Dynamic Interactions of Epidermal Collagen XVII with the Extracellular Matrix
2011; Elsevier BV; Volume: 179; Issue: 2 Linguagem: Inglês
10.1016/j.ajpath.2011.04.019
ISSN1525-2191
AutoresWataru Nishie, Dimitra Kiritsi, Alexander Nyström, Silke Hofmann, Leena Bruckner‐Tuderman,
Tópico(s)Skin and Cellular Biology Research
ResumoTransmembrane collagen XVII, a major component of the hemidesmosomes, is crucial for stable adhesion of the epidermis and dermis in the skin, and its dysfunction results in blistering diseases. The ectodomain of collagen XVII (Ecto-ColXVII) is constitutively shed from the cell surface, but its binding partner(s) in the extracellular matrix (ECM) and the physiologic roles of the ligand interactions remain elusive. Herein, we used a new cleavage site–specific antibody to address the dynamics of collagen XVII shedding and the interactions of Ecto-ColXVII with the ECM. Ecto-ColXVII was present in the migration tracks of primary human keratinocytes and co-localized with laminin 332. The presence of this laminin, but also of collagen IV and Matrigel, in the ECM enhanced shedding and incorporation of Ecto-ColXVII into the matrix. Laminin 332 is a major, but not exclusive, interaction partner in vivo because Ecto-ColXVII deposited in the ECM of laminin 332–deficient keratinocytes was drastically reduced, but Ecto-ColXVII was present in laminin 332–negative human skin. Expression of collagen XVII deletion mutants in HEK 293 cells identified the C-terminal ectodomain stretch Ser978-Pro1497 as necessary for ECM binding. Taken together, migrating keratinocytes shed the Ecto-ColXVII, and this dynamically binds via its C-terminal domain to distinct partners in the ECM. Transmembrane collagen XVII, a major component of the hemidesmosomes, is crucial for stable adhesion of the epidermis and dermis in the skin, and its dysfunction results in blistering diseases. The ectodomain of collagen XVII (Ecto-ColXVII) is constitutively shed from the cell surface, but its binding partner(s) in the extracellular matrix (ECM) and the physiologic roles of the ligand interactions remain elusive. Herein, we used a new cleavage site–specific antibody to address the dynamics of collagen XVII shedding and the interactions of Ecto-ColXVII with the ECM. Ecto-ColXVII was present in the migration tracks of primary human keratinocytes and co-localized with laminin 332. The presence of this laminin, but also of collagen IV and Matrigel, in the ECM enhanced shedding and incorporation of Ecto-ColXVII into the matrix. Laminin 332 is a major, but not exclusive, interaction partner in vivo because Ecto-ColXVII deposited in the ECM of laminin 332–deficient keratinocytes was drastically reduced, but Ecto-ColXVII was present in laminin 332–negative human skin. Expression of collagen XVII deletion mutants in HEK 293 cells identified the C-terminal ectodomain stretch Ser978-Pro1497 as necessary for ECM binding. Taken together, migrating keratinocytes shed the Ecto-ColXVII, and this dynamically binds via its C-terminal domain to distinct partners in the ECM. The extracellular matrix (ECM) is not merely a supporting scaffold for tissue and organ architecture. The ECM can bind and release growth factors, which regulate many biologic processes and cell functions, including migration, differentiation, and proliferation.1Hynes R.O. The extracellular matrix: not just pretty fibrils.Science. 2009; 326: 1216-1219Crossref PubMed Scopus (2290) Google Scholar Interaction of cells with ECM proteins is usually mediated by cell surface receptors of the integrin or syndecan families,1Hynes R.O. The extracellular matrix: not just pretty fibrils.Science. 2009; 326: 1216-1219Crossref PubMed Scopus (2290) Google Scholar, 2Heino J. Kapyla J. Cellular receptors of extracellular matrix molecules.Curr Pharm Des. 2009; 15: 1309-1317Crossref PubMed Scopus (80) Google Scholar, 3Barczyk M. Carracedo S. Gullberg D. Integrins.Cell Tissue Res. 2010; 339: 269-280Crossref PubMed Scopus (1156) Google Scholar but other tissue-specific receptors, such as collagen XVII, also exist.4Franzke C.W. Bruckner P. Bruckner-Tuderman L. Collagenous transmembrane proteins: recent insights into biology and pathology.J Biol Chem. 2005; 280: 4005-4008Crossref PubMed Scopus (147) Google Scholar A major component of epithelial basement membranes, which represent highly specialized ECM, is laminin 332, and its interactions with keratinocyte integrins α3β1 and α6β4 guide cell migration, differentiation, and proliferation.5Sugawara K. Tsuruta D. Ishii M. Jones J.C. Kobayashi H. Laminin-332 and -511 in skin.Exp Dermatol. 2008; 17: 473-480Crossref PubMed Scopus (83) Google Scholar, 6Tsuruta D. Kobayashi H. Imanishi H. Sugawara K. Ishii M. Jones J.C. Laminin-332-integrin interaction: a target for cancer therapy.Curr Med Chem. 2008; 15: 1968-1975Crossref PubMed Scopus (81) Google Scholar In basal keratinocytes of the epidermis, the adhesion receptors are embedded in larger protein complexes, the hemidesmosomes (HDs), which can be observed as electron-dense ultrastructural components facing the basement membrane.7McMillan J.R. Akiyama M. Shimizu H. Epidermal basement membrane zone components: ultrastructural distribution and molecular interactions.J Dermatol Sci. 2003; 31: 169-177Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar HDs play an important role in cell attachment to the epidermal basement membrane, as shown indirectly by abnormalities of HDs in congenital and acquired blistering diseases, such as junctional epidermolysis bullosa and pemphigoid diseases.8Has C. Bruckner-Tuderman L. Molecular and diagnostic aspects of genetic skin fragility.J Dermatol Sci. 2006; 44: 129-144Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 9Van Agtmael T. Bruckner-Tuderman L. Basement membranes and human disease.Cell Tissue Res. 2010; 339: 167-188Crossref PubMed Scopus (102) Google Scholar In addition to integrin α6β4 and laminin 332, HDs contain BP230 and plectin as intracellular components and CD151 and collagen XVII as transmembrane components.7McMillan J.R. Akiyama M. Shimizu H. Epidermal basement membrane zone components: ultrastructural distribution and molecular interactions.J Dermatol Sci. 2003; 31: 169-177Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar Elucidation of biologic consequences of mutations in the genes encoding for these proteins has uncovered both laminin 332 and collagen XVII as vital players in cell-ECM interactions.10Pulkkinen L. Christiano A.M. Gerecke D. Wagman D.W. Burgeson R.E. Pittelkow M.R. Uitto J. A homozygous nonsense mutation in the β3 chain gene of laminin 5 (LAMB3) in Herlitz junctional epidermolysis bullosa.Genomics. 1994; 24: 357-360Crossref PubMed Scopus (206) Google Scholar, 11McGrath J.A. Gatalica B. Christiano A.M. Li K. Owaribe K. McMillan J.R. Eady R.A. Uitto J. Mutations in the 180-kD bullous pemphigoid antigen (BPAG2), a hemidesmosomal transmembrane collagen (COL17A1), in generalized atrophic benign epidermolysis bullosa.Nat Genet. 1995; 11: 83-86Crossref PubMed Scopus (328) Google Scholar However, the interaction mechanisms of collagen XVII with the ECM remain poorly understood. Collagen XVII is a type II integral transmembrane protein with an intracytoplasmic amino terminus and an extracellular carboxyl terminus, which consists of 15 collagenous and 16 noncollagenous subdomains.4Franzke C.W. Bruckner P. Bruckner-Tuderman L. Collagenous transmembrane proteins: recent insights into biology and pathology.J Biol Chem. 2005; 280: 4005-4008Crossref PubMed Scopus (147) Google Scholar, 12Giudice G.J. Emery D.J. Diaz L.A. Cloning and primary structural analysis of the bullous pemphigoid autoantigen BP180.J Invest Dermatol. 1992; 99: 243-250Abstract Full Text PDF PubMed Scopus (486) Google Scholar, 13Li K. Tamai K. Tan E.M. Uitto J. Cloning of type XVII collagen: complementary and genomic DNA sequences of mouse 180-kilodalton bullous pemphigoid antigen (BPAG2) predict an interrupted collagenous domain, a transmembrane segment, and unusual features in the 5′ end of the gene and the 3′ untranslated region of the mRNA.J Biol Chem. 1993; 268: 8825-8834Abstract Full Text PDF PubMed Google Scholar The cytoplasmic domain of collagen XVII has been reported to interact with BP230, plectin, and integrin β4; all of these interactions are important for stabilization of the HDs.14Schaapveld R.Q. Borradori L. Geerts D. van Leusden M.R. Kuikman I. Nievers M.G. Niessen C.M. Steenbergen R.D. Snijders P.J. Sonnenberg A. Hemidesmosome formation is initiated by the β4 integrin subunit, requires complex formation of β4 and HD1/plectin, and involves a direct interaction between β4 and the bullous pemphigoid antigen 180.J Cell Biol. 1998; 142: 271-284Crossref PubMed Scopus (158) Google Scholar, 15Koster J. Geerts D. Favre B. Borradori L. Sonnenberg A. Analysis of the interactions between BP180. BP230, plectin and the integrin α6β4 important for hemidesmosome assembly.J Cell Sci. 2003; 116: 387-399Crossref PubMed Scopus (173) Google Scholar Thus, collagen XVII is thought to be a vital cell surface receptor that links the cytoplasmic structural components with the ECM. Extracellular binding between the noncollagenous 16th domain of collagen XVII and α6 integrin has also been proposed; the α6 integrin binding site in the collagen molecule was presumed to be within the stretch Arg506-Ile519.16Hopkinson S.B. Findlay K. deHart G.W. Jones J.C. Interaction of BP180 (type XVII collagen) and α6 integrin is necessary for stabilization of hemidesmosome structure.J Invest Dermatol. 1998; 111: 1015-1022Crossref PubMed Scopus (64) Google Scholar This would be juxtamembranous and upstream of the recently disclosed major sheddase cleavage sites.17Nishie W. Lamer S. Schlosser A. Licarete E. Franzke C.W. Hofmann S.C. Jackow J. Sitaru C. Bruckner-Tuderman L. Ectodomain shedding generates Neoepitopes on collagen XVII, the major autoantigen for bullous pemphigoid.J Immunol. 2010; 185: 4938-4947Crossref PubMed Scopus (52) Google Scholar Thus, the interaction of the shed ectodomain of collagen XVII (Ecto-ColXVII) with α6β4 integrin still remains elusive. A study of HaCaT keratinocytes proposed a p38MAPK-dependent functional relationship between collagen XVII and collagen IV and/or other components in Matrigel (BD Biosciences, Franklin Lakes, NJ) that regulates cell attachment and migration.18Qiao H. Shibaki A. Long H.A. Wang G. Li Q. Nishie W. Abe R. Akiyama M. Shimizu H. McMillan J.R. Collagen XVII participates in keratinocyte adhesion to collagen IV, and in p38MAPK-dependent migration and cell signaling.J Invest Dermatol. 2009; 129: 2288-2295Crossref PubMed Scopus (25) Google Scholar Furthermore, an in vitro solid-phase protein-protein binding assay using recombinant collagen XVII fragments and laminin 332 indicated an affinity of collagen XVII carboxyl terminus with laminin 332.19Tasanen K. Tunggal L. Chometon G. Bruckner-Tuderman L. Aumailley M. Keratinocytes from patients lacking collagen XVII display a migratory phenotype.Am J Pathol. 2004; 164: 2027-2038Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar Despite the limitations of such assays, the suggestion of a partnership between these two HD components is intriguing and has high pathophysiologic significance because aberrant expression of laminin 332 and collagen XVII has been suggested to be involved in the invasion and proliferation of various cancer cells.6Tsuruta D. Kobayashi H. Imanishi H. Sugawara K. Ishii M. Jones J.C. Laminin-332-integrin interaction: a target for cancer therapy.Curr Med Chem. 2008; 15: 1968-1975Crossref PubMed Scopus (81) Google Scholar, 20Parikka M. Nissinen L. Kainulainen T. Bruckner-Tuderman L. Salo T. Heino J. Tasanen K. Collagen XVII promotes integrin-mediated squamous cell carcinoma transmigration: a novel role for αIIb integrin and tirofiban.Exp Cell Res. 2006; 312: 1431-1438Crossref PubMed Scopus (33) Google Scholar In this context, it is important to note that two different physiologic forms of collagen XVII exist: a full-length transmembrane form and a released ectodomain. Proteinases of the ADAM family cleave the Ecto-ColXVII within the juxtamembranous noncollagenous 16th A (NC16A) domain,4Franzke C.W. Bruckner P. Bruckner-Tuderman L. Collagenous transmembrane proteins: recent insights into biology and pathology.J Biol Chem. 2005; 280: 4005-4008Crossref PubMed Scopus (147) Google Scholar, 17Nishie W. Lamer S. Schlosser A. Licarete E. Franzke C.W. Hofmann S.C. Jackow J. Sitaru C. Bruckner-Tuderman L. Ectodomain shedding generates Neoepitopes on collagen XVII, the major autoantigen for bullous pemphigoid.J Immunol. 2010; 185: 4938-4947Crossref PubMed Scopus (52) Google Scholar yielding a shorter form of the protein, Ecto-ColXVII, which remains stable in the ECM and is likely to interact with other ECM proteins. Thus, functionally, collagen XVII plays a dual role as a cell surface receptor for ECM proteins and as a matrix component. The study of context-dependent regulation of collagen XVII shedding has been impeded by a lack of precise tools to visualize and measure ectodomain shedding. This problem has been overcome by a recently generated novel antibody (Ab), HK139, which specifically detects cleaved Ecto-ColXVII.17Nishie W. Lamer S. Schlosser A. Licarete E. Franzke C.W. Hofmann S.C. Jackow J. Sitaru C. Bruckner-Tuderman L. Ectodomain shedding generates Neoepitopes on collagen XVII, the major autoantigen for bullous pemphigoid.J Immunol. 2010; 185: 4938-4947Crossref PubMed Scopus (52) Google Scholar Herein, we used the Ab HK139 to follow shedding of the Ecto-ColXVII and its deposition in ECM in normal human keratinocytes (NHKs). We show that these processes are dynamic and that distinct ECM proteins play essential roles in interacting with collagen XVII in keratinocytes in vitro and in human skin in vivo. Skin biopsy specimens were obtained from healthy volunteers and from a female neonate with laminin 332–negative Herlitz junctional epidermolysis bullosa after informed consent was provided. The genetic and phenotypic features of patients with Herlitz junctional epidermolysis bullosa have been described elsewhere.21Pulkkinen L. Gerecke D.R. Christiano A.M. Wagman D.W. Burgeson R.E. Uitto J. Cloning of the β3 chain gene (LAMB3) of human laminin 5, a candidate gene in junctional epidermolysis bullosa.Genomics. 1995; 25: 192-198Crossref PubMed Scopus (52) Google Scholar This study was approved by the Ethical Committee of the University of Freiburg. Primary NHKs were isolated from normal and laminin 332–negative skin and were cultured in serum-free keratinocyte growth medium supplemented with bovine pituitary extract and epidermal growth factor (Invitrogen, Carlsbad, CA). Cells up to the fourth passage were used for this study. Immortalized HaCaT keratinocytes, a generous gift from Dr. Norbert E. Fusenig (German Cancer Research Center, Heidelberg, Germany), were cultured under the same conditions. Every 48 hours, freshly prepared ascorbic acid was added to the culture medium to a final concentration of 50 μg/mL to allow hydroxylation of collagens and proper triple helix folding.22Franzke C.W. Tasanen K. Schacke H. Zhou Z. Tryggvason K. Mauch C. Zigrino P. Sunnarborg S. Lee D.C. Fahrenholz F. Bruckner-Tuderman L. Transmembrane collagen XVII, an epithelial adhesion protein, is shed from the cell surface by ADAMs.EMBO J. 2002; 21: 5026-5035Crossref PubMed Scopus (188) Google Scholar, 23Franzke C.W. Tasanen K. Borradori L. Huotari V. Bruckner-Tuderman L. Shedding of collagen XVII/BP180: structural motifs influence cleavage from cell surface.J Biol Chem. 2004; 279: 24521-24529Crossref PubMed Scopus (67) Google Scholar Flp-in 293 host cells (Invitrogen) and transformed 293 cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen) containing 10% fetal bovine serum. For coating coverslips and culture flasks, various ECM proteins, including 50 μg/mL of rat tail collagen I (number 354236, BD Biosciences), 100 μg/mL of mouse collagen IV (number 354233, BD Biosciences), 333 μg/mL of Matrigel, 2 μg/mL of laminin 332 (Oriental Yeast Co., Tokyo, Japan), and 5 μg/mL of mouse laminin 111 (number 354239, BD Biosciences), were used according to the manufacturers' instructions. For collagenase treatment, NHKs grown on coverslips were incubated for 2 hours at 37°C with 100 U/mL of bacterial, chromatographically purified collagenase form III (WAK-Chemie Medical GmbH, Steinbach, Germany) in a buffer containing 50 mmol/L Tris-HCl and 65 mmol/L NaCl (pH 7.4). After careful washing with PBS, the coverslips were subjected to indirect immunofluorescence (IIF) staining. Full-length human collagen XVII cDNA [NM_000494; a gift from Dr. Luca Borradori (Department of Dermatology, University of Bern, Switzerland)] was introduced into the NotI site of pcDNA5/FRT (Invitrogen), designated as COL17 cDNA. To generate different deletion mutants, a 572-bp XbaI-digested fragment was first cut out, and the digested plasmid was self-ligated to obtain XbaI-digested COL17 cDNA. Next, PCR reactions using forward primer ClaI-F 5′-AGGGGTCATCGATGCTCACT-3′ (the ClaI digestion site is shown in italics) and different reverse primers, including COL3-R 5′-CGCTCTAGATCAGTCAGTGCCATAGGGACCCC-3′ (the XbaI digestion site is shown in italics and the stop codon in bold) to generate the Met1-Asp1340 amino acid fragment of collagen XVII designated as COL3, NC6-R 5′-CGCTCTAGATCATCTGAATTCAGACCCTCGCA-3′ (the XbaI digestion site is shown in italics and the stop codon in bold) for Met1-Arg1174 amino acids designated as NC6, and COL11-R 5′-CGCTCTAGATCAAGGAATGCCAAGAGCCCCTG-3′ (the XbaI digestion site is shown in italics and the stop codon in bold) for Met1-Pro977 amino acids designated as COL11, were performed using human collagen cDNA as template. PCR fragments digested with ClaI and XbaI were introduced into the corresponding digested sites of the COL17 cDNA plasmid. To generate a short fragment spanning amino acids Met1-Met816, designated as COL15, linker oligonucleotides 5′-CGATGTGACTCGAGT-3′ and 5′-CTAGACTCGAGTCACAT-3′ (the stop codon is shown in bold) were inserted into the ClaI- and XbaI-digested sites of COL17 cDNA. Finally, the 572-bp XbaI-digested fragment was inserted into each plasmid. To produce another fragment in which sequences from amino acids Pro997-Gly1152 were deleted, designated as ΔPro997-Gly1152, Bluescript SKII vector (Agilent Technologies, Waldbronn, Germany) was digested with ApaI, followed by self-ligation to use a single ApaI site, resulting in ApaI-pBS. Then, a ClaI- and BamHI-digested 1121-bp fragment from COL17 cDNA was inserted into the ClaI- and BamHI-digested sites of ApaI-pBS. Finally, the modified vector was digested with ApaI, followed by self-ligation to obtain ΔPro997-Gly1152. The DNA sequences of all the vectors were confirmed by direct sequencing. COL17 cDNA, COL15, NC6, COL11, and ΔPro997-Gly1152, and the pcDNA5/FRT empty vector as control, were co-transfected with pOG44 (Invitrogen) into Flp-In 293 host cells; this enables stable and isogenic expression of each deletion mutant of collagen XVII. Stably expressing cells were selected under 200 μg/mL of hygromycine B (Invitrogen) as previously described.17Nishie W. Lamer S. Schlosser A. Licarete E. Franzke C.W. Hofmann S.C. Jackow J. Sitaru C. Bruckner-Tuderman L. Ectodomain shedding generates Neoepitopes on collagen XVII, the major autoantigen for bullous pemphigoid.J Immunol. 2010; 185: 4938-4947Crossref PubMed Scopus (52) Google Scholar Abs, including mouse monoclonal Abs NC16A-1 and NC16A-3, directed to distinct epitopes in the NC16A domain of collagen XVII,24Hofmann S.C. Voith U. Schonau V. Sorokin L. Bruckner-Tuderman L. Franzke C.W. Plasmin plays a role in the in vitro generation of the linear IgA dermatosis antigen LADB97.J Invest Dermatol. 2009; 129: 1730-1739Crossref PubMed Scopus (61) Google Scholar were used in a 1:10 dilution for IIF (NC16A-1) and in a 1:2000 dilution for immunoblotting (NC16A-3). The cleavage-site–specific rabbit polyclonal Ab HK139 was prepared as previously described17Nishie W. Lamer S. Schlosser A. Licarete E. Franzke C.W. Hofmann S.C. Jackow J. Sitaru C. Bruckner-Tuderman L. Ectodomain shedding generates Neoepitopes on collagen XVII, the major autoantigen for bullous pemphigoid.J Immunol. 2010; 185: 4938-4947Crossref PubMed Scopus (52) Google Scholar and was used at a concentration of 10 μg/mL for IIF and 1 μg/mL for immunoblotting. For detection of laminin 332 by IIF, the monoclonal Ab 6F12 to the β3 chain25Marinkovich M.P. Lunstrum G.P. Burgeson R.E. The anchoring filament protein kalinin is synthesized and secreted as a high molecular weight precursor.J Biol Chem. 1992; 267: 17900-17906Abstract Full Text PDF PubMed Google Scholar was used in a 1:5000 dilution. For the α3 and γ2 chains, BM165 (a gift from Dr. Johannes A. Eble, Munster, Germany) and GB3 (a gift from Dr. Guerrino Meneguzzi, Nice, France) were used in a dilution of 1:5000 and 1:200, respectively. For the detection of laminin 332 by immunoblotting, a polyclonal Ab to laminin 332 (ab14509, Abcam Inc., Cambridge, MA) was used in a 1:2000 dilution. As an internal control for immunoblotting, anti–β-tubulin Ab (ab6046, Abcam Inc.) was used in a 1:2000 dilution. For staining cytoskeletal actin filaments, rhodamine phalloidin (number R415, Invitrogen) was used. For immunoblotting, subconfluent NHKs, HaCaT cells, or transformed HEK 293 cells were incubated in serum-free medium for 24 hours before harvest. Then, the cells were lysed for 30 minutes on ice in a buffer containing 1% Nonidet P-40 (Caledon Laboratories Ltd., Georgetown, ON, Canada), 0.1 mol/L NaCl, 25 mmol/L Tris-HCl, pH7.4, 10 mmol/L EDTA, and 1 mmol/L Pefabloc (Roche Diagnostics Deutschland GmbH, Mannheim, Germany).22Franzke C.W. Tasanen K. Schacke H. Zhou Z. Tryggvason K. Mauch C. Zigrino P. Sunnarborg S. Lee D.C. Fahrenholz F. Bruckner-Tuderman L. Transmembrane collagen XVII, an epithelial adhesion protein, is shed from the cell surface by ADAMs.EMBO J. 2002; 21: 5026-5035Crossref PubMed Scopus (188) Google Scholar, 23Franzke C.W. Tasanen K. Borradori L. Huotari V. Bruckner-Tuderman L. Shedding of collagen XVII/BP180: structural motifs influence cleavage from cell surface.J Biol Chem. 2004; 279: 24521-24529Crossref PubMed Scopus (67) Google Scholar The medium proteins were precipitated with ethanol before further processing.22Franzke C.W. Tasanen K. Schacke H. Zhou Z. Tryggvason K. Mauch C. Zigrino P. Sunnarborg S. Lee D.C. Fahrenholz F. Bruckner-Tuderman L. Transmembrane collagen XVII, an epithelial adhesion protein, is shed from the cell surface by ADAMs.EMBO J. 2002; 21: 5026-5035Crossref PubMed Scopus (188) Google Scholar Preparation of the ECM proteins was performed as described,26Langhofer M. Hopkinson S.B. Jones J.C. The matrix secreted by 804G cells contains laminin-related components that participate in hemidesmosome assembly in vitro.J Cell Sci. 1993; 105: 753-764PubMed Google Scholar with some modifications. Briefly, after washing with PBS, the cell layer was incubated at room temperature with 20 mmol/L NH4OH solution. After all the cells had detached, the ECM was extensively washed with PBS and dissolved in SDS-PAGE sample buffer. The samples were boiled for 5 minutes and were separated on SDS-PAGE using 7% or 4% to 13% gradient polyacrylamide gels, followed by transfer onto nitrocellulose membrane. After incubation with primary Abs diluted with 2% skim milk in Tris-buffered saline overnight at 4°C, horseradish peroxidase–conjugated secondary Abs in the same buffer were reacted for 1 hour at room temperature. The signals were visualized by ECL Plus (GE Healthcare, Piscataway, NJ), and the intensity of each band was measured using ImageJ software version 1.44 (http://rsbweb.nih.gov/ij, last accessed June 13, 2011). For IIF, normal and laminin 332–deficient human skin was mounted and snap frozen into optimal cutting temperature compound, and 5-μm cryosections were prepared. Cells cultured on coverslips were fixed with 4% paraformaldehyde and were permeabilized with 1% Triton X-100 (Roche Diagnostics GmbH) in PBS. The sections and coverslips were incubated with primary Abs for 1 hour at room temperature, washed with PBS, and detected with Alexa 488/568–conjugated anti-mouse or anti-rabbit IgG (Invitrogen). Mounting medium with DAPI (VECTASHIELD; Vector Laboratories, Burlingame, CA) was used to mount the coverslips. Fluorescent images were obtained using an Axiophot microscope equipped with an AxioCam MRc digital camera or an LSM 510 META laser scanning confocal microscope (Carl Zeiss MicroImaging GmbH, Jena, Germany). IIF staining of cultured NHKs with the Ecto-ColXVII–specific Ab HK139 revealed characteristic “ringlike” or “arclike” staining patterns, combined with numerous small dots around migrating cells (Figure 1A), reflecting dynamic shedding of the Ecto-ColXVII. The ringlike structures also contained laminin 332 (Figure 1B). Collagen XVII staining was sensitive to collagenase digestion, whereas laminin 332 staining was resistant to it (Figure 1B), indicating that the Ab HK139 detected only a collagenous structure. The Ecto-ColXVII was visible in the ECM of NHKs approximately 2 to 4 hours after seeding of the cells (Figure 1C). When Ca2+ concentration in the medium was increased from 0.09 mmol/L to 1.2 mmol/L 4 hours after seeding, the cells started to detach, leaving ringlike deposition of the Ecto-ColXVII in the ECM beneath the cells (Figure 1D). This may suggest that the Ca2+ shift, which is a potent inducer of NHK differentiation,27Stanley J.R. Yuspa S.H. Specific epidermal protein markers are modulated during calcium-induced terminal differentiation.J Cell Biol. 1983; 96: 1809-1814Crossref PubMed Scopus (90) Google Scholar also induced shedding of the Ecto-ColXVII, as observed in calcium influx–stimulated shedding of the Ecto-ColXVII.28Franzke C.W. Bruckner-Tuderman L. Blobel C.P. Shedding of collagen XVII/BP180 in skin depends on both ADAM10 and ADAM9.J Biol Chem. 2009; 284: 23386-23396Crossref PubMed Scopus (80) Google Scholar These results suggest the close relationship between shedding of the Ecto-ColXVII and migration or differentiation of NHKs. The Ab HK139 detected the Ecto-ColXVII usually co-localized with laminin 332, indicating their close relationship (Figure 2A). However, some migrating NHKs left tracks containing normal amounts of laminin 332 but little or no Ecto-ColXVII (Figure 2B). These findings show that the Ecto-ColXVII mostly co-localizes with laminin 332, but not vice versa, and indicates that laminin 332 can undergo different ligand interactions in a stable ECM. Immortalized HaCaT keratinocytes are commonly used in in vitro experiments to substitute for NHKs. Herein, we compared the ECM produced by NHKs and HaCaT keratinocytes for its content of Ecto-ColXVII and laminin 332. In contrast to NHKs (Figure 2A), the amounts of Ecto-ColXVII and laminin 332 were strongly reduced in the ECM of migrating HaCaT keratinocytes, as assessed by IIF (Figure 3A). Immunoblotting showed that HaCaT cells deposited less Ecto-ColXVII in the ECM than did NHKs (Figure 3B). Mean intensity of the HaCaT keratinocyte–derived bands was 38% (n = 3) of that of NHK-derived bands. Ecto-ColXVII in culture medium of HaCaT keratinocytes was also reduced, but to a lesser extent. Mean intensity of the HaCaT medium–derived bands was 80% (n = 3) of that of NHK medium–derived bands (Figure 3C). These results demonstrate that HaCaT cells synthesize and secrete less collagen XVII than do NHKs but also that the incorporation of Ecto-ColXVII into the ECM is different from that into NHKs, at least in quantitative terms in that in HaCaT cell cultures more Ecto-ColXVII remained soluble in the medium. This may reflect reduced laminin 332 and altered composition of the ECM of HaCaT cells (Figure 3D). Thus, these experiments disclosed considerable differences in expression and matrix incorporation of ECM proteins between NHKs and immortalized HaCaT keratinocytes. To depict the influence of different ECM proteins on shedding of collagen XVII and deposition of the Ecto-ColXVII in the ECM, HEK 293 cells were transfected with collagen XVII cDNA. When cultured on plastic, these cells do not deposit significant amounts of Ecto-ColXVII in the ECM (Figure 4). Thus, this cell line is suitable for assessing the role of different ECM proteins, which can interact with collagen XVII and support deposition of the Ecto-ColXVII in the ECM. When the cells were cultured on plastic coated with collagen IV, Matrigel, or laminin 332, the amount of Ecto-ColXVII in the ECM was significantly increased (Figure 4A). Matrigel, which is derived from Engelbreth-Holm-Swarm tumor cells, contains several ECM proteins, among which collagen IV and laminin 111 are major components.29Kleinman H.K. Preparation of basement membrane components from EHS tumors.Curr Protoc Cell Biol. 2001; (10.210)Google Scholar In the context of the present study, it is of interest whether Matrigel also contains laminin 332. Microarray data on Engelbreth-Holm-Swarm tumor cells30Futaki S. Hayashi Y. Yamashita M. Yagi K. Bono H. Hayashizaki Y. Okazaki Y. Sekiguchi K. Molecular basis of constitutive production of basement membrane components: gene expression profiles of Engelbreth-Holm-Swarm tumor and F9 embryonal carcinoma cells.J Biol Chem. 2003; 278: 50691-50701Crossref PubMed Scopus (47) Google Scholar suggest that laminin 111, not laminin 332, is the major laminin in Matrigel. Accordingly, immunoblotting with a polyclonal laminin 332 Ab did not show positive signals with Matrigel (data not shown). Culture on collagen IV moderately increased deposition of Ecto-ColXVII, but laminin 111 failed to generate ECM binding. Culture on laminin 332 enhanced deposition of Ecto-ColXVII strongly (Figure 4A), and, in agreement with the immunoblotting data, IIF with the Ab HK139 revealed increased amounts of Ecto-ColXVII in the environment of cells seeded on laminin 332 (Figure 4B). These observations further corroborate the assumption that laminin 332 is a major binding partner of the Ecto-ColXVII, next to collagen IV and/or other components in Matrigel
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