De Novo Ceramide Synthesis Participates in the Ultraviolet B Irradiation-Induced Apoptosis in Undifferentiated Cultured Human Keratinocytes
2003; Elsevier BV; Volume: 120; Issue: 4 Linguagem: Inglês
10.1046/j.1523-1747.2003.12098.x
ISSN1523-1747
AutoresYoshikazu Uchida, Anna Di Nardo, Vanessa J. Collins, Peter M. Elias, Walter M. Holleran,
Tópico(s)Bioactive Compounds and Antitumor Agents
ResumoUltraviolet irradiation is a major environmental cause of skin cancers, whereas ultraviolet-induced DNA repair and apoptosis are defense mechanisms that rescue and/or protect keratinocytes from this risk. Multiple pathways are involved in ultraviolet-induced keratinocyte apoptosis, including activation of p38-mitogen-activated protein kinase, protein kinase C, and CD95, each of which are associated with caspase activation. Alternatively, ceramides could serve as ultraviolet-induced, second messenger lipids, because they induce cell cycle arrest and apoptosis in a variety of cell types, including keratinocytes. We investigated the role of ceramide versus caspase, and the responsible pathway for ceramide generation in ultraviolet B-induced apoptosis of cultured normal human keratinocytes maintained in low calcium (0.07 mM) medium. Ultraviolet B (40 mJ per cm2) significantly inhibited cultured normal human keratinocyte proliferation, assessed as [3H-methyl]thymidine-thymidine incorporation into DNA, 2 h after irradiation. Terminal nick deoxynucleotide end-labeling-positive apoptotic cells (14.8% at 24 h and 34.4% at 48 h) and trypan blue-positive apoptotic cells (8.4% at 24 h and 28.6% at 48 h) became evident in a time-dependent manner after ultraviolet B irradiation, in parallel with activation of caspase-3. The ceramide content of irradiated cultured normal human keratinocytes increased significantly by 8 h, whereas glucosylceramide only modestly increased, and sphingomyelin content remained unaltered. Metabolic studies with radiolabeled serine, palmitic acid, and phosphorylcholine revealed that the ultraviolet B-induced increase in ceramide results primarily from increased de novo synthesis rather than accelerated sphingomyelin hydrolysis. Increased ceramide synthesis, in turn, could be attributed to increased activity of ceramide synthase (i.e., 1.7-fold increase 8 h after ultraviolet B irradiation), whereas serine palmitoyltransferase activity did not change. Both fumonisin B1, an inhibitor of ceramide synthase, and ISP-1, myriocin an inhibitor of serine palmitoyltransferase, significantly attenuated the ultraviolet B-induced apoptosis in a caspase-3-independent fashion, whereas co-incubation with a caspase-3 inhibitor (Ac-DEVD-chloromethyl-ketone) further attenuated the ultraviolet B-induced apoptosis. Thus, increased de novo ceramide synthesis signals ultraviolet B-induced apoptosis, by a pathway independent of, but in concert with, caspase-3 activation. Ultraviolet irradiation is a major environmental cause of skin cancers, whereas ultraviolet-induced DNA repair and apoptosis are defense mechanisms that rescue and/or protect keratinocytes from this risk. Multiple pathways are involved in ultraviolet-induced keratinocyte apoptosis, including activation of p38-mitogen-activated protein kinase, protein kinase C, and CD95, each of which are associated with caspase activation. Alternatively, ceramides could serve as ultraviolet-induced, second messenger lipids, because they induce cell cycle arrest and apoptosis in a variety of cell types, including keratinocytes. We investigated the role of ceramide versus caspase, and the responsible pathway for ceramide generation in ultraviolet B-induced apoptosis of cultured normal human keratinocytes maintained in low calcium (0.07 mM) medium. Ultraviolet B (40 mJ per cm2) significantly inhibited cultured normal human keratinocyte proliferation, assessed as [3H-methyl]thymidine-thymidine incorporation into DNA, 2 h after irradiation. Terminal nick deoxynucleotide end-labeling-positive apoptotic cells (14.8% at 24 h and 34.4% at 48 h) and trypan blue-positive apoptotic cells (8.4% at 24 h and 28.6% at 48 h) became evident in a time-dependent manner after ultraviolet B irradiation, in parallel with activation of caspase-3. The ceramide content of irradiated cultured normal human keratinocytes increased significantly by 8 h, whereas glucosylceramide only modestly increased, and sphingomyelin content remained unaltered. Metabolic studies with radiolabeled serine, palmitic acid, and phosphorylcholine revealed that the ultraviolet B-induced increase in ceramide results primarily from increased de novo synthesis rather than accelerated sphingomyelin hydrolysis. Increased ceramide synthesis, in turn, could be attributed to increased activity of ceramide synthase (i.e., 1.7-fold increase 8 h after ultraviolet B irradiation), whereas serine palmitoyltransferase activity did not change. Both fumonisin B1, an inhibitor of ceramide synthase, and ISP-1, myriocin an inhibitor of serine palmitoyltransferase, significantly attenuated the ultraviolet B-induced apoptosis in a caspase-3-independent fashion, whereas co-incubation with a caspase-3 inhibitor (Ac-DEVD-chloromethyl-ketone) further attenuated the ultraviolet B-induced apoptosis. Thus, increased de novo ceramide synthesis signals ultraviolet B-induced apoptosis, by a pathway independent of, but in concert with, caspase-3 activation. cultured human keratinocytes serine palmitoyltransferase high-performance thin layer chromatography; UV, ultraviolet Ultraviolet (UV) irradiation is a major cause of epidermal inflammation, immunosuppression, altered epidermal permeability barrier function, premature aging, dyspigmentation, and the development of both nonmelanoma and melanoma skin cancers (Fisher et al., 1996Fisher G.J. Datta S.C. Talwar H.S. Wang Z.Q. Varani J. Kang S. Voorhees J.J. 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Previous studies have demonstrated that multiple signaling pathways are associated with UV-induced apoptosis. Direct activation of CD95, independent of CD95 ligand, by UVB causes cell death in a spontaneous transformed keratinocyte cell line (HaCaT) (Aragane et al., 1998Aragane Y. Kulms D. Metze D. Wilkes G. Poppelmann B. Luger T.A. Schwarz T. Ultraviolet light induces apoptosis via direct activation of CD95 (Fas/APO-1) independently of its ligand CD95L.J Cell Biol. 1998; 140: 171-182Crossref PubMed Scopus (424) Google Scholar). UVB or UVC irradiation also activate caspases that induce keratinocyte apoptosis (Schwarz et al., 1995Schwarz A. Bhardwaj R. Aragane Y. et al.Ultraviolet-B-induced apoptosis of keratinocytes: Evidence for partial involvement of tumor necrosis factor-alpha in the formation of sunburn cells.J Invest Dermatol. 1995; 104: 922-927Crossref PubMed Scopus (245) Google Scholar;Leverkus et al., 1997Leverkus M. Yaar M. 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Protein kinase Cdelta is activated by caspase-dependent proteolysis during ultraviolet radiation-induced apoptosis of human keratinocytes.J Biol Chem. 1998; 273: 29995-30002Crossref PubMed Scopus (202) Google Scholar). Moreover, p38 mitogen-activated protein kinase activation of caspases results in apoptosis in CHK exposed to UVB (Shimizu et al., 1999Shimizu H. Banno Y. Sumi N. Naganawa T. Kitajima Y. Nozawa Y. Activation of p38 mitogen-activated protein kinase and caspases in UVB-induced apoptosis of human keratinocyte HaCaT cells.J Invest Dermatol. 1999; 112: 769-774Crossref PubMed Scopus (112) Google Scholar); however, the UVB induced p38 mitogen-activated protein kinase-stimulated cytochrome c release from mitochondria occurs via a caspase-independent mechanism in CHK (Assefa et al., 2000Assefa Z. Vantieghem A. Garmyn M. et al.p38 mitogen-activated protein kinase regulates a novel, caspase-independent pathway for the mitochondrial cytochrome c release in ultraviolet B radiation-induced apoptosis.J Biol Chem. 2000; 275: 21416-21421Crossref PubMed Scopus (138) Google Scholar). Over the past decade, elevated cellular ceramide levels have been linked to cellular stress resulting from increased levels of reactive oxygen species, cytokines, exposure to chemotherapeutic agents, irradiation, exogenous lipopolysaccharides, etc. (reviewed in:Mathias et al., 1998Mathias S. Pena L.A. Kolesnick R.N. Signal transduction of stress via ceramide.Biochem J. 1998; 335: 465-480Crossref PubMed Scopus (605) Google Scholar;Hannun and Luberto, 2000Hannun Y.A. Luberto C. Ceramide in the eukaryotic stress response.Trends Cell Biol. 2000; 10: 73-80Abstract Full Text Full Text PDF PubMed Scopus (634) Google Scholar;Andrieu-Abadie et al., 2001Andrieu-Abadie N. Gouaze V. Salvayre R. Levade T. Ceramide in apoptosis signaling: Relationship with oxidative stress.Free Radic Biol Med. 2001; 31: 717-728Crossref PubMed Scopus (209) Google Scholar). Increased ceramide, in turn, provokes cell cycle arrest and/or apoptosis in a variety of cell types. Stressors increase ceramide either by (i) acceleration of sphingomyelin hydrolysis resulting from activation of sphingomyelinase (Peña et al., 1997Peña L.A. Fuks Z. Kolesnick R. Stress-induced apoptosis and the sphingomyelin pathway.Biochem Pharmacol. 1997; 53: 615-621Crossref PubMed Scopus (213) Google Scholar;Billis et al., 1998Billis W. Fuks Z. Kolesnick R. Signaling in and regulation of ionizing radiation-induced apoptosis in endothelial cells.Recent Prog Horm Res. 1998; 53: 85-92PubMed Google Scholar;Singh et al., 1998Singh I. Pahan K. Khan M. Singh A.K. Cytokine-mediated induction of ceramide production is redox-sensitive. 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The downstream targets from such stress-induced increases in ceramide include ceramide activated protein kinase, stress-activated protein kinase/c-Jun N-terminal kinase, protein kinase C-α/ζ ceramide activated phosphatase, cathepsin D, phospholipase A2, phospholipase D, and nuclear factor-κB cells, depending on tissue type (Mathias et al., 1998Mathias S. Pena L.A. Kolesnick R.N. Signal transduction of stress via ceramide.Biochem J. 1998; 335: 465-480Crossref PubMed Scopus (605) Google Scholar;Perry and Hannun, 1998Perry D.K. Hannun Y.A. The role of ceramide in cell signaling.Biochim Biophys Acta. 1998; 1436: 233-243Crossref PubMed Scopus (289) Google Scholar;Hannun and Luberto, 2000Hannun Y.A. Luberto C. Ceramide in the eukaryotic stress response.Trends Cell Biol. 2000; 10: 73-80Abstract Full Text Full Text PDF PubMed Scopus (634) Google Scholar;Andrieu-Abadie et al., 2001Andrieu-Abadie N. Gouaze V. Salvayre R. Levade T. Ceramide in apoptosis signaling: Relationship with oxidative stress.Free Radic Biol Med. 2001; 31: 717-728Crossref PubMed Scopus (209) Google Scholar). Keratinocytes are one of several cell types that are susceptible to ceramide challenge (Geilen et al., 1997Geilen C.C. Wieder T. Orfanos C.E. Ceramide signalling: Regulatory role in cell proliferation, differentiation and apoptosis in human epidermis.Arch Dermatol Res. 1997; 289: 559-566Crossref PubMed Scopus (104) Google Scholar, Geilen et al., 2001Geilen C.C. Barz S. Bektas M. Sphingolipid signaling in epidermal homeostasis. Current knowledge and new therapeutic approaches in dermatology.Skin Pharmacol Appl Skin Physiol. 2001; 14: 261-271Crossref PubMed Scopus (29) Google Scholar). In fact, some studies have demonstrated that alterations in cellular ceramide level from either exogenous short chain ceramide (C2–C8 ceramide), or exogenous bacterial sphingomyelinase inhibit DNA synthesis (Wakita et al., 1994Wakita H. Tokura Y. Yagi H. Nishimura K. Furukawa F. Takigawa M. Keratinocyte differentiation is induced by cell-permeant ceramides and its proliferation is promoted by sphingosine.Arch Dermatol Res. 1994; 286: 350-354Crossref PubMed Scopus (88) Google Scholar;Uchida et al., 2002Uchida Y. Murata S. Schmuth M. et al.Glucosylceramide synthesis and synthase expression protect against ceramide-induced stress.J Lipid Res. 2002; 43: 1293-1302Crossref PubMed Google Scholar), and induce CHK apoptosis (Iwasaki-Bessho et al., 1998Iwasaki-Bessho Y. Banno Y. Yoshimura S. Ito Y. Kitajima Y. Nozawa Y. Decreased phospholipase D (PLD) activity in ceramide-induced apoptosis of human keratinocyte cell line HaCaT.J Invest Dermatol. 1998; 110: 376-382Abstract Full Text Full Text PDF PubMed Google Scholar;Wieder et al., 1998Wieder T. Orfanos C.E. Geilen C.C. Induction of ceramide-mediated apoptosis by the anticancer phospholipid analog, hexadecylphosphocholine.J Biol Chem. 1998; 273: 11025-11031Crossref PubMed Scopus (143) Google Scholar;Di Nardo et al., 2000Di Nardo A. Benassi L. Magnoni C. Cossarizza A. Seidenari S. Giannetti A. Ceramide 2 (N-acetyl sphingosine) is associated with reduction in Bcl-2 protein levels by Western blotting and with apoptosis in cultured human keratinocytes.Br J Dermatol. 2000; 143: 491-497Crossref PubMed Google Scholar). We also demonstrated that SPT is upregulated transcriptionally and ceramide synthesis is increased in CHK exposed to UVB (Farrell et al., 1998Farrell A.M. Uchida Y. Nagiec M.M. Harris I.R. Dickson R.C. Elias P.M. Holleran W.M. UVB irradiation up-regulates serine palmitoyltransferase in cultured human keratinocytes.J Lipid Res. 1998; 39: 2031-2038Abstract Full Text Full Text PDF PubMed Google Scholar). AlthoughShimizu et al., 1999Shimizu H. Banno Y. Sumi N. Naganawa T. Kitajima Y. Nozawa Y. Activation of p38 mitogen-activated protein kinase and caspases in UVB-induced apoptosis of human keratinocyte HaCaT cells.J Invest Dermatol. 1999; 112: 769-774Crossref PubMed Scopus (112) Google Scholar concluded that sphingomyelin hydrolysis to ceramide is not involved in the HaCaT keratinocyte response to UVB, a recent study also indicates that the stress/apoptosis signaling pathway in HaCaT cells differs from that in CHK (Chaturvedi et al., 2001Chaturvedi V. Qin J.Z. Denning M.F. Choubey D. Diaz M.O. Nickoloff B.J. Abnormal NF-kappaB signaling pathway with enhanced susceptibility to apoptosis in immortalized keratinocytes.J Dermatol Sci. 2001; 26: 67-78Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). As such, the involvement of de novo synthesized ceramide in UVB-induced CHK cell-cycle arrest and apoptosis has not been adequately addressed. In this study, we directly examined whether ceramide-mediated events are responsible, at least in part, for UVB-induced CHK apoptosis. We demonstrate that ceramide synthesis is activated early following UVB irradiation, an effect attributable to increased ceramide synthase activity, and that blockade of de novo ceramide synthesis inhibits the UVB-induced apoptosis. In addition, the ceramide-associated cell death pathway in response to UVB represents a caspase-3-independent apoptotic pathway in CHK. Ceramides, glucosylceramides, sphingomyelin, fumonisin B1, N-acetyl-Asp-Glu-Val-Asp-amido-4-methyl-coumarin (Ac-DEVD-AMC), and N-acetyl-DEVD-aldehyde (Ac-DEVD-CHO) were purchased from Sigma (St Louis, MO). ISP-1 was from Biomol Research Laboratories Inc. (Plymouth Meeting, PA). Myriocin Ac-DEVD-chloromethyl-ketone (Ac-DEVD-CMK) was from Calbiochem (San Diego, CA). Radiolabeled chemicals were from American Radiolabeled Chemicals Inc. (Arlington Heights, IL). High-performance thin-layer chromatography (HPTLC) plates (Silica Gel 60) were purchased from Merck (Darmstadt, Germany). Normal human keratinocytes were isolated from human neonatal foreskins by a modification of the method ofPittelkow and Scott, 1986Pittelkow M.R. Scott R.E. New techniques for the in vitro culture of human skin keratinocytes and perspectives on their use for grafting of patients with extensive burns.Mayo Clin Proc. 1986; 61: 771-777Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar. Cells were grown in keratinocyte grown medium, supplemented with bovine epidermal growth factor, bovine pituitary extract, insulin, hydrocortisone, and 0.07 mM calcium (Cascade Biologics, Portland, OR). Increasing calcium concentration stimulates keratinocyte differentiation and alters lipid metabolism, including sphingolipids (Ponec et al., 1988Ponec M. Weerheim A. Kempenaar J. Mommaas A.M. Nugteren D.H. Lipid composition of cultured human keratinocytes in relation to their differentiation.J Lipid Res. 1988; 29: 949-961Abstract Full Text PDF PubMed Google Scholar). Although fully differentiated keratinocytes generate a complex and heterogeneous group of ceramide and glucosylceramide molecular species (Lampe et al., 1983aLampe M.A. Burlingame A.L. Whitney J. Williams M.L. Brown B.E. Roitman E. Elias P.M. Human stratum corneum lipids: Characterization and regional variations.J Lipid Res. 1983; 24: 120-130Abstract Full Text PDF PubMed Google Scholar,Lampe et al., 1983bLampe M.A. Williams M.L. Elias P.M. Human epidermal lipids: Characterization and modulations during differentiation.J Lipid Res. 1983; 24: 131-140Abstract Full Text PDF PubMed Google Scholar;Wertz and Downing, 1983aWertz P.W. Downing D.T. Ceramides of pig epidermis: structure determination.J Lipid Res. 1983; 24: 759-765Abstract Full Text PDF PubMed Google Scholar, Wertz and Downing, 1983bWertz P.W. Downing D.T. Glucosylceramides of pig epidermis: Structure determination.J Lipid Res. 1983; 24: 1135-1139Abstract Full Text PDF PubMed Google Scholar), some of which have been implicated in promoting keratinocyte differentiation (Uchida et al., 1990Uchida Y. Iwamori M. Nagai Y. Activation of keratinization of keratinocytes from fetal rat skin with N-(O-linoleoyl) omega-hydroxy fatty acyl sphingosyl glucose (lipokeratinogenoside) as a marker of epidermis.Biochem Biophys Res Commun. 1990; 170: 162-168Crossref PubMed Scopus (24) Google Scholar), the involvement of these more complex sphingolipid species in cell cycle arrest, differentiation, and/or apoptosis remains unknown. As less-differentiated keratinocytes generate a limited number of ceramide and glucosylceramide species (Ponec et al., 1988Ponec M. Weerheim A. Kempenaar J. Mommaas A.M. Nugteren D.H. Lipid composition of cultured human keratinocytes in relation to their differentiation.J Lipid Res. 1988; 29: 949-961Abstract Full Text PDF PubMed Google Scholar), we have employed monolayer-undifferentiated cells cultured in 0.07 mM calcium for these studies. The cultures were maintained at 37°C under 5% CO2 in air, with medium changes performed three times weekly. UVB irradiation was performed as described previously (Farrell et al., 1998Farrell A.M. Uchida Y. Nagiec M.M. Harris I.R. Dickson R.C. Elias P.M. Holleran W.M. UVB irradiation up-regulates serine palmitoyltransferase in cultured human keratinocytes.J Lipid Res. 1998; 39: 2031-2038Abstract Full Text Full Text PDF PubMed Google Scholar). Briefly, cells were seeded (2-4×104 cells per ml) in two well glass chamber slides, 12 multiwell plates, 60 mm or 100 mm dishes and maintained to 80–100% confluence (monolayer cultures). The cells then were rinsed with phosphate-buffered saline containing 0.07 mM calcium, and treated with UVB (emission range 280–340 nm 305 nm max, FS 20/T12 bulbs, National Biological Co., Twinsburg, OH) in phosphate-buffered saline. UVB exposure was measured using a Goldilux Ultraviolet Radiometer (Oriel, Stratford, CT). Cells were exposed to a single dose of UVB (60 mJ per cm2) in most studies, unless indicated otherwise. Immediately after UVB irradiation, phosphate-buffered saline was replaced by culture medium, with or without an added sphingolipid synthetic inhibitor; i.e., fumonisin B1 or ISP-1. Total cellular DNA was determined by the method ofLabarca and Paigen, 1980Labarca C. Paigen K. A simple, rapid, and sensitive DNA assay procedure.Anal Biochem. 1980; 102: 344-352Crossref PubMed Scopus (4495) Google Scholar, using the fluorescent reagent, bis-benzimidazole. Keratinocyte growth was assessed as [3H-methyl]thymidine incorporation into DNA, as described previously (Farrell et al., 1998Farrell A.M. Uchida Y. Nagiec M.M. Harris I.R. Dickson R.C. Elias P.M. Holleran W.M. UVB irradiation up-regulates serine palmitoyltransferase in cultured human keratinocytes.J Lipid Res. 1998; 39: 2031-2038Abstract Full Text Full Text PDF PubMed Google Scholar). At appropriate time points following UVB treatment, cells were incubated with 1 μCi per ml of [3H-methyl]-thymidine for 1 h at 37°C, and the quantity of label in trichloroacetic acid-precipitable macromolecules was determined by liquid scintillation spectrometry. DNA synthesis data are expressed as methyl-[3H]-L-thymidine incorporated per mg DNA. Apoptotic cells were assessed both by terminal deoxynucleotidyltransferase-mediated digoxigenin-deoxyribonucleotide nick-end labeling (TUNEL assay) and by trypan blue-dye exclusion. TUNEL staining was performed using the ApopTag Apoptosis Detection Kit, following the manufacturer's protocol (Intergen Co., Purchase, NY). At least 200 cells were chosen at random on each slide to quantitate apoptosis. As a further measure of apoptosis, caspase-3 activities in cell lysates were determined using a modification of the method ofNicholson et al., 1995Nicholson D.W. Ali A. Thornberry N.A. et al.Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis.Nature. 1995; 376: 37-43Crossref PubMed Scopus (3696) Google Scholar. Briefly, cells were washed with ice-cold phosphate-buffered saline, incubated with 50 mM HEPES buffer, pH 7.4, containing 5 mM CHAPS, 5 mM dithiothreitol, and protease inhibitors (Protease Inhibitor Cocktail for Mammalian Cell and Tissue Extracts, Sigma), for 30 min on ice, followed by separation of supernatants by centrifugation (14,000×g at 4°C). Fifty micrograms of protein was incubated with Ac-DEVD-AMC with or without addition of the caspase-3 inhibitor, N-acetyl-Asp-Glu-Val-Asp-al (Ac-DEVD-CHO), for 30 min at 30°C. Fluorescent product (AMC) was measured by fluorescence spectrophotometry, and activities are reported as AMC generated per min per mg of protein. Protein content was determined by the BCA protein assay method (Pierce, Rockford, IL), using bovine serum albumin as the standard. Total lipids were extracted from CHK by the method ofBligh and Dyer, 1959Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can J Biochem Physiol. 1959; 37: 911-917Crossref PubMed Scopus (40255) Google Scholar, separated into individual lipid species by HPTLC, followed by quantification by scanning densitometry, as described previously (Holleran et al., 1991Holleran W.M. Man M.Q. Gao W.N. Menon G.K. Elias P.M. Feingold K.R. Sphingolipids are required for mammalian epidermal barrier function. Inhibition of sphingolipid synthesis delays barrier recovery after acute perturbation.J Clin Invest. 1991; 88: 1338-1345Crossref PubMed Scopus (204) Google Scholar). To assess sphingomyelin hydrolysis following UVB irradiation, cells (preconfluent) were incubated with [3H-methyl]choline chloride (1 μCi per ml) or [3H]serine (2.5 μCi per ml) for 2 d followed by incubation in a radioisotope-free medium for 16 h. Alternatively, to examine de novo lipid synthesis, cells were cultured with [3H]serine (1.5 μCi per ml) or [9,10-3H]palmitic acid (1.5 μCi per ml) for the final 3 h. Lipids were extracted and analyzed, as described previously (Farrell et al., 1998Farrell A.M. Uchida Y. Nagiec M.M. Harris I.R. Dickson R.C. Elias P.M. Holleran W.M. UVB irradiation up-regulates serine palmitoyltransferase in cultured human keratinocytes.J Lipid Res. 1998; 39: 2031-2038Abstract Full Text Full Text PDF PubMed Google Scholar), and radioisotope incorporated into each lipid fraction was measured by liquid scintillation spectroscopy. The assay employed to measure ceramide synthase activity was a modification of that ofBose et al., 1995Bose R. Verheij M. Haimovitz-Friedman A. Scotto K. Fuks Z. Kolesnick R. Ceramide synthase mediates daunorubicin-induced apoptosis: An alternative mechanism for generating death signals.Cell. 1995; 82: 405-414Abstract Full Text PDF PubMed Scopus (770) Google Scholar. Briefly, microsomal protein (75–100 μg) was incubated with 18 μM sphinganine and 70 μM [1-14C]palmitoyl-coenzyme A (spec. act. 10–15,000 dpm per nmol) at 37°C (60 min). The reaction product, palmitoylsphinganine, was extracted and isolated by HPTLC. The activity was expressed as palmitate equivalents incorporated into sphinganine per min per mg protein. SPT was assayed as previously described (Holleran et al., 1990Holleran W.M. Williams M.L. Gao W.N. Elias P.M. Serine-palmitoyl transferase activity in cultured human keratinocytes.J Lipid Res. 1990; 31: 1655-1661Abstract Full Text PDF PubMed Google Scholar;Weiss and Stoffel, 1997Weiss B. Stoffel W. Human and murine serine-palmitoyl-CoA transferase—cloning, expression and characterization of the key enzyme in sphingolipid synthesis.Eur J Biochem. 1997; 249: 239-247Crossref PubMed Scopus (143) Google Scholar). Microsomal protein (50–75 μg) was incubated with 50 μM pyridoxal phosphate, 300 μM palmitoyl-coenzyme A, 1.0 mM [G-3H] L-serine (spec. act. 45–50,000 dpm per nmol) at 37°C (15 min). The reaction product, 3-ketodihydrosphinganine was then reduced to sphinganine using sodium borohydride and extracted with chloroform–methanol (4:1, v/v) followed by isolation of sphinganine by HPTLC. Radioisotope incorporat
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