Involvement of the Acid Sphingomyelinase Pathway in UVA-induced Apoptosis
2001; Elsevier BV; Volume: 276; Issue: 15 Linguagem: Inglês
10.1074/jbc.m006000200
ISSN1083-351X
AutoresYiguo Zhang, Peter Mattjus, Patricia C. Schmid, Ziming Dong, Shuping Zhong, Weiya Ma, Rhoderick E. Brown, Ann M. Bode, H. Schmid, Zigang Dong,
Tópico(s)Phagocytosis and Immune Regulation
ResumoThe sphingomyelin-ceramide pathway is an evolutionarily conserved ubiquitous signal transduction system that regulates many cell functions including apoptosis. Sphingomyelin (SM) is hydrolyzed to ceramide by different sphingomyelinases. Ceramide serves as a second messenger in mediating cellular effects of cytokines and stress. In this study, we find that acid sphingomyelinase (SMase) activity was induced by UVA in normal JY lymphoblasts but was not detectable in MS1418 lymphoblasts from Niemann-Pick type D patients who have an inherited deficiency of acid SMase. We also provide evidence that UVA can induce apoptosis by activating acid SMase in normal JY cells. In contrast, UVA-induced apoptosis was inhibited in MS1418 cells. Exogenous SMase and its product, ceramide (10–40 μm), induced apoptosis in JY and MS1418 cells, but the substrate of SMase, SM (20–80 μm), induced apoptosis only in JY cells. These results suggest that UVA-induced apoptosis by SM is dependent on acid SMase activity. We also provide evidence that induction of apoptosis by UVA may occur through activation of JNKs via the acid SMase pathway. The sphingomyelin-ceramide pathway is an evolutionarily conserved ubiquitous signal transduction system that regulates many cell functions including apoptosis. Sphingomyelin (SM) is hydrolyzed to ceramide by different sphingomyelinases. Ceramide serves as a second messenger in mediating cellular effects of cytokines and stress. In this study, we find that acid sphingomyelinase (SMase) activity was induced by UVA in normal JY lymphoblasts but was not detectable in MS1418 lymphoblasts from Niemann-Pick type D patients who have an inherited deficiency of acid SMase. We also provide evidence that UVA can induce apoptosis by activating acid SMase in normal JY cells. In contrast, UVA-induced apoptosis was inhibited in MS1418 cells. Exogenous SMase and its product, ceramide (10–40 μm), induced apoptosis in JY and MS1418 cells, but the substrate of SMase, SM (20–80 μm), induced apoptosis only in JY cells. These results suggest that UVA-induced apoptosis by SM is dependent on acid SMase activity. We also provide evidence that induction of apoptosis by UVA may occur through activation of JNKs via the acid SMase pathway. Cell death is an irreversible process that culminates in cessation of biological activity (1Saikumar P. Dong Z. Mikhailov V. Denton M. Weinberg J.M. Venkatachalam M.A. Am. J. Med... 1999; 107: 489-506Google Scholar, 2Darzynkiewicz Z. Juan G. Li X. Gorczyca W. Murakami T. Traganos F. Cytometry.. 1997; 27: 1-20Google Scholar, 3Allen R.T. Hunter W.J.I., II Agrawal D.K. J. Pharmacol. Toxicol. Methods.. 1997; 37: 215-228Google Scholar) and can occur through apoptosis or necrosis (4Kitanaka C. Kuchino Y. Cell Death Differ... 1999; 6: 508-515Google Scholar, 5Tsujimoto Y. Shimizu S. FEBS Lett... 2000; 466: 6-10Google Scholar, 6Fadeel B. Zhivotovsky B. Orrenius S. FASEB J... 1999; 13: 1647-1657Google Scholar, 7Hofmann K. Cell. Mol. Life Sci... 1999; 55: 1113-1128Google Scholar, 8Earnshaw W.C. Martins L.M. Kaufmann S.H. Annu. Rev. Biochem... 1999; 68: 383-424Google Scholar, 9Green D.R. Reed J.C. Science.. 1998; 281: 1309-1312Google Scholar, 10Godar D.E. J. Investig. Dermatol. Symp. Proc... 1999; 4: 17-23Google Scholar, 11Okazaki T. Kondo T. Kitano T. Tashima M. Cell. Signal... 1998; 10: 685-692Google Scholar). Apoptosis is an active and physiological mode of cell death and is well characterized by morphological changes including cell shrinkage, cytoplasmic blebbing, chromatin condensation, and DNA fragmentation (2Darzynkiewicz Z. Juan G. Li X. Gorczyca W. Murakami T. Traganos F. Cytometry.. 1997; 27: 1-20Google Scholar). In recent years, substantial progress has been made in understanding the multistep regulatory mechanisms that are associated with the propensity of a cell to respond to various stimuli with apoptosis (1Saikumar P. Dong Z. Mikhailov V. Denton M. Weinberg J.M. Venkatachalam M.A. Am. J. Med... 1999; 107: 489-506Google Scholar, 2Darzynkiewicz Z. Juan G. Li X. Gorczyca W. Murakami T. Traganos F. Cytometry.. 1997; 27: 1-20Google Scholar, 7Hofmann K. Cell. Mol. Life Sci... 1999; 55: 1113-1128Google Scholar, 8Earnshaw W.C. Martins L.M. Kaufmann S.H. Annu. Rev. Biochem... 1999; 68: 383-424Google Scholar). The regulatory system involves the presence of at least two distinct checkpoints, one controlled by the Bcl-2/Bax family of proteins (5Tsujimoto Y. Shimizu S. FEBS Lett... 2000; 466: 6-10Google Scholar, 6Fadeel B. Zhivotovsky B. Orrenius S. FASEB J... 1999; 13: 1647-1657Google Scholar, 7Hofmann K. Cell. Mol. Life Sci... 1999; 55: 1113-1128Google Scholar) and the other by the cysteine and possibly serine proteases (2Darzynkiewicz Z. Juan G. Li X. Gorczyca W. Murakami T. Traganos F. Cytometry.. 1997; 27: 1-20Google Scholar, 7Hofmann K. Cell. Mol. Life Sci... 1999; 55: 1113-1128Google Scholar, 8Earnshaw W.C. Martins L.M. Kaufmann S.H. Annu. Rev. Biochem... 1999; 68: 383-424Google Scholar, 12Verheij M. Bose R. Lin X.H. Yao B. Jarvis W.D. Grant S. Birrer M.J. Szabo E. Zon L.I. Kyriakis J.M. Haimovitz-Friedman A. Fuks Z. Kolesnick R.N. Nature.. 1996; 380: 75-79Google Scholar, 20Jarvis W.D. Fornari Jr., F.A. Auer K.L. Freemerman A.J. Szabo E. Birrer M.J. Johnson C.R. Barbour S.E. Dent P. Grant S. Mol. Pharmacol... 1997; 52: 935-947Google Scholar, 21Cuvillier O. Pirianov G. Kleuser B. Vanek P.G. Coso O.A. Gutkind J.S. Spiegel S. Nature.. 1996; 381: 800-803Google Scholar, 22Kawano T. Cui J. Koezuka Y. Toura I. Kaneko Y. Motoki K. Ueno H. Nakagawa R. Sato H. Kondo E. Koseki H. Taniguchi M. Science.. 1997; 278: 1626-1629Google Scholar). In addition, mitochondria (9Green D.R. Reed J.C. Science.. 1998; 281: 1309-1312Google Scholar, 13Susin S.A. Lorenzo H.K. Zamzami N. Marzo I. Snow B.E. Brothers G.M. Mangion J. Jacotot E. Costantini P. Loeffler M. Larochette N. Goodlett D.R. Aebersold R. Siderovski D.P. Penninger J.M. Kroemer G. Nature.. 1999; 397: 441-446Google Scholar) and the sphingomyelin (SM)1-ceramide pathway (11Okazaki T. Kondo T. Kitano T. Tashima M. Cell. Signal... 1998; 10: 685-692Google Scholar, 12Verheij M. Bose R. Lin X.H. Yao B. Jarvis W.D. Grant S. Birrer M.J. Szabo E. Zon L.I. Kyriakis J.M. Haimovitz-Friedman A. Fuks Z. Kolesnick R.N. Nature.. 1996; 380: 75-79Google Scholar, 14Hannun Y.A. Science.. 1996; 274: 1855-1859Google Scholar, 15Levade T. Jaffrézou J.P. Biochim. Biophys. Acta.. 1999; 1438: 1-17Google Scholar, 16Mathias S. Peña L.A. Kolesnick R.N. Biochem. J... 1998; 335: 465-480Google Scholar, 17Igarashi Y. J. Biochem. ( Tokyo ).. 1997; 122: 1080-1087Google Scholar) play important roles in apoptotic signal transduction. These systems interact with the machinery regulating cell proliferation and DNA repair through several oncogenes and tumor suppressor genes such as p53 (2Darzynkiewicz Z. Juan G. Li X. Gorczyca W. Murakami T. Traganos F. Cytometry.. 1997; 27: 1-20Google Scholar). Hence, antitumor strategies based on modulation of the propensity of the cell to undergo apoptosis attract great interest in oncology.Sphingolipids such as SM had been previously regarded as metabolically inactive, functioning only as structural components of the membrane (17Igarashi Y. J. Biochem. ( Tokyo ).. 1997; 122: 1080-1087Google Scholar, 18Merrill Jr., A.H. Schmelz E.M. Dillehay D.L. Spiegel S. Shayman J.A. Schroeder J.J. Riley R.T. Voss K.A. Wang E. Toxicol. Appl. Pharmacol... 1997; 142: 208-225Google Scholar). However, besides its structural role in biomembranes, SM plays a pivotal role in signal transduction and regulation of cellular functions including growth, differentiation, proliferation, and apoptosis (14Hannun Y.A. Science.. 1996; 274: 1855-1859Google Scholar, 15Levade T. Jaffrézou J.P. Biochim. Biophys. Acta.. 1999; 1438: 1-17Google Scholar, 16Mathias S. Peña L.A. Kolesnick R.N. Biochem. J... 1998; 335: 465-480Google Scholar, 17Igarashi Y. J. Biochem. ( Tokyo ).. 1997; 122: 1080-1087Google Scholar, 18Merrill Jr., A.H. Schmelz E.M. Dillehay D.L. Spiegel S. Shayman J.A. Schroeder J.J. Riley R.T. Voss K.A. Wang E. Toxicol. Appl. Pharmacol... 1997; 142: 208-225Google Scholar). A number of studies have demonstrated that extracellular cytokines and stress stimuli, such as TNFα, interleukin-1β, FAS ligand, heat shock, γ-radiation (23Santana P. Peña L.A. Haimovitz-Friedman A. Martin S. Green D. McLoughlin M. Cordon-Cardo C. Schuchman E.H. Fuks Z. Kolesnick R. Cell.. 1996; 86: 189-199Google Scholar), and UVC irradiation (12Verheij M. Bose R. Lin X.H. Yao B. Jarvis W.D. Grant S. Birrer M.J. Szabo E. Zon L.I. Kyriakis J.M. Haimovitz-Friedman A. Fuks Z. Kolesnick R.N. Nature.. 1996; 380: 75-79Google Scholar, 14Hannun Y.A. Science.. 1996; 274: 1855-1859Google Scholar), cause the activation of sphingomyelinase (SMase) and the release of ceramide. Ceramide, a product of SMase-catalyzed hydrolysis of SM, was shown to act as a lipid second messenger or biomodulator of diverse stress-related responses including cell cycle arrest, cell senescence, and apoptosis (14Hannun Y.A. Science.. 1996; 274: 1855-1859Google Scholar, 15Levade T. Jaffrézou J.P. Biochim. Biophys. Acta.. 1999; 1438: 1-17Google Scholar, 16Mathias S. Peña L.A. Kolesnick R.N. Biochem. J... 1998; 335: 465-480Google Scholar, 17Igarashi Y. J. Biochem. ( Tokyo ).. 1997; 122: 1080-1087Google Scholar, 18Merrill Jr., A.H. Schmelz E.M. Dillehay D.L. Spiegel S. Shayman J.A. Schroeder J.J. Riley R.T. Voss K.A. Wang E. Toxicol. Appl. Pharmacol... 1997; 142: 208-225Google Scholar, 19Okazaki T. Bell R.M. Hannun Y.A. J. Biol. Chem... 1989; 264: 19076-19080Google Scholar, 20Jarvis W.D. Fornari Jr., F.A. Auer K.L. Freemerman A.J. Szabo E. Birrer M.J. Johnson C.R. Barbour S.E. Dent P. Grant S. Mol. Pharmacol... 1997; 52: 935-947Google Scholar, 21Cuvillier O. Pirianov G. Kleuser B. Vanek P.G. Coso O.A. Gutkind J.S. Spiegel S. Nature.. 1996; 381: 800-803Google Scholar, 22Kawano T. Cui J. Koezuka Y. Toura I. Kaneko Y. Motoki K. Ueno H. Nakagawa R. Sato H. Kondo E. Koseki H. Taniguchi M. Science.. 1997; 278: 1626-1629Google Scholar). This pathway is referred to as the SM cycle, SM-ceramide pathway (11Okazaki T. Kondo T. Kitano T. Tashima M. Cell. Signal... 1998; 10: 685-692Google Scholar, 17Igarashi Y. J. Biochem. ( Tokyo ).. 1997; 122: 1080-1087Google Scholar), or the SMase pathway. To date, at least seven classes of mammalian SMases have been described, differing in subcellular location, pH optimum, cation dependence, and roles in cell regulation (11Okazaki T. Kondo T. Kitano T. Tashima M. Cell. Signal... 1998; 10: 685-692Google Scholar, 15Levade T. Jaffrézou J.P. Biochim. Biophys. Acta.. 1999; 1438: 1-17Google Scholar, 17Igarashi Y. J. Biochem. ( Tokyo ).. 1997; 122: 1080-1087Google Scholar). Two forms of SMases, distinguishable by their pH optima, are capable of initiating signal transduction (12Verheij M. Bose R. Lin X.H. Yao B. Jarvis W.D. Grant S. Birrer M.J. Szabo E. Zon L.I. Kyriakis J.M. Haimovitz-Friedman A. Fuks Z. Kolesnick R.N. Nature.. 1996; 380: 75-79Google Scholar). The acid SMase (pH optimum 4.5–5.0) is activated in cells exposed to ionizing radiation, FAS, CD28, interleukin-1, or TNFα (23Santana P. Peña L.A. Haimovitz-Friedman A. Martin S. Green D. McLoughlin M. Cordon-Cardo C. Schuchman E.H. Fuks Z. Kolesnick R. Cell.. 1996; 86: 189-199Google Scholar, 24Grassmé H. Gulbins E. Brenner B. Ferlinz K. Sandhoff K. Harzer K. Lang F. Meyer T.F. Cell.. 1997; 91: 605-615Google Scholar). The neutral SMase (pH optimum 7.4) has been implicated in mediating apoptosis in cells exposed to serum starvation, anti-FAS antibody, vitamin D, TNFα, or cytosine arabinoside (25Adam D. Wiegmann K. Adam-Klages S. Ruff A. Krönke M. J. Biol. Chem... 1996; 271: 14617-14622Google Scholar, 26Adam-Klages S. Adam D. Wiegmann K. Struve S. Kolanus W. Schneider-Mergener J. Krönke M. Cell.. 1996; 86: 937-947Google Scholar).Solar ultraviolet (UV) radiation is known to be one of the most common environmental carcinogens leading to skin cancer (27De Laat J.M. De Gruijl F.R. Cancer Surv... 1996; 26: 173-191Google Scholar, 28Scharffetter-Kochanek K. Wlaschek M. Brenneisen P. Schauen M. Blaudschun R. Wenk J. Biol. Chem... 1997; 378: 1247-1257Google Scholar, 29Slominski A. Pawelek J. Clin. Dermatol... 1998; 16: 503-515Google Scholar). Also, UV exposure induces apoptosis in cultured cells and in vivo(29Slominski A. Pawelek J. Clin. Dermatol... 1998; 16: 503-515Google Scholar, 30Tada-Oikawa S. Oikawa S. Kawanishi S. Biochem. Biophys. Res. Commun... 1998; 247: 693-696Google Scholar). Most research has focused on the UVC (200–290 nm) and UVB (290–320 nm) induction of apoptosis (31Beissert S. Granstein R.D. Crit. Rev. Biochem. Mol. Biol... 1996; 31: 381-404Google Scholar, 32Bender K. Blattner C. Knebel A. Iordanov M. Herrlich P. Rahmsdorf H.J. J. Photochem. Photobiol. B Biol... 1997; 37: 1-17Google Scholar), and little is known about the effect of UVA (320–400 nm), which comprises over 90% of the solar UV. Here, we observe that acid SMase is activated by UVA, and we provide evidence that UVA-induced apoptosis is dependent on acid SMase activity. Exogenous sphingomyelinase and its product, ceramide, also induce apoptosis independent of activation of intracellular SMase, but induction of apoptosis by SM is dependent on the SMase activity. Our data further indicate that UVA-induced apoptosis may occur through activation of JNKs via the SMase pathway.DISCUSSIONExposure to UV radiation can cause cell cycle arrest (50Herrlich P. Blattner C. Knebel A. Bender K. Rahmsdorf H.J. Biol. Chem... 1997; 378: 1217-1229Google Scholar, 51Tyrrell R.M. Feige U. Morimoto R.I. Yahara I. Polla B. (UV activation of mammalian stress proteins) in Stress-inducible cellular Responses.Birkäuser Verlag. 1996; : 255-271Google Scholar), alterations in mitochondrial membrane permeability (30Tada-Oikawa S. Oikawa S. Kawanishi S. Biochem. Biophys. Res. Commun... 1998; 247: 693-696Google Scholar), and cell death by necrosis or apoptosis (42Obeid L.M. Hannun Y.A. J. Cell. Biochem... 1995; 58: 191-198Google Scholar, 51Tyrrell R.M. Feige U. Morimoto R.I. Yahara I. Polla B. (UV activation of mammalian stress proteins) in Stress-inducible cellular Responses.Birkäuser Verlag. 1996; : 255-271Google Scholar). In the present study, we observed that UVA, like UVB and UVC, induced apoptosis in normal lymphoblast cells (JY) but not in MS1418 lymphoblasts from Niemann-Pick type D patients who have an inherited deficiency of acid SMase. UVA radiation is known to induce an array of stress proteins quite distinct from those induced by UVB or UVC (51Tyrrell R.M. Feige U. Morimoto R.I. Yahara I. Polla B. (UV activation of mammalian stress proteins) in Stress-inducible cellular Responses.Birkäuser Verlag. 1996; : 255-271Google Scholar). UVB and UVC were clearly shown to mimic growth factor responses and stimulate signal transduction including the SMase pathway (12Verheij M. Bose R. Lin X.H. Yao B. Jarvis W.D. Grant S. Birrer M.J. Szabo E. Zon L.I. Kyriakis J.M. Haimovitz-Friedman A. Fuks Z. Kolesnick R.N. Nature.. 1996; 380: 75-79Google Scholar, 20Jarvis W.D. Fornari Jr., F.A. Auer K.L. Freemerman A.J. Szabo E. Birrer M.J. Johnson C.R. Barbour S.E. Dent P. Grant S. Mol. Pharmacol... 1997; 52: 935-947Google Scholar, 32Bender K. Blattner C. Knebel A. Iordanov M. Herrlich P. Rahmsdorf H.J. J. Photochem. Photobiol. B Biol... 1997; 37: 1-17Google Scholar, 43Huang C. Ma W.-Y Ding M. Bowden G.T. Dong Z. J. Biol. Chem... 1997; 272: 27753-27757Google Scholar). Noncytotoxic exposure to UVA can also up-regulate several signal molecules (32Bender K. Blattner C. Knebel A. Iordanov M. Herrlich P. Rahmsdorf H.J. J. Photochem. Photobiol. B Biol... 1997; 37: 1-17Google Scholar, 42Obeid L.M. Hannun Y.A. J. Cell. Biochem... 1995; 58: 191-198Google Scholar), but the role of the SMase pathway in UVA-induced apoptosis is not well understood.In this study, we provide evidence that UVA, UVB, and UVC can all activate acid SMase (Fig. 1) and lead to an increase in ceramide levels (Fig. 2) in normal JY cells. Analysis of DNA fragmentation laddering and morphological changes in apoptotic cells showed that sublethal doses of UVA (20–80 kJ/m2), similar to lethal doses of UVB and UVC, can also induce apoptosis in normal JY cells, but UVA-induced apoptosis is prevented in acid SMase-deficient MS1418 cells. On the other hand, the difference in UVB- or UVC-induced apoptosis was less apparent in normal and SMase-deficient cells. These results suggest that UVA-induced apoptosis occurs through activation of acid SMase, whereas UVB- or UVC-induced apoptosis occurs through SMase-independent pathways. However, additional pathways involved in mediating UVA-induced apoptosis cannot be disregarded, including neutral SMase (25Adam D. Wiegmann K. Adam-Klages S. Ruff A. Krönke M. J. Biol. Chem... 1996; 271: 14617-14622Google Scholar, 26Adam-Klages S. Adam D. Wiegmann K. Struve S. Kolanus W. Schneider-Mergener J. Krönke M. Cell.. 1996; 86: 937-947Google Scholar), stress-activated protein kinase/JNKs (12Verheij M. Bose R. Lin X.H. Yao B. Jarvis W.D. Grant S. Birrer M.J. Szabo E. Zon L.I. Kyriakis J.M. Haimovitz-Friedman A. Fuks Z. Kolesnick R.N. Nature.. 1996; 380: 75-79Google Scholar), caspases (30Tada-Oikawa S. Oikawa S. Kawanishi S. Biochem. Biophys. Res. Commun... 1998; 247: 693-696Google Scholar), and mitochondria (30Tada-Oikawa S. Oikawa S. Kawanishi S. Biochem. Biophys. Res. Commun... 1998; 247: 693-696Google Scholar), because UVA-induced apoptosis is not completely blocked in acid SMase-deficient MS1418 cells.Klotz et al. (44Klotz L.-O. Pelliuex C. Briviba K. Pierlot C. Aubry J.-M. Sies H. Eur. J. Biochem... 1999; 260: 917-922Google Scholar) reported that UVA did not activate ERKs, and we found that UVA, like UVB and UVC, did not induce phosphorylation (Fig. 6) and activation (data not shown) of ERKs in SMase-normal JY cells. However, UVA strongly induced ERKs phosphorylation in SMase-deficient MS1418 cells suggesting that in the absence of SMase, UVA may stimulate alternate pathways (e.g. ERKs) that may protect against UVA-induced apoptosis in these cells. We also found that UVA, as well as UVB and UVC, induced phosphorylation and activation of p38 kinase (Fig. 7). However, UVA-induced phosphorylation of p38 kinase appeared to be less in normal SMase JY cells compared with SMase-deficient MS1418 cells, whereas UVB- or UVC-induced phosphorylation was similar between the two cell lines (Fig. 7). Neither inhibition of UVA-induced ERKs activation by PD98059 (48Deak M. Clifton A.D. Lucocq J.M. Alessi D.R. EMBO J... 1998; 17: 4426-4441Google Scholar) nor inhibition of p38 kinase activation by SB202190 (49Blair A.S. Hajduch E. Litherland G.J. Hundal H.S. J. Biol. Chem... 1999; 274: 36293-36299Google Scholar) blocked UVA-induced apoptosis in normal JY cells (Fig. 8, left panel). However, MS1418 cells pretreated with PD98059 or SB202190 now showed typical apoptosis following UVA irradiation (Fig. 8,right panel). These results further confirm that activation and phosphorylation of ERKs and p38 kinase do not appear to be involved in UVA-induced apoptosis in normal JY cells, but in the absence of SMase, UVA irradiation may induce ERKs and p38 kinase leading to inhibition of apoptosis.Acid SMase was previously shown to be involved in UVB- and UVC-induced activation of JNKs (43Huang C. Ma W.-Y Ding M. Bowden G.T. Dong Z. J. Biol. Chem... 1997; 272: 27753-27757Google Scholar). In the present study, we observed that UVA, like UVB and UVC, induced phosphorylation of JNKs in acid SMase normal JY cells and that the phosphorylation of JNKs was markedly inhibited in SMase-deficient cells (Fig. 5). However, TPA-induced JNKs phosphorylation was not different between the two cell lines. These results suggest that UVA-, UVB-, or UVC-, but not TPA-induced phosphorylation and activation of JNKs, is acid SMase-dependent. In addition, we observed that UVA-induced apoptosis was completely blocked in jnk1−/− andjnk2−/− cells (Fig. 9 B) further indicating that JNKs are a downstream kinase family of the acid SMase pathway (45Ichijo H. Nishida E. Irie K. ten Dijke P. Saitoh M. Moriguchi T. Takagi M. Matsumoto K. Miyazono K. Gotoh Y. Science.. 1997; 275: 90-94Google Scholar, 46Xia Z. Dickens M. Raingeaud J. Davis R.J. Greenberg M.E. Science.. 1995; 270: 1326-1331Google Scholar). Overall, these results strongly suggest that UVA-induced apoptosis in normal SMase JY cells occurs primarily through activation of JNKs via the acid SMase pathway. Cell death is an irreversible process that culminates in cessation of biological activity (1Saikumar P. Dong Z. Mikhailov V. Denton M. Weinberg J.M. Venkatachalam M.A. Am. J. Med... 1999; 107: 489-506Google Scholar, 2Darzynkiewicz Z. Juan G. Li X. Gorczyca W. Murakami T. Traganos F. Cytometry.. 1997; 27: 1-20Google Scholar, 3Allen R.T. Hunter W.J.I., II Agrawal D.K. J. Pharmacol. Toxicol. Methods.. 1997; 37: 215-228Google Scholar) and can occur through apoptosis or necrosis (4Kitanaka C. Kuchino Y. Cell Death Differ... 1999; 6: 508-515Google Scholar, 5Tsujimoto Y. Shimizu S. FEBS Lett... 2000; 466: 6-10Google Scholar, 6Fadeel B. Zhivotovsky B. Orrenius S. FASEB J... 1999; 13: 1647-1657Google Scholar, 7Hofmann K. Cell. Mol. Life Sci... 1999; 55: 1113-1128Google Scholar, 8Earnshaw W.C. Martins L.M. Kaufmann S.H. Annu. Rev. Biochem... 1999; 68: 383-424Google Scholar, 9Green D.R. Reed J.C. Science.. 1998; 281: 1309-1312Google Scholar, 10Godar D.E. J. Investig. Dermatol. Symp. Proc... 1999; 4: 17-23Google Scholar, 11Okazaki T. Kondo T. Kitano T. Tashima M. Cell. Signal... 1998; 10: 685-692Google Scholar). Apoptosis is an active and physiological mode of cell death and is well characterized by morphological changes including cell shrinkage, cytoplasmic blebbing, chromatin condensation, and DNA fragmentation (2Darzynkiewicz Z. Juan G. Li X. Gorczyca W. Murakami T. Traganos F. Cytometry.. 1997; 27: 1-20Google Scholar). In recent years, substantial progress has been made in understanding the multistep regulatory mechanisms that are associated with the propensity of a cell to respond to various stimuli with apoptosis (1Saikumar P. Dong Z. Mikhailov V. Denton M. Weinberg J.M. Venkatachalam M.A. Am. J. Med... 1999; 107: 489-506Google Scholar, 2Darzynkiewicz Z. Juan G. Li X. Gorczyca W. Murakami T. Traganos F. Cytometry.. 1997; 27: 1-20Google Scholar, 7Hofmann K. Cell. Mol. Life Sci... 1999; 55: 1113-1128Google Scholar, 8Earnshaw W.C. Martins L.M. Kaufmann S.H. Annu. Rev. Biochem... 1999; 68: 383-424Google Scholar). The regulatory system involves the presence of at least two distinct checkpoints, one controlled by the Bcl-2/Bax family of proteins (5Tsujimoto Y. Shimizu S. FEBS Lett... 2000; 466: 6-10Google Scholar, 6Fadeel B. Zhivotovsky B. Orrenius S. FASEB J... 1999; 13: 1647-1657Google Scholar, 7Hofmann K. Cell. Mol. Life Sci... 1999; 55: 1113-1128Google Scholar) and the other by the cysteine and possibly serine proteases (2Darzynkiewicz Z. Juan G. Li X. Gorczyca W. Murakami T. Traganos F. Cytometry.. 1997; 27: 1-20Google Scholar, 7Hofmann K. Cell. Mol. Life Sci... 1999; 55: 1113-1128Google Scholar, 8Earnshaw W.C. Martins L.M. Kaufmann S.H. Annu. Rev. Biochem... 1999; 68: 383-424Google Scholar, 12Verheij M. Bose R. Lin X.H. Yao B. Jarvis W.D. Grant S. Birrer M.J. Szabo E. Zon L.I. Kyriakis J.M. Haimovitz-Friedman A. Fuks Z. Kolesnick R.N. Nature.. 1996; 380: 75-79Google Scholar, 20Jarvis W.D. Fornari Jr., F.A. Auer K.L. Freemerman A.J. Szabo E. Birrer M.J. Johnson C.R. Barbour S.E. Dent P. Grant S. Mol. Pharmacol... 1997; 52: 935-947Google Scholar, 21Cuvillier O. Pirianov G. Kleuser B. Vanek P.G. Coso O.A. Gutkind J.S. Spiegel S. Nature.. 1996; 381: 800-803Google Scholar, 22Kawano T. Cui J. Koezuka Y. Toura I. Kaneko Y. Motoki K. Ueno H. Nakagawa R. Sato H. Kondo E. Koseki H. Taniguchi M. Science.. 1997; 278: 1626-1629Google Scholar). In addition, mitochondria (9Green D.R. Reed J.C. Science.. 1998; 281: 1309-1312Google Scholar, 13Susin S.A. Lorenzo H.K. Zamzami N. Marzo I. Snow B.E. Brothers G.M. Mangion J. Jacotot E. Costantini P. Loeffler M. Larochette N. Goodlett D.R. Aebersold R. Siderovski D.P. Penninger J.M. Kroemer G. Nature.. 1999; 397: 441-446Google Scholar) and the sphingomyelin (SM)1-ceramide pathway (11Okazaki T. Kondo T. Kitano T. Tashima M. Cell. Signal... 1998; 10: 685-692Google Scholar, 12Verheij M. Bose R. Lin X.H. Yao B. Jarvis W.D. Grant S. Birrer M.J. Szabo E. Zon L.I. Kyriakis J.M. Haimovitz-Friedman A. Fuks Z. Kolesnick R.N. Nature.. 1996; 380: 75-79Google Scholar, 14Hannun Y.A. Science.. 1996; 274: 1855-1859Google Scholar, 15Levade T. Jaffrézou J.P. Biochim. Biophys. Acta.. 1999; 1438: 1-17Google Scholar, 16Mathias S. Peña L.A. Kolesnick R.N. Biochem. J... 1998; 335: 465-480Google Scholar, 17Igarashi Y. J. Biochem. ( Tokyo ).. 1997; 122: 1080-1087Google Scholar) play important roles in apoptotic signal transduction. These systems interact with the machinery regulating cell proliferation and DNA repair through several oncogenes and tumor suppressor genes such as p53 (2Darzynkiewicz Z. Juan G. Li X. Gorczyca W. Murakami T. Traganos F. Cytometry.. 1997; 27: 1-20Google Scholar). Hence, antitumor strategies based on modulation of the propensity of the cell to undergo apoptosis attract great interest in oncology. Sphingolipids such as SM had been previously regarded as metabolically inactive, functioning only as structural components of the membrane (17Igarashi Y. J. Biochem. ( Tokyo ).. 1997; 122: 1080-1087Google Scholar, 18Merrill Jr., A.H. Schmelz E.M. Dillehay D.L. Spiegel S. Shayman J.A. Schroeder J.J. Riley R.T. Voss K.A. Wang E. Toxicol. Appl. Pharmacol... 1997; 142: 208-225Google Scholar). However, besides its structural role in biomembranes, SM plays a pivotal role in signal transduction and regulation of cellular functions including growth, differentiation, proliferation, and apoptosis (14Hannun Y.A. Science.. 1996; 274: 1855-1859Google Scholar, 15Levade T. Jaffrézou J.P. Biochim. Biophys. Acta.. 1999; 1438: 1-17Google Scholar, 16Mathias S. Peña L.A. Kolesnick R.N. Biochem. J... 1998; 335: 465-480Google Scholar, 17Igarashi Y. J. Biochem. ( Tokyo ).. 1997; 122: 1080-1087Google Scholar, 18Merrill Jr., A.H. Schmelz E.M. Dillehay D.L. Spiegel S. Shayman J.A. Schroeder J.J. Riley R.T. Voss K.A. Wang E. Toxicol. Appl. Pharmacol... 1997; 142: 208-225Google Scholar). A number of studies have demonstrated that extracellular cytokines and stress stimuli, such as TNFα, interleukin-1β, FAS ligand, heat shock, γ-radiation (23Santana P. Peña L.A. Haimovitz-Friedman A. Martin S. Green D. McLoughlin M. Cordon-Cardo C. Schuchman E.H. Fuks Z. Kolesnick R. Cell.. 1996; 86: 189-199Google Scholar), and UVC irradiation (12Verheij M. Bose R. Lin X.H. Yao B. Jarvis W.D. Grant S. Birrer M.J. Szabo E. Zon L.I. Kyriakis J.M. Haimovitz-Friedman A. Fuks Z. Kolesnick R.N. Nature.. 1996; 380: 75-79Google Scholar, 14Hannun Y.A. Science.. 1996; 274: 1855-1859Google Scholar), cause the activation of sphingomyelinase (SMase) and the release of ceramide. Ceramide, a product of SMase-catalyzed hydrolysis of SM, was shown to act as a lipid second messenger or biomodulator of diverse stress-related responses including cell cycle arrest, cell senescence, and apoptosis (14Hannun Y.A. Science.. 1996; 274: 1855-1859Google Scholar, 15Levade T. Jaffrézou J.P. Biochim. Biophys. Acta.. 1999; 1438: 1-17Google Scholar, 16Mathias S. Peña L.A. Kolesnick R.N. Biochem. J... 1998; 335: 465-480Google Scholar, 17Igarashi Y. J. Biochem. ( Tokyo ).. 1997; 122: 1080-1087Google Scholar, 18Merrill Jr., A.H. Schmelz E.M. Dillehay D.L. Spiegel S. Shayman J.A. Schroeder J.J. Riley R.T. Voss K.A. Wang E. Toxicol. Appl. Pharmacol... 1997; 142: 208-225Google Scholar, 19Okazaki T. Bell R.M. Hannun Y.A. J. Biol. Chem... 1989; 264: 19076-19080Google Scholar, 20Jarvis W.D. Fornari Jr., F.A. Auer K.L. Freemerman A.J. Szabo E. Birrer M.J. Johnson C.R. Barbour S.E. Dent P. Grant S. Mol. Pharmacol... 1997; 52: 935-947Google Scholar, 21Cuvillier O. Pirianov G. Kleuser B. Vanek P.G. Coso O.A. Gutkind J.S. Spiegel S. Nature.. 1996; 381: 800-803Google Scholar, 22Kawano T. Cui J. Koezuka Y. Toura I. Kaneko Y. Motoki K. Ueno H. Nakagawa R. Sato H. Kondo E. Koseki H. Taniguchi M. Science.. 1997; 278: 1626-1629Google Scholar). This pathway is referred to as the SM cycle, SM-ceramide pathway (11Okazaki T. Kondo T. Kitano T. Tashima M. Cell. Signal... 1998; 10: 685-692Google Scholar, 17Igarashi Y. J. Biochem. ( Tokyo ).. 1997; 122: 1080-1087Google Scholar), or the SMase pathway. To date, at least seven classes of mammalian SMases have been described, differing in subcellular location, pH optimum, cation dependence, and roles in cell regulation (11Okazaki T. Kondo T. Kitano T. Tashima M. Cell. Signal... 1998; 10: 685-692Google Scholar, 15Levade T. Jaffrézou J.P. Biochim. Biophys. Acta.. 1999; 1438: 1-17Google Scholar, 17Igarashi Y. J. Biochem. ( Tokyo ).. 1997; 122: 1080-1087Google Scholar). Two forms of SMases, distinguishable by their pH optima, are capable of initiating signal transduction (12Verheij M. Bose R. Lin X.H. Yao B. Jarvis W.D. Grant S. Birrer M.J. Szabo E. Zon L.I. Kyriakis J.M. Haimovitz-Friedman A. Fuks Z. Kolesnick R.N. Nature.. 1996; 380: 75-79Google Scholar). The acid SMase (pH optimum 4.5–5.0) is activated in cells exposed to ionizing radiation, FAS, CD28, interleukin-1, or TNFα (23Santana P. Peña L.A. Haimovitz-Friedman A. Martin S. Green D. McLoughlin M. Cordon-Cardo C. Schuchman E.H. Fuks Z. Kolesnick R. Cell.. 1996; 86: 189-199Google Scholar, 24Grassmé H. Gulbins E. Brenner B. Ferlinz K. Sandhoff K. Harzer K. Lang F. Meyer T.F. Cell.. 1997; 91: 605-615Google Scholar). The neutral SMase (pH optimum 7.4) has been implicated in mediating apoptosis in cells exposed to serum starvation, anti-FAS antibody, vitamin D, TNFα, or cytosine arabinoside (25Adam D. Wiegmann K. Adam-Klages S. Ruff A. Krönke M. J. Biol. Chem... 1996; 271: 14617-14622Google Scholar, 26Adam-Klages S. Adam D. Wiegmann K. Struve S. Kolanus W. Schneider-Mergener J. Krönke M. Cell.. 1996; 86: 937-947Google Scholar). Solar ultraviolet (UV) radiation is known to be one of the most common environmental carcinogens leading to skin cancer (27De Laat J.M. De Gruijl F.R. Cancer Surv... 1996; 26: 173-191Google Scholar, 28Scharffetter-Kochanek K. Wlaschek M. Brenneisen P. Schauen M. Blaudschun R. Wenk J. Biol. Chem... 1997; 378: 1247-1257Google Scholar, 29Slominski A. Pawelek J. Clin. Dermatol... 1998; 16: 503-515Google Scholar). Also, UV exposure induces apoptosis in cultured cells and in vivo(29Slominski A. Pawelek J. Clin. Dermatol... 1998; 16: 503-515Google Scholar, 30Tada-Oikawa S. Oikawa S. Kawanishi S. Biochem. Biophys. Res. Commun... 1998; 247: 693-696Google Scholar). Most research has focused on the UVC (200–290 nm) and UVB (290–320 nm) induction of apoptosis (31Beissert S. Granstein R.D. Crit. Rev. Biochem. Mol. Biol... 1996; 31: 381-404Google Scholar, 32Bender K. Blattner C. Knebel A. Iordanov M. Herrlich P. Rahmsdorf H.J. J. Photochem. Photobiol. B Biol... 1997; 37: 1-17Google Scholar), and little is known about the effect of UVA (320–400 nm), which comprises over 90% of the solar UV. Here, we observe that acid SMase is activated by UVA, and we provide evidence that UVA-induced apoptosis is dependent on acid SMase activity. Exogenous sphingomyelinase and its product, ceramide, also induce apoptosis independent of activation of intracellular SMase, but induction of apoptosis by SM is dependent on the SMase activity. Our data further indicate that UVA-induced apoptosis may occur through activation of JNKs via the SMase pathway. DISCUSSIONExposure to UV radiation can cause cell cycle arrest (50Herrlich P. Blattner C. Knebel A. Bender K. Rahmsdorf H.J. Biol. Chem... 1997; 378: 1217-1229Google Scholar, 51Tyrrell R.M. Feige U. Morimoto R.I. Yahara I. Polla B. (UV activation of mammalian stress proteins) in Stress-inducible cellular Responses.Birkäuser Verlag. 1996; : 255-271Google Scholar), alterations in mitochondrial membrane permeability (30Tada-Oikawa S. Oikawa S. Kawanishi S. Biochem. Biophys. Res. Commun... 1998; 247: 693-696Google Scholar), and cell death by necrosis or apoptosis (42Obeid L.M. Hannun Y.A. J. Cell. Biochem... 1995; 58: 191-198Google Scholar, 51Tyrrell R.M. Feige U. Morimoto R.I. Yahara I. Polla B. (UV activation of mammalian stress proteins) in Stress-inducible cellular Responses.Birkäuser Verlag. 1996; : 255-271Google Scholar). In the present study, we observed that UVA, like UVB and UVC, induced apoptosis in normal lymphoblast cells (JY) but not in MS1418 lymphoblasts from Niemann-Pick type D patients who have an inherited deficiency of acid SMase. UVA radiation is known to induce an array of stress proteins quite distinct from those induced by UVB or UVC (51Tyrrell R.M. Feige U. Morimoto R.I. Yahara I. Polla B. (UV activation of mammalian stress proteins) in Stress-inducible cellular Responses.Birkäuser Verlag. 1996; : 255-271Google Scholar). UVB and UVC were clearly shown to mimic growth factor responses and stimulate signal transduction including the SMase pathway (12Verheij M. Bose R. Lin X.H. Yao B. Jarvis W.D. Grant S. Birrer M.J. Szabo E. Zon L.I. Kyriakis J.M. Haimovitz-Friedman A. Fuks Z. Kolesnick R.N. Nature.. 1996; 380: 75-79Google Scholar, 20Jarvis W.D. Fornari Jr., F.A. Auer K.L. Freemerman A.J. Szabo E. Birrer M.J. Johnson C.R. Barbour S.E. Dent P. Grant S. Mol. Pharmacol... 1997; 52: 935-947Google Scholar, 32Bender K. Blattner C. Knebel A. Iordanov M. Herrlich P. Rahmsdorf H.J. J. Photochem. Photobiol. B Biol... 1997; 37: 1-17Google Scholar, 43Huang C. Ma W.-Y Ding M. Bowden G.T. Dong Z. J. Biol. Chem... 1997; 272: 27753-27757Google Scholar). Noncytotoxic exposure to UVA can also up-regulate several signal molecules (32Bender K. Blattner C. Knebel A. Iordanov M. Herrlich P. Rahmsdorf H.J. J. Photochem. Photobiol. B Biol... 1997; 37: 1-17Google Scholar, 42Obeid L.M. Hannun Y.A. J. Cell. Biochem... 1995; 58: 191-198Google Scholar), but the role of the SMase pathway in UVA-induced apoptosis is not well understood.In this study, we provide evidence that UVA, UVB, and UVC can all activate acid SMase (Fig. 1) and lead to an increase in ceramide levels (Fig. 2) in normal JY cells. Analysis of DNA fragmentation laddering and morphological changes in apoptotic cells showed that sublethal doses of UVA (20–80 kJ/m2), similar to lethal doses of UVB and UVC, can also induce apoptosis in normal JY cells, but UVA-induced apoptosis is prevented in acid SMase-deficient MS1418 cells. On the other hand, the difference in UVB- or UVC-induced apoptosis was less apparent in normal and SMase-deficient cells. These results suggest that UVA-induced apoptosis occurs through activation of acid SMase, whereas UVB- or UVC-induced apoptosis occurs through SMase-independent pathways. However, additional pathways involved in mediating UVA-induced apoptosis cannot be disregarded, including neutral SMase (25Adam D. Wiegmann K. Adam-Klages S. Ruff A. Krönke M. J. Biol. Chem... 1996; 271: 14617-14622Google Scholar, 26Adam-Klages S. Adam D. Wiegmann K. Struve S. Kolanus W. Schneider-Mergener J. Krönke M. Cell.. 1996; 86: 937-947Google Scholar), stress-activated protein kinase/JNKs (12Verheij M. Bose R. Lin X.H. Yao B. Jarvis W.D. Grant S. Birrer M.J. Szabo E. Zon L.I. Kyriakis J.M. Haimovitz-Friedman A. Fuks Z. Kolesnick R.N. Nature.. 1996; 380: 75-79Google Scholar), caspases (30Tada-Oikawa S. Oikawa S. Kawanishi S. Biochem. Biophys. Res. Commun... 1998; 247: 693-696Google Scholar), and mitochondria (30Tada-Oikawa S. Oikawa S. Kawanishi S. Biochem. Biophys. Res. Commun... 1998; 247: 693-696Google Scholar), because UVA-induced apoptosis is not completely blocked in acid SMase-deficient MS1418 cells.Klotz et al. (44Klotz L.-O. Pelliuex C. Briviba K. Pierlot C. Aubry J.-M. Sies H. Eur. J. Biochem... 1999; 260: 917-922Google Scholar) reported that UVA did not activate ERKs, and we found that UVA, like UVB and UVC, did not induce phosphorylation (Fig. 6) and activation (data not shown) of ERKs in SMase-normal JY cells. However, UVA strongly induced ERKs phosphorylation in SMase-deficient MS1418 cells suggesting that in the absence of SMase, UVA may stimulate alternate pathways (e.g. ERKs) that may protect against UVA-induced apoptosis in these cells. We also found that UVA, as well as UVB and UVC, induced phosphorylation and activation of p38 kinase (Fig. 7). However, UVA-induced phosphorylation of p38 kinase appeared to be less in normal SMase JY cells compared with SMase-deficient MS1418 cells, whereas UVB- or UVC-induced phosphorylation was similar between the two cell lines (Fig. 7). Neither inhibition of UVA-induced ERKs activation by PD98059 (48Deak M. Clifton A.D. Lucocq J.M. Alessi D.R. EMBO J... 1998; 17: 4426-4441Google Scholar) nor inhibition of p38 kinase activation by SB202190 (49Blair A.S. Hajduch E. Litherland G.J. Hundal H.S. J. Biol. Chem... 1999; 274: 36293-36299Google Scholar) blocked UVA-induced apoptosis in normal JY cells (Fig. 8, left panel). However, MS1418 cells pretreated with PD98059 or SB202190 now showed typical apoptosis following UVA irradiation (Fig. 8,right panel). These results further confirm that activation and phosphorylation of ERKs and p38 kinase do not appear to be involved in UVA-induced apoptosis in normal JY cells, but in the absence of SMase, UVA irradiation may induce ERKs and p38 kinase leading to inhibition of apoptosis.Acid SMase was previously shown to be involved in UVB- and UVC-induced activation of JNKs (43Huang C. Ma W.-Y Ding M. Bowden G.T. Dong Z. J. Biol. Chem... 1997; 272: 27753-27757Google Scholar). In the present study, we observed that UVA, like UVB and UVC, induced phosphorylation of JNKs in acid SMase normal JY cells and that the phosphorylation of JNKs was markedly inhibited in SMase-deficient cells (Fig. 5). However, TPA-induced JNKs phosphorylation was not different between the two cell lines. These results suggest that UVA-, UVB-, or UVC-, but not TPA-induced phosphorylation and activation of JNKs, is acid SMase-dependent. In addition, we observed that UVA-induced apoptosis was completely blocked in jnk1−/− andjnk2−/− cells (Fig. 9 B) further indicating that JNKs are a downstream kinase family of the acid SMase pathway (45Ichijo H. Nishida E. Irie K. ten Dijke P. Saitoh M. Moriguchi T. Takagi M. Matsumoto K. Miyazono K. Gotoh Y. Science.. 1997; 275: 90-94Google Scholar, 46Xia Z. Dickens M. Raingeaud J. Davis R.J. Greenberg M.E. Science.. 1995; 270: 1326-1331Google Scholar). Overall, these results strongly suggest that UVA-induced apoptosis in normal SMase JY cells occurs primarily through activation of JNKs via the acid SMase pathway. Exposure to UV radiation can cause cell cycle arrest (50Herrlich P. Blattner C. Knebel A. Bender K. Rahmsdorf H.J. Biol. Chem... 1997; 378: 1217-1229Google Scholar, 51Tyrrell R.M. Feige U. Morimoto R.I. Yahara I. Polla B. (UV activation of mammalian stress proteins) in Stress-inducible cellular Responses.Birkäuser Verlag. 1996; : 255-271Google Scholar), alterations in mitochondrial membrane permeability (30Tada-Oikawa S. Oikawa S. Kawanishi S. Biochem. Biophys. Res. Commun... 1998; 247: 693-696Google Scholar), and cell death by necrosis or apoptosis (42Obeid L.M. Hannun Y.A. J. Cell. Biochem... 1995; 58: 191-198Google Scholar, 51Tyrrell R.M. Feige U. Morimoto R.I. Yahara I. Polla B. (UV activation of mammalian stress proteins) in Stress-inducible cellular Responses.Birkäuser Verlag. 1996; : 255-271Google Scholar). In the present study, we observed that UVA, like UVB and UVC, induced apoptosis in normal lymphoblast cells (JY) but not in MS1418 lymphoblasts from Niemann-Pick type D patients who have an inherited deficiency of acid SMase. UVA radiation is known to induce an array of stress proteins quite distinct from those induced by UVB or UVC (51Tyrrell R.M. Feige U. Morimoto R.I. Yahara I. Polla B. (UV activation of mammalian stress proteins) in Stress-inducible cellular Responses.Birkäuser Verlag. 1996; : 255-271Google Scholar). UVB and UVC were clearly shown to mimic growth factor responses and stimulate signal transduction including the SMase pathway (12Verheij M. Bose R. Lin X.H. Yao B. Jarvis W.D. Grant S. Birrer M.J. Szabo E. Zon L.I. Kyriakis J.M. Haimovitz-Friedman A. Fuks Z. Kolesnick R.N. Nature.. 1996; 380: 75-79Google Scholar, 20Jarvis W.D. Fornari Jr., F.A. Auer K.L. Freemerman A.J. Szabo E. Birrer M.J. Johnson C.R. Barbour S.E. Dent P. Grant S. Mol. Pharmacol... 1997; 52: 935-947Google Scholar, 32Bender K. Blattner C. Knebel A. Iordanov M. Herrlich P. Rahmsdorf H.J. J. Photochem. Photobiol. B Biol... 1997; 37: 1-17Google Scholar, 43Huang C. Ma W.-Y Ding M. Bowden G.T. Dong Z. J. Biol. Chem... 1997; 272: 27753-27757Google Scholar). Noncytotoxic exposure to UVA can also up-regulate several signal molecules (32Bender K. Blattner C. Knebel A. Iordanov M. Herrlich P. Rahmsdorf H.J. J. Photochem. Photobiol. B Biol... 1997; 37: 1-17Google Scholar, 42Obeid L.M. Hannun Y.A. J. Cell. Biochem... 1995; 58: 191-198Google Scholar), but the role of the SMase pathway in UVA-induced apoptosis is not well understood. In this study, we provide evidence that UVA, UVB, and UVC can all activate acid SMase (Fig. 1) and lead to an increase in ceramide levels (Fig. 2) in normal JY cells. Analysis of DNA fragmentation laddering and morphological changes in apoptotic cells showed that sublethal doses of UVA (20–80 kJ/m2), similar to lethal doses of UVB and UVC, can also induce apoptosis in normal JY cells, but UVA-induced apoptosis is prevented in acid SMase-deficient MS1418 cells. On the other hand, the difference in UVB- or UVC-induced apoptosis was less apparent in normal and SMase-deficient cells. These results suggest that UVA-induced apoptosis occurs through activation of acid SMase, whereas UVB- or UVC-induced apoptosis occurs through SMase-independent pathways. However, additional pathways involved in mediating UVA-induced apoptosis cannot be disregarded, including neutral SMase (25Adam D. Wiegmann K. Adam-Klages S. Ruff A. Krönke M. J. Biol. Chem... 1996; 271: 14617-14622Google Scholar, 26Adam-Klages S. Adam D. Wiegmann K. Struve S. Kolanus W. Schneider-Mergener J. Krönke M. Cell.. 1996; 86: 937-947Google Scholar), stress-activated protein kinase/JNKs (12Verheij M. Bose R. Lin X.H. Yao B. Jarvis W.D. Grant S. Birrer M.J. Szabo E. Zon L.I. Kyriakis J.M. Haimovitz-Friedman A. Fuks Z. Kolesnick R.N. Nature.. 1996; 380: 75-79Google Scholar), caspases (30Tada-Oikawa S. Oikawa S. Kawanishi S. Biochem. Biophys. Res. Commun... 1998; 247: 693-696Google Scholar), and mitochondria (30Tada-Oikawa S. Oikawa S. Kawanishi S. Biochem. Biophys. Res. Commun... 1998; 247: 693-696Google Scholar), because UVA-induced apoptosis is not completely blocked in acid SMase-deficient MS1418 cells. Klotz et al. (44Klotz L.-O. Pelliuex C. Briviba K. Pierlot C. Aubry J.-M. Sies H. Eur. J. Biochem... 1999; 260: 917-922Google Scholar) reported that UVA did not activate ERKs, and we found that UVA, like UVB and UVC, did not induce phosphorylation (Fig. 6) and activation (data not shown) of ERKs in SMase-normal JY cells. However, UVA strongly induced ERKs phosphorylation in SMase-deficient MS1418 cells suggesting that in the absence of SMase, UVA may stimulate alternate pathways (e.g. ERKs) that may protect against UVA-induced apoptosis in these cells. We also found that UVA, as well as UVB and UVC, induced phosphorylation and activation of p38 kinase (Fig. 7). However, UVA-induced phosphorylation of p38 kinase appeared to be less in normal SMase JY cells compared with SMase-deficient MS1418 cells, whereas UVB- or UVC-induced phosphorylation was similar between the two cell lines (Fig. 7). Neither inhibition of UVA-induced ERKs activation by PD98059 (48Deak M. Clifton A.D. Lucocq J.M. Alessi D.R. EMBO J... 1998; 17: 4426-4441Google Scholar) nor inhibition of p38 kinase activation by SB202190 (49Blair A.S. Hajduch E. Litherland G.J. Hundal H.S. J. Biol. Chem... 1999; 274: 36293-36299Google Scholar) blocked UVA-induced apoptosis in normal JY cells (Fig. 8, left panel). However, MS1418 cells pretreated with PD98059 or SB202190 now showed typical apoptosis following UVA irradiation (Fig. 8,right panel). These results further confirm that activation and phosphorylation of ERKs and p38 kinase do not appear to be involved in UVA-induced apoptosis in normal JY cells, but in the absence of SMase, UVA irradiation may induce ERKs and p38 kinase leading to inhibition of apoptosis. Acid SMase was previously shown to be involved in UVB- and UVC-induced activation of JNKs (43Huang C. Ma W.-Y Ding M. Bowden G.T. Dong Z. J. Biol. Chem... 1997; 272: 27753-27757Google Scholar). In the present study, we observed that UVA, like UVB and UVC, induced phosphorylation of JNKs in acid SMase normal JY cells and that the phosphorylation of JNKs was markedly inhibited in SMase-deficient cells (Fig. 5). However, TPA-induced JNKs phosphorylation was not different between the two cell lines. These results suggest that UVA-, UVB-, or UVC-, but not TPA-induced phosphorylation and activation of JNKs, is acid SMase-dependent. In addition, we observed that UVA-induced apoptosis was completely blocked in jnk1−/− andjnk2−/− cells (Fig. 9 B) further indicating that JNKs are a downstream kinase family of the acid SMase pathway (45Ichijo H. Nishida E. Irie K. ten Dijke P. Saitoh M. Moriguchi T. Takagi M. Matsumoto K. Miyazono K. Gotoh Y. Science.. 1997; 275: 90-94Google Scholar, 46Xia Z. Dickens M. Raingeaud J. Davis R.J. Greenberg M.E. Science.. 1995; 270: 1326-1331Google Scholar). Overall, these results strongly suggest that UVA-induced apoptosis in normal SMase JY cells occurs primarily through activation of JNKs via the acid SMase pathway. We thank Drs. Xun Li and Zbigniew Darzynkiewicz at the Cancer Research Institute and Department of Pathology, New York Medical College, Valhalla, NY, for providing some details on a direct DNA strand break labeling (36Li X. Traganos F. Melamed M.R. Darzynkiewicz Z. Cytometry.. 1995; 20: 172-180Google Scholar, 37Li X. Melamed M.R. Darzynkiewicz Z. Exp. Cell Res... 1996; 222: 28-37Google Scholar). We also thank Drs. Nanyue Chen and Qing-Bai She for their help on the assays for the DNA fragmentation ladder assay; Randy Krebsbach for assistance with the ceramide assays, and Andria Hansen for secretarial assistance. sphingomyelin sphingomyelinase ultraviolet light A Dulbecco's modified Eagle's medium fetal bovine serum dimethyl sulfoxide phosphate-buffered saline mitogen-activated protein kinases extracellular signal-regulated kinases c-Jun N-terminal kinases p38 MAPK or p38 kinases 12-O-tetradecanoylphorbol-13-acetate transforming growth factor-α
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