Shedding of Collagen XVII/BP180 in Skin Depends on Both ADAM10 and ADAM9
2009; Elsevier BV; Volume: 284; Issue: 35 Linguagem: Inglês
10.1074/jbc.m109.034090
ISSN1083-351X
AutoresClaus-Werner Franzke, Leena Bruckner‐Tuderman, Carl Blobel,
Tópico(s)Nail Diseases and Treatments
ResumoCollagen XVII is a transmembrane collagen and the major autoantigen of the autoimmune skin blistering disease bullous pemphigoid. Collagen XVII is proteolytically released from the membrane, and the pathogenic epitope harbors the cleavage site for its ectodomain shedding, suggesting that proteolysis has an important role in regulating the function of collagen XVII in skin homeostasis. Previous studies identified ADAMs 9, 10, and 17 as candidate collagen XVII sheddases and suggested that ADAM17 is a major sheddase. Here we show that ADAM17 only indirectly affects collagen XVII shedding and that ADAMs 9 and 10 are the most prominent collagen XVII sheddases in primary keratinocytes because (a) collagen XVII shedding was not stimulated by phorbol esters, known activators of ADAM17, (b) constitutive and calcium influx-stimulated shedding was sensitive to the ADAM10-selective inhibitor GI254023X and was strongly reduced in Adam10−/− cells, (c) there was a 55% decrease in constitutive collagen XVII ectodomain shedding from Adam9−/− keratinocytes, and (d) H2O2 enhanced ADAM9 expression and stimulated collagen XVII shedding in skin and keratinocytes of wild type mice but not of Adam9−/− mice. We conclude that ADAM9 and ADAM10 can both contribute to collagen XVII shedding in skin with an enhanced relative contribution of ADAM9 in the presence of reactive oxygen species. These results provide critical new insights into the identity and regulation of the major sheddases for collagen XVII in keratinocytes and skin and have implications for the treatment of blistering diseases of the skin. Collagen XVII is a transmembrane collagen and the major autoantigen of the autoimmune skin blistering disease bullous pemphigoid. Collagen XVII is proteolytically released from the membrane, and the pathogenic epitope harbors the cleavage site for its ectodomain shedding, suggesting that proteolysis has an important role in regulating the function of collagen XVII in skin homeostasis. Previous studies identified ADAMs 9, 10, and 17 as candidate collagen XVII sheddases and suggested that ADAM17 is a major sheddase. Here we show that ADAM17 only indirectly affects collagen XVII shedding and that ADAMs 9 and 10 are the most prominent collagen XVII sheddases in primary keratinocytes because (a) collagen XVII shedding was not stimulated by phorbol esters, known activators of ADAM17, (b) constitutive and calcium influx-stimulated shedding was sensitive to the ADAM10-selective inhibitor GI254023X and was strongly reduced in Adam10−/− cells, (c) there was a 55% decrease in constitutive collagen XVII ectodomain shedding from Adam9−/− keratinocytes, and (d) H2O2 enhanced ADAM9 expression and stimulated collagen XVII shedding in skin and keratinocytes of wild type mice but not of Adam9−/− mice. We conclude that ADAM9 and ADAM10 can both contribute to collagen XVII shedding in skin with an enhanced relative contribution of ADAM9 in the presence of reactive oxygen species. These results provide critical new insights into the identity and regulation of the major sheddases for collagen XVII in keratinocytes and skin and have implications for the treatment of blistering diseases of the skin. Collagen XVII (also called BP180 or BPAG2) is a hemidesmosomal adhesion component in the skin and mucosa and belongs to the emerging group of collagenous transmembrane proteins (1.Franzke C.W. Tasanen K. Schumann H. Bruckner-Tuderman L. Matrix Biol. 2003; 22: 299-309Crossref PubMed Scopus (75) Google Scholar). This type II oriented transmembrane protein is involved in the molecular pathology of human skin diseases. Mutations in the COL17A1 gene are associated with junctional epidermolysis bullosa, a genetic skin blistering disease (2.Aumailley M. Has C. Tunggal L. Bruckner-Tuderman L. Expert Rev. Mol. Med. 2006; 8: 1-21Crossref PubMed Scopus (66) Google Scholar). Patients with bullous pemphigoid and related autoimmune bullous dermatoses have tissue-bound and circulating autoantibodies targeting collagen XVII (3.Yancey K.B. J. Clin. Investig. 2005; 115: 825-828Crossref PubMed Scopus (80) Google Scholar). Structural and functional changes of collagen XVII play an important role in these diseases, although the molecular pathology is not yet fully understood. The collagen XVII consists of three 180-kDa α1 (XVII) chains, each with an intracellular N-terminal domain, a short transmembrane stretch, and a flexible extracellular C-terminal ectodomain with collagenous (Col) 2The abbreviations used are: ColcollagenousNCnon-collagenousADAMa disintegrin and metalloproteinaseAPalkaline phosphataseIMionomycinmEFmouse embryonic fibroblastEembryonic dayGIGI254023XPMAphorbol 12-myristate 13-acetate. subdomains that are interrupted by short non-collagenous (NC) sequences. The human and murine collagen XVII molecules differ in size and in the number of the Col and NC domains. Human collagen XVII consists of 1497 amino acid residues with 15 Col and 16 NC domains, whereas the murine form, which is 86% identical (4.Li K. Tamai K. Tan E.M. Uitto J. J. Biol. Chem. 1993; 268: 8825-8834Abstract Full Text PDF PubMed Google Scholar), consists of 1433 amino acid residues with 13 Col and 14 NC domains. In humans the extracellular linker domain NC16A between the plasma membrane and the Col15 domain is functionally important because it is believed to play a role in both ectodomain shedding and in the proper folding of the triple helical structure of collagen XVII (5.Areida S.K. Reinhardt D.P. Muller P.K. Fietzek P.P. Kowitz J. Marinkovich M.P. Notbohm H. J. Biol. Chem. 2001; 276: 1594-1601Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 6.McLaughlin S.H. Bulleid N.J. Matrix Biol. 1998; 16: 369-377Crossref PubMed Scopus (98) Google Scholar, 7.Myllyharju J. Kivirikko K.I. Ann. Med. 2001; 33: 7-21Crossref PubMed Scopus (557) Google Scholar). collagenous non-collagenous a disintegrin and metalloproteinase alkaline phosphatase ionomycin mouse embryonic fibroblast embryonic day GI254023X phorbol 12-myristate 13-acetate. Our previous studies revealed two forms of collagen XVII, the 180-kDa membrane-anchored form and the soluble 120-kDa form. The latter represents the extracellular collagenous ectodomain, which is released by cleavage by membrane-anchored metalloproteinases of the adisintegrin and metalloproteinase (ADAM) family (8.Franzke C.W. Tasanen K. Schäcke H. Zhou Z. Tryggvason K. Mauch C. Zigrino P. Sunnarborg S. Lee D.C. Fahrenholz F. Bruckner-Tuderman L. EMBO J. 2002; 21: 5026-5035Crossref PubMed Scopus (189) Google Scholar). The shed ectodomain of collagen XVII is very stable in vivo and in vitro. In wound scratch assays, both addition of the purified soluble ectodomain or overexpression of ADAMs suppressed cell motility (8.Franzke C.W. Tasanen K. Schäcke H. Zhou Z. Tryggvason K. Mauch C. Zigrino P. Sunnarborg S. Lee D.C. Fahrenholz F. Bruckner-Tuderman L. EMBO J. 2002; 21: 5026-5035Crossref PubMed Scopus (189) Google Scholar), indicating that the ectodomain has a role in regulating keratinocyte-matrix interactions. In the context of the known functions of collagen XVII as an adhesion molecule, its shedding could therefore regulate its functions in keratinocyte migration, differentiation, and proliferation. ADAMs are also involved in the release of several other type I or type II transmembrane proteins and are considered to be critical regulators of epidermal growth factor receptor signaling, tumor necrosis factor α release, and Notch signaling to name a few examples (9.Blobel C.P. Nat. Rev. Mol. Cell Biol. 2005; 6: 32-43Crossref PubMed Scopus (928) Google Scholar, 10.Murphy G. Nat. Rev. Cancer. 2008; 8: 929-941Crossref PubMed Scopus (435) Google Scholar). Previously ADAM9, ADAM10, and ADAM17 had been identified as potential sheddases for collagen XVII in keratinocytes by overexpression in cell-based assays (8.Franzke C.W. Tasanen K. Schäcke H. Zhou Z. Tryggvason K. Mauch C. Zigrino P. Sunnarborg S. Lee D.C. Fahrenholz F. Bruckner-Tuderman L. EMBO J. 2002; 21: 5026-5035Crossref PubMed Scopus (189) Google Scholar). Moreover Adam17−/− keratinocytes had 50% diminished collagen XVII shedding, which was interpreted to suggest that ADAM17 represents an important, if not the major, physiological collagen XVII sheddase (8.Franzke C.W. Tasanen K. Schäcke H. Zhou Z. Tryggvason K. Mauch C. Zigrino P. Sunnarborg S. Lee D.C. Fahrenholz F. Bruckner-Tuderman L. EMBO J. 2002; 21: 5026-5035Crossref PubMed Scopus (189) Google Scholar). The major goal of the current study was to further explore the contribution of ADAM17 and other candidate sheddases to the release of collagen XVII from primary keratinocytes and mouse skin. The identification of the major collagen XVII sheddases and their regulation is critical for understanding the role of collagen XVII shedding in the pathogenesis of skin diseases. Adam8−/− (11.Kelly K. Hutchinson G. Nebenius-Oosthuizen D. Smith A.J. Bartsch J.W. Horiuchi K. Rittger A. Manova K. Docherty A.J. Blobel C.P. Dev. Dyn. 2005; 232: 221-231Crossref PubMed Scopus (98) Google Scholar), Adam9−/− (12.Weskamp G. Cai H. Brodie T.A. Higashyama S. Manova K. Ludwig T. Blobel C.P. Mol. Cell. Biol. 2002; 22: 1537-1544Crossref PubMed Scopus (175) Google Scholar), Adam15−/− (13.Horiuchi K. Weskamp G. Lum L. Hammes H.P. Cai H. Brodie T.A. Ludwig T. Chiusaroli R. Baron R. Preissner K.T. Manova K. Blobel C.P. Mol. Cell. Biol. 2003; 23: 5614-5624Crossref PubMed Scopus (154) Google Scholar), Adam17−/− mice (14.Peschon J.J. Slack J.L. Reddy P. Stocking K.L. Sunnarborg S.W. Lee D.C. Russell W.E. Castner B.J. Johnson R.S. Fitzner J.N. Boyce R.W. Nelson N. Kozlosky C.J. Wolfson M.F. Rauch C.T. Cerretti D.P. Paxton R.J. March C.J. Black R.A. Science. 1998; 282: 1281-1284Crossref PubMed Scopus (1371) Google Scholar), and wild type littermate controls were maintained in an accredited animal facility at the Hospital for Special Surgery according to the guidelines of the American Veterinary Association, and all experiments were approved by the Hospital for Special Surgery Institutional Animal Care and Use Committee. The Adam17−/− fibroblast cell line (E2 cells) and the Adam10−/− fibroblast cell line were derived from E13.5 and E9.5 embryos, respectively (15.Reddy P. Slack J.L. Davis R. Cerretti D.P. Kozlosky C.J. Blanton R.A. Shows D. Peschon J.J. Black R.A. J. Biol. Chem. 2000; 275: 14608-14614Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar, 16.Hartmann D. de Strooper B. Serneels L. Craessaerts K. Herreman A. Annaert W. Umans L. Lübke T. Lena Illert A. von Figura K. Saftig P. Hum. Mol. Genet. 2002; 11: 2615-2624Crossref PubMed Google Scholar). Immortalized fibroblasts derived from Adam9−/−, Adam12−/−, and wild type E13.5 embryos were generated by transfecting primary mouse embryonic fibroblast (mEF) cells from the appropriate mouse lines (17.Sahin U. Weskamp G. Kelly K. Zhou H.M. Higashiyama S. Peschon J. Hartmann D. Saftig P. Blobel C.P. J. Cell Biol. 2004; 164: 769-779Crossref PubMed Scopus (794) Google Scholar) with a vector carrying the SV40 large T antigen. Wild type mEFs express ADAMs 9, 10, 12, 15, 17, and 19 (17.Sahin U. Weskamp G. Kelly K. Zhou H.M. Higashiyama S. Peschon J. Hartmann D. Saftig P. Blobel C.P. J. Cell Biol. 2004; 164: 769-779Crossref PubMed Scopus (794) Google Scholar). All cell lines were grown in Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum and antibiotics. The ionophores monensin, valinomycin, and nystatin were purchased from Sigma-Aldrich. Chloride ionophore 1 was purchased from Fluka, and ionomycin was obtained from Calbiochem. Batimastat (BB94) was kindly provided by Dr. D. Becherer (GlaxoSmithKline, Research Triangle Park, NC), and marimastat was provided by Dr. Ouathek Ouerfelli (Memorial Sloan-Kettering Cancer Center). The hydroxamate inhibitors GI254023X (GI) and GW280264X are described elsewhere (18.Ludwig A. Hundhausen C. Lambert M.H. Broadway N. Andrews R.C. Bickett D.M. Leesnitzer M.A. Becherer J.D. Comb. Chem. High Throughput Screen. 2005; 8: 161-171Crossref PubMed Scopus (273) Google Scholar) and were also provided by Dr. Becherer. The following protease inhibitors were used: 4-(2-aminoethyl)benzolsulfonylfluoride hydrochloride (Roche Applied Science) and 1,10-ortho-phenanthroline (Sigma-Aldrich). All other reagents were obtained from Sigma-Aldrich unless otherwise indicated. The expression vector for alkaline phosphatase (AP)-tagged Kit ligand 2 has been described previously (19.Kawaguchi N. Horiuchi K. Becherer J.D. Toyama Y. Besmer P. Blobel C.P. J. Cell Sci. 2007; 120: 943-952Crossref PubMed Scopus (52) Google Scholar). The full-length cDNA for human collagen XVII was generated as described previously (20.Borradori L. Koch P.J. Niessen C.M. Erkeland S. van Leusden M.R. Sonnenberg A. J. Cell Biol. 1997; 136: 1333-1347Crossref PubMed Scopus (114) Google Scholar) and cloned into the NotI site of pcDNA3 (Invitrogen). To produce the C-terminal AP-tagged murine truncated collagen XVII construct, we generated a PCR fragment spanning nucleotides 294–2777 (amino acids 1–828) of murine collagen XVII, including the largest collagenous subdomain Col13 (GenBankTM accession number NM007732) and cloned it into pAPtag5 (Genhunter, Nashville, TN; see Fig. 1B for a diagram of the AP-tagged collagen XVII fragment). For amplification of the fragment we used the forward primer 3′-cgcgggctagccaccatggatgtgaccaagaaaagc-5′ and reverse primer 3′-cgcggaagcttctccgggcacagtgattgttga-5′ with PfuTurbo DNA polymerase (Stratagene) and the Integrated Molecular Analysis of Genomes and their Expression (IMAGE) Consortium mouse cDNA clone 40086691 (Open Biosystems) as template. The proper insertion and the sequence of murine AP-collagen XVII construct (Col13-AP) was verified by DNA sequencing. For real time PCR, total RNA from primary wild type and Adam17−/− keratinocyte cultures was extracted using an RNeasy Mini kit (Qiagen), and 1 μg of total RNA was reverse transcribed using a First Strand cDNA Synthesis kit (Fermentas). Relative quantification of gene expression was performed by real time PCR using iQ SYBR Green Supermix on the iCycler iQ thermal cycler (Bio-Rad) following the manufacturer's protocols. Primer sequences were as follows: mouse glyceraldehyde-3-phosphate dehydrogenase: sense primer, 5′-tggagaaacctgccaagtatg-3′; antisense primer, 5′-gttgaagtcgcaggagacaac-3′; mouse ADAM9: sense primer, 5′-tgaccatcccaacgtacaga-3′; antisense primer, 5′-ttccaaaactggcattctcc-3′; mouse ADAM10: sense primer, 5′-tctccggaatccgtaacatc-3′; antisense primer, 5′-tccaggaacttctccacacc-3′; mouse ADAM17: sense primer, 5′-cagcagcactccataaggaaa-3′; antisense primer, 5′-tttgtaaaagcgttcggta-3′; and mouse collagen XVII: sense primer, 5′-ctggattaggcaaggctgag-3′; antisense primer, 5′-cttgactccccatgtcacct-3′. Relative expression was normalized for levels of glyceraldehyde-3-phosphate dehydrogenase. The generation of amplification products of the correct size was confirmed using agarose gel electrophoresis. Immortalized fibroblast cell lines and COS-7 cells were seeded on 12-well plates at 75% confluency and transfected with 1.5 μg of DNA/well with the indicated plasmids using Lipofectamine 2000 (Invitrogen) according to the manufacturer's recommendations, and 50 μg/ml ascorbic acid was added to the media to allow for hydroxylation of collagen and proper triple helix formation. Fresh Opti-MEM (Invitrogen) medium with or without the indicated reagents was added the next day after transfection and incubated for the designated time period. The AP activity in 100 μl of supernatant and 10 μl of cell lysate was measured by colorimetry as described previously (17.Sahin U. Weskamp G. Kelly K. Zhou H.M. Higashiyama S. Peschon J. Hartmann D. Saftig P. Blobel C.P. J. Cell Biol. 2004; 164: 769-779Crossref PubMed Scopus (794) Google Scholar, 21.Sahin U. Weskamp G. Zheng Y. Chesneau V. Horiuchi K. Blobel C.P. Patel T.B. Bertics P.J. Epidermal Growth Factor: Methods and Protocols. Humana Press Inc., Totowa, NJ2006: 99-113Google Scholar, 22.Zheng Y. Schlondorff J. Blobel C.P. J. Biol. Chem. 2002; 277: 42463-42470Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). The normalized percentage of shedding was calculated as ratio of supernatant AP activity divided by total AP activity (supernatant AP activity + cell lysate AP activity). All experiments were repeated at least three times with similar results. For verification of the expression of the collagen XVII-AP fusion protein and its shedding in murine fibroblasts, the media and cell lysates were processed separately after the indicated times as described earlier (23.Schäcke H. Schumann H. Hammami-Hauasli N. Raghunath M. Bruckner-Tuderman L. J. Biol. Chem. 1998; 273: 25937-25943Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar) and analyzed by Western blot with murine collagen XVII NC14A rabbit antiserum (MO-NC14A (8.Franzke C.W. Tasanen K. Schäcke H. Zhou Z. Tryggvason K. Mauch C. Zigrino P. Sunnarborg S. Lee D.C. Fahrenholz F. Bruckner-Tuderman L. EMBO J. 2002; 21: 5026-5035Crossref PubMed Scopus (189) Google Scholar)). Keratinocytes were isolated from the skin of ADAM-deficient mice and their wild type littermates essentially as described previously (24.Caldelari R. Suter M.M. Baumann D. De Bruin A. Müller E. J. Invest. Dermatol. 2000; 114: 1064-1065Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 25.Echtermeyer F. Streit M. Wilcox-Adelman S. Saoncella S. Denhez F. Detmar M. Goetinck P. J. Clin. Investig. 2001; 107: R9-R14Crossref PubMed Scopus (354) Google Scholar). Briefly the skin was washed with 70% ethanol and phosphate-buffered saline, and epidermis and dermis were detached by overnight digestion with 5 units/ml dispase II (Roche Applied Science) at 4 °C. The epidermis was mechanically separated from the dermis and incubated with 0.25% trypsin (w/v) and 2 mm EDTA for 30 min at 37 °C with vigorous shaking. After stopping the reaction with phosphate-buffered saline containing 10% fetal calf serum, the keratinocyte suspension was passed through a 70-μm sieve, and 105 cells/cm2 were plated in defined serum-free keratinocyte medium supplemented with 100 pm cholera toxin, 100 units/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B (all from Invitrogen) on 6-well plates coated with gelatin (2.5 mg/ml; Sigma-Aldrich). The cells were maintained in defined serum-free keratinocyte medium at 37 °C, 5% CO2, and 95% humidity, and medium was replaced every 48 h. Subconfluent cells derived from passages 2–5 were used for the experiments, and all cells were cultured with medium containing 50 μg/ml ascorbate for 48 h to allow full prolyl and lysyl hydroxylation of newly synthesized collagens. To isolate epidermis sheets from ADAM9-deficient mice and their wild type littermates, the skin was removed from euthanized 2–3-day-old pups, flattened on a Petri dish, and cut into epidermis sheets of equal size using an 8-mm biopsy punch. After 1-h digestion with dispase II at 37 °C, the epidermis was detached from the dermis with tweezers and transferred to a 12-well plate with serum-free keratinocyte growth medium. After a maximum of 2-h incubation at 37 °C, the epidermis sheets were used for the experiments outlined under “Results.” For ectodomain shedding analysis by immunoblotting, cell lysates and media were processed separately as described previously (23.Schäcke H. Schumann H. Hammami-Hauasli N. Raghunath M. Bruckner-Tuderman L. J. Biol. Chem. 1998; 273: 25937-25943Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). Briefly the cells were washed twice with phosphate-buffered saline and lysed on ice for 30 min in lysis buffer (1% Nonidet P-40, 0.1 m NaCl, and 25 mm Tris-HCl, pH 7.4) containing 1 mm Pefabloc (Merck), 2 mm EDTA, 10 mm 1,10-ortho-phenanthroline, and 10 μl/ml protease inhibitor mixture set III (Calbiochem). Then the cell lysate was collected with a rubber cell scraper, centrifuged for 30 min at 13,000 × g at 4 °C, and then used immediately or stored at −80 °C. Total protein content was determined using the microtiter BCATM Protein Assay kit (Pierce), and 30 μg of total protein/sample was used for SDS-PAGE. The medium was collected on ice, 1 mm Pefabloc and 2 mm EDTA were added immediately, and cell debris were then removed by centrifugation. Proteins were precipitated with chloroform-methanol and centrifuged, and the pellets were dissolved in Laemmli sample loading buffer containing 5 mm dithiothreitol and heated at 95 °C for 5 min. Mouse tissues were dissected immediately after euthanasia (performed according to the guidelines of the American Veterinary Association; all animal experiments were approved by the Hospital for Special Surgery Internal Animal Care and Use Committee). We homogenized 0.5 g of each tissue (lung, liver, skeletal muscle, and skin) in 600 μl of lysis buffer consisting of 0.1 m Tris-HCl, pH 6.8, 1 m urea, 1% Nonidet P-40, 10 mm EDTA, 1 mm 4-(2-aminoethyl)benzolsulfonylfluoride hydrochloride, and proteinase inhibitor mixture (26.Bruckner-Tuderman L. Schnyder U.W. Winterhalter K.H. Bruckner P. Eur. J. Biochem. 1987; 165: 607-611Crossref PubMed Scopus (83) Google Scholar) using a Polytron homogenizer for 3 min on ice (Kinematica, Littau, Switzerland). All lysates were centrifuged at 15,000 × g for 30 min to remove debris, and the supernatants were used for microtiter detergent-compatible colorimetric protein detection using the BCA Protein Assay kit (Pierce). Samples of equal protein content (30 μg) were mixed with 5-fold concentrated Laemmli buffer containing 50 mm dithiothreitol, heated at 95 °C for 5 min, and then analyzed by Western blotting (see below). For immunofluorescence microscopy, cryosections of mouse skin were fixed in ice-cold acetone for 10 min, washed in Tris-buffered saline, and blocked with 10% normal goat serum in Tris-buffered saline for 30 min at room temperature. The polyclonal goat anti-ADAM9 antiserum (R&D Systems) was used as primary antibody, and fluorescein isothiocyanate-labeled AffiniPure donkey anti-goat IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) was used as secondary antibody. Mounting medium supplemented with 4′,6-diamidino-2-phenylindole was purchased from Vector Laboratories (Burlingame, CA). For immunoblotting, the proteins were separated by electrophoresis on 7 or 10% SDS-polyacrylamide gels as indicated. Immunoblotting was performed with rabbit polyclonal antiserum against the human collagen XVII NC16A domain (NC16A (27.Schumann H. Baetge J. Tasanen K. Wojnarowska F. Schäcke H. Zillikens D. Bruckner-Tuderman L. Am. J. Pathol. 2000; 156: 685-695Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar) and human collagen XVII cytodomain (Endo-2 (8.Franzke C.W. Tasanen K. Schäcke H. Zhou Z. Tryggvason K. Mauch C. Zigrino P. Sunnarborg S. Lee D.C. Fahrenholz F. Bruckner-Tuderman L. EMBO J. 2002; 21: 5026-5035Crossref PubMed Scopus (189) Google Scholar)), murine collagen XVII NC14A domain (MO-NC14A (8.Franzke C.W. Tasanen K. Schäcke H. Zhou Z. Tryggvason K. Mauch C. Zigrino P. Sunnarborg S. Lee D.C. Fahrenholz F. Bruckner-Tuderman L. EMBO J. 2002; 21: 5026-5035Crossref PubMed Scopus (189) Google Scholar)), murine ADAM17 cytoplasmic domain (28.Schlöndorff J. Blobel C.P. J. Cell Sci. 1999; 112: 3603-3617Crossref PubMed Google Scholar), and murine ADAM9 cytoplasmic domain (29.Weskamp G. Krätzschmar J. Reid M.S. Blobel C.P. J. Cell Biol. 1996; 132: 717-726Crossref PubMed Scopus (185) Google Scholar). Anti-ADAM10 cytoplasmic domain antibodies were from R&D Systems. Immunoblot signals of collagen XVII ectodomain in the media of at least three independent experiments were analyzed and quantified with Quantity One software (Bio-Rad). All values are expressed as means ± S.D. Two populations of data were statistically analyzed using the unpaired two-tailed t test and considered significantly different at p values smaller than 0.05. ADAM10 and ADAM17 have different responses to treatment of cells with phorbol esters or calcium ionophores in cell-based assays. Low concentrations of PMA (20 ng/ml) predominantly activate ADAM17, whereas the calcium ionophore ionomycin stimulates both ADAMs 10 and 17 (30.Horiuchi K. Le Gall S. Schulte M. Yamaguchi T. Reiss K. Murphy G. Toyama Y. Hartmann D. Saftig P. Blobel C.P. Mol. Biol. Cell. 2007; 18: 176-188Crossref PubMed Scopus (182) Google Scholar, 31.Le Gall S.M. Bobé P. Reiss K. Horiuchi K. Niu X.D. Lundell D. Gibb D.R. Conrad D. Saftig P. Blobel C.P. Mol. Biol. Cell. 2009; 20: 1785-1794Crossref PubMed Scopus (58) Google Scholar). Because we had shown previously that ADAMs 9, 10, and 17 have the potential to shed collagen XVII and that these ADAMs were constitutively expressed in COS-7 cells (32.Franzke C.W. Tasanen K. Borradori L. Huotari V. Bruckner-Tuderman L. J. Biol. Chem. 2004; 279: 24521-24529Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), we analyzed the shedding of human full-length collagen XVII in response to PMA, pervanadate, and ionomycin in transiently transfected COS-7 cells. We observed no stimulation of shedding by 20 ng/ml PMA (Fig. 1A) or 100 μm pervanadate for 1 h (not shown), whereas treatment with 2.5 μm ionomycin strongly increased the release of the soluble collagen XVII ectodomain into the culture supernatant (Fig. 1A). For further analysis, a C-terminally truncated murine collagen XVII-alkaline phosphatase fusion protein (Col13-AP) was generated, and its expression and shedding were validated in transfected COS-7 cells. When shedding of the collagen XVII ectodomain-AP fusion protein was assessed by Western blot analysis, the 145-kDa transmembrane form was detected in the cell lysates, and the 90-kDa soluble form was detected in the culture supernatant (Fig. 1B). Stimulation with 20 ng/ml PMA or 100 μm pervanadate for 1 h did not increase the AP activity released into the supernatant, although both stimuli strongly enhanced shedding of the ADAM17 substrate Kit ligand 2 (19.Kawaguchi N. Horiuchi K. Becherer J.D. Toyama Y. Besmer P. Blobel C.P. J. Cell Sci. 2007; 120: 943-952Crossref PubMed Scopus (52) Google Scholar), which was used in parallel as a positive control in transiently transfected COS-7 cells to show that ADAM17 was activated under these conditions (Fig. 1B). Moreover shedding of endogenous collagen XVII from murine keratinocytes was also not stimulated by 20 ng/ml PMA or 100 μm pervanadate for 1 h (Fig. 1C). However, treatment of murine keratinocytes with 1 μm ionomycin for 20 min (Fig. 1C) resulted in strong stimulation of collagen XVII shedding as did stimulation with a 25 μm concentration of the calmodulin inhibitor trifluoroperazine (data not shown). Furthermore collagen XVII shedding from murine keratinocytes was only stimulated by incubation with ionomycin but not with ionophores for sodium (monensin; 20 μm), potassium (valinomycin; 1 μm), monovalent cations (nystatin; 20 μm), or chloride ions (chloride ionophore 1; 1 μm) (data not shown). The lack of stimulation by 1-h treatment with PMA or pervanadate in COS-7 cells and murine keratinocytes (see above) as well as in HaCaT keratinocytes and immortalized mEFs (data not shown) was not consistent with the hypothesis that ADAM17 is a major sheddase for collagen XVII as stimulation of shedding by these agents is a characteristic feature of ADAM17 (30.Horiuchi K. Le Gall S. Schulte M. Yamaguchi T. Reiss K. Murphy G. Toyama Y. Hartmann D. Saftig P. Blobel C.P. Mol. Biol. Cell. 2007; 18: 176-188Crossref PubMed Scopus (182) Google Scholar, 31.Le Gall S.M. Bobé P. Reiss K. Horiuchi K. Niu X.D. Lundell D. Gibb D.R. Conrad D. Saftig P. Blobel C.P. Mol. Biol. Cell. 2009; 20: 1785-1794Crossref PubMed Scopus (58) Google Scholar). Because ADAM10 is activated by the calcium ionophore ionomycin (IM) or the calmodulin inhibitor trifluoroperazine but not by PMA (30.Horiuchi K. Le Gall S. Schulte M. Yamaguchi T. Reiss K. Murphy G. Toyama Y. Hartmann D. Saftig P. Blobel C.P. Mol. Biol. Cell. 2007; 18: 176-188Crossref PubMed Scopus (182) Google Scholar, 31.Le Gall S.M. Bobé P. Reiss K. Horiuchi K. Niu X.D. Lundell D. Gibb D.R. Conrad D. Saftig P. Blobel C.P. Mol. Biol. Cell. 2009; 20: 1785-1794Crossref PubMed Scopus (58) Google Scholar, 33.Nagano O. Murakami D. Hartmann D. De Strooper B. Saftig P. Iwatsubo T. Nakajima M. Shinohara M. Saya H. J. Cell Biol. 2004; 165: 893-902Crossref PubMed Scopus (238) Google Scholar, 34.Weskamp G. Ford J.W. Sturgill J. Martin S. Docherty A.J. Swendeman S. Broadway N. Hartmann D. Saftig P. Umland S. Sehara-Fujisawa A. Black R.A. Ludwig A. Becherer J.D. Conrad D.H. Blobel C.P. Nat. Immunol. 2006; 7: 1293-1298Crossref PubMed Scopus (179) Google Scholar) we further corroborated the involvement of ADAM10 in collagen XVII shedding by testing the inhibition of constitutive collagen XVII shedding in murine keratinocytes by GI, a hydroxamate inhibitor with selectivity for ADAM10 versus ADAM17 at 1 μm (34.Weskamp G. Ford J.W. Sturgill J. Martin S. Docherty A.J. Swendeman S. Broadway N. Hartmann D. Saftig P. Umland S. Sehara-Fujisawa A. Black R.A. Ludwig A. Becherer J.D. Conrad D.H. Blobel C.P. Nat. Immunol. 2006; 7: 1293-1298Crossref PubMed Scopus (179) Google Scholar, 35.Hundhausen C. Misztela D. Berkhout T.A. Broadway N. Saftig P. Reiss K. Hartmann D. Fahrenholz F. Postina R. Matthews V. Kallen K.J. Rose-John S. Ludwig A. Blood. 2003; 102: 1186-1195Crossref PubMed Scopus (558) Google Scholar). Moreover we used GW280264X, which has nearly identical inhibitory activity for ADAM17 and ADAM10 (18.Ludwig A. Hundhausen C. Lambert M.H. Broadway N. Andrews R.C. Bickett D.M. Leesnitzer M.A. Becherer J.D. Comb. Chem. High Throughput Screen. 2005; 8: 161-171Crossref PubMed Scopus (273) Google Scholar). As shown in Fig. 2A, both inhibitors blocked constitutive shedding of collagen XVII at 1 μm by about 80%. In addition, ionomyci
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