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• W2020458656 abstract "Tumor necrosis factor (TNF) has multiple biological effects such as participating in inflammation, apoptosis, and cell proliferation, but the mechanisms of its effects on epithelial cell proliferation have not been examined in detail. At the early stages of liver regeneration, TNF functions as a priming agent for hepatocyte replication and increases the sensitivity of hepatocytes to growth factors such as transforming growth factor α (TGFα); however, the mechanisms by which TNF interacts with growth factors and enhances hepatocyte replication are not known. Using the AML-12 hepatocyte cell line, we show that TNF stimulates proliferation of these cells through transactivation of the epidermal growth factor receptor (EGFR). The transactivation mechanism involves the release of TGFα into the medium through activation of the metalloproteinase TNFα-converting enzyme (also known as ADAM 17). Binding of the ligand to EGFR initiates a mitogenic cascade through extracellular signal-regulated kinases 1 and 2 and the partial involvement of protein kinase B. TNF-induced release of TGFα and activation of EGFR signaling were inhibited by TNFα protease inhibitor-1, an agent that interferes with TNFα-converting enzyme activity. We suggest that TNF-induced transactivation of EGFR may provide an early signal for the entry of hepatocytes into the cell cycle and may integrate proliferative and survival pathways at the start of liver regeneration. Tumor necrosis factor (TNF) has multiple biological effects such as participating in inflammation, apoptosis, and cell proliferation, but the mechanisms of its effects on epithelial cell proliferation have not been examined in detail. At the early stages of liver regeneration, TNF functions as a priming agent for hepatocyte replication and increases the sensitivity of hepatocytes to growth factors such as transforming growth factor α (TGFα); however, the mechanisms by which TNF interacts with growth factors and enhances hepatocyte replication are not known. Using the AML-12 hepatocyte cell line, we show that TNF stimulates proliferation of these cells through transactivation of the epidermal growth factor receptor (EGFR). The transactivation mechanism involves the release of TGFα into the medium through activation of the metalloproteinase TNFα-converting enzyme (also known as ADAM 17). Binding of the ligand to EGFR initiates a mitogenic cascade through extracellular signal-regulated kinases 1 and 2 and the partial involvement of protein kinase B. TNF-induced release of TGFα and activation of EGFR signaling were inhibited by TNFα protease inhibitor-1, an agent that interferes with TNFα-converting enzyme activity. We suggest that TNF-induced transactivation of EGFR may provide an early signal for the entry of hepatocytes into the cell cycle and may integrate proliferative and survival pathways at the start of liver regeneration. Tumor necrosis factor (TNF) 1The abbreviations used are: TNF, tumor necrosis factor; TNFR, TNF receptor; TRADD, TNFR1-associated death domain protein; TRAF2, TNFR-associated factor; FADD, Fas-associated death domain; TGFα, transforming growth factor-α; EGFR, epidermal growth factor receptor; ADAM, a disintegrin and metalloproteinase; TACE, TNFα-converting enzyme; TAPI-1, TNFα protease inhibitor-1; DMEM, Dulbecco's modified Eagle's medium; MP, metalloproteinase; MMP, matrix MP; BrdUrd, bromodeoxyuridine; PBS, phosphate-buffered saline; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase/ERK kinase; HB-EGF, heparin-binding EGF-like growth factor; PI3K, phosphoinositide 3-kinase; PKB, protein kinase B (also known as Akt). is a pleiotropic cytokine that plays a role in diverse cellular responses such as inflammation, apoptosis, and proliferation. It binds to specific type 1 and type 2 receptors (TNFR1 and TNFR2), although TNFR1 is responsible for most of the biological effects of TNF. Binding of the ligand causes activation of the trimeric receptor and recruitment of adapter proteins that interact with specific domains of the receptor, most commonly the death domain (1Wallach D. Varfolomeev E.E. Malinin N.L. Goltsev Y.V. Kovalenko A.V. Boldin M.P. Annu. Rev. Immunol. 1999; 17: 331-367Crossref PubMed Scopus (1131) Google Scholar, 2MacEwan D.J. Cell. Signal. 2002; 14: 477-492Crossref PubMed Scopus (529) Google Scholar, 3Baud V. Karin M. Trends Cell Biol. 2001; 11: 372-377Abstract Full Text Full Text PDF PubMed Scopus (1381) Google Scholar). Specific TNF-induced responses require the association of different adapter proteins to the receptor. For example, the recruitment of adapter proteins such as TRADD and TRAF2 activates multiple signal transduction pathways, including the NFκB pathway, which can protect cells against apoptosis. In contrast, binding of the adapter protein FADD initiates a cascade of events that results in apoptosis. In the liver, signaling through TNFR1 induces cell survival, replication, or apoptosis, depending on the context (4Kirillova I. Chaisson M. Fausto N. Cell Growth & Differ. 1999; 10: 819-828PubMed Google Scholar, 5Pierce R.H. Campbell J.S. Stephenson A.B. Franklin C.C. Chaisson M. Poot M. Kavanagh T.J. Rabinovitch P.S. Fausto N. Am. J. Pathol. 2000; 157: 221-236Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 6Xu Y. Bialik S. Jones B.E. Iimuro Y. Kitsis R.N. Srinivasan A. Brenner D.A. Czaja M.J. Am. J. Physiol. 1998; 275: C1058-C1066Crossref PubMed Google Scholar). We and others have shown that a lack of TNFR1 in knock-out mice inhibits liver regeneration after partial hepatectomy (7Yamada Y. Kirillova I. Peschon J.J. Fausto N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1441-1446Crossref PubMed Scopus (842) Google Scholar) and interferes with hepatic regeneration after acute chemical injury (8Yamada Y. Webber E.M. Kirillova I. Peschon J.J. Fausto N. Hepatology. 1998; 28: 959-970Crossref PubMed Scopus (216) Google Scholar, 9Chiu H. Gardner C.R. Dambach D.M. Durham S.K. Brittingham J.A. Laskin J.D. Laskin D.L. Toxicol. Appl. Pharmacol. 2003; 193: 218-227Crossref PubMed Scopus (70) Google Scholar). We also have shown that TNF functions as a priming agent that sensitizes hepatocytes to the effects of growth factors involved in liver regeneration. Furthermore, in normal liver, TNF synergistically enhances proliferation induced by growth factors TGFα and hepatocyte growth factor (10Webber E.M. Bruix J. Pierce R.H. Fausto N. Hepatology. 1998; 28: 1226-1234Crossref PubMed Scopus (241) Google Scholar). The mechanisms of the proliferative activity of TNF have not been elucidated, although it has been proposed that TNF modulates hepatocyte proliferation through TGFα (11Gallucci R.M. Simeonova P.P. Toriumi W. Luster M.I. J. Immunol. 2000; 164: 872-878Crossref PubMed Scopus (67) Google Scholar). The activation of TNFR1 signaling after partial hepatectomy, sequential activation of NFκB and signal transducer and activator 3 transcription factors, and increased interleukin-6 levels is necessary for hepatocyte replication (7Yamada Y. Kirillova I. Peschon J.J. Fausto N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1441-1446Crossref PubMed Scopus (842) Google Scholar, 12Cressman D.E. Greenbaum L.E. DeAngelis R.A. Ciliberto G. Furth E.E. Poli V. Taub R. Science. 1996; 274: 1379-1383Crossref PubMed Scopus (1325) Google Scholar). Nevertheless, the components of this pathway may act as cell survival factors and may not be directly involved in the triggering of hepatocyte replication (13Kovalovich K. Li W. DeAngelis R. Greenbaum L.E. Ciliberto G. Taub R. J. Biol. Chem. 2001; 276: 26605-26613Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar, 14Blindenbacher A. Wang X. Langer I. Savino R. Terracciano L. Heim M.H. Hepatology. 2003; 38: 674-682Crossref PubMed Scopus (161) Google Scholar, 15Wallenius V. Wallenius K. Hisaoka M. Sandstedt J. Ohlsson C. Kopf M. Jansson J.O. Endocrinology. 2001; 142: 2953-2960Crossref PubMed Scopus (16) Google Scholar). If this is the case, the involvement of TNF in hepatocyte replication requires the activation of additional pathways that are directly linked to cell proliferation. We show here that the proliferative effect of TNF in AML-12 hepatocytes requires transactivation of the epidermal growth factor receptor (EGFR). Transactivation of EGFR by TNF is accomplished through the activity of TNFα-converting enzyme (TACE, also known as ADAM 17) a surface metalloproteinase that releases soluble TGFα from pro-TGFα anchored in the plasma membrane. Released TGFα binds to EGFR, activating a signaling cascade that leads to DNA replication. Materials—Materials were purchased from the following vendors: DMEM/F-12 medium, dexamethasone, and gentamicin from Invitrogen; an insulin:transferrin:selenium mixture from BD Biosciences; fetal bovine serum from Hyclone; recombinant mouse TNF and recombinant human TGFα from R & D Systems; U0126 and anti-rabbit horseradish peroxidase secondary antibody from Promega; AG1478, LY294002, GM6001, GM6001 negative control, MMP-9/MMP-13 inhibitor I, MMP-2/MMP-9 inhibitor II, MMP-8 inhibitor I, and MMP-3 inhibitor IV from Calbiochem; TAPI-1 from Peptides International (Louisville, KY); [3H]thymidine (25 Ci/mmol) and anti-mouse horseradish peroxidase secondary antibody from Amersham Biosciences; BrdUrd labeling reagent and anti-BrdUrd antibody from DAKO; ABC detection kit from Vectastain; ERK1/2 antibody as described previously (16Seger R. Seger D. Reszka A.A. Munar E.S. Eldar-Finkelman H. Dobrowolska G. Jensen A.M. Campbell J.S. Fischer E.H. Krebs E.G. J. Biol. Chem. 1994; 269: 25699-25709Abstract Full Text PDF PubMed Google Scholar); γ-[32P]ATP (3000 Ci/mmol) and Renaissance chemiluminescent reagent from PerkinElmer Life Sciences; Triton X-100, 3,3-diaminobenzidine tablets, phosphoamino acid standards, and protein A-Sepharose from Sigma; Immobilon P polyvinylidene difluoride membrane from Millipore; blotting grade dry milk from Bio-Rad; EGFR (immunoprecipitation) antibody from UBI; EGFR (Western blot) and PY99 antibodies from Santa Cruz Biotechnology; and TGFα-neutralizing antibody from R & D Systems. All other antibodies were purchased from Cell Signaling Technologies. Buffers—The buffers used were Triton lysis buffer (25 mm HEPES, pH 7.4, 5 mm EDTA, 5 mm EGTA, 150 mm NaCl, 10% glycerol, 1% Triton X-100, 50 mm NaF, 4 μg/ml aprotinin, 4 μg/ml leupeptin, 10 μg/ml 4-(2-aminoethyl)-benzenesulfonyl fluoride, 0.1% β-mercaptoethanol, 1 mm Na3VO4), an assay buffer (20 mm HEPES, pH 7.4, 20 mm MgCl2, 0.1% β-mercaptoethanol), and an assay buffer plus ATP (100 μm cold ATP, 20 μCi/ml ATP). Cell Culture—AML-12 hepatocytes, a cell line established from hepatocytes of a TGFα transgenic mouse, were cultured as described previously (17Wu J.C. Merlino G. Fausto N. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 674-678Crossref PubMed Scopus (250) Google Scholar). Unless otherwise indicated, cultures were grown to 60–70% confluence and then serum-starved overnight in complete medium containing no fetal bovine serum. When inhibitors were used, cells were pretreated for 30 min prior to stimulation with the indicated concentration of inhibitor, which remained in the medium for the remainder of the experiment. The final concentration of Me2SO, the diluent for the inhibitors, was present in the culture medium at 0.1% or less. When using the TGFα neutralizing antibody, cultures were preincubated with 3 μg/ml antibody for 2 h prior to stimulation. Aliquots of TNF and TGFα used for stimulation were also preincubated with antibody (3 μg/ml) for 2 h at room temperature. [3H]Thymidine Incorporation Assay—AML-12 cells were plated at 40,000 cells/well in 24-well tissue culture plates and allowed to grow overnight in 10% fetal bovine serum. The following day, the cells were rinsed twice with Hanks' buffered saline solution, and complete medium containing no fetal bovine serum was added to the cells. The cells were stimulated the following day for 24 h unless otherwise noted. [3H]Thymidine (1 μCi/ml final concentration) was added to the medium for the final 3 h of stimulation. The trichloroacetic acid non-precipitable fraction was removed from the cells, and the precipitable fraction was solubilized in NaOH and quantified using a scintillation counter. Each treatment was measured in triplicate, and the data are represented as the average, with the error bars representing the mean ± S.E. BrdUrd Labeling—Cells were plated in 6- or 12-well plates, serum-starved, and stimulated as described previously in the [3H]thymidine incorporation assay. For the last 3 h of stimulation, the cultures were incubated with BrdUrd labeling reagent at 1:1000 dilution. Cells were rinsed and fixed for 30 min at room temperature with acetic alcohol (90% ethanol, 5% acetic acid, 5% distilled H2O). Endogenous peroxidase was blocked by incubation in 1% H2O2 in methanol for 20 min at room temperature. Cells were washed in PBS and incubated in 1.5 n HCl at 37 °C for 15 min followed by extensive washing in PBS. Cells were then incubated in anti-BrdUrd antibody at 1:40 dilution in PBS containing 1% bovine serum albumin for 1 h at 37 °C and washed in PBS. Cells were incubated in anti-mouse secondary antibody at 1:100 dilution in PBS, 1% bovine serum albumin for 30 min at room temperature followed by washing. Cells were incubated in ABC solutions for 30 min at room temperature, washed, and incubated in 50 mm Tris, pH 7.6, for 5 min, and then the 3,3-diaminobenzidine solution was added. Three areas in each well were counted for a total of ∼1000 cells. Proliferation is indicated as a percentage of labeled nuclei. Transforming Growth Factor α Enzyme-linked Immunosorbent Assay—Cells were cultured and stimulated according to [3H]thymidine incorporation protocol. Cell culture medium was collected 21 h after stimulation and frozen in aliquots at –80 °C until analysis. Human TGFα was quantified by enzyme-linked immunosorbent assay (Oncogene Research Products, San Diego, CA) according to the manufacturer's protocol. Preparation of Lysates—To harvest for whole cell protein after the indicated stimulation, cells in 10-cm plates were washed twice with ice-cold PBS and harvested by scraping into 0.5 ml of Triton X-100 lysis buffer. Lysates were sonicated twice for 1–2 s on the lowest setting and then clarified by centrifugation at 16,000 × g for 10 min at 4 °C. Supernatants were frozen in aliquots at –80 °C. Western Blots—Western blots were performed following standard protocols. Briefly, lysates (20 μg) were resolved on SDS polyacrylamide gels and electrotransferred to Immobilon P polyvinylidene difluoride membranes. For anti-EGFR Western blots, 50 μg of lysate was resolved on low bisacrylamide (0.4%) gels (18Aicher L.D. Campbell J.S. Yeung R.S. J. Biol. Chem. 2001; 276: 21017-21021Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar) that were transferred overnight at 4 °C. Membranes were blocked in Tris-buffered saline, 0.1% Tween-20 (TBST) containing 4% milk or bovine serum albumin (anti-phosphotyrosine blots) prior to incubation with primary antibodies. Membranes were then washed well with TBST, incubated with secondary antibody (anti-rabbit horseradish peroxidase at 1:10,000, anti-mouse horseradish peroxidase at 1:5000) for 1 h, washed extensively in TBST, and developed with a chemiluminescent reagent. Epithelial Growth Factor Receptor Immunoprecipitation—AML-12 cells were harvested in Triton X-100 lysis buffer containing no reducing agent (β-mercaptoethanol). Lysate (250 μg) was incubated overnight at 4 °C with 1 μg of antibody and 20 μl of a 50% protein A-Sepharose slurry. The beads were washed once with lysis buffer and once with kinase buffer. The beads were then boiled in 2× Laemmli sample buffer and run on SDS polyacrylamide gels. Gels were transferred to polyvinylidene difluoride membranes overnight at 4 °C and then immunoblotted as described previously. 32P Labeling and Phosphoamino Acid Analysis—10-cm dishes of AML-12 cells were serum-starved overnight in DMEM/hi glucose medium containing the insulin:transferrin:selenium mixture, dexamethasone, and gentamicin. The medium was changed to phosphate-free DMEM/hi glucose, and 1 mCi of [32P]orthophosphate (PerkinElmer Life Sciences) was added to each plate (5 ml). Cells were labeled for 6 h and then stimulated as indicated. Whole cell lysates were prepared as described previously. EGFR was immunoprecipitated overnight (500 μg of lysate, 2 μg of antibody, and protein A-Sepharose). Immunoprecipitates were run on SDS polyacrylamide gels, dried, and exposed to film to visualize 32P incorporation into the receptor. The labeled bands were excised from the gel and quantified by Cerenkov counting. Protein was extracted from the gel and analyzed for phosphoamino acid as described previously (18Aicher L.D. Campbell J.S. Yeung R.S. J. Biol. Chem. 2001; 276: 21017-21021Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). For phosphoamino acid analysis, dried pellets were resuspended in 5 μl of distilled H2O. 1 μlof the phosphoserine, -threonine, and -tyrosine standard and 2 μl of sample were loaded onto the origin of a 10 × 10-cm cellulose TLC plate. First dimension separation was run in ethanol:glacial acetic acid:distilled H2O (1:1:1) for 90 min at room temperature. Second dimension separation was run in butanol:formic acid:distilled H2O (8:3:4, v/v/v) for 40 min. Plates were dried and sprayed with 0.5% ninhydrin in 0.5% acetone and heated to 80 °C for 5 min to visualize standards. Plates were then exposed to film for visualization of 32P incorporation. Induction of Cell Proliferation by Tumor Necrosis Factor—To study the effects of TNF on cell replication, AML-12 cells were maintained in serum-free medium for 24 h, a procedure that reduced DNA replication as determined by BrdUrd labeling to a basal level of less than 10%. Subconfluent serum-starved cells were exposed to 20 ng/ml TNF for 24–72 h (Fig. 1a). Cell labeling by BrdUrd increased to 53% at 24 h and progressively decreased to the same level as non-stimulated cultures by 72 h. In repeated experiments, TNF increased BrdUrd labeling by 2–5-fold at 24 h and also increased the cell number by 2.5-fold at 48 h (data not shown). AML-12 hepatocytes produce membrane-bound pro-TGFα (17Wu J.C. Merlino G. Fausto N. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 674-678Crossref PubMed Scopus (250) Google Scholar). Nevertheless, addition of TGFα to the culture medium enhances DNA replication (Fig. 1b). At the doses used, TGFα increased BrdUrd labeling between 5- and 10-fold 24 h after exposure and increased cell numbers at 48 h. These data indicate that the EGFR is not saturated with endogenous ligand and is receptive to additional ligand stimulation. Consistent with this notion, we found that EGF and HB-EGF also stimulate DNA replication in AML-12 cells at levels comparable with those obtained with TGFα. In contrast, interleukin-6 and oncostatin M had little to no effect (data not shown). Tumor Necrosis Factor-induced Cell Replication Requires Transforming Growth Factor α/Epithelial Growth Factor Receptor Signaling—We have previously shown that TNF functions as a mitogen for intrahepatic stem cells (oval cells) and for primary hepatocytes in serum-containing cultures but is without effect in serum-free cultures (4Kirillova I. Chaisson M. Fausto N. Cell Growth & Differ. 1999; 10: 819-828PubMed Google Scholar). The finding that TNF functions as a complete mitogen for AML-12 hepatocytes maintained in serum-free medium indicated that in these cells a second proliferative signal may be generated by TNF. We hypothesized that signaling from TGFα and EGFR is necessary for TNF stimulation of cell replication. To test this hypothesis, we examined the ability of TNF to stimulate DNA synthesis in cultures in which signaling from both the ligand (TGFα) and its receptor (EGFR) was blocked. Serum-starved AML-12 cells were incubated with TGFα-neutralizing antibody or with the EGFR inhibitor AG1478 before stimulation with TNF or TGFα for 24 h (Fig. 2). As expected, the TGFα-neutralizing antibody almost entirely blocked the stimulation of DNA replication by exogenous TGFα and also decreased basal DNA synthesis by more than 60%. This indicates that TGFα is required for maintenance of the basal level of proliferation in these cells. Most important, exposure of the cells to the TGFα-neutralizing antibody prevented TNF-mediated DNA synthesis (Fig. 2a). The tyrphostin inhibitor AG1478 inhibits EGFR kinase activity in the low nanomolar range. Exposure of the cultured cells to this inhibitor reduced the basal level of DNA replication by 95%. AG1478 blocked the induction of DNA replication by either exogenously added TNF or TGFα (Fig. 2b). Taken together, the results of the experiments presented in Fig. 2 show that TNF-induced DNA replication requires TGFα and tyrosine kinase activity from EGFR. Tumor Necrosis Factor Stimulation Causes Phosphorylation of Epithelial Growth Factor Receptor—We next examined whether exposure to TNF causes phosphorylation of EGFR (Fig. 3). Whole cell lysates from cultures treated with either TNF or TGFα were immunoprecipitated with EGFR antibody and immunoblotted to detect phosphotyrosine. Fig. 3a shows that EGFR is phosphorylated on tyrosine in unstimulated AML-12 cells maintained in serum-free medium as determined by using the anti-phosphotyrosine antibody PY99. As expected, stimulation with TGFα caused increased phosphotyrosine reactivity. The addition of TNF did not cause a detectable increase in phosphotyrosine reactivity over the high basal signal, but it did cause an apparent shift in mobility, suggesting that the receptor might be modified by phosphorylation after TNF stimulation. To analyze the specific residues in the EGFR that may be phosphorylated after exposure to TNF or TGFα, we labeled serum-starved cells with [32P]orthophosphate. Cells were exposed to TNF or TGFα for 15 min and processed for phosphoamino acid analysis of EGFR. Both TGFα and TNF increased EGFR phosphorylation by 2–4-fold. EGFR protein was then extracted from the gel to determine which residues were phosphorylated after TNF and TGFα stimulation. Both agents caused an increase in serine and tyrosine phosphorylation of the receptor but did not appreciably enhance threonine phosphorylation (Fig. 3b). Tumor Necrosis Factor-induced DNA Replication Requires Downstream Signaling from Epithelial Growth Factor Receptor—The MEK1/2-ERK1/2 signaling pathway is activated by multiple tyrosine kinase receptors and is generally involved in cell proliferation. To test whether ERK1/2 is required for TNF-induced DNA replication in AML-12 cells, we inhibited the ERK1/2 pathway by blocking its upstream activator MEK1/2 with U0126 (Fig. 4a). This compound almost entirely blocked DNA replication induced by either TNF or TGFα. Growth factors and cytokines can also activate the phosphoinositide 3-kinase (PI3K) pathway. To determine whether this pathway participates in TNF-induced DNA replication, we blocked PI3K with the specific inhibitor LY294002 (Fig. 4b). DNA replication induced by either TNF or TGFα was blocked by this agent. Inhibition of ERK1/2 and PI3K in TNF-stimulated cells prevented the increase in DNA replication but did not affect cell survival. The MEK/ERK, PI3K, and NFκB pathways are all required for survival and/or proliferation after TNF exposure to AML-12 cells. To determine whether EGFR signaling is required for the activation of these pathways, we blocked EGFR kinase activity with AG1478 and assayed for phosphorylation of ERK1/2, PKB, and IκBα after TNF and TGFα stimulation. In preliminary experiments, we determined that the peak activation of ERK1/2, PKB, and IκBα occurred respectively at 15, 10, and 5 min after TNF exposure (data not shown). Fig. 5 shows that inhibition of EGFR activity during TNF stimulation completely blocked the phosphorylation of ERK1/2 at Thr-202 and Tyr-204 (Fig. 5a), partially blocked phosphorylation of PKB at Ser-473 and Thr-308 (Fig. 5b), and had no effect on the phosphorylation of IκBα at Ser-32 (Fig. 5c). AG1478 had similar effects on the activation of ERK1/2 and PKB by TGFα, but this growth factor failed to induce IκBα phosphorylation. We conclude that 1) activation of ERK1/2 by TNF is entirely dependent on EGFR signaling; 2) activation of PKB by TNF is only partially dependent on EGFR signaling; and 3) activation of IκBα by TNF does not involve EGFR signaling and takes place solely through TNF receptors. Tumor Necrosis Factor-induced Proliferation Requires Protease Activity—Although the experiments presented so far clearly indicate that TNF induces cell proliferation through the TGFα/EGFR signaling pathway, the mechanisms responsible for this effect have not yet been examined. One possibility is that there is intracellular cross-talk between TNF receptors and EGFR. The experiments with TGFα neutralizing antibody shown in Fig. 1, however, suggest that extracellular TGFα is required for the TNF effect. We therefore investigated whether EGFR could be transactivated by TNF in a manner analogous to the cross-talk between G-protein-coupled receptors and EGFR (19Daub H. Wallasch C. Lankenau A. Herrlich A. Ullrich A. EMBO J. 1997; 16: 7032-7044Crossref PubMed Scopus (588) Google Scholar). In this case, EGFR transactivation requires the cleavage of membrane-bound EGFR ligands by a metalloproteinase, with subsequent binding of the released factor to EGFR. We hypothesized that TNF-induced proliferation of AML-12 cells requires the activity of a metalloproteinase (MP) that cleaves membrane-bound TGFα. To test this hypothesis, we first examined whether TNF caused the release of TGFα into the culture medium and whether MP inhibitors had an effect on TGFα release and DNA replication in cells exposed to TNF (Fig. 6). Exposure to TNF caused an ∼5-fold increase in the amounts of TGFα released in the culture medium (Fig. 6a). The broad specificity MP inhibitor GM6001 prevented this increase at concentrations of 10 and 50 μm, whereas an inactive form of GM6001 (negative control) had no effect (Fig. 6a). The inhibitor also blocked TNF-induced DNA replication by more than 50% (Fig. 6b) but had no effect on DNA synthesis induced by TGFα. This suggests that MPs act in TNF-mediated cell proliferation but do not participate in EGFR activation by exogenous TGFα. To determine what types of MPs may be involved in this process, we tested the effect of MP inhibitors with more defined specificity. TACE is a metalloproteinase disintegrin that cleaves proteins anchored in the cell membrane (20Massague J. Pandiella A. Annu. Rev. Biochem. 1993; 62: 515-541Crossref PubMed Scopus (600) Google Scholar, 21Black 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 (2728) Google Scholar). TNF, TGFα, and other ligands of the EGF family can serve as substrates for this enzyme (22Lee D.C. Sunnarborg S.W. Hinkle C.L. Myers T.J. Stevenson M.Y. Russell W.E. Castner B.J. Gerhart M.J. Paxton R.J. Black R.A. Chang A. Jackson L.F. Ann. N. Y. Acad. Sci. 2003; 995: 22-38Crossref PubMed Scopus (154) Google Scholar, 23Borrell-Pages M. Rojo F. Albanell J. Baselga J. Arribas J. EMBO J. 2003; 22: 1114-1124Crossref PubMed Scopus (240) Google Scholar). TAPI-1, a specific inhibitor of TACE, blocked TNF-induced DNA replication and the release of TGFα into the medium (Fig. 7, a and b). In marked contrast, inhibitors of matrix metalloproteinases 2, 3, 8, 9, and 13 had no effect on TNF-induced DNA replication (Fig. 7c).Fig. 7TACE activity is required for TNF-induced proliferation.a, serum-starved AML-12 cells were pretreated with Me2SO (Control), increasing concentrations of TAPI-1, or MMP-9/13 inhibitor for 30 min. Cells were then left untreated (Untx, hatched bars) or stimulated with 20 ng/ml TNF (filled bars). TAPI-1 concentrations were 0.1, 0.5, 1.0, 5.0, 10, and 50 μm, and MMP-9/13 inhibitor concentration was 50 nm. Medium was collected from cultured AML-12 cells 21 h after treatment, and TGFα levels were measured by enzyme-linked immunosorbent assay as described under “Experimental Procedures.” b, serum-starved AML-12 cells were pretreated with increasing concentrations of TAPI-1. Cells were then left untreated (squares) or treated with 20 ng/ml TNF (triangles) for 24 h. DNA replication was measured by [3H]thymidine incorporation as described under “Experimental Procedures.” c, AML-12 cells were pretreated with either 0.1% Me2SO (Control) or the MMP inhibitors indicated. Cells were then left untreated (hatched bars) or stimulated with 20 ng/ml TNF (filled bars) for 24 h. MMP inhibitors were used at the following concentrations: MMP-2/9, 200 nm; MMP-9/13, 50 nm; MMP-3, 5 μm; and MMP-8, 100 nm (final). DNA replication was measured by [3H]thymidine incorporation as described under “Experimental Procedures.” Results are representative of at least two independent experiments.View Large Image Figure ViewerDownload (PPT) We next determined whether TACE blockage, shown to inhibit TNF-induced DNA replication, would interfere with EGFR signaling. Cells treated with TAPI-1 or Me2SO (as control) were stimulated with TNF or TGFα. Immunoblot analysis (Fig. 8) showed that TAPI-1 completely blocked the phosphorylation of ERK1/2 induced by TNF (Fig. 8a) and partially prevented the phosphorylation of PKB by TNF (Fig. 8b). The TACE inhibitor had no effect on ERK1/2 or PKB phosphorylation induced by exogenous TGFα. These experiments suggest that TNF stimulates cell proliferation in AML-12 hepatocytes through the activation of TACE, which cleaves membrane-bound TGFα. This ligand in turn binds to EGFR to initiate a mitogenic cascade that involves ERK1/2 and PKB. TNF induces multiple responses in the liver, including cytokine activation, cell death, and cell proliferation (24Fausto N. J. Hepatol. 2000; 32: 19-31Abstract Full Text PDF PubMed Google Scholar). Although the mechanisms by which TNF causes cytokine stimulation and apoptosis have been well studied, much less is known about the mechanisms by which TNF stimulates hepatocyte replication. Activation of NFκB and signal transducer and activator 3 and increases in interleukin-6 are associated with hepatocyte proliferation in the regenerating liver in vivo and in cell cultures, but it is still unresolved whether these agents function to maintain cell survival or are direct participants in signaling events that culminate in DNA replication. We have shown that TNF acts in conjunction with growth factors to stimulate hepatocyte replication (10Webber E.M. Bruix J. Pierce R.H. Fausto N. Hepatology. 1998; 28: 1226-1234Crossref PubMed Scopus (241) Google Scholar). This conclusion is based on the following observations: 1) TNF injections cause little stimulation of hepatocyte replication in mouse liver; 2) in primary cell cultures, TNF stimulates hepatocyte DNA replication in serum-containing cultures but has little activity in cultures maintained in serum-free medium, suggesting that it is not a complete mitogen; and 3) a single injection of 10 μg of TNF enhances the proliferative activity of TGFα and hepatocyte growth factor in normal livers. To investigate the relationship between TNF and mitogenic growth factors for hepatocytes, we examined the proliferative effects of TNF on AML-12 cells, a differentiated, non-transformed hepatocyte cell line developed in this laboratory. These cells produce precursor TGFα, which is anchored in the cell membrane. We show that in these cells TNF stimulates DNA replication by causing release of TGFα into the culture medium through the metalloproteinase disintegrin TACE. TGFα then activates EGFR and multiple downstream intracellular signaling cascades that are required for DNA replication. The EGFR ligands TGFα and HB-EGF are synthesized as transmembrane precursor molecules (20Massague J. Pandiella A. Annu. Rev. Biochem. 1993; 62: 515-541Crossref PubMed Scopus (600) Google Scholar). Cleavage of these molecules by limited proteolysis releases soluble growth factors, a process known as ectodomain shedding (25Kheradmand F. Werb Z. BioEssays. 2002; 24: 8-12Crossref PubMed Scopus (111) Google Scholar, 26Moss M.L. Lambert M.H. Essays Biochem. 2002; 38: 141-153Crossref PubMed Scopus (73) Google Scholar). The soluble factors cleaved from the precursor molecules bind to and activate EGFR signaling. Examples of this type of mechanism include HB-EGF-mediated EGFR transactivation by stimulation of G-protein-coupled receptors (27Prenzel N. Zwick E. Daub H. Leserer M. Abraham R. Wallasch C. Ullrich A. Nature. 1999; 402: 884-888Crossref PubMed Scopus (1501) Google Scholar, 28Kalmes A. Daum G. Clowes A.W. Ann. N. Y. Acad. Sci. 2001; 947 (discussion 54–45): 42-54Crossref PubMed Scopus (85) Google Scholar, 29Yan Y. Shirakabe K. Werb Z. J. Cell Biol. 2002; 158: 221-226Crossref PubMed Scopus (279) Google Scholar) and TGFα-mediated EGFR transactivation in colonic and gastric mucosal cells (30McCole D.F. Keely S.J. Coffey R.J. Barrett K.E. J. Biol. Chem. 2002; 277: 42603-42612Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 31Xiao Z.Q. Majumdar A.P. Am. J. Physiol. 2001; 281: G111-G116Crossref PubMed Google Scholar). Although the mechanisms of EGFR signaling triggered by ligand binding are well described, much less is known about the source and generation of these ligands. Several lines of evidence suggest that the cell surface proteinases known as ADAM (adisintegrin and metalloproteinase), which contain a zinc-dependent catalytic domain, play an important role in ectodomain shedding of adhesion molecules and EGFR ligands (22Lee D.C. Sunnarborg S.W. Hinkle C.L. Myers T.J. Stevenson M.Y. Russell W.E. Castner B.J. Gerhart M.J. Paxton R.J. Black R.A. Chang A. Jackson L.F. Ann. N. Y. Acad. Sci. 2003; 995: 22-38Crossref PubMed Scopus (154) Google Scholar, 32Kheradmand F. Rishi K. Werb Z. J. Cell Sci. 2002; 115: 839-848PubMed Google Scholar, 33Gschwind A. Hart S. Fischer O.M. Ullrich A. EMBO J. 2003; 22: 2411-2421Crossref PubMed Scopus (287) Google Scholar, 34Garton 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 (222) Google Scholar). In particular, TACE has been implicated in TGFα-mediated EGFR activation (22Lee D.C. Sunnarborg S.W. Hinkle C.L. Myers T.J. Stevenson M.Y. Russell W.E. Castner B.J. Gerhart M.J. Paxton R.J. Black R.A. Chang A. Jackson L.F. Ann. N. Y. Acad. Sci. 2003; 995: 22-38Crossref PubMed Scopus (154) Google Scholar, 23Borrell-Pages M. Rojo F. Albanell J. Baselga J. Arribas J. EMBO J. 2003; 22: 1114-1124Crossref PubMed Scopus (240) Google Scholar). In the work described here, we show that TACE activity is involved in TNF-induced TGFα shedding and EGFR transactivation in AML-12 hepatocytes. The TACE inhibitor TAPI-1 interferes with TGFα release into the culture medium and subsequent EGFR signaling through ERK1/2 and PKB, thereby blocking DNA replication. Cleavage of membrane proteins by TACE generally occurs at very low levels in unstimulated cells but is greatly enhanced by exposure of cells to various agents such as phorbol 12-myristate 13-acetate (35Doedens J.R. Mahimkar R.M. Black R.A. Biochem. Biophys. Res. Commun. 2003; 308: 331-338Crossref PubMed Scopus (90) Google Scholar). We demonstrate that in AML-12 hepatocytes TGFα is released into the culture medium after TNF treatment and that this release is blocked by TAPI-1, suggesting that TACE is activated by TNF. We do not know how TNF activates TACE, but our data suggest that ERK1/2 may not be involved in ligand shedding as has been proposed in other systems (22Lee D.C. Sunnarborg S.W. Hinkle C.L. Myers T.J. Stevenson M.Y. Russell W.E. Castner B.J. Gerhart M.J. Paxton R.J. Black R.A. Chang A. Jackson L.F. Ann. N. Y. Acad. Sci. 2003; 995: 22-38Crossref PubMed Scopus (154) Google Scholar, 36Zheng Y. Schlondorff J. Blobel C.P. J. Biol. Chem. 2002; 277: 42463-42470Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Transactivation of EGFR by TNF has been described in the human endometrium (37Chobotova K. Muchmore M.E. Carver J. Yoo H.J. Manek S. Gullick W.J. Barlow D.H. Mardon H.J. J. Clin. Endocrinol. Metab. 2002; 87: 5769-5777Crossref PubMed Scopus (40) Google Scholar), mammary epithelial cells (38Chen W.N. Woodbury R.L. Kathmann L.E. Opresko L.K. Zangar R.C. Wiley H.S. Thrall B.D. J. Biol. Chem. 2004; 279: 18488-18496Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), and squamous carcinoma cells (39Donato N.J. Gallick G.E. Steck P.A. Rosenblum M.G. J. Biol. Chem. 1989; 264: 20474-20481Abstract Full Text PDF PubMed Google Scholar), but in squamous carcinoma cells, EGFR transactivation involves the up-regulation of EGFR, a mechanism that does not occur in hepatocytes (data not shown). At another level of regulation, it has also been shown that in the liver TNF may enhance TGFα transcription (11Gallucci R.M. Simeonova P.P. Toriumi W. Luster M.I. J. Immunol. 2000; 164: 872-878Crossref PubMed Scopus (67) Google Scholar). Our results identify an important mode of interaction between cytokines and growth factors in hepatocytes. We suggest that in the regenerating liver, TNF provides the conditions for cell survival through NFκB activation and also triggers an initial stage for cell replication that involves the rapid transactivation of EGFR through metalloproteinase-mediated ligand release. At later times during liver regeneration, sustained production of these ligands is mediated by transcriptional mechanisms (24Fausto N. J. Hepatol. 2000; 32: 19-31Abstract Full Text PDF PubMed Google Scholar). We thank Alyssa Stephenson-Famy, Jessica Foraker, and Mary Nivison for technical assistance and Guenter Daum and Andreas Kalmes for helpful discussions." @default.
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• W2020458656 title "Epidermal Growth Factor Receptor Transactivation Mediates Tumor Necrosis Factor-induced Hepatocyte Replication" @default.
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