinogenesis, and suggest potential clinical applications of that understanding. Mol Cancer Res; 10(4); 1–9. Ó2012 AACR. Introduction epithelial organs, such as the liver, lung, kidney, pancreas, The intermediate filaments consist of a large number of gastrointestinal tract, and mammary gland, and are also nuclear and cytoplasmic proteins that are expressed in a expressed by cancers that arise from these tissues (7). In the tissue- and differentiation-dependent manner. The compo- absence of CK8, the CK18 protein is degraded and keratin fi intermediate filaments are not formed (8).
    [Show full text]
  • Deciphering the Mechanoresponsive Role of Β-Catenin in Keratoconus
    bioRxiv preprint doi: https://doi.org/10.1101/603738; this version posted April 9, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 Deciphering the mechanoresponsive role of β-catenin in Keratoconus 2 epithelium 3 4 Chatterjee Amit 1,2, Prema Padmanabhan3, Janakiraman Narayanan#1 5 1 Department of Nanobiotechnology, Vision Research Foundation,Sankara Nethralaya campus, 6 Chennai, Tamil Nadu, India 7 2 School of Chemical and Biotechnology, SASTRA University, Tanjore, Tamil Nadu, India 8 3 Department of Cornea, Medical Research Foundation, Sankara Nethralaya campus, Chennai, 9 Tamil Nadu ,India 10 Running Title: β-catenin mechanotransduction in kerataconus 11 Correspondence: #Dr. Janakiraman Narayanan, Department of Nanobiotechnology, KNBIRVO 12 Block, Vision Research Foundation, Sankara Nethralaya campus 18/41, College road 13 Nungambakkam, Chennai, Tamil Nadu, India 600006, Tel-+91-44-28271616, (Ext) 1358, Fax- 14 +91-44-28254180, Email: [email protected] 15 16 17 18 19 20 21 22 23 1 bioRxiv preprint doi: https://doi.org/10.1101/603738; this version posted April 9, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 24 25 Abstract 26 Keratoconus (KC) a disease with established biomechanical instability of the corneal stroma, is an 27 ideal platform to identify key proteins involved in mechanosensing.
    [Show full text]
  • Types I and II Keratin Intermediate Filaments
    Downloaded from http://cshperspectives.cshlp.org/ on October 10, 2021 - Published by Cold Spring Harbor Laboratory Press Types I and II Keratin Intermediate Filaments Justin T. Jacob,1 Pierre A. Coulombe,1,2 Raymond Kwan,3 and M. Bishr Omary3,4 1Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland 21205 2Departments of Biological Chemistry, Dermatology, and Oncology, School of Medicine, and Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, Maryland 21205 3Departments of Molecular & Integrative Physiologyand Medicine, Universityof Michigan, Ann Arbor, Michigan 48109 4VA Ann Arbor Health Care System, Ann Arbor, Michigan 48105 Correspondence: [email protected] SUMMARY Keratins—types I and II—are the intermediate-filament-forming proteins expressed in epithe- lial cells. They are encoded by 54 evolutionarily conserved genes (28 type I, 26 type II) and regulated in a pairwise and tissue type–, differentiation-, and context-dependent manner. Here, we review how keratins serve multiple homeostatic and stress-triggered mechanical and nonmechanical functions, including maintenance of cellular integrity, regulation of cell growth and migration, and protection from apoptosis. These functions are tightly regulated by posttranslational modifications and keratin-associated proteins. Genetically determined alterations in keratin-coding sequences underlie highly penetrant and rare disorders whose pathophysiology reflects cell fragility or altered
    [Show full text]
  • Apoptosis-Induced Cleavage of Keratin 15 and Keratin 17 in a Human Breast Epithelial Cell Line
    Cell Death and Differentiation (2001) 8, 308 ± 315 ã 2001 Nature Publishing Group All rights reserved 1350-9047/01 $15.00 www.nature.com/cdd Apoptosis-induced cleavage of keratin 15 and keratin 17 in a human breast epithelial cell line V Badock1, U Steinhusen2, K Bommert*,2, ment (NF) proteins, and glial fibrillary acidic protein (GFAP). IF B Wittmann-Liebold1 and A Otto1 proteins are expressed in a tissue-specific manner: keratins in epithelial cells, vimentin in mesenchymal cells, desmin in 1 Department of Protein Chemistry, Max-DelbruÈck-Center for Molecular muscle, and neurofilaments in neuronal cells. The largest and Medicine, Robert-RoÈssle-Str. 10, D-13092 Berlin, Germany most complex group of IF proteins are keratins. At least 20 2 Department of Medical Oncology and Tumorimmunology, different keratin proteins have been described (K1 ± K20), that Robert-RoÈssle-Str.10, D-13092 Berlin, Germany are subdivided into the acidic type I keratins 9 ± 20 and the * Corresponding author: K Bommert, Max-DelbruÈck-Center for Molecular basic type II keratins 1 ± 8. Keratin filaments assemble as Medicine, Department of Medical Oncology and Tumorimmunology, Robert-RoÈssle-Str. 10, D-13092 Berlin, Germany. obligate heteropolymers consisting of a 1 : 1 molar ratio of Tel: ++49-30-94063817; Fax: ++49-30-94063124; types I and II monomers. Mutation studies in keratins indicate e-mail: [email protected] that one function of the keratin network is to maintain the mechanical integrity in epithelial cells and to resist shear Received 7.8.00; revised 2.11.00; accepted 9.11.00 stress applied to the cell.1±4Keratin 17 is associated with type Edited by SJ Martin II keratin 6 and is normally expressed in the basal cells of complex epithelia but not in stratified or simple epithelia.
    [Show full text]
  • Frequency and Extent of Cytokeratin Expression in Paraganglioma: an Immunohistochemical Study of 60 Cases from 5 Anatomic Sites
    Human Pathology (2019) 93,16–22 www.elsevier.com/locate/humpath Original contribution Frequency and extent of cytokeratin expression in paraganglioma: an immunohistochemical study of 60 cases from 5 anatomic sites and review of the literature Josephine Kamtai Dermawan MD, PhD⁎, Sanjay Mukhopadhyay MD, Akeesha Alia Shah MD Department of Pathology, Pathology and Laboratory Medicine Institute, Cleveland Clinic, Cleveland, OH, 44195, USA Received 24 June 2019; revised 9 August 2019; accepted 13 August 2019 Keywords: Summary The absence of cytokeratin expression in paraganglioma helps to differentiate it from other neu- Paraganglioma; roendocrine neoplasms such as carcinoid tumor. Although rare cytokeratin positive paragangliomas have Cytokeratin; been reported, there are no large systematic studies of this phenomenon. The aim of this study was to deter- Immunohistochemistry; mine the frequency and extent of cytokeratin expression in paragangliomas using a large cohort of cases Keratin; from multiple anatomic sites. Immunohistochemical staining for keratin AE1/AE3 (mouse monoclonal, Lumbar; MAB3412; Millipore) and CAM 5.2 (mouse monoclonal, 349 205; Becton-Dickinson) was performed on Mediastinal whole-tissue sections from 60 resected paragangliomas from the head and neck (36), thorax (10), abdomen (8), intradural/epidural spine (5) and bone, left iliac (1). Cytokeratin expression was identified in only 2/60 (3.3%) cases. One was a mediastinal paraganglioma with moderate to strong expression of keratin AE1/AE3 and CAM 5.2 in b5% tumor cells. The other was a lumbar intradural paraganglioma positive for CAM 5.2 (moderate to strong, 80% of tumor cells) but negative for keratin AE1/AE3. All other paragangliomas (58/ 60, 96.7%) were negative for keratin AE1/AE3 and CAM 5.2.
    [Show full text]
  • Inducible Expression of Filaggrin Increases Keratinocyte Susceptibility to Apoptotic Cell Death
    Cell Death and Differentiation (2000) 7, 566 ± 573 ã 2000 Macmillan Publishers Ltd All rights reserved 1350-9047/00 $15.00 www.nature.com/cdd Inducible expression of filaggrin increases keratinocyte susceptibility to apoptotic cell death MK Kuechle*1,2, RB Presland1,2, SP Lewis1, P Fleckman2 and diamine tetra-acetic acid; GFP, green ¯uorescent protein; PBS, BA Dale1,2,3,4 phosphate buffered saline; REK, rat epidermal keratinocyte; TBS, tris buffered saline; TUNEL, terminal deoxytransferase-mediated 1 Department of Oral Biology, University of Washington, Seattle, Washington, dUTP-biotin nick end labeling WA 98195, USA 2 Department of Medicine (Dermatology), University of Washington, Seattle, Washington, WA 98195, USA 3 Department of Periodontics, University of Washington, Seattle, Washington, Introduction WA 98195, USA The stratum corneum is the thin (12 ± 15 mm) tough, outer- 4 Department of Biochemistry, University of Washington, Seattle, Washington, WA 98195, USA most layer of the epidermis composed of overlapping, 1 * Corresponding author: MK Kuechle, Departments of Oral Biology/Medicine flattened, corneocytes and lipid-rich, intercellular lamellae. (Dermatology), Box 357132, University of Washington, Seattle, Washington, The stratum corneum functions as a barrier both to keep WA 98185-7132, USA. Tel: (206) 543-1595; Fax: (206) 685-3162; environmental insults out and to prevent water loss. Many E-mail: [email protected] morphologic and biochemical changes occur during the formation of the stratum corneum. Loricrin, involucrin,
    [Show full text]
  • The Cytokeratin Filament-Aggregating Protein Filaggrin Is the Target of the So-Called "Antikeratin Antibodies," Autoantibodies Specific for Rheumatoid Arthritis
    The cytokeratin filament-aggregating protein filaggrin is the target of the so-called "antikeratin antibodies," autoantibodies specific for rheumatoid arthritis. M Simon, … , G Salama, G Serre J Clin Invest. 1993;92(3):1387-1393. https://doi.org/10.1172/JCI116713. Research Article In rheumatoid arthritis (RA), the high diagnostic value of serum antibodies to the stratum corneum of rat esophagus epithelium has been widely reported. These so-called "antikeratin antibodies," detected by indirect immunofluorescence, were found to be autoantibodies since they also labeled human epidermis. Despite their name, the actual target of these autoantibodies was not known. In this study, a 40-kD protein (designated as 40K), extracted from human epidermis and specifically immunodetected by 75% of RA sera, was purified and identified as a neutral/acidic isoform of basic filaggrin, a cytokeratin filament-aggregating protein, by peptide mapping studies and by the following evidences: (a) mAbs specific for filaggrin reacted with the 40K protein; (b) the autoantibodies, affinity-purified from RA sera on the 40K protein, immunodetected purified filaggrin; (c) the reactivity of RA sera to the 40K protein was abolished after immunoadsorption with purified filaggrin; (d) the 40K protein and filaggrin had similar amino acid compositions. Furthermore, autoantibodies against the 40K protein and the so-called "antikeratin antibodies" were shown, by immunoadsorption experiments, to be largely the same. The identification of filaggrin as a RA-specific autoantigen could
    [Show full text]
  • Mathematical Modeling of the Impact of Actin and Keratin Filaments On&Nbsp;Keratinocyte Cell Spreading
    1828 Biophysical Journal Volume 103 November 2012 1828–1838 Mathematical Modeling of the Impact of Actin and Keratin Filaments on Keratinocyte Cell Spreading Jin Seob Kim,†* Chang-Hun Lee,† Baogen Y. Su,† and Pierre A. Coulombe†‡§* †Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland; ‡Department of Biological Chemistry and §Department of Dermatology, School of Medicine, Johns Hopkins University, Baltimore, Maryland ABSTRACT Keratin intermediate filaments (IFs) form cross-linked arrays to fulfill their structural support function in epithelial cells and tissues subjected to external stress. How the cross-linking of keratin IFs impacts the morphology and differentiation of keratinocytes in the epidermis and related surface epithelia remains an open question. Experimental measurements have established that keratinocyte spreading area is inversely correlated to the extent of keratin IF bundling in two-dimensional culture. In an effort to quantitatively explain this relationship, we developed a mathematical model in which isotropic cell spreading is considered as a first approximation. Relevant physical properties such as actin protrusion, adhesion events, and the corresponding response of lamellum formation at the cell periphery are included in this model. Through optimization with experimental data that relate time-dependent changes in keratinocyte surface area during spreading, our simulation results confirm the notion that the organization and mechanical properties of cross-linked keratin filaments affect cell spreading; in addi- tion, our results provide details of the kinetics of this effect. These in silico findings provide further support for the notion that differentiation-related changes in the density and intracellular organization of keratin IFs affect tissue architecture in epidermis and related stratified epithelia.
    [Show full text]
  • Keratin Filaments Have Similar Structures (Electron Microscopy/Spectropolarimetry/Wide-Angle X-Ray Diffraction) PETER M
    Proc. Nati. Acad. Sci. USA Vol. 75, No. 12, pp. 6098-6101, December 1978 Cell Biology Ten-nanometer filaments of hamster BHK-21 cells and epidermal keratin filaments have similar structures (electron microscopy/spectropolarimetry/wide-angle x-ray diffraction) PETER M. STEINERT"t, STEVEN B. ZIMMERMAN*, JUDITH M. STARGER§, AND ROBERT D. GOLDMAN§ *Dermatology Branch, National Cancer Institute; *Laboratory of Molecular Biology, National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health, Bethesda, Maryland 20014; and §Department of Biological Sciences, Carnegie-Mellon University, 4400 Fifth-Avenue, Pittsburgh, Pennsylvania 15213 Communicated by David R. Davies, September 22,1978 ABSTRACT The 10-nm filaments of baby hamster kidney structure of keratin filaments from both hair (and wool) and (BHK-21) cells, when examined either in the form of native epidermis is now well advanced (26, 27). Thus consideration ilanent caps or polymerized in vitro, are long tubes of protein 8-10 nm in diameter. They contain about 42% a-helix, which, and comparison of the properties-of keratin filaments with those on the basis of x-ray diffraction data, is arranged n a coiled-coil of the similar-sized filaments from other cell types may be conformation characteristic of proteins of the a type. The known useful. Keratin filaments are a-helix-rich fibrous proteins that structural properties such as morphology, dimensions, subunit exhibit an a type x-ray diffraction pattern characteristic of composition, and ultrastructure of this fibrous protein are very proteins of the k ("hard" keratin)-m (myosin)-e (epidermin similar to those of the mammalian epidermal keratin filament, = epidermal keratin)-f (fibrin) class (19, 28).
    [Show full text]