The membrane-anchored heparin-binding EGF-like growth factor precursor (proHB-EGF)/diphtheria toxin receptor (DTR) belongs to a class of transmembrane growth factors and physically associates with CD9/DRAP27 which is also a transmembrane protein. To evaluate the biological activities of proHB-EGF/DTR as a juxtacrine growth factor and the biological significance of its association with CD9/DRAP27, the mitogenic activity of proHB-EGF/DTR was analyzed using stable transfectants of mouse L cells expressing both human proHB-EGF/DTR and monkey CD9/DRAP27, or either one alone. Juxtacrine activity was assayed by measuring the ability of cells in co-culture to stimulate DNA synthesis in an EGF receptor ligand dependent cell line, EP170.7. LH-2 cells expressing human proHB-EGF/DTR stimulated EP170.7 cell growth moderately. However, LCH-1 cells, a stable co-transfectant expressing both human proHB-EGF/DTR and monkey CD9/DRAP27 cDNAs, dramatically unregulated the juxtacrine growth factor activity of proHB-EGF/DTR approximately 25 times over that of LH-2 cells even though both cell types expressed similar levels of proHB-EGF/DTR on the cell surface. Anti-CD9/DRAP27 antibodies which were not able to neutralize the mitogenic activity of soluble HB-EGF suppressed LCH-1 cell juxtacrine growth activity to the same extent as did anti-HB-EGF neutralizing antibodies and CRM 197, specific inhibitors of human HG-EGF. These findings suggest that optimal expression of the juxtacrine growth activity of proHB-EGF/DTR requires co-expression of CD9/DRAP27. These studies also indicate that growth factor potentiation effects which have been observed previously for soluble growth factors also occurs at the level of cell surface associated growth factors.
Heparin-binding epidermal-like growth factor (HB-EGF) is synthesized as a transmembrane precursor (HB-EGFTM). The addition of phorbol ester (PMA, phorbol 12-myristate 13-acetate) to cells expressing HB-EGFTM results in the metalloproteinase-dependent release (shedding) of soluble HB-EGF. To analyze mechanisms that regulate HB-EGF shedding, a stable cell line was established expressing HB-EGFTM in which the ectodomain and the cytoplasmic tail were tagged with hemagglutinin (HA) and Myc epitopes, respectively (HB-EGFTMHA/Myc). HB-EGFTMHA/Myc cleavage was followed by the appearance of soluble HB-EGFHA in conditioned medium, the loss of biotinylated cell-surface HB-EGFTMHA/Myc, and the appearance of a Myc-tagged cytoplasmic tail fragment in cell lysates. By using this approach, several novel metalloproteinase-dependent regulators of HB-EGFTM shedding were identified as follows. (i) HB-EGFTMHA/Myc shedding induced by PMA was blocked by the mitogen-activated protein (MAP) kinase kinase inhibitor, PD98059. PMA activated MAP kinase within 5 min, but HB-EGFTMHA/Myc shedding did not occur until 20 min, suggesting that MAP kinase activation was a necessary step in the pathway of PMA-induced HB-EGFTM cleavage. (ii) Activation of an inducible Raf-1 kinase, ΔRaf-1:estrogen receptor, resulted in a rapid MAP kinase activation within 10 min and shedding of HB-EGFTMHA/Myc within 20–40 min. (iii) Serum induced MAP kinase activation and HB-EGFTMHA/Myc shedding that were inhibited by PD98059. (iv) Whereas PMA induced HB-EGFTMHA/Myc shedding in attached cells, no shedding occurred when the cells were placed in suspension. Shedding was fully restored shortly after cells were allowed to spread on fibronectin, and the extent of PMA-induced shedding increased with the extent of cell spreading. PMA induced the same level of MAP kinase activation whether the cells were attached or in suspension suggesting that although MAP kinase activation might be necessary for shedding, it was not sufficient. Taken together, these results suggest that there are two components of cell regulation that contribute to the shedding process, not previously recognized, the Raf-1/MAP kinase signal transduction pathway and cell adhesion and spreading. Heparin-binding epidermal-like growth factor (HB-EGF) is synthesized as a transmembrane precursor (HB-EGFTM). The addition of phorbol ester (PMA, phorbol 12-myristate 13-acetate) to cells expressing HB-EGFTM results in the metalloproteinase-dependent release (shedding) of soluble HB-EGF. To analyze mechanisms that regulate HB-EGF shedding, a stable cell line was established expressing HB-EGFTM in which the ectodomain and the cytoplasmic tail were tagged with hemagglutinin (HA) and Myc epitopes, respectively (HB-EGFTMHA/Myc). HB-EGFTMHA/Myc cleavage was followed by the appearance of soluble HB-EGFHA in conditioned medium, the loss of biotinylated cell-surface HB-EGFTMHA/Myc, and the appearance of a Myc-tagged cytoplasmic tail fragment in cell lysates. By using this approach, several novel metalloproteinase-dependent regulators of HB-EGFTM shedding were identified as follows. (i) HB-EGFTMHA/Myc shedding induced by PMA was blocked by the mitogen-activated protein (MAP) kinase kinase inhibitor, PD98059. PMA activated MAP kinase within 5 min, but HB-EGFTMHA/Myc shedding did not occur until 20 min, suggesting that MAP kinase activation was a necessary step in the pathway of PMA-induced HB-EGFTM cleavage. (ii) Activation of an inducible Raf-1 kinase, ΔRaf-1:estrogen receptor, resulted in a rapid MAP kinase activation within 10 min and shedding of HB-EGFTMHA/Myc within 20–40 min. (iii) Serum induced MAP kinase activation and HB-EGFTMHA/Myc shedding that were inhibited by PD98059. (iv) Whereas PMA induced HB-EGFTMHA/Myc shedding in attached cells, no shedding occurred when the cells were placed in suspension. Shedding was fully restored shortly after cells were allowed to spread on fibronectin, and the extent of PMA-induced shedding increased with the extent of cell spreading. PMA induced the same level of MAP kinase activation whether the cells were attached or in suspension suggesting that although MAP kinase activation might be necessary for shedding, it was not sufficient. Taken together, these results suggest that there are two components of cell regulation that contribute to the shedding process, not previously recognized, the Raf-1/MAP kinase signal transduction pathway and cell adhesion and spreading. The extracellular domains of many membrane-anchored proteins are proteolytically cleaved from the cell surface in a process termed as shedding. Shedding is an irreversible post-translational modification that regulates biological function by releasing growth factors, enzymes, and soluble receptors (1Bosenberg M.W. Massague J. Curr. Opin. Cell Biol. 1993; 5: 832-838Crossref PubMed Scopus (66) Google Scholar, 2Blobel C.P. Cell. 1997; 90: 589-592Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar, 3Werb Z. Yan Y. Science. 1998; 282: 1279-1280Crossref PubMed Google Scholar). For example, shedding converts a juxtacrine growth factor such as the membrane-anchored TGF-α 1The abbreviations used are:TGF-αtransforming growth factor-αTNF-αtumor necrosis factor-αHB-EGFheparin-binding EGF-like growth factorEGFepidermal growth factorPMAphorbol 12-myristate 13-acetateMAP kinasemitogen-activated protein kinaseERKextracellular signal-regulated kinaseMDC9metalloproteinase/disintegrin/cysteine-rich protein 9ADAMadisintegrinand metalloproteinaseβ-APPβ-amyloid precursor proteinα-MEMα-minimal essential mediumCHOChinese hamster ovaryHAhemagglutininCMconditioned mediumMEKMAP kinase/ERKPBSphosphate-buffered salineBSAbovine serum albuminERestrogen receptorCAPS3-(cyclohexylamino)propanesulfonic acidCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid 1The abbreviations used are:TGF-αtransforming growth factor-αTNF-αtumor necrosis factor-αHB-EGFheparin-binding EGF-like growth factorEGFepidermal growth factorPMAphorbol 12-myristate 13-acetateMAP kinasemitogen-activated protein kinaseERKextracellular signal-regulated kinaseMDC9metalloproteinase/disintegrin/cysteine-rich protein 9ADAMadisintegrinand metalloproteinaseβ-APPβ-amyloid precursor proteinα-MEMα-minimal essential mediumCHOChinese hamster ovaryHAhemagglutininCMconditioned mediumMEKMAP kinase/ERKPBSphosphate-buffered salineBSAbovine serum albuminERestrogen receptorCAPS3-(cyclohexylamino)propanesulfonic acidCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid precursor into a potent paracrine growth factor (4Brachmann R. 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Chem. 1991; 266: 5769-5773Abstract Full Text PDF PubMed Google Scholar). It has been suggested that all the components required for TGF-α shedding are located at or close to the cell surface (9Bosenberg M.W. Pandiella A. Massague J. J. Cell Biol. 1993; 122: 95-101Crossref PubMed Scopus (54) Google Scholar). There may be a common mechanism for PMA-induced shedding since a mutant CHO cell line isolated for its inability to cleave TGF-α was unable to cleave β-APP and a variety of other cell-surface molecules in response to PMA (6Arribas J. Massague J. J. Cell Biol. 1995; 128: 433-441Crossref PubMed Scopus (130) Google Scholar). transforming growth factor-α tumor necrosis factor-α heparin-binding EGF-like growth factor epidermal growth factor phorbol 12-myristate 13-acetate mitogen-activated protein kinase extracellular signal-regulated kinase metalloproteinase/disintegrin/cysteine-rich protein 9 adisintegrinand metalloproteinase β-amyloid precursor protein α-minimal essential medium Chinese hamster ovary hemagglutinin conditioned medium MAP kinase/ERK phosphate-buffered saline bovine serum albumin estrogen receptor 3-(cyclohexylamino)propanesulfonic acid 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid transforming growth factor-α tumor necrosis factor-α heparin-binding EGF-like growth factor epidermal growth factor phorbol 12-myristate 13-acetate mitogen-activated protein kinase extracellular signal-regulated kinase metalloproteinase/disintegrin/cysteine-rich protein 9 adisintegrinand metalloproteinase β-amyloid precursor protein α-minimal essential medium Chinese hamster ovary hemagglutinin conditioned medium MAP kinase/ERK phosphate-buffered saline bovine serum albumin estrogen receptor 3-(cyclohexylamino)propanesulfonic acid 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid In PMA-induced shedding, the enzymes responsible for proteolytic cleavage and release appear to be metalloproteinases since shedding is blocked by synthetic hydroxamic acid-based compounds that are metalloproteinase inhibitors (10Mullberg J. 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Among the metalloproteinases, the disintegrin metalloproteinases known as ADAMs (ADisintegrin and aMetalloproteinase) have been strongly implicated in shedding (2Blobel C.P. Cell. 1997; 90: 589-592Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar, 3Werb Z. Yan Y. Science. 1998; 282: 1279-1280Crossref PubMed Google Scholar). ADAM17 had been cloned and identified as the TNF-α-converting enzyme (16Black R.A. Rauch C.T. Kozlosky C.J. Peschon J.J. Slack J.L. Wolfson M.F. Castner B.J. Stocking K.L. Reddy P. Srinivasan S. Nelson N. Boiani N. Schooley K.A. Gerhart M. Davis R. Fitzner J.N. Johnson R.S. Paxton R.J. March C.J. Cerretti D.P. Nature. 1997; 385: 729-733Crossref PubMed Scopus (2690) Google Scholar, 17Moss M.L. Jin S.L. Milla M.E. Bickett D.M. Burkhart W. Carter H.L. Chen W.J. Clay W.C. Didsbury J.R. Hassler D. Hoffman C.R. Kost T.A. Lambert M.H. Leesnitzer M.A. McCauley P. McGeehan G. Mitchell J. Moyer M. Pahel G. Rocque W. Overton L.K. Schoenen F. Seaton T. 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Another ADAM family member, MDC9 (ADAM9/Meltrin γ), has been recently shown to be involved in the shedding of HB-EGFTM (21Izumi Y. Hirata M. Hasuwa H. Iwamoto R. Umata T. Miyado K. Tamai Y. Kurisaki T. Sehara-Fujisawa A. Ohno S. Mekada E. EMBO J. 1998; 17: 7260-7272Crossref PubMed Scopus (474) Google Scholar). Protein phosphorylation may be involved in the regulation of shedding. The PMA-induced shedding of TGF-α, β-APP (8Pandiella A. Massague J. J. Biol. Chem. 1991; 266: 5769-5773Abstract Full Text PDF PubMed Google Scholar, 14Arribas J. Coodly L. Vollmer P. Kishimoto T.K. Rose-John S. Massague J. J. Biol. Chem. 1996; 271: 11376-11382Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar),l-selectin (22Frey M. Appenheimer M.M. Evans S.S. J. Immunol. 1997; 158: 5424-5434PubMed Google Scholar), TNF-α and its receptors (7Pradines F.A. Raetz C.R. J. Biol. Chem. 1992; 267: 23261-23268Abstract Full Text PDF PubMed Google Scholar, 23Crowe P.D. VanArsdale T.L. Goodwin R.G. Ware C.F. J. Immunol. 1993; 151: 6882-6890PubMed Google Scholar, 24Zhang L. Higuchi M. Totpal K. Chaturvedi M.M. Aggarwal B.B. J. Biol. Chem. 1994; 269: 10270-10279Abstract Full Text PDF PubMed Google Scholar), HER-4/ErbB4 (25Vecchi M. Baulida J. Carpenter G. J. Biol. Chem. 1996; 271: 18989-18995Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar), and HB-EGFTM (26Goishi K. Higashiyama S. Klagsbrun M. Nakano N. Umata T. Ishikawa M. Mekada E. Taniguchi N. Mol. Biol. Cell. 1995; 6: 967-980Crossref PubMed Scopus (252) Google Scholar, 27Dethlefsen S.M. Raab G. Moses M.A. Adam R.M. Klagsbrun M. Freeman M.R. J. Biol. Chem. 1998; 69: 143-153Google Scholar) are all inhibited by the relatively nonspecific protein kinase inhibitor staurosporin. Tyrosine phosphorylation (28Slack B.E. Breu J. Petryniak M.A. Srivastava K. Wurtman R.J. J. Biol. Chem. 1995; 270: 8337-8344Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar) and phosphatase inhibitors promote shedding. For example, the shedding of β-APP (29Buxbaum J.D. Gandy S.E. Cicchetti P. Ehrlich M.E. Czernik A.J. Fracasso R.P. Ramabhadran T.V. Unterbeck A.J. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6003-6006Crossref PubMed Scopus (427) Google Scholar) and TNF-α receptors (30Higuchi M. Aggarwal B.B. J. Biol. Chem. 1993; 268: 5624-5631Abstract Full Text PDF PubMed Google Scholar) is induced by okadaic acid, and the shedding of syndecan-1 (31Reiland J. Ott V.L. Lebakken C.S. Yeaman C. McCarthy J. Rapraeger A.C. Biochem. J. 1996; 319: 39-47Crossref PubMed Scopus (86) Google Scholar), ErbB4/HER-4 and amphiregulin (32Vecchi M. Rudolph-Owen L.A. Brown C.L. Dempsey P.J. Carpenter G. J. Biol. Chem. 1998; 273: 20589-20595Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) is induced by pervanadate. The mechanisms by which PMA induces shedding are still for the most part unknown. To address this question we examined possible mechanisms involved in the PMA-induced shedding of HB-EGF. HB-EGF is a member of the EGF family of growth factors (33Higashiyama S. Abraham J.A. Miller J. Fiddes J.C. Klagsbrun M. Science. 1991; 251: 936-939Crossref PubMed Scopus (1042) Google Scholar) that is synthesized as a membrane-anchored molecule (HB-EGFTM), capable of supporting cell-cell adhesion (34Raab G. Kover K. Paria B.C. Dey S.K. Ezzell R.M. Klagsbrun M. Development. 1996; 122: 637-645Crossref PubMed Google Scholar) and juxtacrine stimulation (26Goishi K. Higashiyama S. Klagsbrun M. Nakano N. Umata T. Ishikawa M. Mekada E. Taniguchi N. Mol. Biol. Cell. 1995; 6: 967-980Crossref PubMed Scopus (252) Google Scholar, 35Higashiyama S. Iwamoto R. Goishi K. Raab G. Taniguchi N. Klagsbrun M. Mekada E. J. Cell Biol. 1995; 128: 929-938Crossref PubMed Scopus (279) Google Scholar). HB-EGFTM is also the receptor for diphtheria toxin (36Naglich J.G. Metherall J.E. Russel D.W. Eidels L. Cell. 1992; 69: 1051-1061Abstract Full Text PDF PubMed Scopus (465) Google Scholar). PMA treatment induces cleavage of HB-EGFTM within 15 min in a number of cell lines (15Suzuki M. Raab G. Moses M.A. Fernandez C.A. Klagsbrun M. J. Biol. Chem. 1997; 272: 31730-31737Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 26Goishi K. Higashiyama S. Klagsbrun M. Nakano N. Umata T. Ishikawa M. Mekada E. Taniguchi N. Mol. Biol. Cell. 1995; 6: 967-980Crossref PubMed Scopus (252) Google Scholar, 27Dethlefsen S.M. Raab G. Moses M.A. Adam R.M. Klagsbrun M. Freeman M.R. J. Biol. Chem. 1998; 69: 143-153Google Scholar, 37Raab G. Higashiyama S. Hetelekidis S. Abraham J.A. Damm D. Ono M. Klagsbrun M. Biochem. Biophys. Res. Commun. 1994; 204: 592-597Crossref PubMed Scopus (71) Google Scholar). There is a loss of cell-surface associated HB-EGFTM, acquisition of cell resistance to diphtheria toxin (37Raab G. Higashiyama S. Hetelekidis S. Abraham J.A. Damm D. Ono M. Klagsbrun M. Biochem. Biophys. Res. Commun. 1994; 204: 592-597Crossref PubMed Scopus (71) Google Scholar) and release of the mature soluble form of HB-EGF into conditioned medium (CM) (15Suzuki M. Raab G. Moses M.A. Fernandez C.A. Klagsbrun M. J. Biol. Chem. 1997; 272: 31730-31737Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 27Dethlefsen S.M. Raab G. Moses M.A. Adam R.M. Klagsbrun M. Freeman M.R. J. Biol. Chem. 1998; 69: 143-153Google Scholar, 37Raab G. Higashiyama S. Hetelekidis S. Abraham J.A. Damm D. Ono M. Klagsbrun M. Biochem. Biophys. Res. Commun. 1994; 204: 592-597Crossref PubMed Scopus (71) Google Scholar). Cleavage of HB-EGFTM is inhibited by metalloproteinase inhibitors (15Suzuki M. Raab G. Moses M.A. Fernandez C.A. Klagsbrun M. J. Biol. Chem. 1997; 272: 31730-31737Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar,27Dethlefsen S.M. Raab G. Moses M.A. Adam R.M. Klagsbrun M. Freeman M.R. J. Biol. Chem. 1998; 69: 143-153Google Scholar, 38Lanzrein M. Garred O. Olsnes S. Sandvig K. Biochem. J. 1995; 310: 285-289Crossref PubMed Scopus (46) Google Scholar). Mature soluble HB-EGF is a potent stimulator of cell proliferation and migration, for example of smooth muscle cells (SMC), fibroblasts, and keratinocytes (39Raab G. Klagsbrun M. Biochem. Biophys. Acta. 1997; 1333: 179-199PubMed Google Scholar, 40Higashiyama S. Abraham J.A. Klagsbrun M. J. Cell Biol. 1993; 122: 933-940Crossref PubMed Scopus (313) Google Scholar, 41Hashimoto K. Higashiyama S. Asada H. Hashimura E. Kobayashi T. Sudo K. Nakagawa T. Damm D. Yoshikawa K. Taniguchi N. J. Biol. Chem. 1994; 269: 20060-20066Abstract Full Text PDF PubMed Google Scholar). HB-EGF may play a role in SMC hyperplasia (39Raab G. Klagsbrun M. Biochem. Biophys. Acta. 1997; 1333: 179-199PubMed Google Scholar). Its expression is up-regulated in the neointima following balloon injury to rat carotid arteries (42Igura T. Kawata S. Miyagawa J. Inui Y. Tamura S. Fukuda K. Isozaki K. Yamamori K. Taniguchi N. Higashiyama S. Matsuzawa Y. Arterioscler. Thromb. Vasc. Biol. 1996; 16: 1524-1531Crossref PubMed Scopus (59) Google Scholar) and in rat models of pulmonary hypertension (43Powell P.P. Klagsbrun M. Abraham J.A. Jones R.C. Am. J. Pathol. 1993; 143: 784-793PubMed Google Scholar). In addition, it has been detected in medial SMC and in foamy macrophages found in human atherosclerotic plaques (44Miyagawa J. Higashiyama S. Kawata S. Inui Y. Tamura S. Yamamoto K. Nishida M. Nakamura T. Yamashita S. Matsuzawa Y. Taniguchi J. J. Clin. Invest. 1995; 95: 404-411Crossref PubMed Google Scholar). It may be that aberrant shedding of HB-EGF may contribute to these pathologies. Since the conversion of HB-EGFTM to mature soluble HB-EGF has possible physiological and pathological implications, we have further analyzed mechanisms of PMA-induced shedding. In this report we identify several previously unrecognized regulators of HB-EGFTM shedding. These are the Raf-1/MAP kinase cascade and cell adhesion and spreading. All cell culture reagents were purchased from Life Technologies, Inc. Anti-phospho-ERK1/2 antibodies and PD98059 were purchased from Calbiochem. Polyclonal goat anti-ERK1/2, polyclonal rabbit anti-Raf-1, and monoclonal anti-Myc antibodies 9E10 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Tamoxifen (4-hydroxy) was purchased from Research Biochemicals International (Natick, MA). Fibronectin was purchased from Becton Dickinson (Franklin Lakes, NJ). Heparin-agarose was purchased from Sigma. EZ-link-sulfo-NHS-Biotin was purchased from Pierce. Gamma bind protein G-Sepharose, was purchased from Amersham Pharmacia Biotech. Horseradish peroxidase-conjugated streptavidin, horseradish peroxidase-conjugated anti-rabbit IgG, and CompleteTMmixture of protease inhibitors were purchased from Roche Molecular Biochemicals. Horseradish peroxidase-conjugated anti-mouse IgG was purchased from Promega (Madison, WI). The hydroxamic acid-based metalloproteinase inhibitor, BB3489, was kindly provided by British Biotech (Oxford, UK). Chinese hamster ovary (CHO-K1) cells were purchased from American Type Culture Collection (ATCC, Manassas, VA) and maintained in α-minimal essential medium (α-MEM) supplemented with 10% fetal calf serum, 1% glutamine, 1% penicillin and streptomycin, in 5% CO2. CHO-HB-EGFTMHA/Myc cells were grown to 90–95% confluence in 10-cm dishes (1.8 × 106 cells/dish). HB-EGFTMHA/Myc was constructed so that the hemagglutinin (HA) epitope was inserted in the N-terminal extracellular domain between amino acids Leu83 and Thr85, and the Myc epitope was placed at the C terminus (Fig. 1 A). The doubled-tagged construct was prepared as follows. First HB-EGFTM/myc was prepared by polymerase chain reaction amplification of the complete open reading frame of HB-EGF cDNA (33Higashiyama S. Abraham J.A. Miller J. Fiddes J.C. Klagsbrun M. Science. 1991; 251: 936-939Crossref PubMed Scopus (1042) Google Scholar) using synthetic DNA oligonucleotide primers: a forward primer, 5′-GCTCTAGAGCATGAAGCTGCTGCCGTCG-3′ corresponding to the 5′ end of the full-length HB-EGF open reading frame, and a reverse primer, 5′-GCTCACAGATCCTCTTCTGAGATGAGTTTTTGTTCGTGGGAATTAGTCATGCC-3′, complementary to the nucleotide sequence of a Myc tag followed by the 3′ end of the full-length HB-EGF. The polymerase chain reaction product was ligated into a pCR3.1 mammalian expression vector using the TA cloning kit (Invitrogen, Carlsbad, CA). HB-EGFTM ha/myc was prepared using two complementary oligonucleotides 3′-CCTACCCATACGACGTCCCAGACTACG-5′ and 5′-CGTAGTCGTGGACGTCGTATGGGTAGG-3′ encoding the HA epitope flanked by an MscI site. The oligonucleotides were synthesized and annealed to each other and then inserted into a uniqueMscI site in HB-EGFTM (between amino acids Leu83 and Thr85). The correct sequence of HB-EGFTM ha/myc was confirmed by DNA sequence analysis. For expression, the HB-EGFTM ha/mycinsert was excised from the pCR3.1 vector after an EcoRI digest and subcloned into the EcoRI site of the pIRES/neo mammalian expression vector (CLONTECH, Palo Alto, CA). The resulting plasmid pIRES/neo-HB-EGFTM ha/myc was transfected into CHO-K1 cells using LipofectAMINE and opti-MEM transfection medium (Life Technologies, Inc.) according to the manufacturer's instructions. Twenty four hours post-transfection, cells were passaged 1:25 and plated on 10-cm tissue culture dishes. They were grown in α-MEM supplemented with 10% fetal calf serum, 1% penicillin/streptomycin, and 1 mg/ml G418 in 5% CO2. After 12 days stable clones were selected, expanded, and conditioned media (CM) were collected and tested for the presence of HA-tagged soluble HB-EGF ectodomain by Western blotting with anti-HA antibodies. Five independent clones that overexpressed HB-EGFTMHA/Myc were expanded and characterized. CHO-HB-EGFTMHA/Myc cells were grown overnight to approximately 65% confluence in 10-cm dishes. They were transfected transiently using LipofectAMINE as above with ΔRaf-1:ER plasmid DNA (16 μg/10-cm dish) alone (provided by Dr. Martin McMahon, University of California, San Francisco/Mt. Zion Cancer Center, San Francisco) (45Samuels M.L. Weber M.J. Bishop J.M. McMahon M. Mol. Cell. Biol. 1993; 13: 6241-6252Crossref PubMed Scopus (322) Google Scholar) or co-transfected with HA-ERK1 cDNA (provided by Dr. John Blenis, Harvard Medical School) (46Meloche S. Pages G. Pouyssegur J. Mol. Biol. Cell. 1992; 3: 63-71Crossref PubMed Scopus (131) Google Scholar) and ΔRaf-1:ER cDNA. In co-transfection experiments, the ratio of ΔRaf-1:ER cDNA to HA-ERK1 cDNA was 10-fold. The total amount of cDNA did not exceed 16 μg/10-cm dish. CHO-HB-EGFTMHA/Myc analysis was carried out 22–24 h post-transfection. For stable expression, the ΔRaf-1:ER cDNA construct was transfected into CHO-HB-EGFTMHA/Myc cells as above. Twenty-four hours post-transfection, cells were passaged 1:25 and plated on 10-cm tissue culture dishes. Cells were grown in α-MEM supplemented with 10% fetal calf serum, 1% penicillin/streptomycin, 0.5 mg/ml G418, and 5 μg/ml puromycin (CLONTECH, Palo Alto, CA) in 5% CO2. After 9 days stable clones were selected, expanded, and assayed for activation of MAP kinase in response to tamoxifen. Five independent clones were chosen for further studies. Cells were washed twice with 20 mm Hepes buffer, pH 7.2, 150 mm NaCl (HBS), and incubated on ice with EZ-link-NHS-sulfo-biotin (Pierce, 0.1 mg/ml) in HBS, for 10 min in order to minimize the internalization of cell-surface HB-EGFTM. After aspiration, the cells were washed twice with 20 mm Tris-HCl, pH 7.2, 150 mm NaCl to quench the biotinylation reaction. The cells were washed with HBS, and serum-free α-MEM supplemented with 0.05% BSA (5 ml/plate) was added to cells prior to use. Cells from a 10-cm dish were harvested by scraping into 1 ml of phospho-homogenization buffer that contained 20 mm sodium phosphate, pH 7.2, 50 mm NaCl, 250 mm sucrose, 2 mm EDTA, 0.5 mmsodium orthovanadate, 10 mm NaF, 5 mm sodium pyrophosphate, and a mixture of protease inhibitors (SPH buffer), and then homogenized by passing six times through a 26.5-gauge needle. The nuclei were pelleted by centrifugation at 400 × g. Fractions containing HB-EGFTMHA/Myc were obtained by centrifugation of the post-nuclear supernatants at 15,800 ×g (P2). HB-EGFTMHA/Myc was solubilized by resuspending the P2 pellets in SPH buffer supplemented with Triton X-100 (1% final concentration) and incubating on ice for 10 min. The Triton X-100-insoluble material was pelleted by brief centrifugation at 15,800 × g. Biotinylation studies have shown that virtually all cell-surface HB-EGFTMHA/Myc is contained in P2. Cells grown overnight were washed once with phosphate-buffered saline (PBS) and then detached by incubation with PBS supplemented with 5 mm EDTA for 5 min at 37 °C, 5% CO2. Cells were washed with Hepes-buffered serum-free α-MEM supplemented with 0.1% BSA and resuspended in serum-free medium containing 0.1% BSA. Cells were either maintained in suspension for 30 min or re-plated after 30 min on bacterial dishes precoated with fibronectin at various densities (0–2500 cm2), as described previously (47Ingber D.E. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3579-3583Crossref PubMed Scopus (408) Google Scholar). Proteins were resolved on 10 or 15% SDS-PAGE for MAP kinase/ERK or HB-EGF detection, respectively. Proteins were electroblotted for 1.5 h onto a polyvinylidene difluoride membranes (Bio-Rad) in 40 mm CAPS buffer, pH 10.5, in 15% methanol. A constant 24 V was applied. For detection of HB-EGFTMHA/Myc and HB-EGFHA, the membranes were blocked with 3% bovine serum albumin in PBS, 0.25% Tween 20 (PBST). The blots were first incubated with anti-HA or anti-Myc monoclonal antibodies (1:5000) and then with anti-mouse IgG conjugated to horseradish peroxidase (1:5000). For detection of phospho-ERK and total ERKs in the blotting, Tris-buffered saline was substituted for PBS. The blots were incubated with anti-phospho-ERK or anti-ERK1/2 antibodies in (1:2000), followed by anti-rabbit or anti-goat IgG, respectively, conjugated to horseradish peroxidase (1:5000). To detect cell-surface biotinylation after immunoprecipitation with anti-HA, biotinylated proteins were detected using horseradish peroxidase-coupled streptavidin (1:5000). The blots were developed using an enhanced chemiluminescence (ECL) kit according to the manufacturer's instructions (NEN Life Science Products). Cells from a 10-cm dish were scraped into 1 ml of SPH buffer. Triton X-100 was added to 1% final concentration, and the cells were lysed for 20 min on ice. The insoluble material was pelleted by centrifugation at 14,000 ×g at 4 °C for 10 min. Supernatants were precleared by incubation with 40 μl of protein G-Sepharose (50% v/v slurry), for 1 h at 4 °C and incubated overnight with 0.2 μg of the appropriate antibody. The immune complexes were collected by incubating the samples with 40 μl of protein G-Sepharose (50% v/v slurry) for 1.5 h at 4 °C, washed four times with lysis buffer, and boiled in 2× Laemmli's sample buffer. The extent of the PMA-induced HB-EGFTMHA/Myc cleavage and MAP kinase activation was quantified by densitometric scanning of films obtained after ECL using a UMAX PowerLookII scanner and the NIH Image program. The extent of cleavage was calculated by dividing the amount of the intact HB-EGFTMHA/Myc prior to PMA treatment by the amount of intact HB-EGFTMHA/Myc after PMA treatment and corrected for loading. The extent of HB-EGFTM cleavage and MAP kinase activation is expressed in arbitrary units. Cells were transferred a
ABSTRACT Previous studies have shown that heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) mRNA is synthesized in the mouse uterine luminal epithelium, temporally, just prior to implantation, and spatially, only at the site of blastocyst apposition (Das, S. K., Wang, X. N., Paria, B. C., Damm, D., Abraham, J. A., Klagsbrun, M., Andrews, G. K. and Dey, S. K. (1994) Development 120, 1071–1083). HB-EGF is synthesized as a transmembrane protein (HB-EGFTM) that can be processed to release the soluble growth factor. An antibody that cross-reacts only with the transmembrane form detected HB-EGFTM in uterine luminal epithelium in a spatial manner similar to that of HB-EGF mRNA. HB-EGFTM is a juxtacrine growth factor that mediates cell-cell contact. To ascertain if HB-EGFTM could be an adhesion factor for blastocysts, a mouse cell line synthesizing human HB-EGFTM was co-cultured with mouse blastocysts. Cells synthesizing HB-EGFTM adhered to day-4 mouse blasto-cysts more extensively than parental cells or cells synthesizing a constituitively secreted form of HB-EGF. Adhesion of cells synthesizing HB-EGFTM to blastocysts was inhibited by excess recombinant HB-EGF but less so by TGF-α. Adhesion was also inhibited by the synthetic peptide P21 corresponding to the HB-EGF heparin binding domain, and by incubating the blastocysts with heparinase. In addition, adhesion to delayed implanting dormant blastocysts, which lack EGF receptor (EGFR), was diminished relative to normal blastocysts. These results suggested that adhesion between blastocysts and cells synthesizing HB-EGFTM was mediated via interactions with both blastocyst EGFR and heparan sulfate proteoglycan (HSPG). It was concluded that HB-EGFTM, which is synthesized exclusively in the luminal epithelium at the site of blastocyst apposition, and which is a juxtacrine adhesion factor for blastocysts, could be one of the mediators of blastocyst adhesion to the uterus in the process of implantation.
The epidermal growth factor receptor (HER1) has been implicated in regenerative growth and proliferative diseases of the human bladder epithelium (urothelium), however a cognate HER1 ligand that can act as a growth factor for normal human urothelial cells (HUC) has not been identified. Here we show that heparin-binding EGF-like growth factor (HB-EGF), an activating HER1 ligand, is an autocrine regulator of HUC growth. This conclusion is based on demonstration of HB-EGF synthesis and secretion by primary culture HUC, identification of HER1 as an activatable HB-EGF receptor on HUC surfaces, stimulation of HUC clonal growth by HB-EGF, inhibition of HB-EGF-stimulated growth by heparin and of log-phase growth by CRM 197, a specific inhibitor of HB-EGF/HER1 interaction, and identification of human urothelium as a site of HB-EGF precursor (proHB-EGF) synthesis in vivo. ProHB-EGF expression was also detected in the vascular and detrusor smooth muscle of the human bladder. These data suggest a physiologic role for HB-EGF in the regulation of urothelial proliferation and regeneration subsequent to mucosal injury. Expression of proHB-EGF is also a feature of differentiated vascular and detrusor smooth muscle in the bladder. Because proHB-EGF is known to be the high affinity diphtheria toxin (DT) receptor in human cells, synthesis of the HB-EGF precursor by human urothelium also suggests the possibility of using the DT-binding sites of proHB-EGF as an in vivo target for the intraluminal treatment of urothelial diseases.
Airway smooth muscle constriction leads to the development of compressive stress on bronchial epithelial cells. Normal human bronchial epithelial cells exposed to an apical-to-basal transcellular pressure difference equivalent to the computed stress in the airway during bronchoconstriction demonstrate enhanced phosphorylation of extracellular signal-regulated kinase (ERK). The response is pressure dependent and rapid, with phosphorylation increasing 14-fold in 30 min, and selective, since p38 and c-Jun NH 2 -terminal kinase phosphorylation remains unchanged after pressure application. Transcellular pressure also elicits a ninefold increase in expression of mRNA encoding heparin-binding epidermal growth factor-like growth factor (HB-EGF) after 1 h, followed by prominent immunostaining for pro-HB-EGF after 6 h. Inhibition of the ERK pathway with PD-98059 results in a dose-dependent reduction in pressure-induced HB-EGF gene expression. The magnitude of the HB-EGF response to transcellular pressure and tumor necrosis factor (TNF)-α (1 ng/ml) is similar, and the combined mechanical and inflammatory stimulus is more effective than either stimulus alone. These results demonstrate that compressive stress is a selective and potent activator of signal transduction and gene expression in bronchial epithelial cells.
The heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) is a member of the EGF family of growth factors that binds to and activates the EGF receptor (EGFR) and the related receptor tyrosine kinase, ErbB4. HB-EGF-null mice (HB del/del ) were generated to examine the role of HB-EGF in vivo . More than half of the HB del/del mice died in the first postnatal week. The survivors developed severe heart failure with grossly enlarged ventricular chambers. Echocardiographic examination showed that the ventricular chambers were dilated and that cardiac function was diminished. Moreover, HB del/del mice developed grossly enlarged cardiac valves. The cardiac valve and the ventricular chamber phenotypes resembled those displayed by mice lacking EGFR, a receptor for HB-EGF, and by mice conditionally lacking ErbB2, respectively. HB-EGF–ErbB interactions in the heart were examined in vivo by administering HB-EGF to WT mice. HB-EGF induced tyrosine phosphorylation of ErbB2 and ErbB4, and to a lesser degree, of EGFR in cardiac myocytes. In addition, constitutive tyrosine phosphorylation of both ErbB2 and ErbB4 was significantly reduced in HB del/del hearts. It was concluded that HB-EGF activation of receptor tyrosine kinases is essential for normal heart function.
Several cell functions related to growth and survival regulation have been attributed specifically to the membrane form of heparin-binding EGF-like growth factor (proHB-EGF), rather than to the diffusible, processed HB-EGF isoform. These findings suggest the existence of a functional binding partner specifically for the membrane form of the growth factor. In this study we have identified the prosurvival cochaperone, BAG-1, as a protein that interacts with the cytoplasmic tail domain of proHB-EGF. Interaction between BAG-1 and the 24-amino acid proHB-EGF cytoplasmic tail was initially identified in a yeast two-hybrid screen and was confirmed in mammalian cells. The proHB-EGF tail bound BAG-1 in an hsp70-independent manner and within a 97-amino acid segment that includes the ubiquitin homology domain in BAG-1 but does not include the hsp70 binding site. Effects of BAG-1 and proHB-EGF co-expression were demonstrated in cell adhesion and cell survival assays and in quantitative assays of regulated secretion of soluble HB-EGF. Because the BAG-1 binding site is not present on the mature, diffusible form of the growth factor, these findings suggest a new mechanism by which proHB-EGF, in isolation from the diffusible form, can mediate cell signaling events. In addition, because effects of BAG-1 on regulated secretion of soluble HB-EGF were also identified, this interaction has the potential to alter the signaling capabilities of both the membrane-anchored and the diffusible forms of the growth factor.
