Mannose 6-Phosphorylated Proteins Are Required for Tumor Necrosis Factor-induced Apoptosis
2004; Elsevier BV; Volume: 279; Issue: 51 Linguagem: Inglês
10.1074/jbc.m408261200
ISSN1083-351X
AutoresClaudine Tardy, Hélène Autefage, Virginie Garcia, Thierry Levade, Nathalie Andrieu‐Abadie,
Tópico(s)Immune Response and Inflammation
ResumoWhereas caspases are essential components in apoptosis, other proteases seem to be involved in programmed cell death. This study investigated the role of lysosomal mannose 6-phosphorylated proteins in tumor necrosis factor (TNF)-induced apoptosis. We report that fibroblasts isolated from patients affected with inclusion-cell disease (ICD), having a deficient activity of almost all lysosomal hydrolases, are resistant to the toxic effect of TNF. These mutant cells exhibited a defect in TNF-induced caspase activation, Bid cleavage, and release of cytochrome c. In contrast, TNF-induced p42/p44 MAPK activation and CD54 expression remained unaltered. Human ICD lymphoblasts and fibroblasts derived from mice nullizygous for Igf2 and the two mannose 6-phosphate (M6P) receptors, Mpr300 and Mpr46, which develop an ICD-like phenotype, were also resistant to CD95 ligand and TNF, respectively. Moreover, correction of the lysosomal enzyme defect of ICD fibroblasts, using a medium enriched in M6P-containing proteins, enabled restoration of sensitivity to TNF. This effect was blocked by exogenous M6P but not by cathepsin B or L inhibitors. Altogether, these findings suggest that some M6P-bearing glycoproteins modulate the susceptibility to TNF-induced apoptosis. As a matter of fact, exogenous tripeptidyl peptidase 1, a lysosomal carboxypeptidase, could sensitize ICD fibroblasts to TNF. These observations highlight the hitherto unrecognized role of some mannose 6-phosphorylated proteins such as tripeptidyl peptidase 1 in the apoptotic cascade triggered by TNF. Whereas caspases are essential components in apoptosis, other proteases seem to be involved in programmed cell death. This study investigated the role of lysosomal mannose 6-phosphorylated proteins in tumor necrosis factor (TNF)-induced apoptosis. We report that fibroblasts isolated from patients affected with inclusion-cell disease (ICD), having a deficient activity of almost all lysosomal hydrolases, are resistant to the toxic effect of TNF. These mutant cells exhibited a defect in TNF-induced caspase activation, Bid cleavage, and release of cytochrome c. In contrast, TNF-induced p42/p44 MAPK activation and CD54 expression remained unaltered. Human ICD lymphoblasts and fibroblasts derived from mice nullizygous for Igf2 and the two mannose 6-phosphate (M6P) receptors, Mpr300 and Mpr46, which develop an ICD-like phenotype, were also resistant to CD95 ligand and TNF, respectively. Moreover, correction of the lysosomal enzyme defect of ICD fibroblasts, using a medium enriched in M6P-containing proteins, enabled restoration of sensitivity to TNF. This effect was blocked by exogenous M6P but not by cathepsin B or L inhibitors. Altogether, these findings suggest that some M6P-bearing glycoproteins modulate the susceptibility to TNF-induced apoptosis. As a matter of fact, exogenous tripeptidyl peptidase 1, a lysosomal carboxypeptidase, could sensitize ICD fibroblasts to TNF. These observations highlight the hitherto unrecognized role of some mannose 6-phosphorylated proteins such as tripeptidyl peptidase 1 in the apoptotic cascade triggered by TNF. Lysosomes are cytoplasmic organelles that form part of a complex intracellular recycling system involved in the degradation of macromolecules. Interest in lysosomes and lysosomal enzymes is justified by the fact that over 50 lysosomal storage diseases have been described in humans, resulting from the lack or deficient activity of lysosomal hydrolases, transporters, or integral membrane proteins (1Neufeld E.F. Annu. Rev. Biochem. 1991; 60: 257-280Crossref PubMed Scopus (485) Google Scholar, 2Winchester B. Vellodi A. Young E. Biochem. Soc. 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A series of studies failed to show a requirement for cathepsins or other lysosomal proteases in the cell death machinery but rather suggested a role of these enzymes in cell growth and tissue homeostasis. Indeed, homozygous cathepsin D-deficient mice exhibit increased apoptosis in the thymus (32Saftig P. Hetman M. Schmahl W. Weber K. Heine L. Mossmann H. Koster A. Hess B. Evers M. von Figura K. Peters C. EMBO J. 1995; 14: 3599-3608Crossref PubMed Scopus (368) Google Scholar), thalamus (33Nakanishi H. Zhang J. Koike M. Nishioku T. Okamoto Y. Kominami E. von Figura K. Peters C. Yamamoto K. Saftig P. Uchiyama Y. J. Neurosci. 2001; 21: 7526-7533Crossref PubMed Google Scholar), and retina (34Koike M. Shibata M. Ohsawa Y. Nakanishi H. Koga T. Kametaka S. Waguri S. Momoi T. Kominami E. Peters C. Figura K. Saftig P. Uchiyama Y. Mol. Cell. Neurosci. 2003; 22: 146-161Crossref PubMed Scopus (126) Google Scholar). 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This disorder, also called inclusion-cell disease (ICD), is characterized by the deficient activity of UDP-N-acetylglucosamine: glycoprotein N-acetylglucosamine-1-phosphotransferase, resulting in the synthesis of lysosomal enzymes that lack mannose 6-phosphate (M6P) residues (40Hasilik A. Waheed A. von Figura K. Biochem. Biophys. Res. Commun. 1981; 98: 761-767Crossref PubMed Scopus (189) Google Scholar, 41Reitman M.L. Varki A. Kornfeld S. J. Clin. Invest. 1981; 67: 1574-1579Crossref PubMed Scopus (215) Google Scholar). These lysosomal enzymes fail to bind to the M6P receptors (MPRs) in the trans-Golgi network and are therefore not targeted to the lysosomal compartment but rather quantitatively secreted (42Wiesmann U. Vassella F. Herschkowitz N. N. Engl. J. Med. 1971; 285: 1090-1091Crossref PubMed Scopus (80) Google Scholar). 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The present study evaluated the importance of lysosomal proteases in TNF-induced apoptosis of human or murine ICD cells. We show that TNF-induced cell death was strongly inhibited in ICD fibroblasts as compared with control cells. This phenomenon is likely due to functional inefficiency of some lysosomal hydrolases resulting from their nonlysosomal compartmentalization. Indeed, the apoptosis defect could be partially corrected when the activity of lysosomal hydrolases was restored in ICD fibroblasts, after binding to the MPRs, indicating the importance of these enzymes in cell death. The participation of tripeptidyl peptidase 1, a lysosomal serine-carboxypeptidase, in TNF-induced apoptosis is demonstrated. Reagents—Human recombinant TNF was purchased from Peprotech-Tebu (Le-Perray-en-Yvelines, France). Anti-CD95 (clone CH-11) was from Beckman-Coulter (Marseille, France), and CD95 ligand was recovered in the culture medium of transfected Neuro2a cells, which overexpress a murine CD95 ligand (47Shimizu M. Fontana A. Takeda Y. Yagita H. Yoshimoto T. Matsuzawa A. J. Immunol. 1999; 162: 7350-7357PubMed Google Scholar). Ac-Asp-Glu-Val-Asp-aminomethylcoumarin (Ac-DEVD-AMC) was from Bachem (Voisins-Le-Breton-neux, France). RPMI 1640 Glutamax, DMEM, trypsin-EDTA, fetal calf serum (FCS), penicillin, and streptomycin were from Invitrogen. Other reagents, including 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and d-mannose 6-phosphate, were supplied from Sigma (Lisle d'Abeau, France). Cell Culture—Human SV40-transformed skin fibroblasts were derived from normal individuals or from patients affected with ICD (48Chatelut M. Harzer K. Christomanou H. Feunteun J. Pieraggi M.T. Paton B.C. Kishimoto Y. O'Brien J.S. Basile J.P. Thiers J.C. Salvayre R. Levade T. Clin. Chim. Acta. 1997; 262: 61-76Crossref PubMed Scopus (29) Google Scholar). Murine fibroblasts derived from MPR46-deficient, MPR300-deficient (49Pohlmann R. Boeker M.W. von Figura K. J. Biol. Chem. 1995; 270: 27311-27318Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar), or triple-deficient mice (null alleles for Igf2, Mpr300, and Mpr46) (45Dittmer F. Hafner A. Ulbrich E.J. Moritz J.D. Schmidt P. Schmahl W. Pohlmann R. von Figura K. Transgenic Res. 1998; 7: 473-483Crossref PubMed Scopus (26) Google Scholar) were kindly provided by Dr. K. von Figura (Göttingen, Germany). These cells were grown in a humidified 5% CO2 atmosphere at 37 °C in DMEM containing Glutamax (2 mm), penicillin (100 units/ml), streptomycin (100 μg/ml), and heat-inactivated FCS (10%) (48Chatelut M. Harzer K. Christomanou H. Feunteun J. Pieraggi M.T. Paton B.C. Kishimoto Y. O'Brien J.S. Basile J.P. Thiers J.C. Salvayre R. Levade T. Clin. Chim. Acta. 1997; 262: 61-76Crossref PubMed Scopus (29) Google Scholar). Human Epstein-Barr virus-transformed lymphoid cell lines were derived from control subjects or from patients affected with ICD and were grown in RPMI 1640 medium containing 10% FCS. CHO cells engineered to overexpress and secrete TPP1 (CHO-TPP1) (50Lin L. Lobel P. Biochem. J. 2001; 357: 49-55Crossref PubMed Scopus (75) Google Scholar) were kindly provided by Dr. P. Lobel (Piscataway, NJ) and cultured in DMEM. Cytotoxicity Assay—Cell viability was evaluated by using the tetrazolium-based MTT assay (51Denizot F. Lang R. J. Immunol. Methods. 1986; 89: 271-277Crossref PubMed Scopus (4357) Google Scholar). Fluorogenic DEVD Cleavage Enzyme Assay—After incubation with TNF and cycloheximide, cells were sedimented and washed with phosphate-buffered saline. Cell pellets were homogenized in 10 mm HEPES (pH 7.4), 42 mm KCl, 5 mm MgCl2, 0.5% CHAPS, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, and 2 μg/ml leupeptin. Reaction mixtures contained 100 μl of cell lysates and 100 μlof40 μm Ac-DEVD-AMC. After 30-min incubation at room temperature, the amount of the released fluorescent product aminomethylcoumarin (AMC) was determined at 351 and 430 nm for the excitation and emission wavelengths, respectively. Release of Cytochrome c—After incubation with TNF and cycloheximide, cells were sedimented and washed with phosphate-buffered saline. Cell pellets were resuspended in 5 volumes of ice-cold homogenization buffer (20 mm HEPES/KOH (pH 7.4), 1 mm EDTA, 0.1% fatty acid-free bovine serum albumin, 250 mm sucrose, 1 mm dithiothreitol, 0.1 mm phenylmethylsulfonyl fluoride, 20 μg/ml leupeptin, 10 μg/ml aprotinin and 10 μg/ml pepstatin A). After swelling for 10 min on ice, cells were homogenized by 15 strokes of a loose-fitting Dounce homogenizer. The suspension was then centrifuged at 750 × g for 5 min at 4 °C, and post-nuclear supernatants were centrifuged at 10,000 × g for 15 min at 4 °C. Equal amounts of protein were then analyzed by SDS-PAGE (15% gel) and Western blotting by using 1 μg/ml anti-cytochrome c mAb (BD Biosciences). Western Blot Analyses—Equal amounts of proteins were separated in a 10–15% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane (Bio-Rad). Proteins were detected using an ECL detection system (Pierce). Caspase-3 was detected by using a rabbit polyclonal antiserum (a gift from Dr. Nicholson, Merk-Frosst Centre for Therapeutic Research, Pointe Claire-Dorval, Quebec, Canada); caspase-8 with a rabbit polyclonal antiserum given by Dr. Peter, The Ben May Institute for Cancer Research (Chicago, IL); Bid and PARP by using rabbit polyclonal antibodies (Cell Signaling, Le-Perray-en-Yvelines, France); p42/p44-phosphorylated MAPK by using a mouse monoclonal antiserum (Cell Signaling); and XIAP by using a mouse monoclonal antiserum (BD Biosciences). Goat anti-rabbit and anti-mouse secondary antibodies were from Cell Signaling. An anti-β-actin (Sigma) or an anti-ERK2 (Santa Cruz Biotechnology-Tebubio) were used as controls for protein loading. Lysosomal Enzyme Assays—β-Hexosaminidase, β-galactosidase, and β-glucuronidase activities were determined using 0.5 mg/ml of the appropriate 4-methylumbelliferyl derivative substrate (Sigma) at pH 4.5 (β-hexosaminidase) or 3.7 (β-galactosidase and β-glucuronidase), in the presence of 0.1% Triton X-100 (52Little L. Alcouloumre M. Drotar A.M. Herman S. Robertson R. Yeh R.Y. Miller A.L. Biochem. J. 1987; 248: 151-159Crossref PubMed Scopus (22) Google Scholar). Protein concentration was measured with the Bio-Rad dye reagent using bovine serum albumin as a standard. Cathepsin B activity was assayed with 5 μm Z-Arg-Arg-AMC (Calbiochem) in the presence or absence of 5 μm CA-074Me (Calbiochem), a specific inhibitor of cathepsin B. Cell pellets were homogenized in 250 μl of sodium acetate buffer (0.3 m, pH 5) with 0.1% Triton X-100, 60 mm acetic acid, 4 mm EDTA, and 8 mm dithiothreitol and incubated for 15 min at 4 °C. Reaction mixtures contained 100 μl of cell suspension and 100 μl of substrate preparation. After 30-min incubation at 37 °C, the reaction was stopped by adding 1 ml of 100 mm sodium chloroacetate. The amount of the released fluorescent product AMC was determined by fluorometry. Cathepsin L activity was assayed with Z-Phe-Arg-AMC 20 μm (Bachem) in the presence of 5 μm CA-074Me. Cell pellets were homogenized in 500 μl of sodium acetate buffer (0.1 m, pH 4.5) with 2 mm EDTA and 10 mm dithiothreitol and incubated for 15 min at 4 °C. Reaction mixtures contained 100 μl of cell suspension and 100 μl of substrate preparation. After 30-min incubation at 37 °C, the reaction was stopped and the amount of AMC was determined as described above for cathepsin B. Tripeptidyl peptidase 1 (TPP1) activity was assayed with 100 μm H-Ala-Ala-Phe-AMC (Bachem). Cell pellets were homogenized in 500 μl of sodium acetate buffer (50 mm, pH 4) with 0.1% Triton X-100, briefly sonicated, and centrifuged at 10,000 × g for 5 min at 4 °C. Reaction mixtures contained 150 μl of supernatant and 50 μl of substrate preparation. After 30-min incubation at 37 °C, the reaction was stopped and the amount of the released fluorescent product AMC was determined by fluorometry. Flow Cytometry Analyses—TNFR1 expression was analyzed on resting cells. For the analysis of CD54 expression, cells were incubated in medium containing 1% FCS for 24 h in the presence or absence of 50 ng/ml TNF. Then, cells were detached using phosphate-buffered saline containing 10 mm EDTA, sedimented at 4 °C, and washed with phosphate-buffered saline. Cells were incubated at 4 °C in the dark for 30 min with fluorescein isothiocyanate-conjugated mouse anti-human TNF-R1 (R&D Systems, Lille, France) or with fluorescein isothiocyanate-conjugated mouse anti-human CD54 (Immunotech, Marseille, France). Isotype control monoclonal antibody (Immunotech) was used as a negative control. Cytometric analyses were performed on a FAC-Scan (BD Biosciences) cytometer. Enzymatic Correction Assay—Fibroblasts from wild-type or triple-deficient mice were incubated for 72 h in DMEM containing 1% FCS. Then, the culture medium of these cells was harvested (centrifuged and filtrated) and added to ICD fibroblasts for 48 h. Similar experiments were carried out using the medium of CHO or CHO-TPP1 cells. Statistical Analyses—Data are presented as means ± S.E. Student's t test was used for statistical analysis (*, p < 0.05; **, p < 0.01; and ***, p < 0.001). I-cell Disease Fibroblasts Are Partially Resistant to TNF Toxicity—To investigate the role of lysosomal hydrolases in TNF-induced cell death, we compared the effects of TNF on human SV40-transformed (Fig. 1, A–C) or untransformed (Fig. 1D) fibroblasts derived from normal individuals with those on fibroblasts isolated from ICD patients, in which the activity of almost all lysosomal enzymes is severely impaired (53Tardy C. Tyynela J. Hasilik A. Levade T. Andrieu-Abadie N. Cell Death Differ. 2003; 10: 1090-1100Crossref PubMed Scopus (29) Google Scholar). The cytotoxic effect of TNF on control cells, in the presence of cycloheximide, was time (Fig. 1A)- and dose (Fig. 1C)-dependent. Under all conditions tested, ICD fibroblasts were more resistant to the lethal effect of TNF than their normal counterparts. This finding was confirmed on several cell lines derived from different normal subjects or patients affected with ICD (Fig. 1, B and D), suggesting that some lysosomal enzymes are key effectors of TNF-induced cytotoxicity in human fibroblasts. In addition, ICD fibroblasts were also partially resistant to staurosporine-induced cell death (53Tardy C. Tyynela J. Hasilik A. Levade T. Andrieu-Abadie N. Cell Death Differ. 2003; 10: 1090-1100Crossref PubMed Scopus (29) Google Scholar). Because TNFR1 is a well known death receptor able to activate the caspase cascade (54Beyaert R. Van Loo G. Heyninck K. Vandenabeele P. Int. Rev. Cytol. 2002; 214: 225-272Crossref PubMed Scopus (43) Google Scholar, 55Micheau O. Tschopp J. Cell. 2003; 114: 181-190Abstract Full Text Full Text PDF PubMed Scopus (2026) Google Scholar), the implication of lysosomal proteases in the apoptotic cell death induced by TNF in ICD transformed fibroblasts was examined. Executioner caspase activity, as measured by the cleavage of the fluorogenic tetrapeptide substrate DEVD-AMC, increased in normal cells within 2 h, peaking at 4 h post-treatment (Fig. 2). Activation of caspase-3 (i.e. the decrease in the procaspase form and appearance of cleaved forms) was further demonstrated by Western blot analysis (Fig. 3D) and by measuring the cleavage of PARP, a well known substrate of caspase-3 (Fig. 3E). Of interest was the finding that these TNF-induced apoptotic events were impaired in ICD cells. TNF treatment of normal cells also resulted in cleavage of the initiator caspase-8 (Fig. 3A), which occurred quite concomitantly to that of effector caspases (Fig. 3D). The cleavage of caspase 8, as monitored by the disappearance of the proform (not shown) and increase in the cleaved forms, was accompanied by the disappearance of Bid, a member of the Bcl-2 family known as a substrate of caspase-8 (Fig. 3B). Progressive release of cytochrome c was also seen in cytosolic, mitochondria-free extracts from TNF-treated control cells (Fig. 3C). Processing of both caspase-8 and Bid, as well as the release of cytochrome c in the cytosol, were considerably reduced in ICD fibroblasts, further supporting the role of some lysosomal proteins in the apoptotic cascade initiated by TNF with a site of action likely lying upstream of mitochondria. Finally, TNF-induced apoptosis in control fibroblasts was accompanied by a decrease in the content of X-linked inhibitor of apoptosis proteins (XIAPs), which are endogenous inhibitors of the terminal caspase cascade. This phenomenon was not observed in mutant cells derived from ICD patients (Fig. 3E).Fig. 3The TNF-induced apoptosis cascade is altered in I-cell disease fibroblasts. Control and ICD transformed fibroblasts were treated for the indicated periods with 50 μg/ml cycloheximide and 50 ng/ml TNF. Cell lysates were prepared and analyzed by Western blot for caspase-8 cleavage (A), Bid proform disappearance (B), release of cytochrome c in the cytosol (C), caspase-3 cleavage (D), XIAP proform disappearance (E), and PARP cleavage (F). An anti-β-actin was used as a control for protein loading. Results are representative of three to six independent experiments. Similar results were obtained with the ICD2 cell line.View Large Image Figure ViewerDownload Hi-res image Download (PPT) TNF-induced p42/p44 MAPK Activation and TNFR1 or CD54 Expression Are Not Impaired in I-cell Disease Fibroblasts—To rule out the possibility that all TNF-induced signaling pathways were affected in ICD cells in an unspecific manner, other biological activities of this cytokine on fibroblasts, such as p42/p44 MAPK activation and CD54 expression, two well established effects of TNF (56Chen G. Goeddel D.V. Science. 2002; 296: 1634-1635Crossref PubMed Scopus (1505) Google Scholar), were assessed. Fig. 4 shows that p42/p44 MAPK activation, observed after 15 min of exposure to TNF, appeared unaffected in ICD cells as compared with normal cells (Fig. 4A). Similarly, the expression of adhesion molecules such as CD54 triggered by TNF on fibroblasts was not abolished in deficient cells (Fig. 4B). Of note, ICD and normal cells expressed similar levels of TNF receptor 1 at the plasma membrane (44% versus 41% in ICD and control cells, respectively) (Fig. 4C). These data indicate that lysosomal enzymes are preferentially implicated in TNF-induced apoptotic pathways but do not interfere with other TNF activities. I-cell Disease Human Lymphoblasts and Murine Fibroblasts Are Also Resistant to Cell Death—To further evaluate the role of lysosomes in apoptosis, we investigated the lethal effect of CD95, a member of the TNF receptor superfamily (57Barnhart B.C. Alappat E.C. Peter M.E. Semin. Immunol. 2003; 15: 185-193Crossref PubMed Scopus (378) Google Scholar), in Epstein-Barr virus-transformed lymphoid cells derived from patients affected with ICD. As illustrated in Fig. 5A, the dose-dependent cytotoxic effect of an agonistic anti-CD95 antibody was considerably reduced in ICD lymphoblasts. The resistance of ICD lymphoblasts was further confirmed by examining the cytotoxic effect of recombinant CD95 ligand (Fig. 5A). These data suggest that the resistance of ICD cells to death inducers is not cell-type specific. We then analyzed the respons
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