Glucosylceramide synthesis and synthase expression protect against ceramide-induced stress
2002; Elsevier BV; Volume: 43; Issue: 8 Linguagem: Inglês
10.1194/jlr.m100442-jlr200
ISSN1539-7262
AutoresYoshikazu Uchida, Satoru Murata, Matthias Schmuth, Martin J. Behne, Jeong Deuk Lee, Shinichi Ichikawa, Peter M. Elias, Yoshio Hirabayashi, Walter M. Holleran,
Tópico(s)Erythrocyte Function and Pathophysiology
ResumoCeramides (Cers), critical for epidermal barrier function, also can inhibit keratinocyte proliferation, while glucosylceramides (GlcCers) exert pro-mitogenic effects. Since alterations in Cer-to-GlcCer ratios appear to modulate cellular growth versus apoptosis, we assessed whether keratinocytes up-regulate GlcCer synthesis as a protective mechanism against Cer-induced stress. Exogenous sphingomyelinase (SMase) treatment of cultured human keratinocytes (CHK) initially decreased proliferation and cellular sphingomyelin (50–60% decrease; P < 0.001), and increased Cer levels (6.1- to 6.8-fold; P < 0.001). Proliferation recovered to normal rates by 24 h, in parallel with increased cellular GlcCer. Both GlcCer synthesis and GlcCer synthase activity increased significantly by 8 h following SMase (8.2- and 2.4-fold, respectively; P < 0.01 each vs. control), attributed to antecedent increases in GlcCer synthase mRNA and protein expression. Further evidence that GlcCer production is responsible for normalized CHK proliferation includes: a) attenuation of SMase-induced inhibition of proliferation by exogenous GlcCer; and b) enhancement of the SMase effect in cells cotreated with the GlcCer synthase inhibitor, PDMP (d-threo-1-phenyl-2(decanoylamino)-3-morpholino-1-propanol). Finally, although proliferation in immortalized, nontransformed keratinocytes (HaCaT) also was inhibited by SMase, HaCaT cells that overexpress GlcCer synthase were resistant to this effect.Thus, SMase-induced stress initiates a response in keratinocytes that includes upregulation of GlcCer synthesis which may protect against the deleterious effects of excess Cer. Ceramides (Cers), critical for epidermal barrier function, also can inhibit keratinocyte proliferation, while glucosylceramides (GlcCers) exert pro-mitogenic effects. Since alterations in Cer-to-GlcCer ratios appear to modulate cellular growth versus apoptosis, we assessed whether keratinocytes up-regulate GlcCer synthesis as a protective mechanism against Cer-induced stress. Exogenous sphingomyelinase (SMase) treatment of cultured human keratinocytes (CHK) initially decreased proliferation and cellular sphingomyelin (50–60% decrease; P < 0.001), and increased Cer levels (6.1- to 6.8-fold; P < 0.001). Proliferation recovered to normal rates by 24 h, in parallel with increased cellular GlcCer. Both GlcCer synthesis and GlcCer synthase activity increased significantly by 8 h following SMase (8.2- and 2.4-fold, respectively; P < 0.01 each vs. control), attributed to antecedent increases in GlcCer synthase mRNA and protein expression. Further evidence that GlcCer production is responsible for normalized CHK proliferation includes: a) attenuation of SMase-induced inhibition of proliferation by exogenous GlcCer; and b) enhancement of the SMase effect in cells cotreated with the GlcCer synthase inhibitor, PDMP (d-threo-1-phenyl-2(decanoylamino)-3-morpholino-1-propanol). Finally, although proliferation in immortalized, nontransformed keratinocytes (HaCaT) also was inhibited by SMase, HaCaT cells that overexpress GlcCer synthase were resistant to this effect. Thus, SMase-induced stress initiates a response in keratinocytes that includes upregulation of GlcCer synthesis which may protect against the deleterious effects of excess Cer. The epidermis of terrestrial mammals generates large quantities of ceramides (Cers) largely destined for the extracellular domains of the outermost epidermal layer, the stratum corneum, where they are critical components of the permeability barrier (1Holleran 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 (213) Google Scholar, 2Elias P.M. Menon G.K. Structural and lipid biochemical correlates of the epidermal permeability barrier.Adv. Lipid Res. 1991; 24: 1-26Crossref PubMed Google Scholar). In contrast, additional studies implicate Cer generation in the induction of cellular stress responses, including accelerated differentiation, apoptosis, and senescence (3Merrill Jr., A.H. Cell regulation by sphingosine and more complex sphingolipids.J. Bioenerg. Biomembr. 1991; 23: 83-104Crossref PubMed Scopus (214) Google Scholar, 4Hannun Y.A. Bell R.M. The sphingomyelin cycle: a prototypic sphingolipid signaling pathway.Adv. Lipid Res. 1993; 25: 27-41PubMed Google Scholar, 5Hakomori S. Igarashi Y. Gangliosides and glycosphingolipids as modulators of cell growth, adhesion, and transmembrane signaling.Adv. Lipid Res. 1993; 25: 147-162PubMed Google Scholar, 6Wakita 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 (94) Google Scholar). In an established stress model, increased Cer can result from activation of endogenous sphingomyelinase (SMase) following several types of cellular stress, such as γ irradiation, heat shock, and inflammation, leading to hydrolysis of sphingomyelin (SM) to Cer (7Kim M.Y. Linardic C. Obeid L. Hannun Y. Identification of sphingomyelin turnover as an effector mechanism for the action of tumor necrosis factor alpha and gamma-interferon. Specific role in cell differentiation.J. Biol. Chem. 1991; 266: 484-489Abstract Full Text PDF PubMed Google Scholar, 8Kolesnick R.N. Sphingomyelin and derivatives as cellular signals.Prog. Lipid Res. 1991; 30: 1-38Crossref PubMed Scopus (258) Google Scholar, 9Jayadev S. Liu B. Bielawska A.E. Lee J.Y. Nazaire F. Pushkareva M. Obeid L.M. Hannun Y.A. Role for ceramide in cell cycle arrest.J. Biol. Chem. 1995; 270: 2047-2052Abstract Full Text Full Text PDF PubMed Scopus (469) Google Scholar, 10Pronk G.J. Ramer K. Amiri P. Williams L.T. Requirement of an ICE-like protease for induction of apoptosis and ceramide generation by REAPER.Science. 1996; 271: 808-810Crossref PubMed Scopus (260) Google Scholar). Similarly, exogenous SMase induces increased Cer levels, resulting in cell-specific responses including apoptosis and differentiation (11Okazaki T. Bell R.M. Hannun Y.A. Sphingomyelin turnover induced by vitamin D3 in HL-60 cells. Role in cell differentiation.J. Biol. Chem. 1989; 264: 19076-19080Abstract Full Text PDF PubMed Google Scholar, 12Haimovitz-Friedman A. Kan C.C. Ehleiter D. Persaud R.S. McLoughlin M. Fuks Z. Kolesnick R.N. Ionizing radiation acts on cellular membranes to generate ceramide and initiate apoptosis.J. Exp. Med. 1994; 180: 525-535Crossref PubMed Scopus (851) Google Scholar, 13Santana P. Peña L.A. Haimovitz-Friedman A. Martin S. Green D. McLoughlin M. Cordon-Cardo C. Schuchman E.H. Fuks Z. Kolesnick R. Acid sphingomyelinase-deficient human lymphoblasts and mice are defective in radiation-induced apoptosis.Cell. 1996; 86: 189-199Abstract Full Text Full Text PDF PubMed Scopus (727) Google Scholar, 14Mathias S. Pena L.A. Kolesnick R.N. Signal transduction of stress via ceramide.Biochem. J. 1998; 335: 465-480Crossref PubMed Scopus (619) Google Scholar). Finally, increased de novo Cer synthesis from activation of either Cer synthase (15Bose 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 (784) Google Scholar, 16Liao W.C. Haimovitz-Friedman A. Persaud R.S. McLoughlin M. Ehleiter D. Zhang N. Gatei M. Lavin M. Kolesnick R. Fuks Z. Ataxia telangiectasia-mutated gene product inhibits DNA damage-induced apoptosis via ceramide synthase.J. Biol. Chem. 1999; 274: 17908-17917Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar) and/or serine palmitoyltransferase (17Perry D.K. Carton J. Shah A.K. Meredith F. Uhlinger D.J. Hannun Y.A. Serine palmitoyltransferase regulates de novo ceramide generation during etoposide-induced apoptosis.J. Biol. Chem. 2000; 275: 9078-9084Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar, 18Lehtonen J.Y. Horiuchi M. Daviet L. Akishita M. Dzau V.J. Activation of the de novo biosynthesis of sphingolipids mediates angiotensin II type 2 receptor-induced apoptosis.J. Biol. Chem. 1999; 274: 16901-16906Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 19Herget T. Esdar C. Oehrlein S.A. Heinrich M. Schütze S. Maelicke A. van Echten-Deckert G. Production of ceramides causes apoptosis during early neural differentiation in vitro.J. Biol. Chem. 2000; 275: 30344-30354Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar) can induce apoptosis in some cell types following exposure to stress. Cer generated by any of these mechanisms, in turn, appear to signal divergent downstream pathways, including activation of either a Cer-activated kinase, a cytosolic Cer-activated protein phosphatase, protein kinase C-zeta, and/or NF-kB [(20Mathias S. Dressler K.A. Kolesnick R.N. Characterization of a ceramide-activated protein kinase: stimulation by tumor necrosis factor alpha.Proc. Natl. Acad. Sci. USA. 1991; 88: 10009-10013Crossref PubMed Scopus (349) Google Scholar, 21Westwick J.K. Bielawska A.E. Dbaibo G. Hannun Y.A. Brenner D.A. Ceramide activates the stress-activated protein kinases.J. Biol. Chem. 1995; 270: 22689-22692Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar, 22Dobrowsky R.T. Hannun Y.A. Ceramide stimulates a cytosolic protein phosphatase.J. Biol. Chem. 1992; 267: 5048-5051Abstract Full Text PDF PubMed Google Scholar, 23Wolff R.A. Dobrowsky R.T. Bielawska A. Obeid L.M. Hannun Y.A. Role of ceramide-activated protein phosphatase in ceramide-mediated signal transduction.J. Biol. Chem. 1994; 269: 19605-19609Abstract Full Text PDF PubMed Google Scholar, 24Galadari S. Kishikawa K. Kamibayashi C. Mumby M.C. Hannun Y.A. Purification and characterization of ceramide-activated protein phosphatases.Biochemistry. 1998; 37: 11232-11238Crossref PubMed Scopus (72) Google Scholar, 25Machleidt T. Wiegmann K. Henkel T. Schütze S. Baeuerle P. Krönke M. Sphingomyelinase activates proteolytic I kappa B-alpha degradation in a cell-free system.J. Biol. Chem. 1994; 269: 13760-13765Abstract Full Text PDF PubMed Google Scholar); reviewed in (14Mathias S. Pena L.A. Kolesnick R.N. Signal transduction of stress via ceramide.Biochem. J. 1998; 335: 465-480Crossref PubMed Scopus (619) Google Scholar, 26Hannun Y.A. Luberto C. Ceramide in the eukaryotic stress response.Trends Cell Biol. 2000; 10: 73-80Abstract Full Text Full Text PDF PubMed Scopus (646) Google Scholar)]. In contrast to the growth-inhibitory effects of Cer, glucosylceramide (GlcCer) exert pro-mitogenic properties in a variety of tissues and cell types (27Datta S.C. Radin N.S. Stimulation of liver growth and DNA synthesis by glucosylceramide.Lipids. 1988; 23: 508-510Crossref PubMed Scopus (43) Google Scholar, 28Shayman J.A. Deshmukh G.D. Mahdiyoun S. Thomas T.P. Wu D. Barcelon F.S. Radin N.S. Modulation of renal epithelial cell growth by glucosylceramide. Association with protein kinase C, sphingosine, and diacylglycerol.J. Biol. Chem. 1991; 266: 22968-22974Abstract Full Text PDF PubMed Google Scholar, 29Marsh N.L. Elias P.M. Holleran W.M. Glucosylceramides stimulate murine epidermal hyperproliferation.J. Clin. Invest. 1995; 95: 2903-2909Crossref PubMed Scopus (73) Google Scholar, 30Marchell N.L. Uchida Y. Brown B.E. Elias P.M. Holleran W.M. Glucosylceramides stimulate mitogenesis in aged murine epidermis.J. Invest. Dermatol. 1998; 110: 383-387Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). For example, treatment of MDCK cells with a β-glucocerebrosidase inhibitor simultaneously increases cellular GlcCer levels and stimulates cellular proliferation (28Shayman J.A. Deshmukh G.D. Mahdiyoun S. Thomas T.P. Wu D. Barcelon F.S. Radin N.S. Modulation of renal epithelial cell growth by glucosylceramide. Association with protein kinase C, sphingosine, and diacylglycerol.J. Biol. Chem. 1991; 266: 22968-22974Abstract Full Text PDF PubMed Google Scholar). Increased endogenous GlcCer also may account, in part, for the organomegaly associated with β-glucocerebrosidase deficiency (Gaucher disease) (31Barranger J.A. Ginns E.I. Glucosylceramide lipidoses: Gaucher disease. McGraw-Hill, New York1989: 1677-1698Google Scholar). Furthermore, administration of exogenous GlcCer directly into either liver (27Datta S.C. Radin N.S. Stimulation of liver growth and DNA synthesis by glucosylceramide.Lipids. 1988; 23: 508-510Crossref PubMed Scopus (43) Google Scholar) or skin (29Marsh N.L. Elias P.M. Holleran W.M. Glucosylceramides stimulate murine epidermal hyperproliferation.J. Clin. Invest. 1995; 95: 2903-2909Crossref PubMed Scopus (73) Google Scholar, 30Marchell N.L. Uchida Y. Brown B.E. Elias P.M. Holleran W.M. Glucosylceramides stimulate mitogenesis in aged murine epidermis.J. Invest. Dermatol. 1998; 110: 383-387Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar) stimulates growth. These studies suggest either: 1) that Cer and GlcCer exert opposing effects on cellular growth; or 2) that GlcCer synthesis protects against the negative consequences of increased Cer. Accordingly, Hirabayashi et al. showed that exogenous SMase-induced increases in cellular Cer inhibit proliferation in UDP-glucose:Cer β-d-glucosyltransferase (GlcT-1 or GlcCer synthase)-deficient GM-95 cells, but not in GlcCer synthase-replete B16 melanoma cells (32Komori H. Ichikawa S. Hirabayashi Y. Ito M. Regulation of intracellular ceramide content in B16 melanoma cells. Biological implications of ceramide glycosylation.J. Biol. Chem. 1999; 274: 8981-8987Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Similarly, Cabot et al. showed that conversion of Cer to GlcCer confers resistance to chemotherapy-induced cytotoxicity, i.e., anthracycline-sensitive MCF-7 cells became resistant when induced to overexpress GlcCer synthase (33Liu Y.Y. Han T.Y. Giuliano A.E. Cabot M.C. Expression of glucosylceramide synthase, converting ceramide to glucosylceramide, confers adriamycin resistance in human breast cancer cells.J. Biol. Chem. 1999; 274: 1140-1146Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). Recently, we showed that GlcCer attenuates the growth inhibitory effect of Cer in cultured human keratinocytes (CHK) (Y. Uchida and W. Holleran, unpublished observations). Yet, overexpression of GlcCer synthase in Jurkat cells failed to protect cells from apoptosis induced by CD95, etoposide, or γ irradiation (34Tepper A.D. Diks S.H. van Blitterswijk W.J. Borst J. Glucosylceramide synthase does not attenuate the ceramide pool accumulating during apoptosis induced by CD95 or anti-cancer regimens.J. Biol. Chem. 2000; 275: 34810-34817Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Thus, a balance of Cer versus GlcCer appears to be critical for maintaining cellular homeostasis; i.e., between apoptosis/cytotoxicity versus proliferation/cell survival in a variety of cell types. To further explore whether diversion of Cer into GlcCer is a general, protective pathway, with important implications for cutaneous and extracutaneous tissue homeostasis, we investigated cellular responses to increased Cer using a well-established stress model, i.e., treatment of CHK with exogenous neutral SMase. SM levels decreased while Cer levels increased in keratinocytes following exposure to exogenous bacterial SMase, changes that correlated with inhibition of keratinocyte proliferation. Yet, DNA synthesis recovered concurrent with: a) increased GlcCer levels; b) increased GlcCer synthase expression; and c) overexpression of GlcCer synthase in cells exposed to SMase-induced stress. These results are consistent with the hypothesis that the Cer-to-GlcCer synthetic pathway helps to protect keratinocytes from Cer-induced stress. Bacterial SMase (both recombinant and from Staphylococcus aureus) were purchased from Funakoshi (Tokyo, Japan) and Sigma (St. Louis, MO), respectively. Conduritol B-epoxide (CBE) and N-octanoylsphingosine were obtained from Toronto Research Chemicals, Inc. (Toronto, Ontario, Canada) and Matreya Inc. (Philadelphia, PA), respectively. β-d-glucosyl-(N-acetyl)-sphingosine (GlcC2Cer) was prepared by acetylation of glucosylsphingosine (Sigma) with acetic anhydride and subsequent purification. Other lipids were from Sigma. d-threo-PDMP (d-threo-1-phenyl-2(decanoylamino)-3-morpholino-1-propanol) was a gift from Dr. Norman Radin (Emeritus; Mental Health Research Institute, University of Michigan, MI). [Methyl-3H] thymidine (85 Ci/mmole), l-[3H]serine (147 Ci/mmole), [3H]galactose (40–60 Ci/mmole), and [3H]UDP-glucose (60 Ci/mmole) were from American Radiolabeled Chemical Inc. (St. Louis, MO) and Amersham Life Sciences (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 the method of Pittelkow and Scott (35Pittelkow 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 (199) Google Scholar), as modified (36Pillai S. Bikle D.D. Hincenbergs M. Elias P.M. Biochemical and morphological characterization of growth and differentiation of normal human neonatal keratinocytes in a serum-free medium.J. Cell. Physiol. 1988; 134: 229-237Crossref PubMed Scopus (150) Google Scholar). Second-passage cells were grown in keratinocyte growth medium, supplemented with bovine epidermal growth factor, bovine pituitary extract, insulin, hydrocortisone, and 0.07 mM calcium (KGM; Clonetics, San Diego, CA; or Cascade Biologics, Portland, OR) to 80–95% confluence. Immortalized, nontransformed, low-passage number HaCaT cells, derived from human epidermis, were a gift from Dr. N. Fusenig (Heidelberg, Germany). The cultures were maintained at 37°C under 5% CO2 in air. GlcCer synthase (1.2 kB): pLNCX2 Retroviral vector or empty vector, were transfected into PT-67 cell-packaging cell line. HaCaT cells were incubated with virus-containing cultured cell supernatant in the presence of 8 μg/ml of polybrene 32°C for 24 h. Selection of transduced cells was performed following the addition of G418 (0.8 mg/ml). Cells were seeded in twelve or twenty four-well multiwell plates, cultured to 80–100% confluence, and then treated with SMase and/or GlcC2Cer. GlcC2Cer was complexed with fatty-acid-free BSA (1:1 molar ratio) prior to addition to the medium. Aqueous soluble SMase was dissolved directly in the medium. Stock solutions were prepared immediately prior to use. Total cellular DNA was assayed by the method of Labarca and Paigan (37Labarca C. Paigen K. A simple, rapid, and sensitive DNA assay procedure.Anal. Biochem. 1980; 102: 344-352Crossref PubMed Scopus (4552) Google Scholar). Keratinocyte growth was assessed by measuring [methyl-3H]thymidine incorporation into DNA. After appropriate incubations with the test compounds, cells were incubated with 1 μCi/ml of [3H]thymidine for 1 h at 37°C, and the quantity of label in TCA-precipitable macromolecules was determined by liquid scintillation spectroscopy. DNA synthesis was expressed as [3H]thymidine incorporated per microgram DNA. Lipid extracts were prepared from cellular homogenates by the method of Bligh and Dyer (38Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42828) Google Scholar). Separation of individual lipid species was achieved by HPTLC and quantitated by densitometry as previously described (1Holleran 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 (213) Google Scholar). GlcCers and Cers were separated first using chloroform-methanol-water (40:10:1, v/v/v) to 2.0 cm and to 5.0 cm, then chloroform-methanol-acetic acid (94:4:1, v/v) to 8.5 cm, followed by n-hexane-diethyl acetic acid (65:35:1, v/v/v) to the top of the plate. SM was separated using chloroform-methanol-acetic acid-water (50:30:8:4, v/v/v/v). To assess the fate of preformed sphingolipids, keratinocytes were incubated with [3H]serine (1 μCi/ml) for 2 days and washed with phosphate-buffered saline. Cells were incubated in fresh medium with or without SMase. To then examine de novo lipid synthesis, cells were cultured with SMase and then incubated with [3H]serine (1.5 μCi/ml) or [3H]galactose (2.0 μCi/ml) during the final 3 h of incubation. Lipids were extracted and analyzed as described above. Radioactivity in each lipid fraction was determined using a Beckman LS-1800 scintillation counter. Lipid synthesis was expressed as cpm per mg DNA. The assay for GlcCer synthase activity was performed according to the method of Shukla and Radin (39Shukla G.S. Radin N.S. Glucosyceramide synthase of mouse kidney: further characterization with an improved assay method.Arch. Biochem. Biophys. 1990; 283: 372-378Crossref PubMed Scopus (33) Google Scholar) with modification (40Chujor C.S. Feingold K.R. Elias P.M. Holleran W.M. Glucosylceramide synthase activity in murine epidermis: quantitation, localization, regulation, and requirement for barrier homeostasis.J. Lipid Res. 1998; 39: 277-285Abstract Full Text Full Text PDF PubMed Google Scholar). Keratinocyte homogenates (150–200 μg protein) were incubated with 100 μM [3H]UDP-glucose (Spec. Act., 20,000 dpm/nmole), 1 mM dithiothreitol, 2 mM EDTA, 10 mM MgCl2, 2.5 mM NADH, 20 mM Tris-HCl buffer, pH 7.4, and liposomal substrate (42.5 μg N-octanoylsphingosine, 250 μg dipalmitoylphosphatidylcholine, and 50 μg sulfatide) in a total volume of 0.2 ml at 32°C (60 min). The reaction was terminated by adding 500 μl of chloroform-methanol (1:1, v/v). The mixture was centrifuged for 5 min at 3,000 rpm. The lower organic phase was washed twice with water saturated with chloroform-methanol, and the incorporation determined by liquid scintillation spectrometry, as above. Each DNase I-treated total RNA sample was prepared from cells using the RN-easy mini kit and RNase-free DNase (Qiagen, Valencia, CA). cDNA was reverse transcribed from DNase I-treated total RNA with random hexamer using the SuperScript Preamplification System for First Strand cDNA Synthesis (GIBCO-BRL, Grand Island, MD). An aliquot of the first strand cDNA was used to PCR amplify both the 400 bp GlcCer synthase and the 801 bp GAPDH gene fragments using Taq DNA polymerase (Qiagen). The following sets of primer pairs were used: GlcCer synthase-F1 (derived from GlcT-1) (42Farrell 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): 144,5′-CAAGCTCCCAGGTGTCTCTCTT-3′, 165; GlcCer synthase-R1 (derived from GlcT-1): 543, 5′-AGCAAAGCCCTGTCTGTCTGCT-3′, 522; GPDH-F1 (derived from GAPDH): 317, 5′-AGAAGGCTGGGGCTCATTTGCA-3′, 338; GPDH-R1 (derived from GAPDH): 1117, 5′-AGGAGGGGAGATTCAGTGTGGT-3′, 1096. Confirmation of PCR products was performed following electrophoresis (1.5% of agarose) with the size of resultant products compared with a coamplified control template(s). In addition, fragments obtained by HindIII restriction endonuclease digestion were compared with those from control templates. The 400 bp GlcT-1 fragment was amplified from human GlcCer synthase (GlcT-1) cDNA (pCG-2) (41Ichikawa S. Sakiyama H. Suzuki G. Hidari K.I. Hirabayashi Y. Expression cloning of a cDNA for human ceramide glucosyltransferase that catalyzes the first glycosylation step of glycosphingolipid synthesis.Proc. Natl. Acad. Sci. USA. 1996; 93: 12654Crossref PubMed Scopus (220) Google Scholar) using GlcCer synthase-F1 and GlcCer synthase-R1 primers. The PCR product was ligated with T7 promoter using the Lig'nScribe Kit (Ambion, Austin TX) and subjected to in vitro transcription to yield digoxigenin-labeled RNA probe using an RNA labeling kit (Roche Molecular Biochemicals, Mannheim, Germany). Poly(A)+ RNA was prepared as described previously (42Farrell 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, 43Harris I.R. Farrell A.M. Holleran W.M. Jackson S. Grunfeld C. Elias P.M. Feingold K.R. Parallel regulation of sterol regulatory element binding protein-2 and the enzymes of cholesterol and fatty acid synthesis but not ceramide synthesis in cultured human keratinocytes and murine epidermis.J. Lipid Res. 1998; 39: 412-422Abstract Full Text Full Text PDF PubMed Google Scholar). Three micrograms of poly(A)+ RNA were electrophoresed on a 1.5% agarose gel containing 0.4 M formaldehyde, followed by capillary transfer and UV cross-linking to a positively charged nylon filter (Roche Molecular Biochemicals). The membrane was hybridized with both the digoxigenin-labeled anti-sense GlcCer synthase probe in Dig Easy Hyb solution (Roche) and the Dig-labeled actin RNA probe (Roche). The membrane was washed in 2× SSC containing 0.1% SDS, and then in 0.1× SSC containing 0.1% SDS. Detection was performed by addition of CSPD ready-to-use solution (Roche) to the membrane, followed by exposure to X-ray film and development. Western blot analysis was performed as described previously (42Farrell 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, 44Watanabe R. Wu K. Paul P. Marks D.L. Kobayashi T. Pittelkow M.R. Pagano R.E. Up-regulation of glucosylceramide synthase expression and activity during human keratinocyte differentiation.J. Biol. Chem. 1998; 273: 9651-9655Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Briefly, keratinocytes were solubilized with 50 mM Tris-HCl, pH 7.5, containing 150 mM NaCl, 1% Nonidet P40, 0.5% sodium deoxycholate, and protease inhibitors (CompeteTM, Roche Biochemical) and centrifuged to remove cell debris. Supernatant in sample buffer (60 mM Tris-HCl, pH 6.8, containing 2% SDS, 10% glycerol, 5% β-mercaptoethanol and 0.005% bromophenol blue) was resolved by electrophoresis on 12% SDS-polyacrylamide gel. Resultant bands were electrophoretically transferred to polyvinylidene difluoride membranes (Biorad, Hercules, CA), probed with anti-GlcCer synthase antiserum (kind gift of Dr. Richard Pagano, Rochester MN), and detected using an enhanced chemiluminescence system (NEN, Wilmington, DE). Statistical analyses were performed using an unpaired Student t-test. Previous studies have shown that either exogenous Cer administration or increased endogenous Cer resulting from inhibition of GlcCer synthase, results in a decline in CHK proliferation that reaches its nadir between 18 and 24 h (6Wakita 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 (94) Google Scholar) (Y. Uchida and W. Holleran, unpublished observations). To ascertain whether Cer mobilized from the membrane pool of SM exerts similar effects, we first measured incorporation of [3H]thymidine into DNA (DNA synthesis) after addition of exogenous SMase. Between SMase concentrations of 0.01–0.1 U/ml, no direct toxicity to cells was noted, i.e., trypan blue uptake remained unchanged during incubation period (data not shown). Yet, DNA synthesis decreased significantly 1 to 3 h following SMase treatment of CHK, returning toward normal by 8 h and thereafter (Fig. 1;P < 0.001, at 1, 2, and 3 h, respectively). These results indicate that SMase treatment causes a rapid decrease in CHK proliferation. To assess whether the effects of SMase could be mediated by changes in cellular sphingolipid content, we next measured cellular SM, Cer, and GlcCer levels before and after SMase exposure, as well as in vehicle-treated controls. Whereas SM content decreased precipitously between 1 and 8 h following SMase treatment (i.e., >50–60% decrease) (Fig. 2A), levels of other phospholipids, including phosphatidylcholine, remained unchanged (data not shown). Conversely, Cer levels increased rapidly (i.e., >6-fold; P < 0.001) over vehicle-treated controls between 1 and 3 h, remaining elevated up to 8 h (i.e., 4.4-fold; P < 0.001) (Fig. 2B). In contrast, GlcCer levels increased more slowly over time, reaching a 2.1-fold increase 8 h after SMase exposure (Fig. 2C). These results show that the rebound in CHK proliferation parallels divergent changes in SM, Cer, and GlcCer content. We next assessed whether the rebound in DNA synthesis and increase in GlcCer content that occur after SMase treatment are linked to increased GlcCer synthesis. Two approaches were employed; first, we assessed the fate of prelabeled ([3H]serine) sphingolipids. While the level of labeled SM decreased significantly, as expected at 8 h following SMase (i.e., to 51.6% of untreated controls), the levels of labeled Cer and GlcCer both increased markedly (i.e., to 822% and 573%, respectively; P < 0.01 in SMase-treated vs. control cells) (Fig. 3A). The increase in labeled Cer plus GlcCer (i.e., +56,100 cpm/
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