Induction of Unresponsiveness to Tumor Necrosis Factor (TNF) after Autocrine TNF Expression Requires TNF Membrane Retention
1998; Elsevier BV; Volume: 273; Issue: 6 Linguagem: Inglês
10.1074/jbc.273.6.3271
ISSN1083-351X
AutoresEls Decoster, Bart Vanhaesebroeck, Elke Boone, Stéphane Plaisance, Kurt J. De Vos, Guy Haegeman, Johan Grooten, Walter Fiers,
Tópico(s)Glycosylation and Glycoproteins Research
ResumoTumor necrosis factor (TNF) has a specific gene-inducing activity on many cell types and exerts a cytotoxic effect on a number of tumor cell lines. However, several tumor cell types are resistant to TNF-induced effects, and some of these produce TNF. We previously demonstrated that introduction of an exogenous TNF gene in the TNF-sensitive cell line L929sA induced autocrine TNF production and unresponsiveness to the cytotoxic activity of TNF. This resistance required biologically active TNF and was correlated with complete down-modulation of the TNF receptors on the cell surface. We have now characterized this process in more detail. The role of expression of the membrane-bound TNF proform and its subsequent proteolytic processing in the induction of TNF unresponsiveness was investigated. Exchange of the TNF presequence for the signal sequence of interleukin-6 resulted in production of secreted TNF, but not in induction of TNF resistance. On the other hand, expression of non-secretable, membrane-bound TNF generated complete TNF unresponsiveness. To explore whether the requirement for anchoring reflected a specific functional role of the TNF presequence, the latter was replaced by the membrane anchor of trimeric chicken hepatic lectin. Expression of this construct induced complete TNF unresponsiveness. Hence, the role of the TNF presequence in the induction of TNF unresponsiveness only involves its function as a membrane anchor, which permits oligomerization of the TNF molecule into a biologically active homotrimer. Tumor necrosis factor (TNF) has a specific gene-inducing activity on many cell types and exerts a cytotoxic effect on a number of tumor cell lines. However, several tumor cell types are resistant to TNF-induced effects, and some of these produce TNF. We previously demonstrated that introduction of an exogenous TNF gene in the TNF-sensitive cell line L929sA induced autocrine TNF production and unresponsiveness to the cytotoxic activity of TNF. This resistance required biologically active TNF and was correlated with complete down-modulation of the TNF receptors on the cell surface. We have now characterized this process in more detail. The role of expression of the membrane-bound TNF proform and its subsequent proteolytic processing in the induction of TNF unresponsiveness was investigated. Exchange of the TNF presequence for the signal sequence of interleukin-6 resulted in production of secreted TNF, but not in induction of TNF resistance. On the other hand, expression of non-secretable, membrane-bound TNF generated complete TNF unresponsiveness. To explore whether the requirement for anchoring reflected a specific functional role of the TNF presequence, the latter was replaced by the membrane anchor of trimeric chicken hepatic lectin. Expression of this construct induced complete TNF unresponsiveness. Hence, the role of the TNF presequence in the induction of TNF unresponsiveness only involves its function as a membrane anchor, which permits oligomerization of the TNF molecule into a biologically active homotrimer. Tumor necrosis factor (TNF) 1The abbreviations used are: TNF, tumor necrosis factor; ActD, actinomycin D; CHL, chicken hepatic lectin; CHX, cycloheximide; IFN, interferon; IL, interleukin; mTNF, murine tumor necrosis factor; neo r, neomycin-resistant; NF, nuclear factor; TNF-R55, 55-kDa TNF receptor; TNF-R75, 75-kDa TNF receptor; wt, wild-type. is a pleiotropic cytokine that is primarily produced by activated macrophages and some T-lymphocyte subsets. TNF exerts a wide range of biological activities related to inflammation, mitogenesis, differentiation, immune modulation, and antitumor immunity (1Vassalli P. Annu. Rev. Immunol. 1992; 10: 411-452Crossref PubMed Scopus (1810) Google Scholar, 2Fiers W. DeVita Jr., V.T. Hellman S. Rosenberg S.A. Biologic Therapy of Cancer. 2nd Ed. J. B. Lippincott, Philadelphia1995: 295-327Google Scholar, 3Aggarwal B.B. Natarajan K. Eur. Cytokine Netw. 1996; 7: 93-124PubMed Google Scholar). These activities are induced through interaction with specific cell surface receptors expressed on almost every cell type, except unstimulated T-lymphocytes and erythrocytes. In man and mouse, two types of TNF receptor have been characterized, namely TNF-R55 and TNF-R75, with molecular masses of 55 and 75 kDa, respectively. TNF effects are mainly mediated by TNF-R55, whereas the role of TNF-R75 as a signal transducer is mostly confined to T-lymphocytes (4Vandenabeele P. Declercq W. Beyaert R. Fiers W. Trends Cell Biol. 1995; 5: 392-399Abstract Full Text PDF PubMed Scopus (742) Google Scholar). TNF is synthesized as a 26-kDa, type II transmembrane proform (5Kriegler M. Perez C. DeFay K. Albert I. Lu S.D. Cell. 1988; 53: 45-53Abstract Full Text PDF PubMed Scopus (935) Google Scholar), which is biologically active (6Perez C. Albert I. DeFay K. Zachariades N. Gooding L. Kriegler M. Cell. 1990; 63: 251-258Abstract Full Text PDF PubMed Scopus (371) Google Scholar). This precursor is processed by a metalloprotease (7Gearing A.J.H. Beckett P. Christodoulou M. Churchill M. Clements J. Davidson A.H. Drummond A.H. Galloway W.A. Gilbert R. Gordon J.L. Leber T.M. Mangan M. Miller K. Nayee P. Owen K. Patel S. Thomas W. Wells G. Wood L.M. Woolley K. Nature. 1994; 370: 555-557Crossref PubMed Scopus (1111) Google Scholar, 8McGeehan G.M. Becherer J.D. Bast Jr., R.C. Boyer C.M. Champion B. Connolly K.M. Conway J.G. Furdon P. Karp S. Kidao S. McElroy A.B. Nichols J. Pryzwansky K.M. Schoenen F. Sekut L. Truesdale A. Verghese M. Warner J. Ways J.P. Nature. 1994; 370: 558-561Crossref PubMed Scopus (545) Google Scholar, 9Mohler K.M. Sleath P.R. Fitzner J.N. Cerretti D.P. Alderson M. Kerwar S.S. Torrance D.S. Otten-Evans C. Greenstreet T. Weerawarna K. Kronheim S.R. Petersen M. Gerhart M. Kozlosky C.J. March C.J. Black R.A. Nature. 1994; 370: 218-220Crossref PubMed Scopus (573) Google Scholar), which results in release of mature, trimeric TNF consisting of 17-kDa subunits. Membrane-bound TNF mediates TNF effects at the local, paracrine level via cell-cell contact (10Grell M. Douni E. Wajant H. Löhden M. Clauss M. Maxeiner B. Georgopoulos S. Lesslauer W. Kollias G. Pfizenmaier K. Scheurich P. Cell. 1995; 83: 793-802Abstract Full Text PDF PubMed Scopus (1170) Google Scholar), whereas diffusible TNF acts at longer distances, generating systemic responses to this cytokine. Both TNF forms induce killing of TNF-sensitive target cells (6Perez C. Albert I. DeFay K. Zachariades N. Gooding L. Kriegler M. Cell. 1990; 63: 251-258Abstract Full Text PDF PubMed Scopus (371) Google Scholar, 11Decoster E. Vanhaesebroeck B. Vandenabeele P. Grooten J. Fiers W. J. Biol. Chem. 1995; 270: 18473-18478Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). TNF-producing cells are completely resistant to TNF-induced cytotoxicity (12Rubin B.Y. Anderson S.I. Sullivan S.A. Williamson B.D. Carswell E.A. Old L.J. J. Exp. Med. 1986; 164: 1350-1355Crossref PubMed Scopus (92) Google Scholar, 13Spriggs D. Imamura K. Rodriguez C. Horiguchi J. Kufe D.W. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 6563-6566Crossref PubMed Scopus (123) Google Scholar, 14Himeno T. Watanabe N. Yamauchi N. Maeda M. Tsuji Y. Okamoto T. Neda H. Niitsu Y. Cancer Res. 1990; 50: 4941-4945PubMed Google Scholar, 15Okamoto T. Watanabe N. Yamauchi N. Tsuji Y. Tsuji N. Itoh Y. Neda H. Niitsu Y. Cancer Res. 1992; 52: 5278-5281PubMed Google Scholar). Remarkably, transfection with an exogenous TNF gene under a constitutive promoter converts even very sensitive cell lines, like L929, to TNF production and to complete resistance to TNF-induced cytotoxicity; this resistance correlates with and can be explained by the absence of TNF receptors on these cells (16Vanhaesebroeck B. Decoster E. Van Ostade X. Van Bladel S. Lenaerts A. Van Roy F. Fiers W. J. Immunol. 1992; 148: 2785-2794PubMed Google Scholar). We have now further analyzed this system of complete unresponsiveness. Expression of a TNF gene leads to disappearance from the plasma membrane of TNF-R55 and TNF-R75, despite unaltered levels of corresponding mRNAs. The TNF presequence fulfills a crucial role in this process by its function as a membrane anchor. The fibrosarcoma cell lines L929sA (16Vanhaesebroeck B. Decoster E. Van Ostade X. Van Bladel S. Lenaerts A. Van Roy F. Fiers W. J. Immunol. 1992; 148: 2785-2794PubMed Google Scholar), L929r2 (17Vanhaesebroeck B. Van Bladel S. Lenaerts A. Suffys P. Beyaert R. Lucas R. Van Roy F. Fiers W. Cancer Res. 1991; 51: 2469-2477PubMed Google Scholar), and WEHI 164 cl 13 (18Espevik T. Nissen-Meyer J. J. Immunol. Methods. 1986; 95: 99-105Crossref PubMed Scopus (1279) Google Scholar) were cultured as described previously. These cell lines were repeatedly screened forMycoplasma by a DNA-fluorochrome assay and were found to be negative. Purified Escherichia coli-derived murine (m) TNF was produced in our laboratory and had a specific biological activity of 2 × 108 IU/mg (international standard according to the National Institute for Biological Standards and Control, Potters Bar, UK). Recombinant murine interferon (IFN)-β was also produced in our laboratory and had a specific activity of 3 × 108 units/ml, as determined on murine cells in an L929/vesicular stomatitis virus assay. Polyclonal rabbit antiserum against mTNF was provided by J. Van der Heyden (Roche Research, Ghent, Belgium). Polyclonal rabbit antiserum directed against amino acid sequence 99–115 (tip region of mTNF; Fig. 2) (19Lucas R. Magez S. De Leys R. Fransen L. Scheerlinck J.-P. Rampelberg M. Sablon E. De Baetselier P. Science. 1994; 263: 814-817Crossref PubMed Scopus (173) Google Scholar) was a generous gift of Dr. R. Lucas (University Medical Center, Geneva, Switzerland). Polyclonal rabbit antiserum against mTNF-R55 and mTNF-R75 was a kind gift of Dr. W. Buurman (State University of Limburg, Maastricht, The Netherlands). Site-directed mutagenesis was carried out with the pMa phasmid, which contains a chloramphenicol-sensitive gene (20Stanssens P. Opsomer C. McKeown Y.M. Kramer W. Zabeau M. Fritz H.-J. Nucleic Acids Res. 1989; 17: 4441-4453Crossref PubMed Scopus (259) Google Scholar). Using two oligonucleotides containing the mutations of interest, a plasmid conferring chloramphenicol resistance was obtained. Mutagenesis was performed with a kit from CLONTECH. All mutations were verified by DNA sequencing. The interleukin (IL)-6.TNF fusion product containing the signal sequence of murine IL-6 (a gift from Dr. J. Van Snick, Ludwig Institute, Brussels, Belgium) and mature mTNF was constructed as follows. First, a unique StuI restriction site was introduced by site-directed mutagenesis in the signal sequence of murine IL-6 (amino acids −24 to −1) at positions −10/−9/−8. The mutated IL-6 gene was then inserted as a bluntedEcoRI-SalI fragment into pSV23s, a eukaryotic expression vector under control of the SV40 early promoter (21Huylebroeck D. Maertens G. Verhoeyen M. Lopez C. Raeymaekers A. Min Jou W. Fiers W. Gene (Amst.). 1988; 66: 163-181Crossref PubMed Scopus (35) Google Scholar), which was cleaved with StuI-SalI. A uniqueBglII restriction site was introduced by site-directed mutagenesis at amino acid positions 2 and 3 of mature mTNF; this mutated TNF gene was inserted as a SalI fragment intoSalI-cleaved pSV23s. The pSV23s vector containing the IL-6.TNF chimeric gene was constructed as follows. TheBglII-SalI fragment (containing mature mTNF) was ligated to the StuI-SalI fragment of pSV23smIL6 (containing only the signal sequence of IL-6); in-phase fusion of the two gene parts was achieved by insertion of a synthetic linker coding for amino acids −7 to −1 of the IL-6 signal sequence and the first two amino acids of mature mTNF. The chicken hepatic lectin (CHL).TNF fusion product containing the membrane anchor structure of CHL (a gift from Dr. K. Drickamer, Columbia University, New York, NY) and mature mTNF was constructed as follows. A unique BclI restriction site was introduced by site-directed mutagenesis in pMaCHL at the last amino acid of the neck region of CHL. Then, the CHL membrane anchor was cloned as anEcoRI-BclI fragment into theEcoRI-BglII-cleaved pMacmTNFmBglII vector fragment and fused in-phase with a synthetic linker (containing the last amino acid of the neck region of CHL, the last four amino acids of the TNF presequence, and the first amino acid of mature mTNF) to mature mTNF. The CHL.TNF gene was inserted as a SalI fragment in the SalI site of pSV23s (21Huylebroeck D. Maertens G. Verhoeyen M. Lopez C. Raeymaekers A. Min Jou W. Fiers W. Gene (Amst.). 1988; 66: 163-181Crossref PubMed Scopus (35) Google Scholar). The construction of pMx-mTNF was achieved by insertion of the mTNF gene as a SalI fragment in a SalI-cleaved pSP64MxpA vector containing the Mx promoter inducible by IFN type I, either IFN-α or IFN-β (22Lleonart R. Näf D. Browning H. Weissmann C. Bio/Technology. 1990; 8: 1263-1267Crossref PubMed Scopus (39) Google Scholar). The pSV2neo plasmid encoding the neomycin-resistant (neo r) gene under control of the SV40 early promoter was used as a selection marker in L929 transfections (23Southern P.J. Berg P. J. Mol. Appl. Genet. 1982; 1: 327-341PubMed Google Scholar). The plasmid used for transfection was purified with columns from Qiagen (Chatsworth, CA). Stable transfection was achieved by an improved DNA-calcium phosphate co-precipitation method as described previously (16Vanhaesebroeck B. Decoster E. Van Ostade X. Van Bladel S. Lenaerts A. Van Roy F. Fiers W. J. Immunol. 1992; 148: 2785-2794PubMed Google Scholar). Supernatants of transfected cell lines were concentrated 100-fold by centrifugation in Centriprep-10 and Centricon-10 micro-separation devices (Amicon, Danvers, MA). TNF was quantified in an 18-h cytotoxicity assay using WEHI 164 cl 13 cells in the presence of 1 μg of actinomycin D (ActD)/ml (18Espevik T. Nissen-Meyer J. J. Immunol. Methods. 1986; 95: 99-105Crossref PubMed Scopus (1279) Google Scholar). The detection limit of this assay was about 1 pg/ml. These procedures were performed as described (11Decoster E. Vanhaesebroeck B. Vandenabeele P. Grooten J. Fiers W. J. Biol. Chem. 1995; 270: 18473-18478Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 17Vanhaesebroeck B. Van Bladel S. Lenaerts A. Suffys P. Beyaert R. Lucas R. Van Roy F. Fiers W. Cancer Res. 1991; 51: 2469-2477PubMed Google Scholar). Membrane-bound TNF was analyzed using polyclonal rabbit antiserum directed against the tip region as described previously (11Decoster E. Vanhaesebroeck B. Vandenabeele P. Grooten J. Fiers W. J. Biol. Chem. 1995; 270: 18473-18478Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). The presence of TNF-R55 and TNF-R75 was determined by staining for 1 h at 4 °C with polyclonal rabbit antiserum against TNF-R55 or TNF-R75 (1 μg of antiserum/4 × 105 cells in 200 μl), followed by incubation for 1 h at 4 °C with biotinylated donkey anti-rabbit polyclonal antiserum (Amersham Life Science, Amersham, UK), and by incubation for 1 h at 4 °C with phycoerythrin-conjugated streptavidin. Analyses were performed by flow fluorocytometry using a Coulter Epics 753 fluorocytometer equipped with an argon-ion laser (Coulter, Hialeah, FL). TNF sensitivity was determined as described previously (17Vanhaesebroeck B. Van Bladel S. Lenaerts A. Suffys P. Beyaert R. Lucas R. Van Roy F. Fiers W. Cancer Res. 1991; 51: 2469-2477PubMed Google Scholar). The presence of IL-6 in unconcentrated supernatants was determined by its capacity to induce proliferation of 7TD1 cells (24Van Snick J. Cayphas S. Vink A. Uyttenhove C. Coulie P.G. Rubira M.R. Simpson R.J. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 9679-9683Crossref PubMed Scopus (677) Google Scholar). Activation of NF-κB was measured by an electrophoretic mobility shift assay. Nuclear extracts and binding reactions were carried out as described previously (25Patestos N.P. Haegeman G. Vandevoorde V. Fiers W. Biochimie. 1993; 75: 1007-1018Crossref PubMed Scopus (20) Google Scholar). The double-stranded oligonucleotide containing the NF-κB site from the IL-6 promoter was 32P-labeled; after purification, 50,000 cpm was used for the binding assay. Poly(A)+ mRNA was isolated using a FastTrack mRNA Isolation Kit (Invitrogen, San Diego, CA). 5 μg of poly(A)+ mRNA was separated by electrophoresis through a 1.4% agarose-formaldehyde gel and transferred to a Hybond-N+ membrane (Amersham Life Science). The RNA was UV-fixed, and hybridization of the membrane was achieved at 42 °C in the presence of formamide. A 656-base pairBstEII-BamHI fragment of pBLUmTNFR55 and a 1300-base pair BamHI-BglII fragment of pUCmTNFR75 were used as probes. As control for the quantity of RNA loaded, a probe for glyceraldehyde-3-phosphate dehydrogenase was used. Probes were32P-labeled with a Random Primed labeling kit (Boehringer, Mannheim, Germany). Transfection of the TNF-sensitive L929 cell line with an exogenous TNF gene induces TNF production and TNF unresponsiveness (16Vanhaesebroeck B. Decoster E. Van Ostade X. Van Bladel S. Lenaerts A. Van Roy F. Fiers W. J. Immunol. 1992; 148: 2785-2794PubMed Google Scholar). To determine whether proteolytic processing of the membrane-bound TNF proform is necessary for the induction of this resistance, we assayed two membrane-bound, non-secretable TNF mutants, which were described previously (11Decoster E. Vanhaesebroeck B. Vandenabeele P. Grooten J. Fiers W. J. Biol. Chem. 1995; 270: 18473-18478Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). TNFΔ1–9K11E, in which, in addition to deletion of the first nine amino acids of mature TNF, Lys at position 11 was replaced by Glu, is a biologically active mutein. The TNF mutant TNFΔ1–12, with deletion of the first 12 amino acids of mature TNF, is considerably less biologically active (11Decoster E. Vanhaesebroeck B. Vandenabeele P. Grooten J. Fiers W. J. Biol. Chem. 1995; 270: 18473-18478Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). As shown in Table I, L929sA cells transfected with TNFΔ1–12 were partially TNF-resistant, whereas TNFΔ1–9K11E-producing L929sA transfectants became fully resistant to TNF cytotoxicity. This resistance is similar to that observed in L929sA cells transfected with wild-type (wt) TNF, which produce both membrane-bound and secreted TNF. L929sA cells transfected only with aneo r selection marker retained their sensitivity to the cytotoxic activity of TNF. Since TNF also mediates a gene-inducing activity in L929 cells, the latter activity was analyzed by assaying IL-6 production and NF-κB activation. TNF stimulation could not induce IL-6 (Table I) or activate NF-κB (Fig. 1) in wtTNF-producing or TNFΔ1–9K11E-producing L929sA cells, whereas addition of exogenous TNF could still induce IL-6 production in TNFΔ1–12 transfectants (Table I). Given these results, we conclude that TNF does not need to be processed into a soluble, secreted form to induce unresponsiveness to TNF-mediated effects.Table IResponsiveness to TNF of L929sA cells transfected with wtTNF, TNFΔ1–12, or TNFΔ1–9K11ETransfected geneColonyTNF production1-aAssessed in unconcentrated culture supernatant from the cytotoxic activity on the highly TNF-sensitive cell line WEHI 164 cl 13. Membrane-bound TNF was detected by flow cytometry; ND, not detectable.TNF sensitivity1-bUnits required to obtain 50% cell death within 18 h in the presence of ActD; > means that 50% cell death was not observed with the indicated TNF concentration.IL-6 production1-cAssessed in the supernatant of L929sA transfectants after 24 h of culture either untreated or treated with 40,000 IU/ml TNF.SupernatantMembraneNone+ TNFIU/mlpg/mlneo rB1<0.1ND2<106000B2<0.1ND2 40,000<10 40,000<10<10neo r + TNFΔ1–12F3<0.1+4444<101226F6<0.1+187<102220neo r + TNFΔ1–9K11EG1 40,000<10<10G7 40,000<10 means that 50% cell death was not observed with the indicated TNF concentration.1-c Assessed in the supernatant of L929sA transfectants after 24 h of culture either untreated or treated with 40,000 IU/ml TNF. Open table in a new tab To express TNF exclusively as a secreted protein, we exchanged the TNF presequence for the classical signal sequence of IL-6 (Fig. 2). Cotransfection of this chimeric IL-6.TNF gene with the neo r gene in L929sA cells and G418 selection yielded only marginal numbers of G418-resistant colonies, none of which produced TNF (data not shown). This failure to isolate IL-6.TNF transfectants suggested that the transfectants were counter-selected, which means that expression of the IL-6.TNF gene resulted in cell death. To verify this hypothesis, the IL-6.TNF gene was transfected in L929r2, a TNF-resistant and non-TNF-producing cell line derived from TNF-sensitive L929 s cells (17Vanhaesebroeck B. Van Bladel S. Lenaerts A. Suffys P. Beyaert R. Lucas R. Van Roy F. Fiers W. Cancer Res. 1991; 51: 2469-2477PubMed Google Scholar). After transfection with the IL-6.TNF gene and G418 selection, a normal number of G418-resistant L929r2 colonies were obtained. These transfectants released between 50 and 350 IU/ml TNF in the culture medium as detected by a cytotoxicity assay. This is higher than observed with L929r2 cells transfected with the wtTNF gene, which constitutively secrete between 1 and 40 IU/ml TNF. Unlike wtTNF-transfected L929r2 cells, no membrane-bound TNF form could be detected in the IL-6.TNF transfectants by flow fluorocytometric analysis (Fig. 3). These results demonstrate that expression of the IL-6.TNF chimeric gene gives rise to biologically active TNF, which is produced exclusively as a secretory protein. Furthermore, since IL-6.TNF-producing transfectants were isolated only after transfection of the TNF-resistant L929r2 variant, and not with the TNF-sensitive L929sA cell line, the TNF-mediated counter-selection was presumably the direct cause of the negligible transfection efficiency obtained with L929sA cells. Although L929r2 cells are resistant to the cytotoxic activity of TNF, the cells become sensitive in the presence of ActD or cycloheximide (CHX), inhibitors of RNA or protein synthesis, respectively (17Vanhaesebroeck B. Van Bladel S. Lenaerts A. Suffys P. Beyaert R. Lucas R. Van Roy F. Fiers W. Cancer Res. 1991; 51: 2469-2477PubMed Google Scholar). This feature allowed us to study whether the secreted IL-6.TNF chimera and wtTNF expressed in L929r2 differ in the induction of resistance to the cytotoxic combination of TNF and ActD, CHX, or staurosporine. The latter is a nonspecific protein kinase C inhibitor, which also sensitizes L929r2 cells to TNF effects (26Beyaert R. Vanhaesebroeck B. Heyninck K. Boone E. De Valck D. Schulze-Osthoff K. Haegeman G. Van Roy F. Fiers W. Cancer Res. 1993; 53: 2623-2630PubMed Google Scholar). As shown in Table II, IL-6.TNF transfectants exhibited extensive cell death after treatment with ActD, CHX, or staurosporine alone. Apparently, these cells were killed as a result of the combined activity of autocrine-produced TNF and sensitizing agents. In contrast to this, wtTNF transfectants, producing both membrane-bound and secreted TNF, exhibited full resistance to the sensitizing drugs alone or supplemented with exogenous TNF (Table II). A similar differential responsiveness was observed by following the induction of IL-6. In agreement with the observation that L929r2 cells still respond to TNF by producing IL-6, introduction of the constitutively expressed IL-6.TNF chimeric gene induced high levels of IL-6 production in the transfectants (an autocrine stimulation), whereas introduction of the wtTNF gene eliminated the inducible IL-6 response observed in the control transfectants (Table II). Clearly, omission of the intermediary step of the membrane-bound proform in the course of TNF biosynthesis abolished the capacity of endogenously produced TNF to generate unresponsiveness.Table IIResponsiveness to TNF of L929r2 cells transfected with wtTNF or chimeric IL-6.TNFTransfected geneColonySurvival2-aCells were treated with ActD (1 μg/ml), CHX (25 μg/ml), or staurosporine (STS, 1.5 μm) in the absence of exogenous TNF. Cell survival in control cultures was 100% for all colonies.TNF sensitivity2-bUnits required to obtain 50% cell death in the presence of the indicated inhibitor; > means that 50% cell death was not observed with the TNF concentration mentioned; NR, not relevant.IL-6 production2-cAssessed in the supernatant of L929sA transfectants after 24 h of culture either untreated or treated with 40,000 IU/ml TNF.ActDCHXSTSActDCHXSTSNone+ TNF%IU/mlpg/mlneo rB17575716211002000B2808067.5220.62084000neo r + wtTNFC3807061>40,00020,000>40,00095120C9708075>40,00040,000>40,000150200neo r + IL-6.TNFE2104.54.5NRNRNR53007000E3153.55.5NRNRNR700080002-a Cells were treated with ActD (1 μg/ml), CHX (25 μg/ml), or staurosporine (STS, 1.5 μm) in the absence of exogenous TNF. Cell survival in control cultures was 100% for all colonies.2-b Units required to obtain 50% cell death in the presence of the indicated inhibitor; > means that 50% cell death was not observed with the TNF concentration mentioned; NR, not relevant.2-c Assessed in the supernatant of L929sA transfectants after 24 h of culture either untreated or treated with 40,000 IU/ml TNF. Open table in a new tab The requirement for membrane retention in the induction of unresponsiveness to TNF implies an involvement of the TNF presequence either structurally, as a membrane anchor, or functionally, involving (signal-transducing) amino acid sequence information. To answer this question, we exchanged the TNF presequence for the membrane anchor of CHL (27Mellow T.E. Halberg D. Drickamer K. J. Biol. Chem. 1988; 263: 5468-5473Abstract Full Text PDF PubMed Google Scholar). CHL is a trimeric, type II transmembrane liver glycoprotein receptor (28Chiacchia K.B. Drickamer K. J. Biol. Chem. 1984; 259: 15440-15446Abstract Full Text PDF PubMed Google Scholar, 29Steer C.J. Osborne Jr., J.C. Kempner E.S. J. Biol. Chem. 1990; 265: 3744-3749Abstract Full Text PDF PubMed Google Scholar), which contains an anchor allowing trimerization of the extracellular domains. Transfection of L929sA cells with the CHL.TNF chimeric construct (Fig. 2) yielded a normal number of colonies, all of which secreted high amounts of biologically active TNF, varying within the range of 30–300 IU/ml (Table III). These L929sA transfectants were completely unresponsive to the cytotoxic and gene-inducing activity of TNF, which means that they behaved in the same way as L929sA cells transfected with the wtTNF gene (Table III).Table IIICharacterization of L929sA cells transfected with wtTNF or CHL.TNFTransfected geneColonyTNF production3-aAssessed in unconcentrated culture supernatant.TNF sensitivity3-bUnits required to obtain 50% cell death within 18 h in the presence of ActD; > means that 50% cell death was not observed with the indicated TNF concentration.IL-6 production3-cAssessed in the supernatant of L929sA transfectants after 24 h of culture either untreated or treated with 40,000 IU/ml TNF.None+ TNFIU/mlIU/mlpg/mlneo rB1<0.12<106000B2<0.12 40,000<10 40,000<10 40,000<10 40,000<10 means that 50% cell death was not observed with the indicated TNF concentration.3-c Assessed in the supernatant of L929sA transfectants after 24 h of culture either untreated or treated with 40,000 IU/ml TNF. Open table in a new tab Immunoprecipitation and immunoblotting of cell lysates revealed a 27.8-kDa protein band corresponding to the expected CHL.TNF proform and several higher M r forms (Fig. 4 A), which presumably correspond to heterogeneity in glycosylation. The presence of membrane-bound fusion protein was also confirmed by flow fluorocytometric analysis, where the levels of immunofluorescence remained unchanged after acidic treatment, which removes receptor-bound, soluble TNF (Fig. 5). In the culture supernatant, mature TNF as well as several highM r forms were detected (Fig. 4 B). The latter bands were due to N-glycosylation, as they disappeared after glycopeptidase F treatment (data not shown).Figure 5Flow-cytometric analysis of expression of membrane-bound TNF by L929sA transfectants. Cells were transfected with neo r alone (A), or co-transfected with wtTNF (B) or CHL.TNF (C). Cells were treated with secondary antibody alone (dashed curve) or with antiserum to the tip region of TNF (amino acids 99–115) and secondary antibody (solid curve).View Large Image Figure ViewerDownload (PPT) TNF exerts its cytotoxic and gene-inducing activity through binding and subsequent clustering of its receptors TNF-R55 and/or TNF-R75. We previously described the down-modulation of TNF receptor molecules on the cell surface after autocrine production of TNF (16Vanhaesebroeck B. Decoster E. Van Ostade X. Van Bladel S. Lenaerts A. Van Roy F. Fiers W. J. Immunol. 1992; 148: 2785-2794PubMed Google Scholar). Therefore, the expression level of TNF receptors on the plasma membrane of L929sA transfectants expressing wtTNF, membrane-bound TNFΔ1–9K11E, TNFΔ1–12, or CHL.TNF fusion gene was determined. As shown in Fig. 6, using 125I-TNF, no specific TNF binding could be demonstrated on L929sA cells expressing wtTNF, TNFΔ1–9K11E, or C
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