Title: ADAM10 Mediates Ectodomain Shedding of the Betacellulin Precursor Activated by p-Aminophenylmercuric Acetate and Extracellular Calcium Influx
Abstract: Betacellulin belongs to the family of epidermal growth factor-like growth factors that are expressed as transmembrane precursors and undergo proteolytic ectodomain shedding to release a soluble mature growth factor. In this study, we investigated the ectodomain shedding of the betacellulin precursor (pro-BTC) in conditionally immortalized wild-type (WT) and ADAM-deficient cell lines. Sequential ectodomain cleavage of the predominant cell-surface 40-kDa form of pro-BTC generated a major (26–28 kDa) and two minor (20 and 15 kDa) soluble forms and a cellular remnant lacking the ectodomain (12 kDa). Pro-BTC shedding was activated by calcium ionophore (A23187) and by the metalloprotease activator p-aminophenylmercuric acetate (APMA), but not by phorbol esters. Culturing cells in calcium-free medium or with the protein kinase Cδ inhibitor rottlerin, but not with broad-based protein kinase C inhibitors, blocked A23187-activated pro-BTC shedding. These same treatments were without effect for constitutive and APMA-induced cleavage events. All pro-BTC shedding was blocked by treatment with a broad-spectrum metalloprotease inhibitor (GM6001). In addition, constitutive and activated pro-BTC shedding was differentially blocked by TIMP-1 or TIMP-3, but was insensitive to treatment with TIMP-2. Pro-BTC shedding was functional in cells from ADAM17- and ADAM9-deficient mice and in cells overexpressing WT or catalytically inactive ADAM17. In contrast, overexpression of WT ADAM10 enhanced constitutive and activated shedding of pro-BTC, whereas overexpression of catalytically inactive ADAM10 reduced shedding. These results demonstrate, for the first time, activated pro-BTC shedding in response to extracellular calcium influx and APMA and provide evidence that ADAM10 mediates constitutive and activated pro-BTC shedding. Betacellulin belongs to the family of epidermal growth factor-like growth factors that are expressed as transmembrane precursors and undergo proteolytic ectodomain shedding to release a soluble mature growth factor. In this study, we investigated the ectodomain shedding of the betacellulin precursor (pro-BTC) in conditionally immortalized wild-type (WT) and ADAM-deficient cell lines. Sequential ectodomain cleavage of the predominant cell-surface 40-kDa form of pro-BTC generated a major (26–28 kDa) and two minor (20 and 15 kDa) soluble forms and a cellular remnant lacking the ectodomain (12 kDa). Pro-BTC shedding was activated by calcium ionophore (A23187) and by the metalloprotease activator p-aminophenylmercuric acetate (APMA), but not by phorbol esters. Culturing cells in calcium-free medium or with the protein kinase Cδ inhibitor rottlerin, but not with broad-based protein kinase C inhibitors, blocked A23187-activated pro-BTC shedding. These same treatments were without effect for constitutive and APMA-induced cleavage events. All pro-BTC shedding was blocked by treatment with a broad-spectrum metalloprotease inhibitor (GM6001). In addition, constitutive and activated pro-BTC shedding was differentially blocked by TIMP-1 or TIMP-3, but was insensitive to treatment with TIMP-2. Pro-BTC shedding was functional in cells from ADAM17- and ADAM9-deficient mice and in cells overexpressing WT or catalytically inactive ADAM17. In contrast, overexpression of WT ADAM10 enhanced constitutive and activated shedding of pro-BTC, whereas overexpression of catalytically inactive ADAM10 reduced shedding. These results demonstrate, for the first time, activated pro-BTC shedding in response to extracellular calcium influx and APMA and provide evidence that ADAM10 mediates constitutive and activated pro-BTC shedding. Betacellulin (BTC) 1The abbreviations used are: BTC, betacellulin; EGF, epidermal growth factor; ADAM, a disintegrin and metalloproteinase; TGFα, transforming growth factor-α; HB-EGF, heparin-binding EGF-like growth factor; MMP, matrix metalloprotease; APMA, p-aminophenylmercuric acetate; TIMP, tissue inhibitor of metalloproteases; HA, hemagglutinin; IFN-γ, interferon-γ; PMA, phorbol 12-myristate 13-acetate; ConA, concanavalin A; DMEM, Dulbecco's modified Eagle's medium; CM, conditioned medium/media; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; PKCδ, protein kinase Cδ; WT, wild-type; SH3, Src homology 3. belongs to the epidermal growth factor (EGF)-like family of cytokines that are expressed as membrane-anchored precursor proteins containing an extracellular N-terminal ectodomain, a transmembrane domain, and a cytoplasmic domain. The extracellular N-terminal ectodomain can be proteolytically cleaved to release the mature active growth factor, which can then bind ErbB receptors in an autocrine or paracrine fashion, resulting in the activation of multiple signal transduction cascades affecting cell proliferation, differentiation, migration, and survival (1Holbro T. Hynes N.E. Annu. Rev. Pharmacol. Toxicol. 2004; 44: 195-217Crossref PubMed Scopus (507) Google Scholar, 2Yarden Y. Sliwkowski M.X. Nat. Rev. Mol. Cell. Biol. 2001; 2: 127-137Crossref PubMed Scopus (5772) Google Scholar). Although a number of in vitro studies have indicated that the membrane-anchored precursors may also activate ErbB receptor signaling in a juxtacrine manner (3Wong S.T. Winchell L.F. McCune B.K. Earp H.S. Teixido J. Massagué J. Herman B. Lee D.C. Cell. 1989; 56: 495-506Abstract Full Text PDF PubMed Scopus (414) Google Scholar, 4Brachmann R. Lindquist P.B. Nagashima M. Kohr W. Lipari T. Napier M. Derynck R. 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Genes Dev. 2002; 16: 307-323Crossref PubMed Scopus (387) Google Scholar). In contrast to other members of the EGF-like growth factor family, two recent reports have shown that ADAM17 does not appear to be the primary sheddase responsible for shedding of the BTC precursor (pro-BTC). Rather, a role for ADAM10 at least in the basal shedding of pro-BTC has been proposed (30Hinkle C.L. Sunnarborg S.W. Loiselle D. Parker C.E. Stevenson M. Russell W.E. Lee D.C. J. Biol. Chem. 2004; 279: 24179-24188Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 31Sahin 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 (809) Google Scholar). The ectodomain shedding of pro-BTC has not been extensively characterized. Analysis of the pro-BTC cDNA suggests that the primary translation product encodes a signal sequence, N- and O-linked glycosylation sites, and a hydrophobic transmembrane region followed by a hydrophilic C terminus (32Dunbar A.J. Goddard C. Int. J. Biochem. Cell Biol. 2000; 32: 805-815Crossref PubMed Scopus (94) Google Scholar). The ectodomain shedding of pro-BTC has been demonstrated to involve a single C-terminal proteolytic cleavage between Tyr111 and Leu112 proximal to the transmembrane region (30Hinkle C.L. Sunnarborg S.W. Loiselle D. Parker C.E. Stevenson M. Russell W.E. Lee D.C. J. Biol. Chem. 2004; 279: 24179-24188Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar) and multiple N-terminal cleavage events, resulting in variable N-terminal ectodomain forms (33Watanabe T. Shintani A. Nakata M. Shing Y. Folkman J. Igarashi K. Sasada R. J. Biol. Chem. 1994; 269: 9966-9973Abstract Full Text PDF PubMed Google Scholar). In this study, we describe a detailed map of the molecular sizes and identity of BTC protein fragments generated by ectodomain shedding and demonstrate for the first time that the metalloprotease-dependent shedding of pro-BTC is activated by extracellular calcium influx and p-aminophenylmercuric acetate (APMA). We also demonstrate that activated and constitutive pro-BTC shedding is sensitive to TIMP-1 (tissue inhibitor of metalloproteases) and TIMP-3, but not to TIMP-2, suggesting the involvement of one or more ADAM proteases and not MMPs. Constitutive and activated pro-BTC shedding was unaffected by ADAM17 overexpression or by an ADAM17 null mutation. Consistent with a possible role of ADAM10 in pro-BTC shedding (31Sahin 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 (809) Google Scholar), we show that overexpression of wild-type or catalytically inactive ADAM10 modulates constitutive and stimulus-induced pro-BTC shedding. Antibodies and Reagents—The following antibody reagents were used: goat anti-human BTC ectodomain (Asp32–Tyr111) antibody, biotinylated goat anti-human BTC ectodomain antibody, mouse anti-human BTC ectodomain antibody, rat anti-mouse ADAM10 antibody, and rabbit anti-human ADAM17 cytoplasmic domain antibody (R&D Systems); rabbit anti-hemagglutinin (HA) epitope tag antibody (Zymed Laboratories Inc.); horseradish peroxidase-conjugated donkey anti-rabbit IgG F(ab′)2 fragment (Amersham Biosciences); and phycoerythrin-conjugated donkey anti-goat IgG F(ab′)2 fragment (Jackson ImmunoResearch Laboratories, Inc.). Recombinant human BTC and mouse interferon-γ (IFN-γ) were purchased from R&D Systems. Calcium ionophore (A23187), phorbol 12-myristate 13-acetate (PMA), APMA, concanavalin A (ConA)-agarose, and protease inhibitor mixture were purchased from Sigma. Sulfosuccinimidyl 6-(biotinamido)hexanoate, SuperSignal West Pico substrate, heparin-agarose, streptavidin-agarose, and protein G- and A-agarose were obtained from Pierce. Horseradish peroxidase-conjugated streptavidin was purchased from Jackson ImmunoResearch Laboratories, Inc. SP600123 was obtained from BIOMOL Research Labs Inc. GM6001 was obtained from Chemicon International, Inc. All other inhibitors were purchased from Calbiochem. TIMP-1 and TIMP-2 were generous gifts from Chris Overall (University of British Colombia), and TIMP-3 was a generous gift from John Doedens and Roy Black (Amgen). Cell Culture—The isolation and characterization of conditionally immortalized wild-type, ADAM17ΔZn/ΔZn, and ADAM9–/– dermal fibroblast and stomach epithelial cell lines have been described previously (34Garton K.J. Gough P.J. Blobel C.P. Murphy G. Greaves D.R. Dempsey P.J. Raines E.W. J. Biol. Chem. 2001; 276: 37993-38001Abstract Full Text Full Text PDF PubMed Google Scholar, 35Garton K.J. Gough P.J. Philalay J. Wille P.T. Blobel C.P. Whitehead R.H. Dempsey P.J. Raines E.W. J. Biol. Chem. 2003; 278: 37459-37464Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). Cells were cultured at 33 °C in Dulbecco's modified Eagle's medium (DMEM) plus 10% fetal bovine serum/penicillin/streptomycin/nonessential amino acids and 5 units/ml IFN-γ. Prior to experiments, cells were cultured for 36 h at 37 °C in medium lacking IFN-γ to arrest immortalization by the SV40 large T antigen. All treatments were performed at 37 °C. Generation of Expression Constructs and Retroviral Transduction—A cDNA encoding human BTC containing a C-terminal HA epitope tag was generated by PCR amplification using flanking primers (5′-GGCCATTCTGGCCCGCCACCATGGACCGGGCCGCCCGGTGC and 3′-GGCCGCTGCGGCCCCGTAAAACAAGTCAACTCTCTC) containing unique SfiI restriction sites. For stable expression of BTC-HA alone, the full-length pro-BTC cDNA fragment was directionally subcloned into the first cistron of the pBM-IRES-PURO retroviral vector, which contains an HA epitope tag cassette flanked by 5′-SfiI and 3′-NotI restriction sites (34Garton K.J. Gough P.J. Blobel C.P. Murphy G. Greaves D.R. Dempsey P.J. Raines E.W. J. Biol. Chem. 2001; 276: 37993-38001Abstract Full Text Full Text PDF PubMed Google Scholar). For coexpression with different ADAM cDNAs, BTC-HA was cloned into the pBM-IRES-EGFP vector (36Hitoshi Y. Lorens J. Kitada S.I. Fisher J. LaBarge M. Ring H.Z. Francke U. Reed J.C. Kinoshita S. Nolan G.P. Immunity. 1998; 8: 461-471Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). C-terminally HA-tagged murine wild-type ADAM17 and catalytically inactive ADAM17(E406A) (34Garton K.J. Gough P.J. Blobel C.P. Murphy G. Greaves D.R. Dempsey P.J. Raines E.W. J. Biol. Chem. 2001; 276: 37993-38001Abstract Full Text Full Text PDF PubMed Google Scholar) and wild-type murine ADAM10 and catalytically inactive ADAM10(E385A) (37Gough P.J. Garton K.J. Wille P.T. Rychlewski M. Dempsey P.J. Raines E.W. J. Immunol. 2004; 172: 3678-3685Crossref PubMed Scopus (221) Google Scholar) were cloned into the pBM-IRES-PURO retroviral vector. All cDNA constructs were verified by DNA sequencing. High titer retroviral supernatants were generated as described previously (36Hitoshi Y. Lorens J. Kitada S.I. Fisher J. LaBarge M. Ring H.Z. Francke U. Reed J.C. Kinoshita S. Nolan G.P. Immunity. 1998; 8: 461-471Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). For retroviral transduction, 4 × 105 wild-type or ADAM9- or ADAM17-deficient cells were seeded into 25-cm2 tissue culture flasks and cultured for 24 h prior to infection. Cells were incubated with 5 ml of virus stock for 12 h in the presence of 4 μg/ml Polybrene and then replenished with fresh medium. For PURO vectors, cells were then grown for 48 h prior to passage into medium containing 2–5 μg/ml puromycin. Resistant cells were used in subsequent experiments. For ADAM coexpression experiments, cells were initially transduced with pBM-BTC-HA-IRES-EGFP retrovirus, and a pooled population of enhanced green fluorescent protein-positive cells was isolated by fluorescence-activated cell sorting using a MoFlo cytometer (Cytomation, Inc.). Indirect immunofluorescence was used to confirm BTC cell-surface expression. Subsequently, sorted cells were superinfected with pBM-IRES-PURO retroviruses expressing different ADAM constructs and then selected with puromycin as described above. BTC Cleavage Assays—Cells were seeded either at 1 × 105 cells/well in 6-well plates or at 3 × 105 cells in 100-mm dishes. Cells were grown for 48 h to confluence at 33 °C in medium containing IFN-γ and then for an additional 36 h at 37 °C in medium lacking IFN-γ. For analysis of constitutive shedding, cells were cultured for 24 h in serum-free DMEM plus 50 μm GM6001 or vehicle (Me2SO). To evaluate stimulated pro-BTC shedding, cells were cultured for 1 h in serum-free DMEM with or without APMA, A23187, or PMA. Preincubation of cells with GM6001, kinase inhibitors, calcium-free DMEM, or TIMPs was performed for 30 min prior to addition of shedding activators. For TIMP inhibition experiments, 4 × 104 cells were seeded into 24-well plates, and cells were used in shedding assays as described above. Conditioned media (CM) and cell lysates were then harvested. Biochemical Characterization of Cellular and Soluble BTC Forms— CM was harvested from cell cultures, and phenylmethylsulfonyl fluoride was added to a final concentration of 2 mm. Cell monolayers were washed twice with cold phosphate-buffered saline (PBS) and then incubated in lysis buffer (25 mm Tris, 50 mm NaCl, 0.5% Nonidet P-40, 0.5% sodium deoxycholate (pH 8.0), and protease inhibitor mixture) for 30 min at 4 °C. Subsequently, CM and cell lysates were centrifuged at 10,000 × g for 10 min to remove cell debris. CM and cell lysates were used directly in the BTC enzyme-linked immunosorbent assay (ELISA) and/or in immunoprecipitation and Western blot experiments. All samples were precleared prior to immunoprecipitation with appropriate antibodies. Protein concentrations were determined by the BCA protein assay (Pierce). For BTC ectodomain immunoprecipitation, CM and cell lysates were incubated overnight with affinity-purified goat anti-BTC antibody (2 μg), followed by 30 μl of a 50% slurry of protein G-agarose. For HA immunoprecipitation, CM and cell lysates were incubated with rabbit anti-HA antibody (2 μg), followed by 30 μl of a 50% slurry of protein A-agarose. For cell-surface biotinylation studies, cell monolayers were washed three times with ice-cold PBS and incubated with 0.5 mg/ml sulfosuccinimidyl 6-(biotinamido)hexanoate for 30 min at 4 °C. Labeling reagent was quenched by successive washes with PBS containing 0.1 m glycine and with PBS containing 0.2% bovine serum albumin. Subsequently, cells were lysed in lysis buffer containing 0.2% bovine serum albumin, and biotinylated proteins were precipitated by incubation with 30 μl of a 50% slurry of streptavidin-agarose. For detection of BTC forms that bind to heparin, CM or cell lysates were incubated overnight with 50 μl of a 50% slurry of heparin-agarose and washed twice with 20 mm Tris-HCl (pH 6.8). All CM, cell lysates, and precipitates were combined with sample buffer and heated for 5 min at 95 °C. All samples were then separated on 10–20% gradient Tris/Tricine gels, transferred to Hybond nitrocellulose (Amersham Biosciences), and subsequently immunoblotted with specific antibodies. After incubation with the appropriate horseradish peroxidase-conjugated secondary antibody, blots were developed using SuperSignal West Pico substrate. Direct detection of BTC in CM and cell lysates was performed by incubating immunoblots with biotinylated goat anti-BTC antibody (0.1 μg/ml), followed by horseradish peroxidase-conjugated streptavidin, and immunoreactive proteins were visualized as described above. For ADAM coexpression experiments, the amounts of CM and cell lysates of each cell line loaded onto gels were normalized to the cellular BTC content in the presence of GM6001 as determined by ELISA. In some cases, nitrocellulose membranes were stripped by immersing in Restore™ Western blot stripping buffer (Pierce) according to the manufacturer's instructions. Membranes were then reblocked and probed with another antibody. For metabolic labeling, cells were washed twice and incubated for 30 min with RPMI 1640 medium (lacking l-methionine) supplemented with 5% dialyzed fetal bovine serum. Cells were then pulse-labeled with 250 μCi/ml l-[35S]methionine (Amersham Biosciences) for 20 min and chased in DMEM containing a 10-fold excess of unlabeled l-methionine. At different chase times, cells were washed twice with ice-cold PBS and lysed, and BTC was immunoprecipitated by overnight incubation with 2 μg of anti-HA antibody and 30 μl of a 50% slurry of protein A-agarose as described above. BTC immunoprecipitates were separated on 10–20% Tris/Tricine gels under reducing conditions and visualized by autoradiography. BTC ELISA—A specific human BTC sandwich ELISA (R&D Systems) was used to quantify BTC levels in CM and cell lysates according to the manufacturer's instructions. The recombinant human BTC ectodomain (R&D Systems) was used as a standard. ADAM Western Blotting—For detection of ADAM17 and ADAM10 in coexpression experiments, cells were lysed with lysis buffer containing 5 mm o-phenanthroline. ConA-agarose precipitates were prepared from cell lysates, separated by 10% SDS-PAGE, and then transferred to nitrocellulose as described above. Data Analysis—All experiments were repeated at least three times with similar results, and a representative figure is presented. Values for each experiment are expressed as the means ± S.D. of quadruplicate determinations. Identification of Multiple Cellular and Shed Forms of BTC— For several EGF-like growth factors, including TGFα, HB-EGF, and amphiregulin, cell-surface ectodomain shedding and generation of soluble ligand are thought to be critical steps for functional ErbB receptor signaling. However, only limited knowledge about the sequential and regulated processing of EGF-like growth factors and the molecular mechanisms that control these shedding events is currently available. Soluble BTC forms have been detected in CM from BTC-expressing cell lines (7Tada H. Sasada R. Kawaguchi Y. Kojima I. Gullick W.J. Salomon D.S. Igarashi K. Seno M. Yamada H. J. Cell. Biochem. 1999; 72: 423-434Crossref PubMed Scopus (45) Google Scholar, 33Watanabe T. Shintani A. Nakata M. Shing Y. Folkman J. Igarashi K. Sasada R. J. Biol. Chem. 1994; 269: 9966-9973Abstract Full Text PDF PubMed Google Scholar, 38Shing Y. Christofori G. Hanahan D. Ono Y. Sasada R. Igarashi K. Folkman J. Science. 1993; 259: 1604-1607Crossref PubMed Scopus (379) Google Scholar), indicating that these cells possess the appropriate proteolytic machinery to shed BTC from the cell surface. In this study, we have further characterized the shedding of BTC using mouse dermal fibroblasts and stomach epithelial cells transduced with a retroviral BTC-HA expression construct (Fig. 1A).Table IPKCδ inhibitor rottlerin can inhibit A23187-induced pro-BTC sheddingTreatmentBTC sheddingConstitutiveA23187APMAVehicle100100100GM6001 (50 μm)211618Calcium-freeND4107Rottlerin (10 μm)1272397 Open table in a new tab Immunoprecipitation and Western blotting using the anti-HA antibody detected five cellular BTC fragments of 40, 30, 25, 19, and 12 kDa in cell lysates from dermal fibroblasts transduced with BTC-HA (Fig. 1B, left panel). Immunoprecipitation with the anti-BTC ectodomain (Asp32–Tyr111) antibody (referred to as the anti-BTC antibody) or affinity purification with heparin-agarose and Western blotting with the anti-HA antibody did not detect the 12-kDa fragment, indicating that it did not contain the BTC ectodomain. This suggests that the 12-kDa form is the BTC cytoplasmic remnant generated by ectodomain shedding. The larger fragments, which contained the BTC ectodomain, represent candidate pro-BTC forms that undergo shedding. No additional bands were detected by immunoprecipitation with the anti-BTC antibody, indicating that there are only five cellular BTC species that all share the same C terminus. Analysis of CM from dermal fibroblasts expressing BTC-HA using the anti-BTC antibody or affinity purification with heparin-agarose detected one major but diffuse BTC species of ∼26–28 kDa (Fig. 1B, right panel). In agreement with previous results that the major soluble BTC forms detected in CM have variable N- and O-linked glycosylation (7Tada H. Sasada R. Kawaguchi Y. Kojima I. Gullick W.J. Salomon D.S. Igarashi K. Seno M. Yamada H. J. Cell. Biochem. 1999; 72: 423-434Crossref PubMed Scopus (45) Google Scholar, 32Dunbar A.J. Goddard C. Int. J. Biochem. Cell Biol. 2000; 32: 805-815Crossref PubMed Scopus (94) Google Scholar, 33Watanabe T. Shintani A. Nakata M. Shing Y. Folkman J. Igarashi K. Sasada R. J. Biol. Chem. 1994; 269: 9966-9973Abstract Full Text PDF PubMed Google Scholar), both of these BTC species were precipitated with ConA-agarose (data not shown). The heterogeneity of the 26–28-kDa shed species is therefore likely to result from differences in post-translational glycosylation, although confirmation of this will require further analysis (7Tada H. Sasada R. Kawaguchi Y. Kojima I. Gullick W.J. Salomon D.S. Igarashi K. Seno M. Yamada H. J. Cell. Biochem. 1999; 72: 423-434Crossref PubMed Scopus (45) Google Scholar, 32Dunbar A.J. Goddard C. Int. J. Biochem. Cell Biol. 2000; 32: 805-815Crossref PubMed Scopus (94) Google Scholar, 33Watanabe T. Shintani A. Nakata M. Shing Y. Folkman J. Igarashi K. Sasada R. J. Biol. Chem. 1994; 269: 9966-9973Abstract Full Text PDF PubMed Google Scholar). In other experiments, two additional minor BTC ectodomains of ∼20 and 15 kDa that may have resulted from the shedding of smaller pro-BTC forms were detected (