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• W1985110367 abstract "Owing to its ability to induce growth arrest and differentiation of keratinocytes, 1α,25-dihydroxyvitamin D3 and its analogs are useful for the treatment of hyperproliferative skin diseases, such as psoriasis vulgaris. It has been implicated that the 1α,25-dihydroxyvitamin D3-induced differentiation of keratinocytes is mediated, at least in part, by the formation of ceramides; however, ceramides have also been identified to induce apoptosis in many cells, including keratinocytes. Therefore, it was of interest to investigate the influence of 1α,25-dihydroxyvitamin D3 on apoptosis in keratinocytes. Most interestingly, physiological concentrations of 1α,25-dihydroxyvitamin D3 did not induce apoptosis in keratinocytes, despite the formation of ceramides. Moreover, 1α,25-dihydroxyvitamin D3 appeared cytoprotective and made keratinocytes resistant to apoptosis induced by ceramides, ultraviolet irradiation, or tumor necrosis factor-α. The cytoprotective effect was accompanied by the formation of the sphingolipid breakdown product sphingosine-1-phosphate, which prevented apoptosis in analogy to 1α,25-dihydroxyvitamin D3. The effect of 1α,25-dihydroxyvitamin D3 was specific as the almost inactive precursor cholecalciferol neither induced sphingosine-1-phosphate formation nor prevented cells from apoptosis. Besides this, the cytoprotective aptitude of 1α,25-dihydroxyvitamin D3 was completely abolished by the sphingosine kinase inhibitor N, N-dimethylsphingosine, which blocked sphingosine-1-phosphate formation. Moreover, sphingosine-1-phosphate was able to restore the cytoprotective effect of 1α,25-dihydroxyvitamin D3 in the presence of N, N-dimethylsphingosine. Taken together, here we report for the first time that 1α,25-dihydroxyvitamin D3 protects keratinocytes from apoptosis and additionally this cytoprotection is mediated via the formation of sphingosine-1-phosphate. Owing to its ability to induce growth arrest and differentiation of keratinocytes, 1α,25-dihydroxyvitamin D3 and its analogs are useful for the treatment of hyperproliferative skin diseases, such as psoriasis vulgaris. It has been implicated that the 1α,25-dihydroxyvitamin D3-induced differentiation of keratinocytes is mediated, at least in part, by the formation of ceramides; however, ceramides have also been identified to induce apoptosis in many cells, including keratinocytes. Therefore, it was of interest to investigate the influence of 1α,25-dihydroxyvitamin D3 on apoptosis in keratinocytes. Most interestingly, physiological concentrations of 1α,25-dihydroxyvitamin D3 did not induce apoptosis in keratinocytes, despite the formation of ceramides. Moreover, 1α,25-dihydroxyvitamin D3 appeared cytoprotective and made keratinocytes resistant to apoptosis induced by ceramides, ultraviolet irradiation, or tumor necrosis factor-α. The cytoprotective effect was accompanied by the formation of the sphingolipid breakdown product sphingosine-1-phosphate, which prevented apoptosis in analogy to 1α,25-dihydroxyvitamin D3. The effect of 1α,25-dihydroxyvitamin D3 was specific as the almost inactive precursor cholecalciferol neither induced sphingosine-1-phosphate formation nor prevented cells from apoptosis. Besides this, the cytoprotective aptitude of 1α,25-dihydroxyvitamin D3 was completely abolished by the sphingosine kinase inhibitor N, N-dimethylsphingosine, which blocked sphingosine-1-phosphate formation. Moreover, sphingosine-1-phosphate was able to restore the cytoprotective effect of 1α,25-dihydroxyvitamin D3 in the presence of N, N-dimethylsphingosine. Taken together, here we report for the first time that 1α,25-dihydroxyvitamin D3 protects keratinocytes from apoptosis and additionally this cytoprotection is mediated via the formation of sphingosine-1-phosphate. N-acetylsphingosine N, N-dimethylsphingosine 1α,25-dihydroxyvitamin D3 propidium iodide sphingosine-1-phosphate Besides its long recognized role in calcium homeostasis, 1α,25-dihydroxyvitamin D3 (1,25-(OH)2D3) is known to inhibit proliferation and to promote differentiation in a variety of cell types, including breast and colon carcinoma cells as well as leukemic and epidermal cells (Bouillon et al., 1995Bouillon R. Okamura W.H. Norman A.W. Structure-function relationships in the vitamin D endocrine system.Endocr Rev. 1995; 16: 200-257Crossref PubMed Google Scholar;Kobayashi et al., 1998Kobayashi T. Okumura H. Hashimoto K. Asada H. Inui S. Yoshikawa K. Synchronization of normal human keratinocytes in culture: its application to the analysis of 1,25-dihydroxyvitamin D3 effects on cell cycle.J Dermatol Sci. 1998; 17: 108-114Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar;Nolan et al., 1998Nolan E. Donepudi M. VanWeelden K. Flanagan L. Welsh J. Dissociation of vitamin D3 and anti-estrogen mediated growth regulation in MCF-7 breast cancer cells [In Process Citation].Mol Cell Biochem. 1998; 188: 13-20https://doi.org/10.1023/a:1006879213501Crossref PubMed Google Scholar). Therefore, the therapeutic potential of 1,25-(OH)2D3 and its derivatives (e.g., calcipotriol) has been investigated for the treatment of cancer, acute myeloid leukemia, and psoriasis. 1,25-(OH)2D3 exerts its effects by binding to the vitamin D3 receptor, which belongs to the nuclear hormone-receptor gene family (Baker et al., 1988Baker A.R. McDonnell D.P. Hughes M. et al.Cloning and expression of full-length cDNA encoding human vitamin D receptor.Proc Natl Acad Sci USA. 1988; 85: 3294-3298Crossref PubMed Scopus (820) Google Scholar). The hormone-receptor complex heterodimerizes with the retinoid X receptor and binds to cognate response elements in the promoters of target genes involved in the regulation of cell growth and differentiation (Minghetti and Norman, 1988Minghetti P.P. Norman A.W. 1,25(OH)2-vitamin D3 receptors: gene regulation and genetic circuitry.FASEB J. 1988; 2: 3043-3053Crossref PubMed Scopus (429) Google Scholar;Kuroki et al., 1995Kuroki Y. Shiozawa S. Kano J. Chihara K. Competition between c-fos and 1,25(OH)2 vitamin D3 in the transcriptional control of type I collagen synthesis in MC3T3–E1 osteoblastic cells.J Cell Physiol. 1995; 164: 459-464Crossref PubMed Scopus (19) Google Scholar). Recent studies have also indicated that 1,25-(OH)2D3 induces several rapid, apparently nongenomic biologic effects influencing a number of signal transduction pathways, including phosphoinositide signaling, intracellular calcium increase, and protein kinase C activation (Wali et al., 1990Wali R.K. Baum C.L. Sitrin M.D. Brasitus T.A. 1,25(OH)2 vitamin D3 stimulates membrane phosphoinositide turnover, activates protein kinase C, and increases cytosolic calcium in rat colonic epithelium.J Clin Invest. 1990; 85: 1296-1303Crossref PubMed Scopus (165) Google Scholar). 1,25-(OH)2D3 was the first compound identified as an inducer of neutral Mg2+-dependent sphingomyelinase leading to the hydrolysis of the membrane lipid sphingomyelin and a concomitant increase of intracellular ceramide levels. Cell-permeable ceramide analogs or bacterial sphingomyelinase mimic the effects of 1,25-(OH)2D3 on cell differentiation (Bielawska et al., 1992bBielawska A. Linardic C.M. Hannun Y.A. Modulation of cell growth and differentiation by ceramide.FEBS Lett. 1992; 307: 211-214Abstract Full Text PDF PubMed Scopus (88) Google Scholar) indicating an important role for ceramides in the actions of 1,25-(OH)2D3. Moreover, ceramide has been identified as a crucial component in the induction of apoptosis. A variety of proapoptotic stimuli, including tumor necrosis factor (TNF)-α, Fas-ligand, growth factor withdrawal, anticancer drugs, oxidative stress, heat shock, ionizing radiation as well as ultraviolet (UV) light have been found to stimulate sphingomyelinase activity leading to an enhanced cellular level of ceramide (Hannun and Obeid, 1995Hannun Y.A. Obeid L.M. Ceramide an intracellular signal for apoptosis.Trends Biochem Sci. 1995; 20: 73-77Abstract Full Text PDF PubMed Scopus (561) Google Scholar;Hannun, 1996Hannun Y.A. Functions of ceramide in coordinating cellular responses to stress.Science. 1996; 274: 1855-1859https://doi.org/10.1126/science.274.5294.1855Crossref PubMed Scopus (1457) Google Scholar;Jarvis et al., 1996Jarvis W.D. Grant S. Kolesnick R.N. Ceramide and the induction of apoptosis.Clin Cancer Res. 1996; 2: 1-6PubMed Google Scholar;Spiegel et al., 1996Spiegel S. Foster D. Kolesnick R. Signal transduction through lipid second messengers.Curr Opin Cell Biol. 1996; 8: 159-167Crossref PubMed Scopus (467) Google Scholar;Kolesnick and Kronke, 1998Kolesnick R.N. Kronke M. Regulation of ceramide production and apoptosis.Annu Rev Physiol. 1998; 60: 643-665Crossref PubMed Scopus (712) Google Scholar). Indeed, in a number of cancer cells 1,25-(OH)2D3 has also been recognized to induce apoptosis (Diaz et al., 2000Diaz G.D. Paraskeva C. Thomas M.G. Binderup L. Hague A. Apoptosis is induced by the active metabolite of vitamin D3 and its analogue EB1089 in colorectal adenoma and carcinoma cells: possible implications for prevention and therapy.Cancer Res. 2000; 60: 2304-2312PubMed Google Scholar;van den Bemd et al., 2000van den Bemd G.J. Pols H.A. van Leeuwen J.P. Anti-tumor effects of 1,25-dihydroxyvitamin D3 and vitamin D analogs.Curr Pharm Des. 2000; 6: 717-732Crossref PubMed Scopus (57) Google Scholar;Wang et al., 2000Wang Q. Yang W. Uytingco M.S. Christakos S. Wieder R. 1,25-Dihydroxyvitamin D3 and all-trans-retinoic acid sensitize breast cancer cells to chemotherapy-induced cell death.Cancer Res. 2000; 60: 2040-2048PubMed Google Scholar). In contrast 1,25-(OH)2D3 fails to induce programmed cell death in HL-60 cells as well as in human thyrocytes despite ceramide formation (Studzinski et al., 1986Studzinski G.P. Bhandal A.K. Brelvi Z.S. Potentiation by 1-α,25-dihydroxyvitamin D3 of cytotoxicity to HL-60 cells produced by cytarabine and hydroxyurea.J Natl Cancer Inst. 1986; 76: 641-648PubMed Google Scholar;Xu et al., 1992Xu H.M. Kolla S.S. Goldenberg N.A. Studzinski G.P. Resistance to 1,25-dihydroxyvitamin D3 of a deoxycytidine kinase-deficient variant of human leukemia HL60 cells.Exp Cell Res. 1992; 203: 244-250Crossref PubMed Scopus (22) Google Scholar,Xu et al., 1993Xu H.M. Tepper C.G. Jones J.B. Fernandez C.E. Studzinski G.P. 1,25-Dihydroxyvitamin D3 protects HL60 cells against apoptosis but down-regulates the expression of the Bcl-2 gene.Exp Cell Res. 1993; 209: 367-374Crossref PubMed Scopus (89) Google Scholar;Wang and Studzinski, 1997Wang X. Studzinski G.P. Antiapoptotic action of 1,25-dihydroxyvitamin D3 is associated with increased mitochondrial Mcl-1 and Raf-1 proteins and reduced release of cytochrome c.Exp Cell Res. 1997; 235: 210-217Crossref PubMed Scopus (90) Google Scholar). In fact, prolonged incubation of HL-60 cells and thyrocytes with 1,25-(OH)2D3 prevents the appearance of apoptotic cell death induced by calcium ionophores, anticancer drugs, and even cell-permeable ceramide analogs. Anti-sense inhibition of vitamin D3 receptor expression revealed that this protective effect is mediated via 1,25-(OH)2D3 binding to its nuclear receptor (Hewison et al., 1996Hewison M. Dabrowski M. Vadher S. et al.Antisense inhibition of vitamin D receptor expression induces apoptosis in monoblastoid U937 cells.J Immunol. 1996; 156: 4391-4400PubMed Google Scholar). Recently, we demonstrated that 1,25-(OH)2D3 enhances sphingosine kinase activity in HL-60 cells leading to a concomitant increase of sphingosine-1-phosphate (SPP), which prevents ceramide-induced apoptosis (Kleuser et al., 1998Kleuser B. Cuvillier O. Spiegel S. 1α,25-dihydroxyvitamin D3 inhibits programmed cell death in HL-60 cells by activation of sphingosine kinase.Cancer Res. 1998; 58: 1817-1824PubMed Google Scholar). Hydrolysis of sphingomyelin after stimulation with 1,25-(OH)2D3 or calcipotriol has also been documented in keratinocytes. Here, 1,25-(OH)2D3 increases expression of TNF-α, which induces ceramide formation via an autocrine mechanism (Geilen et al., 1996Geilen C.C. Bektas M. Wieder T. Orfanos C.E. The vitamin D3 analogue, calcipotriol, induces sphingomyelin hydrolysis in human keratinocytes.FEBS Lett. 1996; 378: 88-92Abstract Full Text PDF PubMed Scopus (55) Google Scholar,Geilen et al., 1997Geilen C.C. Bektas M. Wieder T. Kodelja V. Goerdt S. Orfanos C.E. 1α,25-dihydroxyvitamin D3 induces sphingomyelin hydrolysis in HaCaT cells via tumor necrosis factor α.J Biol Chem. 1997; 272: 8997-9001Crossref PubMed Scopus (63) Google Scholar). As TNF-α is in analogy to ceramides in a classical inductor of apoptosis, it was of interest to investigate the effect of 1,25-(OH)2D3 on keratinocyte survival. We found that 1,25-(OH)2D3 in physiological concentrations did not enhance apoptosis in human keratinocytes despite the expression of TNF-α and the subsequent formation of ceramide. Moreover, our studies demonstrate for the first time that 1,25-(OH)2D3 even protects keratinocytes from apoptosis and this resistance is a consequence of SPP formation. 1,25-(OH)2D3 and calcipotriol were kindly donated by Dr. Lise Binderup (Leo-Pharmaceutical Products, Ballerup, Denmark). [methyl-3H]thymidine (35 Ci per mmol), [3H]putrescine (80 Ci per mmol), and [γ-32P]adenosine triphosphate (4500 Ci per mmol) were purchased from ICN Biomedicals (Costa Mesa, CA). Cardiolipin and standard phospholipids were from Avanti Polar Lipids (Birmingham, AL). SPP, N, N-dimethylsphingosine (DMS), sphingosine, and N-acetylsphingosine (C2-cer) were purchased from Biomol Research Laboratory (Plymouth Meeting, PA). Annexin V-fluorescein isothiocyanate (Annexin V-FITC) was obtained from Bender (Vienna, Austria). Dimethylcasein, putrescine, propidium iodide (PI), ceramides (bovine brain, type III), leupeptin, aprotinin, dithiothreitol, phenylmethylsulfonylfluoride, o-phthaldialdehyde, cholecalciferol, sodium orthovanadate, deoxypyridoxine, bovine serum albumin, HEPES, Triton X-100, actinomycin D, and Dulbecco's modified Eagle's medium were purchased from Sigma (St. Louis, MO). TNF-α was from Seromed Biochrom (Berlin, Germany). Escherichia coli diacylglycerol kinase and octyl-β-D-glycopyranosides were obtained from Calbiochem (La Jolla, CA). Keratinocyte basal medium, epidermal growth factor, insulin, hydrocortisone, bovine pituitary extract, gentamicin sulfate, and amphotericin B were purchased from Clonetics (San Diego, CA). All high-performance liquid chromatography solvents were obtained from Merck (Darmstadt, Germany). To isolate human keratinocytes juvenile foreskin from surgery was incubated at 4°C in a solution of 0.25% trypsin and 0.2% ethylenediamine tetraacetic acid (EDTA) for 20 h. Trypsinization was terminated by the addition of ice-cold Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Cells were washed with phosphate-buffered saline (PBS) and centrifuged at 250 × g for 5 min. The pellet was resuspended in keratinocyte growth medium that was prepared from keratinocyte basal medium by the addition of 0.1 ng per ml recombinant epidermal growth factor, 5.0 µg insulin per ml, 0.5 µg hydrocortisone per ml, 0.15 mM Ca2+, 30 µg bovine pituitary extract per ml, 50 µg gentamicin sulfate per ml, 50 ng amphotericin B per ml. Keratinocytes were pooled from several donors and cultured at 37°C in 5% CO2. For all experiments only cells of the second or third passage were used. Keratinocytes (4 × 104 cells per well) were grown in 24-well plates for 24 h. Then medium was replaced by fresh keratinocyte growth medium and cells were incubated with 1,25-(OH)2D3 for 72 h. Keratinocytes were pulsed with 1 µCi of [methyl-3H]thymidine per well and incubated for 23 h. The medium was removed and cells were washed twice each with PBS and ice-cold trichloroacetic acid (5%). The precipitated material was dissolved in 0.3 M NaOH solution and incorporated [methyl-3H]thymidine was determined in a scintillation counter (MicroBeta Plus, Wallac Oy, Turku, Finland). Human keratinocytes grown in 100 mm dishes until a confluence of approximately 60% were treated with 1,25(OH)2D3 or the indicated agents for various incubation periods. Cells were then washed twice with ice-cold PBS and suspended in 200 µl of kinase buffer [20 mM Tris buffer (pH 7.4) containing 20% (vol/vol) glycerol, 1 mM β-mercaptoethanol, 1 mM EDTA, 1 mM sodium orthovanadate, 15 mM NaF, 10 µg per ml leupeptin and aprotinin, 1 mM phenylmethylsulfonylfluoride, and 0.5 mM 4-deoxypyridoxine] as described previously (Olivera and Spiegel, 1993Olivera A. Spiegel S. Sphingosine-1-phosphate as second messenger in cell proliferation induced by PDGF and FCS mitogens.Nature. 1993; 365: 557-560Crossref PubMed Scopus (793) Google Scholar). Cells were disrupted by freeze-thawing, and the cytosolic fraction was prepared by centrifugation at 13,000 × g for 30 min at 4°C. The cytosolic fraction (120 µl) was incubated with 10 µl of sphingosine (1 mM), delivered as a sphingosine–bovine serum albumin complex (4 mg bovine serum albumin per ml). Kinase buffer was added to a final volume of 190 µl, and reactions were started by adding 10 µl of [γ-32P]adenosine triphosphate (1–2 µCi, 20 mM) containing 100 mM MgCl2. Samples were incubated for 30 min at 37°C, followed by the addition of 20 µl of 1 M HCl. Lipids were extracted by the addition of 0.8 ml chloroform/methanol/concentrated HCl (100:200:1, vol/vol/vol). After vigorous vortexing, 240 µl of chloroform and 240 µl of 2 M KCl were added for phase separation. The samples were vortexed and centrifuged. Labeled lipids in the organic phase were separated by thin-layer chromatography on silica gel G60 using 1-butanol/methanol/acetic acid/water (80:20:10:20, vol/vol/vol/vol) as the solvent. The radioactive spots corresponding to authentic SPP were visualized by autoradiography, scraped from the plates, and counted in a scintillation counter. SPP was determined as recently described (Ruwisch et al., 2001Ruwisch L. Schaefer-Korting M. Kleuser B. An improved high-performance liquid chromatographic method for the determination of sphingosine-1-phosphate in complex biological materials.Naunyn Schmiedebergs Arch Pharmacol. 2001; 363: 358-363Crossref PubMed Scopus (64) Google Scholar). Briefly, keratinocytes were washed twice with PBS and scraped in 1 ml of methanol containing 2.5 µl concentrated HCl. Lipids were extracted by addition of 1 ml chloroform and 1 ml 1 M NaCl. For alkalization, 100 µl of a 3 M NaOH solution were added. After centrifugation (300 × g, 5 min), the alkaline aqueous phase containing SPP was transferred into a siliconized glass tube, the organic phase was re-extracted with 0.5 ml methanol, 0.5 ml 1 M NaCl and 50 µl 3 M NaOH. The aqueous phases were acidified with 100 µl concentrated HCl and extracted twice with 1.5 ml chloroform. The combined organic phases were evaporated using a vacuum system (Savant, Bethesda, MD). The dried lipids were resolved in 275 µl methanol/0.07 M K2HPO4 (9:1) by rigorous vortexing and sonication on ice for 5 min. A derivatization mixture of 10 mg of o-phthaldialdehyde, 200 µl of ethanol, 10 µl of β-mercaptoethanol, and 10 ml of a 3% boric acid solution adjusted to pH 10.5 with potassium hydroxide was prepared. 25 µl of this derivatization mixture were added to the resolved lipids for 15 min at room temperature. The derivatives were analyzed by a Merck Hitachi LaChrom high-performance liquid chromatography system (Merck Hitachi, Darmstadt, Germany). Fluorescence was measured at an emission wavelength of 455 nm and an excitation wavelength of 340 nm after separation on a RP 18 Kromasil column (Chromatographie Service, Langerwehe, Germany) kept at 35°C. The flow rate was adjusted to 1.3 ml per min, methanol and 0.07 M K2HPO4 were used as eluents. Resulting profiles were evaluated using the Merck system manager software. Keratinocytes, cultured in 100 mm dishes, were treated with vehicle or 100 nM of 1,25-(OH)2D3 in the presence or absence of 5 µM DMS. Then lipids were extracted by the addition of 3 ml of methanol/chloroform/water (1:1:1, vol/vol/vol) and an aliquot of 200 µl of the chloroform phase was dried under a nitrogen stream. The lipids or standard bovine brain type III ceramides were suspended in 40 µl of 7.5% (wt/vol) octyl-β-D-glycopyranoside, 5 mM cardiolipin in 1 mM diethylenetriaminepentaacetic acid, 10 mM imidazole (pH 6.6) and then solubilized by freeze-thawing followed by sonication. The enzymatic reaction was started by the addition of 20 µl of dithiothreitol (20 mM), 20 µl of Escherichia coli diacylglycerol kinase (0.88 units per ml), 20 µl of [γ-32P]adenosine triphosphate (10 mM, 1 µCi per nmol), and 100 µl of reaction buffer [100 mM imidazole (pH 6.6), 100 mM NaCl, 25 mM MgCl2, and 2 mM ethyleneglycol-bis-(β-aminoethylether)-N,N,N′,N′-tetraacetic acid]. Lipids were incubated for 1 h at room temperature and then extracted by the addition of 1 ml of chloroform/methanol/concentrated HCl (100:200:1, vol/vol/vol) and 170 µl of PBS containing 10 mM EDTA. An aliquot of 50 µl of the organic phase was analyzed by thin-layer chromatography (Silica Gel G60) with chloroform/acetone/methanol/acetic acid/water (10:4:3:2:1, vol/vol/vol/vol/vol) as the solvent. Radioactive spots corresponding to ceramide-1-phosphate (Rf = 0.23 ± 0.08) were counted. Keratinocytes (1.7 × 105 cells per well) were cultured in keratinocyte basal medium containing 5.0 µg insulin per ml, 0.5 µg hydrocortisone per ml, 50 µg gentamicin sulfate per ml, 50 ng amphotericin B per ml and incubated with the indicated agents for 24 h. Then cells were trypsinized and washed twice with binding buffer (10 mM HEPES/NaOH pH 7.4, 140 mM NaCl, 2.5 mM CaCl2). Apoptosis was determined by flow cytometric detection of phosphatidylserine translocation using fluorescein-labeled Annexin V (Vermes et al., 1997Vermes I. Haanen C. Richel D.J. Schaafsma M.R. Kalsbeek-Batenburg E. Reutelingsperger C.P. Apoptosis and secondary necrosis of lymphocytes in culture.Acta Haematol. 1997; 98: 8-13Crossref PubMed Scopus (55) Google Scholar). To discriminate between early apoptotic cells (Annexin V+/PI−) as well as late apoptotic and necrotic cells (Annexin V+/PI+), dye exclusion of the nonvital dye PI was simultaneously measured. Therefore, cells were resuspended in binding buffer followed by the addition of Annexin V-FITC (final concentration 0.5 µg per ml). The mixture was incubated for 10 min in the dark at room temperature, washed, and resuspended in binding buffer. Then PI was added (1 µg per ml) and samples were analyzed by bivariate flow cytometry. Keratinocytes were directly stained on chamber slides by the in situ cell death detection kit (Roche Diagnostics, Mannheim, Germany), as recommended by the manufacturer. Cell layers were fixed with a 4% buffered paraformaldehyde solution and then blocked with 3% H2O2 in methanol before permeabilization with 0.1% Triton X-100 in 0.1% sodium citrate. Cells were incubated with fluorescein-labeled nucleotides (fluorescein-deoxyuridine triphosphate) and terminal deoxynucleotidyl transferase at 37°C for 1 h. Cells were washed with PBS and incorporated fluorescein at the damaged sites of the DNA was detected by an anti-fluorescein antibody conjugated horseradish peroxidase. After substrate reaction using diaminobenzidine, approximately 100 cells were evaluated in randomly selected high-power fields by light microscopy. Negative control was obtained by replacing the primary incubation with a nucleotide mixture without terminal deoxynucleotidyl transferase. Transglutaminase activity was determined by the method described byWakita 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. Cells were cultured in keratinocyte growth medium and incubated with the test substances for 96 h. Keratinocytes were collected with a rubber policeman in 20 mM Tris–HCl buffer containing 2 mM EDTA (pH 8.0) and homogenized by freeze-thawing. After centrifugation at 600 × g for 10 min, 100 µl of the supernatant were mixed with 600 µl 50 mM Tris–HCl buffer (pH 8.0) containing 10 mM CaCl2, 5 mM dithiothreitol, 540 µg dimethylcasein, 1 mM putrescine, and 2.5 µCi [3H]putrescine (80 Ci per mmol). The mixture was incubated for 1 h at 37°C and the enzymatic reaction was stopped by the addition of 600 µl ice-cold trichloroacetic acid (10%). The protein precipitate was washed three times with ice-cold trichloroacetic acid (5%) containing 10 mM putrescine and once with ethanol (95%). The pellet was solubilized in 200 µl 1 M NaOH solution and radioactivity was determined in the scintillation counter. After treatment of keratinocytes with the indicated agents for 24 h, cells were washed twice with PBS, scraped from dishes, and suspended in kinase buffer [0.1 M Tris–HCl (pH 7.4) containing 20% glycerol, 1 mM β-mercaptoethanol, 1 mM EDTA, 1 mM sodium orthovanadate, 15 mM NaF, 10 mg leupeptin per ml and aprotinin each, 1 mM phenylmethylsulfonylfluoride, and 0.5 mM 4-deoxypyridoxine]. Cells were lyzed by freeze-thawing and centrifuged at 140,000 × g for 30 min. The supernatant, containing the cytosolic fraction, was collected, whereas the pellet was resuspended by passing through a 27 gauge needle 10 times in kinase buffer containing 0.1% Triton X-100. After centrifugation at 140,000 × g for 30 min, supernatant was saved and designated as the membrane fraction. Protein concentrations were determined and equal amounts from cytosolic and membrane fractions were assayed for protein kinase C (PKC) activity, using a commercial assay kit (Upstate Biotechnology, Inc, Lake Placid, NY). The procedure was performed as described in the manufacturer's instructions. The expression of Bcl-2 was determined by western blot analysis using a mouse monoclonal antibody to human Bcl-2 (PharMingen, San Diego, CA). Lysates of keratinocytes were prepared by scraping cells from plates and suspended in PBS. The cells were collected by centrifugation and the resulting pellets were suspended in ice-cold lysis buffer [20 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1 mM Na2EDTA, 1 mM ethyleneglycol-bis-(β-aminoethylether)-N,N,N′,N′-tetraacetic acid, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM sodium orthovanadate and 1 µg leupeptin per ml). After incubation on ice for 30 min, samples were centrifuged at 13,000 × g for 20 min. The Triton-soluble fraction was collected, and 15 µg of protein were subjected to a 12.5% polyacrylamide gel and transferred to nitrocellulose membranes. The blots were blocked in Tris-buffered saline/Tween 20 (0.1%) with 5% nonfat dry milk for 1 h, incubated with the primary antibody for 3 h at 37°C and a horseradish peroxidase-conjugated second antibody (New England Biolabs, Beverly, MA) for 1 h at room temperature. Immune complexes were detected with an enhanced chemoluminescence detection method (Santa Cruz Biotechnology, CA). Data are the mean from triplicate assays and are expressed as mean ± SD. All experiments were repeated at least three times independently. Statistics were performed using Student's t test, with p ≤ 0.05 considered significant. 1,25-(OH)2D3 has been well established to inhibit cell growth of human keratinocytes. In agreement with many previous studies a slight but significant anti-proliferative effect was visible with 1 nM of 1,25-(OH)2D3, whereas a concentration of 100 nM of 1,25-(OH)2D3 reduced [methyl-3H]thymidine incorporation by more than 60% Table I. To prove whether the anti-proliferative effect in this concentration range is accompanied by the induction of apoptosis, we used TUNEL staining and additionally measured the translocation of phosphatidylserine, an early event in the apoptotic process, by flow cytometry using Annexin V-FITC. To distinguish between early apoptotic as well as late apoptotic and necrotic cells, dye exclusion of the nonvital dye PI was simultaneously measured. It is of interest that 1–100 nM of 1,25-(OH)2D3 neither increased Annexin V binding nor PI uptake indicating that 1,25-(OH)2D3 did not possess apoptotic or necrotic actions itself in this concentration range Table I. But it should be noted that 1,25-(OH)2D3 concentrations exceeding 1 µM, however, resulted in an increased detachment of the cells from the dishes. Indeed, measurement of apoptosis/necrosis demonstrated an increase of Annexin V+/PI+-cells suggesting a cytotoxic effect of 1,25-(OH)2D3 at concentrations above 1 µM Table I. This result was confirmed by TUNEL staining and is in agreement with other studies (Benassi et al., 1997Benassi L. Ottani D. Fantini F. Marconi A. Chiodino C. Giannetti A. Pincelli C. 1,25-dihydroxyvitamin D3, transforming growth factor beta1, calcium, and ultraviolet B radiation induce apoptosis in cultured human keratinocytes.J Invest Dermatol. 1997; 109: 276-282Crossref PubMed Scopus (75) Google Scholar;Bektas et al., 2000Bektas M. Orfanos C.E. Geilen C.C. Different vitamin D analogues induce sphingomyelin hydrolysis and apoptosis in the human keratinocyte cell line HaCaT.Cell Mol Biol. 2000; 46: 111-119PubMed Google Scholar)Table IEffect of 1,25-(OH)2D3 on proliferation, apoptosis and necrosis in human keratinocytesaKeratinocytes were treated with the indicated concentrations of 1,25-(OH)2D3 for 3 d. Proliferation was measured by [methyl-3H]thymidine incorporation as described in Materials and Methods. Annexin V+/PI− and Annexin V+/PI+ cells were determined by flow cytometric detection of phosphatidylserine translocation and PI uptake. Additionally, TUNEL staining was performed. Data are mean ± SD of triplicate determinations.1,25-(OH)2D3ProliferationApoptosis/necrosis[methyl-3H] thymidine incorporationTUNEL-positive cells (%)Annexin V+/PI- cells (%)Annexin V+/PI+ cells (%)(cpm × 103)(% of control)Control53.3 ± 5.510014.7 ± 2.53.4 ± 0.77.5 ± 2.10.1 nM51.7 ± 6.29616.3 ± 3.14.2 ± 1" @default.
• W1985110367 created "2016-06-24" @default.
• W1985110367 creator A5002454933 @default.
• W1985110367 creator A5036135633 @default.
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• W1985110367 date "2001-11-01" @default.
• W1985110367 modified "2023-10-12" @default.
• W1985110367 title "1α,25-Dihydroxyvitamin D3 Protects Human Keratinocytes from Apoptosis by the Formation of Sphingosine-1-Phosphate" @default.
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