Natural Ceramide Reverses Fas Resistance of Acid Sphingomyelinase −− Hepatocytes
2001; Elsevier BV; Volume: 276; Issue: 11 Linguagem: Inglês
10.1074/jbc.m008732200
ISSN1083-351X
AutoresFrançois Paris, Heike Grassmé, Aida Cremesti, Jonathan S. Zager, Yuman Fong, Adriana Haimovitz–Friedman, Zvi Fuks, Erich Gulbins, Richard Kolesnick,
Tópico(s)Erythrocyte Function and Pathophysiology
ResumoThe role of the second messenger ceramide in Fas-mediated death requires clarification. To address this issue, we generated hepatocytes from paired acid sphingomyelinase(ASMase; asmase) +/+ andasmase −/− mice.asmase −/− hepatocytes, derived from 8-week-old mice, manifested normal sphingomyelin content and normal morphological, biochemical, and biologic features. Nonetheless, ASMase-deficient hepatocytes did not display rapid ceramide elevation or apoptosis in response to Jo2 anti-Fas antibody.asmase −/− hepatocytes were not inherently resistant to apoptosis because staurosporine, which did not induce early ceramide elevation, stimulated a normal apoptotic response. The addition of low nanomolar quantities of natural C16-ceramide, which by itself did not induce apoptosis, completely restored the apoptotic response to anti-Fas inasmase −/− hepatocytes. Other sphingolipids did not replace natural ceramide and restore Fas sensitivity. Overcoming resistance to Fas inasmase −/− hepatocytes by natural ceramide is evidence that it is the lack of ceramide and not ASMase which determines the apoptotic phenotype. The ability of natural ceramide to rescue the phenotype without reversing the genotype provides evidence that ceramide is obligate for Fas induction of apoptosis in hepatocytes. The role of the second messenger ceramide in Fas-mediated death requires clarification. To address this issue, we generated hepatocytes from paired acid sphingomyelinase(ASMase; asmase) +/+ andasmase −/− mice.asmase −/− hepatocytes, derived from 8-week-old mice, manifested normal sphingomyelin content and normal morphological, biochemical, and biologic features. Nonetheless, ASMase-deficient hepatocytes did not display rapid ceramide elevation or apoptosis in response to Jo2 anti-Fas antibody.asmase −/− hepatocytes were not inherently resistant to apoptosis because staurosporine, which did not induce early ceramide elevation, stimulated a normal apoptotic response. The addition of low nanomolar quantities of natural C16-ceramide, which by itself did not induce apoptosis, completely restored the apoptotic response to anti-Fas inasmase −/− hepatocytes. Other sphingolipids did not replace natural ceramide and restore Fas sensitivity. Overcoming resistance to Fas inasmase −/− hepatocytes by natural ceramide is evidence that it is the lack of ceramide and not ASMase which determines the apoptotic phenotype. The ability of natural ceramide to rescue the phenotype without reversing the genotype provides evidence that ceramide is obligate for Fas induction of apoptosis in hepatocytes. neutral sphingomyelinase acid sphingomyelinase Niemann-Pick disease fluorescence-activated cell sorting 7- amino-4-trifluro-coumarin Engagement of Fas/CD95/APO-1 receptors by Fas ligand or anti-Fas antibody results in formation of a death-inducing signaling complex comprised of the adaptor molecules FADD/MORT-1 and caspase 8, resulting in release of active caspase 8 to initiate the apoptotic process (1Walczak H. Krammer P.H. Exp. Cell Res. 2000; 256: 58-66Crossref PubMed Scopus (552) Google Scholar). Apoptosis, however, proceeds only after an ensuing commitment step, which allows effector caspase activation. Recently, Peter and co-workers (2Scaffidi C. Fulda S. Srinivasan A. Friesen C. Li F. Tomaselli K.J. Debatin K.M. Krammer P.H. Peter M.E. EMBO J. 1998; 17: 1675-1687Crossref PubMed Scopus (2633) Google Scholar) provided evidence that the quality and quantity of the activated caspase 8 signal are regulated in a cell type-specific fashion and suggested that Fas initiates apoptosis via two different mechanisms. In type I cells, apoptosis occurs after high caspase 8 activation, which signals exclusively via a hierarchical caspase cascade, independent of mitochondrial dysfunction. In contrast, apoptosis in type II cells proceeds after minimal caspase 8 activation through an amplification cascade involving mitochondrial dysfunction, release of mitochondrial cytochrome c, and activation of Apaf-1 and caspase 9. Although many of the upstream elements in type II apoptosis are unknown, this event is inhibited by Bcl-2 and can be mimicked by exogenous addition of short chain ceramides. In both mechanisms, apoptosis eventually ensues after effector caspases, such as 3 and 7, are activated. There are numerous reports of early ceramide elevation upon Fas activation (3Hannun Y.A. Science. 1996; 274: 1855-1859Crossref PubMed Scopus (1500) Google Scholar, 4Mathias S. Pena L.A. Kolesnick R.N. Biochem. J. 1998; 335: 465-480Crossref PubMed Scopus (621) Google Scholar). Ceramide is a second messenger in an evolutionarily conserved stress response pathway that, in different cells, signals events as diverse as differentiation, proliferation, and apoptosis (3Hannun Y.A. Science. 1996; 274: 1855-1859Crossref PubMed Scopus (1500) Google Scholar). Ceramide is generated from sphingomyelin by the action of a neutral or acid sphingomyelinase (NSMase1 or ASMase) or byde novo synthesis coordinated through the enzyme ceramide synthase (5Kolesnick R.N. Kronke M. Annu. Rev. Physiol. 1998; 60: 643-665Crossref PubMed Scopus (731) Google Scholar). In response to Fas ligation, ceramide elevation is often biphasic, a rapid elevation with return to baseline is followed by a more prolonged elevation that is progressive. Evidence has been provided that both ASMase and NSMase might contribute to Fas-mediated ceramide elevation (6Cifone M.G. De Maria R. Roncaioli P. Rippo M.R. Azuma M. Lanier L.L. Santoni A. Testi R. J. Exp. Med. 1994; 180: 1547-1552Crossref PubMed Scopus (598) Google Scholar, 7Lin T. Genestier L. Pinkoski M.J. Castro A. Nicholas S. Mogil R. Paris F. Fuks Z. Schuchman E.H. Kolesnick R.N. Green D.R. J. Biol. Chem. 2000; 275: 8657-8663Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar), with ASMase involvement in the acute event. Recent investigations titrated down the amount of FADD or procaspase 8 transfected into HeLa and 293T cells to the point where they no longer induced apoptosis (8Grullich C. Sullards M.C. Fuks Z. Merrill Jr., A.H. Kolesnick R. J. Biol. Chem. 2000; 275: 8650-8656Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Under these conditions, FADD and procaspase 8 nonetheless induced maximal ceramide generation, which was inhibitable by the initiator caspase inhibitor CrmA. These experiments provided evidence that ceramide generation is initiator caspase-dependent and occurs prior to commitment to the effector phase of the apoptotic process. Whether the ceramide generated in response to Fas ligation is involved in the apoptotic process or is an epiphenomenon is a matter of ongoing debate. Testi and co-workers (9De Maria R. Rippo M.R. Schuchman E.H. Testi R. J. Exp. Med. 1998; 187: 897-902Crossref PubMed Scopus (138) Google Scholar) claimed that Epstein-Barr virus-transformed lymphoblasts from Niemann-Pick disease (NPD) patients, which have an inherited deficiency of ASMase activity, displayed deficits in ceramide generation and apoptosis, whereas Borst and co-workers (10Cock J.G. Tepper A.D. de Vries E. van Blitterswijk W.J. Borst J. J. Biol. Chem. 1998; 273: 7560-7565Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar) found no differences. Recently, Green and co-workers (7Lin T. Genestier L. Pinkoski M.J. Castro A. Nicholas S. Mogil R. Paris F. Fuks Z. Schuchman E.H. Kolesnick R.N. Green D.R. J. Biol. Chem. 2000; 275: 8657-8663Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar) reported partial resistance to Jo2 anti-Fas-induced liver failure and death inasmase −/− mice in vivo. Furthermore, the establishment of primary cultures of several cell types derived from asmase −/− mice showed that hepatocytes (11Kirschnek S. Paris F. Weller M. Grassme H. Ferlinz K. Riehle A. Fuks Z. Kolesnick R. Gulbins E. J. Biol. Chem. 2000; 275: 27316-27323Abstract Full Text Full Text PDF PubMed Google Scholar) but not thymocytes (7Lin T. Genestier L. Pinkoski M.J. Castro A. Nicholas S. Mogil R. Paris F. Fuks Z. Schuchman E.H. Kolesnick R.N. Green D.R. J. Biol. Chem. 2000; 275: 8657-8663Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar) displayed resistance to Fas-mediated death. Although these genetic models tended to support a possible cell type-specific role for ASMase, and indirectly for ceramide, in Fas-induced apoptosis, other interpretations are possible. For instance, the NPD lymphoblasts described above were transformed with Epstein-Barr virus and the transformation process itself, as well as known Epstein-Barr virus strain-specific phenotypic differences (12Miller C.L. Lee J.H. Kieff E. Longnecker R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 772-776Crossref PubMed Scopus (216) Google Scholar, 13Miller C.L. Burkhardt A.L. Lee J.H. Stealey B. Longnecker R. Bolen J.B. Kieff E. Immunity. 1995; 2: 155-166Abstract Full Text PDF PubMed Scopus (270) Google Scholar, 14Khanna K.K. Yan J. Watters D. Hobson K. Beamish H. Spring K. Shiloh Y. Gatti R.A. Lavin M.F. J. Biol. Chem. 1997; 272: 9489-9495Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar), might have affected the outcome. Further, it remains possible that the resistance of cells from asmase −/− mice to Fas-induced apoptotic death might result from subtle alterations in membrane structure/function independent of ceramide itself. To address directly the issue of the involvement of ceramide in Fas-mediated death, we examined the ability of primary cultures of hepatocytes derived from asmase +/+ andasmase −/− mice to undergo apoptosis in response to anti-Fas antibody. Like embryonic fibroblasts and thymocytes from asmase −/− mice (15Lozano J. Morales A. Cremesti A. Fuks Z. Tilly J.L. Schuchman E. Gulbins E. Kolesnick R. Cell Death Differ. 2001; 8: 100-102Crossref PubMed Scopus (26) Google Scholar), asmase −/− hepatocytes displayed normal sphingomyelin content and were indistinguishable fromasmase +/+ hepatocytes by numerous morphological, biochemical, and biologic criteria. In contrast to wild type hepatocytes, however, ASMase-deficient hepatocytes failed to generate ceramide upon activation of Fas and were markedly resistant to Fas-mediated apoptotic death. Further, the addition of low nanomolar quantities of natural C16-ceramide, which by itself does not effect apoptosis, restored Fas-mediated apoptosis inasmase −/− hepatocytes. Overcoming apoptosis resistance by provision of natural ceramide is evidence that it is the lack of ceramide and not ASMase that determines the Fas sensitivity. The ability of natural ceramide to rescue the phenotype without reversing the genotype provides evidence that ceramide is obligate for efficient Fas induction of apoptosis in hepatocytes. ASMase knockout mice, maintained in a sv129 × C57BL/6 background, are propagated using heterozygous breeding pairs and genotyped as described (16Horinouchi K. Erlich S. Perl D.P. Ferlinz K. Bisgaier C.L. Sandhoff K. Desnick R.J. Stewart C.L. Schuchman E.H. Nat. Genet. 1995; 10: 288-293Crossref PubMed Scopus (417) Google Scholar). Experimental mice were 8–12 weeks old and sacrificed by carbon dioxide asphyxiation. Harvested livers were washed three times with phosphate-buffered saline at 37 °C, cut into small pieces, and separated into small clumps with a spatula. Individual hepatocytes were dispersed mechanically by passage through an 18-gauge needle, filtered through a 100-μm cell strainer, washed once with RPMI 1640 complete medium, and resuspended into the same medium containing 10% fetal bovine serum, as described (11Kirschnek S. Paris F. Weller M. Grassme H. Ferlinz K. Riehle A. Fuks Z. Kolesnick R. Gulbins E. J. Biol. Chem. 2000; 275: 27316-27323Abstract Full Text Full Text PDF PubMed Google Scholar). Viability was always greater than 95% as defined by trypan blue exclusion analysis. After 1 h, cells (5 × 106/ml) were placed in 24-well plates pretreated with 10 mg/ml bovine serum albumin fraction V (Sigma) in an incubator at 37 °C with 5% CO2 atmosphere. Cells were treated with anti-Fas Jo2 antibody (Pharmingen) or staurosporine (Sigma) for the indicated times while under constant agitation using a nutator (Becton Dickinson). Hepatocytes were isolated by cannulation of the portal vein and retrograde in situ collagenase perfusion according the method described previously by Picardo et al. (17Picardo A. Fong Y. Karpoff H.M. Yeh S. Blumgart L.H. Brennan M.F. J. Surg. Res. 1996; 63: 452-456Abstract Full Text PDF PubMed Scopus (5) Google Scholar). Briefly, livers were perfused under sterile conditions at 40 °C consecutively with Leffert's buffer (100 mm HEPES, pH 7.4, 30 mm KCl, 1.3 m NaCl, 10 mmNaH2PO4, 100 mmd-glucose) with 0.5 mm EGTA for 4 min, without EGTA for 2 min, and then with 33% (w/v) collagenase type V (Roche) and 5 mm CaCl2 for 12 min using an Easy Load perfusion pump (Masterflex, Cole Parmer; flow rate of 16 ml/min). The liver was then minced in cold Leffert's buffer containing 5 mm CaCl2, and isolated hepatocytes were filtered through a 100-μm cell strainer. Isolated hepatocytes were washed twice at 50 × g and resuspended into RPMI 1640 complete medium containing 10% fetal bovine serum. Viability was always greater than 95% using this method. Cells (5 × 106/ml) were pretreated for 10 min with C16-ceramide, sphingosine, sphingomyelin, sphinganine, sphingosine 1-phosphate (Biomol), or C16 -dihydroceramide (Toronto Research Chemicals, Inc.) in dodecane:ethanol (2:98, v/v; 0.05% final concentration) or diluent alone prior to the addition of Jo2 antibody. A single cell suspension of hepatocytes was washed twice and resuspended into 100 μl of H/S (132 mm NaCl, 20 mm HEPES, 5 mm KCl, 1 mm CaCl2, 0.7 mm MgCl2, 0.8 mm MgSO4) containing 2% fetal calf serum and 0.2% NaN3 supplemented with 1 μg/ml anti-Fas Jo2 antibody or an isotype-specific control immunoglobulin. After 45 min on ice, cells were washed with the same buffer and stained with a fluorescein isothiocyanate-coupled anti-hamster antibody (Pharmingen 12084D). Cell surface Fas was determined in labeled cells by flow cytometry, as described (11Kirschnek S. Paris F. Weller M. Grassme H. Ferlinz K. Riehle A. Fuks Z. Kolesnick R. Gulbins E. J. Biol. Chem. 2000; 275: 27316-27323Abstract Full Text Full Text PDF PubMed Google Scholar). Hepatocytes, stimulated for the indicated times with Jo2, were lysed in 25 mm HEPES, pH 7.4, 0.5% SDS, 1% Triton X-100, 10 mm EDTA, 10 mm each sodium pyrophosphate and sodium fluoride, 10 μg/ml each aprotinin and leupeptin, 125 mm NaCl. Lipids were extracted, and ceramide was quantified by the diacylglycerol kinase reaction as described previously (18Bose R. Chen P. Loconti A. Grullich C. Abrams J.M. Kolesnick R.N. J. Biol. Chem. 1998; 273: 28852-28859Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Hepatocytes were trypsinized after treatment with anti-Fas antibody or staurosporine and then permeabilized with a solution of 0.1% Triton X-100 and 0.1% sodium citrate at 4 °C for 5 min. Apoptosis was assessed by terminal nucleotidyl transferase according to the manufacturer's instructions (Roche, Indianapolis, IN). At least 200 cells were counted for each point. Cells were stained with 4 μg/ml each ethidium bromide and acridine orange for 10 min and analyzed by fluorescence microscopy as described (19Coligan J.E. Dunn B.M. Ploegh H.L. Speicher D.W. Wingfield P.T. Current Protocols in Immunology. Wiley-Interscience, New York1991: 3.17.2-3.17.4Google Scholar). The fluorogenic substrate Ac-DEVD-AFC was used to measure caspase 3-like activity according to the manufacturer's instructions (Kamiya, Seattle, WA). Statistical analysis was performed by Student's t test and t test for correlation coefficient. We reported recently that 8–10 week oldasmase −/− mice derived from our colony display normal sphingomyelin content in multiple tissues (15Lozano J. Morales A. Cremesti A. Fuks Z. Tilly J.L. Schuchman E. Gulbins E. Kolesnick R. Cell Death Differ. 2001; 8: 100-102Crossref PubMed Scopus (26) Google Scholar,20Lozano J. Menendez S. Morales A. Ehleiter D. Liao W.-C. Wagman R. Haimovitz-Friedman A. Fuks Z. Kolesnick R. J. Biol. Chem. 2001; 276: 442-448Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Consequently, these animals are physiologically normal and do not manifest NPD, as NPD requires sphingomyelin accumulation (21Brady R.O. Arch. Neurol. 1998; 55: 1055-1056Crossref PubMed Scopus (23) Google Scholar). In the present studies, we generated primary cultures of hepatocytes from livers of asmase +/+ andasmase −/− mice. Livers from 8-week-old ASMase-deficient mice displayed normal architecture, and isolated hepatocytes manifested physiologic morphology. No measurable ASMase activity was detected in theasmase −/− hepatocytes (TableI); NSMase activity was unchanged. Further, asmase −/− hepatocytes manifested normal sphingomyelin and ceramide content (Table I), consistent with the observation that these animals develop normally into adulthood (15Lozano J. Morales A. Cremesti A. Fuks Z. Tilly J.L. Schuchman E. Gulbins E. Kolesnick R. Cell Death Differ. 2001; 8: 100-102Crossref PubMed Scopus (26) Google Scholar, 22Pena L.A. Fuks Z. Kolesnick R.N. Cancer Res. 2000; 60: 321-327PubMed Google Scholar, 23Santana P. Pena L.A. Haimovitz-Friedman A. Martin S. Green D. McLoughlin M. Cordon-Cardo C. Schuchman E.H. Fuks Z. Kolesnick R. Cell. 1996; 86: 189-199Abstract Full Text Full Text PDF PubMed Scopus (729) Google Scholar). The distribution of glycosphingolipid-enriched microdomains, a compartment putatively involved in ceramide signaling (24Okamoto T. Schlegel A. Scherer P.E. Lisanti M.P. J. Biol. Chem. 1998; 273: 5419-5422Abstract Full Text Full Text PDF PubMed Scopus (1347) Google Scholar) and their content was similar inasmase +/+ andasmase −/− hepatocytes (Table I), as was the expression of Fas on the cell surface, measured by FACS using the Jo2 anti-Fas antibody (Fig. 1). Similarly, the levels of factors known to participate in Fas-mediated apoptosis in hepatocytes including FADD/MORT-1, Bid, Bcl-2, Apaf-1, and caspases 3, 8, and 9 were identical in asmase +/+and asmase −/− hepatocytes (TableI). Moreover, the activation patterns of p38, c-Jun terminal, and extracellular signal-regulated kinases in response epidermal growth factor, tumor necrosis factor-α, heat shock, and hyperosmolarity were also similar in asmase +/+ andasmase −/− hepatocytes (Table I). Thus, asmase +/+ andasmase −/− hepatocytes appeared similar as assessed by numerous criteria.Table IComparison of asmase+/+ and asmase−/− hepatocytesasmase+/+asmase−/−Biochemical properties 1-aASMase and NSMase activities were measured as in (84). Sphingomyelin levels were determined by charring as described in (85). Ceramide levels were quantitated by the diacylglycerol kinase assay. The data (mean ± S.D.) are compiled from two studies performed in duplicate. Note that the residual "ASMase" activity detected in the asmase −/−hepatocytes likely represents NSMase activity manifest at pH 5.0.ASMase activity (pmol/μg × h)15 ± 2<1.0NSMase activity (pmol/μg × h)49 ± 446 ± 4Sphingomyelin (pmol/106 cells)1,150 ± 801,200 ± 85Ceramide (pmol/106cells)170 ± 10175 ± 5Physiological properties 1-bFor morphology and structure studies, livers were fixed in 4% paraformaldehyde overnight, and 5-μm sections, stained with hematoxylin/eosin, were observed by light microscopy.Hepatocyte morphologyNormalNormalLiver structureNormalNormalGEM properties1-cGEMs (glycolipid-enriched microdomains) were isolated as a Triton X-100-insoluble fraction by sucrose gradient density centrifugation as in (86). The fractional distribution of protein was determined by the Bio-Rad Dc assay, and the distribution of the GEM marker protein Src was measured as in (15).Isolation++Distribution++Components of the Fas pathway 1-dExcept for Fas, which was detected by FACS (see Fig. 1), components of the Fas pathway were evaluated by Western blot using the following antibodies: FADD (Santa Cruz sc-6035); Bcl-2 (Santa Cruz sc-509); Bcl-XL (Santa Cruz sc-8392); Bax (Santa Cruz sc-493); Bid (Santa Cruz sc-6290), BAD (Santa Cruz sc-7869), FLIPL (Santa Cruz sc-7109); Apaf-1 (Santa Cruz sc-7232); caspase 3 (Santa Cruz sc-7148); anti-caspase 8 (a kind gift of Dr. Klaus Schulze-Osthoff) (87); caspase 9 (Calbiochem 218794); inhibitor of caspase-activated deoxyribonuclease (ICAD) (Chemicon AB16966). Levels of these proteins in asmase −/−hepatocytes, determined by densitometry of Western blots, are expressed in parentheses as fold of the levels in asmase +/+hepatocytes: FADD/MORT-1 (0.98), Bcl-2 (1.02), Bcl-XL (1.01), Bax (0.91), Bid (1.05), BAD (1.07), FLIPL (1.01), Apaf-1 (0.96), caspase 3 (0.99), caspase 8 (0.97), caspase 9 (0.93) and ICAD (0.96).Fas++FADD/MORT-1++Bcl-2++Bcl-XL++Bax++Bid++BAD++FLIPL++Apaf-1++Caspase 3++Caspase 8++Caspase 9++ICAD++Signaling properties 1-eExtracellular signal-regulated kinases (ERK1/2), c-Jun terminal kinase (JNK), and p38 activation were measured in response to epidermal growth factor (10–100 ng/ml), tumor necrosis factor-α (10–100 ng/ml), heat shock (42 °C for 20 min) and hyperosmolarity (0.4 m sucrose for 20 min) as described (20).ERK1/2 activation++P38 activation++JNK activation++The "+" symbol is used to indicate optimal detection of an event.1-a ASMase and NSMase activities were measured as in (84Lin X. Hengartner M.O. Kolesnick R. J. Biol. Chem. 1998; 273: 14374-14379Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Sphingomyelin levels were determined by charring as described in (85Boucher L.M. Wiegmann K. Futterer A. Pfeffer K. Machleidt T. Schutze S. Mak T.W. Kronke M. J. Exp. Med. 1995; 181: 2059-2068Crossref PubMed Scopus (190) Google Scholar). Ceramide levels were quantitated by the diacylglycerol kinase assay. The data (mean ± S.D.) are compiled from two studies performed in duplicate. Note that the residual "ASMase" activity detected in the asmase −/−hepatocytes likely represents NSMase activity manifest at pH 5.0.1-b For morphology and structure studies, livers were fixed in 4% paraformaldehyde overnight, and 5-μm sections, stained with hematoxylin/eosin, were observed by light microscopy.1-c GEMs (glycolipid-enriched microdomains) were isolated as a Triton X-100-insoluble fraction by sucrose gradient density centrifugation as in (86Lisanti M.P. Tang Z. Scherer P.E. Sargiacomo M. Methods Enzymol. 1995; 250: 655-668Crossref PubMed Scopus (117) Google Scholar). The fractional distribution of protein was determined by the Bio-Rad Dc assay, and the distribution of the GEM marker protein Src was measured as in (15Lozano J. Morales A. Cremesti A. Fuks Z. Tilly J.L. Schuchman E. Gulbins E. Kolesnick R. Cell Death Differ. 2001; 8: 100-102Crossref PubMed Scopus (26) Google Scholar).1-d Except for Fas, which was detected by FACS (see Fig. 1), components of the Fas pathway were evaluated by Western blot using the following antibodies: FADD (Santa Cruz sc-6035); Bcl-2 (Santa Cruz sc-509); Bcl-XL (Santa Cruz sc-8392); Bax (Santa Cruz sc-493); Bid (Santa Cruz sc-6290), BAD (Santa Cruz sc-7869), FLIPL (Santa Cruz sc-7109); Apaf-1 (Santa Cruz sc-7232); caspase 3 (Santa Cruz sc-7148); anti-caspase 8 (a kind gift of Dr. Klaus Schulze-Osthoff) (87Belka C. Marini P. Lepple-Wienhues A. Budach W. Jekle A. Los M. Lang F. Schulze-Osthoff K. Gulbins E. Bamberg M. Oncogene. 1999; 18: 4983-4992Crossref PubMed Scopus (78) Google Scholar); caspase 9 (Calbiochem 218794); inhibitor of caspase-activated deoxyribonuclease (ICAD) (Chemicon AB16966). Levels of these proteins in asmase −/−hepatocytes, determined by densitometry of Western blots, are expressed in parentheses as fold of the levels in asmase +/+hepatocytes: FADD/MORT-1 (0.98), Bcl-2 (1.02), Bcl-XL (1.01), Bax (0.91), Bid (1.05), BAD (1.07), FLIPL (1.01), Apaf-1 (0.96), caspase 3 (0.99), caspase 8 (0.97), caspase 9 (0.93) and ICAD (0.96).1-e Extracellular signal-regulated kinases (ERK1/2), c-Jun terminal kinase (JNK), and p38 activation were measured in response to epidermal growth factor (10–100 ng/ml), tumor necrosis factor-α (10–100 ng/ml), heat shock (42 °C for 20 min) and hyperosmolarity (0.4 m sucrose for 20 min) as described (20Lozano J. Menendez S. Morales A. Ehleiter D. Liao W.-C. Wagman R. Haimovitz-Friedman A. Fuks Z. Kolesnick R. J. Biol. Chem. 2001; 276: 442-448Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Open table in a new tab The "+" symbol is used to indicate optimal detection of an event. To investigate whether ASMase-deficient cells displayed defects in Fas-mediated signaling through the sphingomyelin pathway, we treated primary cultures of asmase +/+ andasmase −/− hepatocytes with increasing concentrations of Jo2 anti-Fas antibody. Hepatocytes were treated in suspension, as Kass and co-workers (25Jones R.A. Johnson V.L. Buck N.R. Dobrota M. Hinton R.H. Chow S.C. Kass G.E. Hepatology. 1998; 27: 1632-1642Crossref PubMed Scopus (89) Google Scholar) showed that plating hepatocytes as monolayers conferred Fas resistance, i.e. the development of a requirement for cycloheximide for death induction (25Jones R.A. Johnson V.L. Buck N.R. Dobrota M. Hinton R.H. Chow S.C. Kass G.E. Hepatology. 1998; 27: 1632-1642Crossref PubMed Scopus (89) Google Scholar). In contrast, hepatocytes in suspension retained sensitivity to Fas-mediated death. Fig. 2 Ashows that asmase +/+ hepatocytes displayed dose-dependent elevation in ceramide content when measured at 5 min of treatment. As little as 50 ng/ml Jo2 increased ceramide content from a baseline of 170 ± 20 pmol/106 cells to 210 ± 20 pmol/106 cells, and a maximal increase to 515 ± 15 pmol/106 cells (p < 0.01) was achieved with 1,000 ng/ml. In contrast,asmase −/− hepatocytes failed to manifest increased ceramide levels at any Jo2 dose up to 8,000 ng/ml. Studies were also performed to determine the time course of ceramide elevation after Jo2 treatment using a 500-ng/ml dose (Fig.2 B). In asmase +/+ hepatocytes, ceramide levels increased to 1.6-fold of control by 30 s (p < 0.01), peaked at 5 min, and remained elevated for at least 20 min. asmase −/−hepatocytes, however, did not demonstrate elevation of ceramide levels at any time up to 20 min. Thus,asmase −/− hepatocytes display a complete defect in the early phase of ceramide generation upon Jo2 treatment. To evaluate whetherasmase −/− hepatocytes also displayed defects in Fas-mediated apoptosis, hepatocytes were rested for 1 h in RPMI 1640 complete medium containing 10% fetal bovine serum prior to treatment with Jo2 anti-Fas antibody. Prior investigations showed that Jo2 antibody induced time- and dose-dependent apoptosis in wild type hepatocytes in vivo and ex vivo (7Lin T. Genestier L. Pinkoski M.J. Castro A. Nicholas S. Mogil R. Paris F. Fuks Z. Schuchman E.H. Kolesnick R.N. Green D.R. J. Biol. Chem. 2000; 275: 8657-8663Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 11Kirschnek S. Paris F. Weller M. Grassme H. Ferlinz K. Riehle A. Fuks Z. Kolesnick R. Gulbins E. J. Biol. Chem. 2000; 275: 27316-27323Abstract Full Text Full Text PDF PubMed Google Scholar, 26de la Coste A. Fabre M. McDonell N. Porteu A. Gilgenkrantz H. Perret C. Kahn A. Mignon A. Am. 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Subsequent investigations measured the dose dependence of Jo2-induced apoptosis in asmase +/+hepatocytes after 8 h of treatment. As shown in Fig.3 A, apoptosis occurred over 2–3 logs of Jo2 doses; as little as 100 ng/ml Jo2 was effective; a maximal apoptotic effect was achieved using 2,000 ng/ml. Thus, the dose dependence for Jo2-induced ceramide generation is slightly lower than that for apoptosis. asmase −/−hepatocytes, which contain normal numbers of cell surface Fas receptors (Fig. 1), were nonetheless resistant to treatment with anti-Fas antibody. In contrast to the total resistance to ceramide generation, apoptosis was inducible by increasing the dose of Jo2 antibody about 1 log. Thus, apoptosis was elicited with a 2,000-ng/ml dose of Jo2 inasmase −/− hepatocytes and increased to a maximum at 8,000 ng/ml Jo2. Similar results were obtained using ethidium bromide and acridine orange staining to detect morphological changes of apoptosis (Fig. 3 B). These studies indicate that asmase −/−hepatocytes can undergo ASMase-dependent and -independent apoptosis after Jo2 treatment. Because the hepatocytes used in these studies were obtained by mechanical dispersion, we compared the effect of Jo2 to induce death using asmase +/+ andasmase −/− hepatocytes obtained by an alternative technique, in vivo collagenase treatment. For these studies, collagenase was delivered to intact livers after cannulation of the inferior hepatic vein and isolated hepatocytes derived as described (17Picardo A. Fong Y. Karpoff H.M. Yeh S. Blumgart L.H. Brennan M.F. J. Surg. Res. 1996; 63: 452-456Abstract Full Text PDF PubMed Scopus (5) Google Scholar). Using this approach, we again obs
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