Sphingosine-1-Phosphate and Its Potentially Paradoxical Effects on Critical Parameters of Cutaneous Wound Healing
2003; Elsevier BV; Volume: 120; Issue: 4 Linguagem: Inglês
10.1046/j.1523-1747.2003.12096.x
ISSN1523-1747
AutoresRüdiger Vogler, Bettina Sauer, Dong-Seok Kim, Monika Schäfer‐Korting, Burkhard Kleuser,
Tópico(s)Pharmacological Effects of Natural Compounds
ResumoThe sphingolipid metabolite sphingosine-1-phosphate has emerged as a new bioactive molecule involved in the regulation of cell growth, differentiation, survival, and chemotaxis as well as angiogenesis and embryogenesis. These effects are mediated either via G-protein-coupled receptors or through intracellular actions. The most prominent sources of sphingosine-1-phosphate are human platelets suggesting its potential role in wound healing. In agreement with a positive function on reconstruction of wounded skin, we identified sphingosine-1-phosphate as a potent chemoattractant for keratinocytes as well as an activator of extracellular matrix production by fibroblasts. An unexpected finding is a strong cell growth arrest of keratinocytes after exposure to sphingosine-1-phosphate, as keratinocyte proliferation is critical for re-epithelialization of the wound. Most interestingly, the anti-proliferative effect of sphingosine-1-phosphate is not a result of cytotoxicity or apoptosis as sphingosine-1-phosphate even protects these cells from programmed cell death. Moreover, sphingosine-1-phosphate enhances differentiation of keratinocytes. To investigate further by which signaling pathway cell growth inhibition is mediated expression of the mRNA of all sphingosine-1-phosphate receptors (S1P1–5) was identified. 1 (Edg 1), 2 (Edg 5), 3 (Edg 3), 4 (Edg 6), and 5 (Edg 8) mRNA in keratinocytes was identified. As demonstrated in guanosine 5-[γ-35S] triphosphate-γS binding assays, these G-protein-coupled receptors are functional at nanomolar concentrations. As the anti-proliferative effect of sphingosine-1-phosphate is only partially inhibited in the presence of pertussis toxin, it was investigated if intracellular actions are also involved. Microinjections of sphingosine-1-phosphate in keratinocytes also reduce proliferation suggesting that both sphingosine-1-phosphate receptors as well as intracellular actions mediate sphingosine-1-phosphate- induced cell growth arrest. The sphingolipid metabolite sphingosine-1-phosphate has emerged as a new bioactive molecule involved in the regulation of cell growth, differentiation, survival, and chemotaxis as well as angiogenesis and embryogenesis. These effects are mediated either via G-protein-coupled receptors or through intracellular actions. The most prominent sources of sphingosine-1-phosphate are human platelets suggesting its potential role in wound healing. In agreement with a positive function on reconstruction of wounded skin, we identified sphingosine-1-phosphate as a potent chemoattractant for keratinocytes as well as an activator of extracellular matrix production by fibroblasts. An unexpected finding is a strong cell growth arrest of keratinocytes after exposure to sphingosine-1-phosphate, as keratinocyte proliferation is critical for re-epithelialization of the wound. Most interestingly, the anti-proliferative effect of sphingosine-1-phosphate is not a result of cytotoxicity or apoptosis as sphingosine-1-phosphate even protects these cells from programmed cell death. Moreover, sphingosine-1-phosphate enhances differentiation of keratinocytes. To investigate further by which signaling pathway cell growth inhibition is mediated expression of the mRNA of all sphingosine-1-phosphate receptors (S1P1–5) was identified. 1 (Edg 1), 2 (Edg 5), 3 (Edg 3), 4 (Edg 6), and 5 (Edg 8) mRNA in keratinocytes was identified. As demonstrated in guanosine 5-[γ-35S] triphosphate-γS binding assays, these G-protein-coupled receptors are functional at nanomolar concentrations. As the anti-proliferative effect of sphingosine-1-phosphate is only partially inhibited in the presence of pertussis toxin, it was investigated if intracellular actions are also involved. Microinjections of sphingosine-1-phosphate in keratinocytes also reduce proliferation suggesting that both sphingosine-1-phosphate receptors as well as intracellular actions mediate sphingosine-1-phosphate- induced cell growth arrest. 1α,25-dihydroxyvitamin D3 bromodesoxyuridine endothelial differentiation gene N-formyl-methionyl-leucyl-phenylalanine keratinocyte basal medium keratinocyte growth medium lysophosphatidic acid 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide plasminogen activator inhibitor-1 phenylmethylsulfonyl fluoride pertussis toxin sphingosine-1-phosphate The bioactive sphingolipid metabolite sphingosine-1-phosphate (S1P) plays a prominent part as a signaling molecule to elicit a variety of physiologic and pathophysiologic responses. In particular, S1P is involved in the regulation of cell proliferation, differentiation, survival, and motility (Goetzl and An, 1998Goetzl E.J. An S. Diversity of cellular receptors and functions for the lysophospholipid growth factors lysophosphatidic acid and sphingosine 1-phosphate.FASEB J. 1998; 12: 1589-1598Crossref PubMed Scopus (486) Google Scholar;Spiegel et al., 1998Spiegel S. Cuvillier O. Edsall L.C. et al.Sphingosine-1-phosphate in cell growth and cell death.Ann N Y Acad Sci. 1998; 845: 11-18Crossref PubMed Scopus (182) Google Scholar;Pyne and Pyne, 2000Pyne S. Pyne N.J. Sphingosine 1-phosphate signalling in mammalian cells.Biochem J. 2000; 349: 385-402Crossref PubMed Scopus (648) Google Scholar;Spiegel and Milstien, 2000Spiegel S. Milstien S. Sphingosine-1-phosphate. signaling inside and out.FEBS Lett. 2000; 476: 55-57Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar). A well known source of S1P are human platelets, from which it is released upon activation by physiologic stimuli, suggesting that S1P is a significant factor involved in endothelial injury, inflammation, thrombosis, angiogenesis, and wound healing by increasing migration and proliferation of endothelial cells (Yatomi et al., 1997Yatomi Y. Igarashi Y. Yang L. et al.Sphingosine 1-phosphate, a bioactive sphingolipid abundantly stored in platelets, is a normal constituent of human plasma and serum.J Biochem (Tokyo). 1997; 121: 969-973Crossref PubMed Scopus (402) Google Scholar;Ruwisch et al., 2001Ruwisch L. Schafer-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). Growing interest in S1P has been increased by the discovery of a family of distinct G-protein-coupled receptors, which originally were designated endothelial differentiation gene (Edg) receptors (Verlinden et al., 1998Verlinden L. Verstuyf A. Convents R. Marcelis S. Van Camp M. Bouillon R. Action of 1,25(OH)2D3 on the cell cycle genes, cyclin D1, 21 and p27 in MCF-7 cells.Mol Cell Endocrinol. 1998; 142: 57-65Crossref PubMed Scopus (139) Google Scholar). Most recently, a nomenclature subcommittee of the International Union of Pharmacologists has renamed these receptors according to the binding ligand and the order of discovery. To date, five S1P receptors and three lysophosphatidic acid (LPA) receptors have been identified, which are named S1P1 (Edg 1), S1P2 (Edg 5), S1P3 (Edg 3), S1P4 (Edg 6), S1P5 (Edg 8); and LPA1 (Edg 2), LPA2 (Edg 4), LPA3 (Edg 7) (Fukushima et al., 2001Fukushima N. Ishii I. Contos J.J. Weiner J.A. Chun J. Lysophospholipid receptors.Annu Rev Pharmacol Toxicol. 2001; 41: 507-534Crossref PubMed Scopus (314) Google Scholar;Chun et al., 2002Chun J. Goetzl E.J. Hla T. et al.International Union of Pharmacology. XXXIV. Lysophospholipid receptor nomenclature.Pharmacol Rev. 2002; 54: 265-269Crossref PubMed Scopus (439) Google Scholar). Intracellular signaling pathways activated by the cloned S1P receptors have been characterized in heterologous expression systems indicating an influence on cyclic adenosine monophosphate levels as well as activation of phospholipase C, Ras, mitogen-activated protein kinase, Rho, and several protein tyrosine kinases (An et al., 1998An S. Goetzl E.J. Lee H. Signaling mechanisms and molecular characteristics of G protein-coupled receptors for lysophosphatidic acid and sphingosine 1-phosphate.J Cell Biochem Suppl. 1998; 31: 147-157Crossref Google Scholar;Lee et al., 1998Lee M.J. Van Brocklyn J.R. Thangada S. et al.Sphingosine-1-phosphate as a ligand for the G protein-coupled receptor EDG-1.Science. 1998; 279: 1552-1555Crossref PubMed Scopus (857) Google Scholar;Okamoto et al., 1998Okamoto H. Takuwa N. Gonda K. et al.EDG1 is a functional sphingosine-1-phosphate receptor that is linked via a Gi/o to multiple signaling pathways, including phospholipase C activation, Ca2+ mobilization, Ras-mitogen-activated protein kinase activation, and adenylate cyclase inhibition.J Biol Chem. 1998; 273: 27104-27110Crossref PubMed Scopus (233) Google Scholar,Okamoto et al., 1999Okamoto H. Takuwa N. Yatomi Y. Gonda K. Shigematsu H. Takuwa Y. EDG3 is a functional receptor specific for sphingosine 1-phosphate and sphingosylphosphorylcholine with signaling characteristics distinct from EDG1 and AGR16.Biochem Biophys Res Commun. 1999; 260: 203-208Crossref PubMed Scopus (147) Google Scholar;Van Brocklyn et al., 1998Van Brocklyn J.R. Lee M.J. Menzeleev R. et al.Dual actions of sphingosine-1-phosphate: extracellular through the Gi-coupled receptor Edg-1 and intracellular to regulate proliferation and survival.J Cell Biol. 1998; 142: 229-240Crossref PubMed Scopus (440) Google Scholar;Zondag et al., 1998Zondag G.C. Postma F.R. Etten I.V. Verlaan I. Moolenaar W.H. Sphingosine 1-phosphate signalling through the G-protein-coupled receptor Edg-1.Biochem J. 1998; 330: 605-609Crossref PubMed Scopus (232) Google Scholar;Gonda et al., 1999Gonda K. Okamoto H. Takuwa N. et al.The novel sphingosine 1-phosphate receptor AGR16 is coupled via pertussis toxin-sensitive and -insensitive G-proteins to multiple signalling pathways.Biochem J. 1999; 337: 67-75Crossref PubMed Scopus (173) Google Scholar). Nevertheless, several lines of incidence indicate that S1P also acts as an intracellular second messenger. As sphingosine kinase is the crucial enzyme for the formation of S1P, numerous stimuli have been identified to increase its activity and subsequent intracellular S1P levels. These stimuli comprise platelet-derived growth factor, nerve growth factor, 1α,25-dihydroxyvitamin D3 (1,25-(OH)2D3), phorbol myristate acetate, activation of the formyl peptide receptor by N-formyl-methionyl-leucyl-phenylalanine, and cross-linking of the FcεRI receptors by antigens (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;Mazurek et al., 1994Mazurek N. Megidish T. Hakomori S. Igarashi Y. Regulatory effect of phorbol esters on sphingosine kinase in BALB/C 3T3 fibroblasts (variant A31): demonstration of cell type-specific response—a preliminary note.Biochem Biophys Res Commun. 1994; 198: 1-9Crossref PubMed Scopus (89) Google Scholar;Choi et al., 1996Choi O.H. Kim J.H. Kinet J.P. Calcium mobilization via sphingosine kinase in signalling by the Fc epsilon RI antigen receptor.Nature. 1996; 380: 634-636Crossref PubMed Scopus (376) Google Scholar;Edsall et al., 1997Edsall L.C. Pirianov G.G. Spiegel S. Involvement of sphingosine 1-phosphate in nerve growth factor-mediated neuronal survival and differentiation.J Neurosci. 1997; 17: 6952-6960Crossref PubMed Google Scholar;Kleuser et al., 1998Kleuser B. Cuvillier O. Spiegel S. 1Alpha,25-dihydroxyvitamin D3 inhibits programmed cell death in HL-60 cells by activation of sphingosine kinase.Cancer Res. 1998; 58: 1817-1824PubMed Google Scholar;Melendez et al., 1998Melendez A. Floto R.A. Cameron A.J. Gillooly D.J. Harnett M.M. Allen J.M. A molecular switch changes the signalling pathway used by the Fc gamma RI antibody receptor to mobilise calcium.Curr Biol. 1998; 8: 210-221Abstract Full Text Full Text PDF PubMed Google Scholar;Alemany et al., 1999Alemany R. Meyer zu Heringdorf D. van Koppen C.J. Jakobs K.H. Formyl peptide receptor signaling in HL-60 cells through sphingosine kinase.J Biol Chem. 1999; 274: 3994-3999Crossref PubMed Scopus (94) Google Scholar). In agreement, inhibition of sphingosine kinase activity utilizing N,N-dimethylsphingosine prevents cellular proliferation induced by mitogenic stimuli and revokes the cytoprotective effects of protein kinase C, nerve growth factor, and 1,25-(OH)2D3 (Cuvillier et al., 1996Cuvillier O. Pirianov G. Kleuser B. Vanek P.G. Coso O.A. Gutkind S. Spiegel S. Suppression of ceramide-mediated programmed cell death by sphingosine-1-phosphate.Nature. 1996; 381: 800-803Crossref PubMed Scopus (1290) Google Scholar;Edsall et al., 1997Edsall L.C. Pirianov G.G. Spiegel S. Involvement of sphingosine 1-phosphate in nerve growth factor-mediated neuronal survival and differentiation.J Neurosci. 1997; 17: 6952-6960Crossref PubMed Google Scholar;Kleuser et al., 1998Kleuser B. Cuvillier O. Spiegel S. 1Alpha,25-dihydroxyvitamin D3 inhibits programmed cell death in HL-60 cells by activation of sphingosine kinase.Cancer Res. 1998; 58: 1817-1824PubMed Google Scholar). In addition, increase of intracellular S1P levels by microinjection mobilizes calcium from internal stores and induces proliferation of Swiss 3T3 cells (Van Brocklyn et al., 1998Van Brocklyn J.R. Lee M.J. Menzeleev R. et al.Dual actions of sphingosine-1-phosphate: extracellular through the Gi-coupled receptor Edg-1 and intracellular to regulate proliferation and survival.J Cell Biol. 1998; 142: 229-240Crossref PubMed Scopus (440) Google Scholar). Furthermore, sphingosine kinase has been overexpressed in NIH 3T3 and HEK 293 cells leading to an increase of intracellular S1P levels and a subsequent promotion of cell growth and survival (Olivera et al., 1999Olivera A. Kohama T. Edsall L. Nava V. Cuvillier O. Poulton S. Spiegel S. Sphingosine kinase expression increases intracellular sphingosine-1-phosphate and promotes cell growth and survival.J Cell Biol. 1999; 147: 545-558Crossref PubMed Scopus (447) Google Scholar). In these studies an export of S1P from the cytoplasm to the extracellular space was not observed. On the contrary in a further study it has been reported that transfection of HEK 293 with sphingosine kinase leads to a constitutive export of the enzyme into the extracellular environment suggesting an external formation of S1P (Ancellin et al., 2002Ancellin N. Colmont C. Su J. et al.Extracellular export of sphingosine kinase-1 enzyme.J Biol Chem. 2002; 277: 6667-6675Crossref PubMed Scopus (248) Google Scholar). Interestingly, S1P is stored in human platelets and released at wounded sites suggesting a positive role of this sphingolipid metabolite in the process of wound healing of the skin (Lee et al., 2000Lee H. Goetzl E.J. An S. Lysophosphatidic acid and sphingosine 1-phosphate stimulate endothelial cell wound healing.Am J Physiol Cell Physiol. 2000; 278: C612-C618PubMed Google Scholar). Contrary to these expectations, most recently we found that S1P is increased in keratinocytes after treatment with those doses of 1,25-(OH)2D3, which inhibit keratinocyte proliferation (Manggau et al., 2001Manggau M. Kim D.S. Ruwisch L. Vogler R. Korting H.C. Schäfer-Korting M. Kleuser B. 1alpha,25-dihydroxyvitamin D3 protects human keratinocytes from apoptosis by the formation of sphingosine-1-phosphate.J Invest Dermatol. 2001; 117: 1241-1249Crossref PubMed Scopus (109) Google Scholar). This is of interest as a cell growth inhibitory effect of keratinocytes opposes re-epithelialization of the skin. Therefore, the influence of S1P on critical parameters of wound healing such as proliferation and migration of keratinocytes as well as proliferation and matrix formation of fibroblasts was investigated. In agreement with results of several fibroblast cell lines S1P induced proliferation of primary fibroblasts. Moreover, matrix protein formation by S1P was observed. In keratinocytes S1P enhanced migration but induced a significant cell growth arrest of human keratinocytes not due to toxic or apoptotic effects. The latter finding was unexpected as inhibition of cell growth of keratinocytes decelerates re-epithelialization of the cutaneous barrier. Additionally, despite expression of functional S1P receptors, the anti-proliferative effect was also visible after microinjection of S1P. 1,25-(OH)2D3 was a generous gift from Leo Pharmaceuticals (Ballerup, Denmark). [methyl-3H]Thymidine (35 Ci per mmol), [3H]putrescine (80 Ci per mmol), [35S]guanosine 5-[y-35S] triphosphate (GTPγS) (1000 Ci per mmol), and gelatin Sepharose 4B were purchased from Amersham Pharmacia Biotech (Freiburg, Germany). [35S]Methionine (1175 Ci per mmol) was from Perkin Elmer (Boston, MA). S1P was purchased from Biomol Research Laboratory Inc. (Plymouth Meeting, PA). Transforming growth factor (TGF)-β was from Calbiochem (Bad Soden, Germany). Mouse monoclonal anti-human plasminogen activator inhibitor 1 (PAI-1) and mouse monoclonal anti-human fibronectin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Dimethylcasein, putrescine, propidium iodide, leupeptin, aprotinin, phenylmethylsulfonyl fluoride (PMSF), sodium orthovanadate, deoxypyridoxine, bovine serum albumin (BSA), Triton X-100, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), ethidium bromide, GIEMSA, Dulbecco's modified Eagle's medium (DMEM), aprotinin, pepstatin, and leupeptin were purchased from Sigma-Aldrich (Taufkirchen, Germany). Fetal bovine serum was from Seromed Biochrom (Berlin, Germany). Keratinocyte growth medium (KGM), keratinocyte basal medium (KBM), epidermal growth factor, insulin, hydrocortisone, bovine pituitary extract, gentamicin sulfate, and amphotericin B were purchased from Cell Systems (St Katharinen, Germany). Human keratinocytes and fibroblasts were isolated from juvenile foreskin from surgery. Skin was incubated at 4°C in a solution of 0.25% trypsin and 0.2% ethylenediamine tetraacetic acid for 20 h and trypsinization was terminated by addition of ice-cold DMEM containing 10% fetal bovine serum. Keratinocytes were scraped from the epidermis, washed with phosphate-buffered saline (PBS) and centrifuged at 250×g for 5 min. The pellet was resuspended in KGM that was prepared from KBM by the addition of 0.1 ng recombinant epidermal growth factor per ml, 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, and 50 ng amphotericin B per ml. The remaining skin was trypsinized for another 10 min at 37°C. The enzymatic reaction was terminated by the addition of ice-cold DMEM containing 10% fetal bovine serum. Fibroblasts were scraped from the dermis, filtered, centrifuged, and resuspended in DMEM containing 10% fetal bovine serum. Both keratinocytes and fibroblasts 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 (8×104 cells per well), seeded into 24-well plates for 24 h, were incubated with test substances for 24 h at 37°C in 5% CO2. After the addition of 100 μl MTT solution (5 mg per ml) per well, the plates were incubated for another 4 h. The supernatants were removed and the formazan crystals were solubilized in 1 ml of dimethyl sulfoxide. The optical density was determined at 540 nm using a scanning microplate spectrophotometer (Multiscan® Plus, Labsystems, Helsinki, Finland). Cells (4×104 cells per well) were grown in 24-well plates for 24 h. Then medium was replaced by fresh KBM or KGM for keratinocytes and by serum-free DMEM for fibroblasts. Cells were incubated with the indicated substances for 72 h and pulsed with 1 μCi of [methyl-3H]thymidine per well. After 23 h 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). Cell cycle analysis was performed using a cycle test plus DNA reagent kit (Becton and Dickinson, Heidelberg, Germany). Keratinocytes were fixed, RNA was digested and DNA was labeled with propidium iodide according to the manufacturer's instructions. Propidium iodide staining was determined by flow cytometry using a FACScalibur™ (Becton and Dickinson). 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 KGM 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 ethylenediamine tetraacetic acid (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 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. Measurement of fibronectin and PAI-1 synthesis was assayed as previously described (Wrana et al., 1992Wrana J.L. Attisano L. Carcamo J. et al.TGF beta signals through a heteromeric protein kinase receptor complex.Cell. 1992; 71: 1003-1014Abstract Full Text PDF PubMed Scopus (1340) Google Scholar). Briefly, fibroblasts (1×105 cells per well) were incubated overnight in serum-free DMEM, followed by 12 h stimulation with the indicated concentrations of S1P. For control assays cells were stimulated with TGF-β (2 ng per ml). Cultures were labeled with 50 μCi per ml [35S]methionine for 4 h. For measurement of newly synthesized secreted fibronectin, an aliquot of labeled media was incubated with gelatin-Sepharose in the presence of 0.5% Triton X-100. The beads were washed once with Tris-buffered saline (50 mM Tris–HCl, pH 7.4, 150 mM NaCl), once with 0.5 M NaCl, once with 50 mM Tris–HCl (pH 7.4), and once again with Tris-buffered saline. Fibronectin was eluated by boiling in electrophoresis sample buffer containing dithiothreitol and samples were analyzed by sodium dodecyl sulfate–gel electrophoresis. Fibronectin was identified as a band of 250 kDa. For measurement of PAI-1 labeled cells were removed by washing once with PBS, three times with 10 mM Tris–HCl (pH 8.0), 0.5% sodium deoxycholate, 1 mM PMSF, once with 10 mM Tris–HCl (pH 8.0), and once again with PBS. Matrix proteins were extracted by scraping into electrophoresis sample buffer containing dithiothreitol. PAI-1 was identified as a characteristic protein band of 45 kDa. For western blot analysis of fibronectin and PAI-1, treated or control cells were washed twice with PBS before being lyzed on ice in lysis buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1 mM ethylenediamine tetraacetic acid, 1% Nonidet P-40, 1 mM PMSF, and 1 μg per ml each of leupeptin, pepstatin, and aprotinin). The cell lysates were centrifuged at 12,000×g for 5 min at 4°C and the supernatant was collected. Samples were prepared in Laemmli reducing buffer, boiled for 10 min, and analyzed by sodium dodecyl sulfate–gel electrophoresis. Gels were blotted on to PDVF-membranes. After blocking with 5% nonfat dry milk overnight at 4°C membranes were incubated with the primary antibodies (mouse monoclonal anti-human PAI-1, mouse monoclonal anti-human fibronectin) for 2 h. The blots were washed three times in PBS-Tween (0.1%) followed by incubation with the secondary antibodies (rabbit anti-mouse IgG-horseradish peroxidase, rabbit anti-mouse IgG-AP alkaline phosphatase) for 1 h. After washing, the blots were developed according to the manufacturer's protocol. Chemotactic migration of cells in response to a gradient of S1P or TGF-β was measured in a modified Boyden chamber as described (Wang et al., 1999Wang F. Nohara K. Olivera A. Thompson E.W. Spiegel S. Involvement of focal adhesion kinase in inhibition of motility of human breast cancer cells by sphingosine 1-phosphate.Exp Cell Res. 1999; 247: 17-28Crossref PubMed Scopus (61) Google Scholar). Cells were added to the upper well of the chamber. The lower chamber, separated by a fibronectin-coated membrane, contained S1P in the indicated concentration. As control for the chemotactic response TGF-β (1 ng per ml) was used. Cells that had migrated through the membrane were fixed and stained by GIEMSA. The migrated cells were quantified by light microscopy at a magnification of×150 by counting the stained cells from four randomly selected fields. The mRNA of human keratinocytes or intact skin, and Jurkat cells were isolated by QuickPrep Micro mRNA Purification Kit (Amersham Biosciences, Freiburg, Germany) according to the manufacturer's instructions. Aliquots of mRNA preparation were frozen at-80°C until use. One microgram of mRNA was reverse transcribed (Superscript reverse transcriptase, Invitrogen, Karlsruhe, Germany) in the presence of 1 pmol of a 25–30 mer oligo(dT) primer. Based upon the nucleotide sequences of the human S1P receptors in the database, oligonucleotide primer pairs were prepared: S1P1: 5′-CCC AAG CTT ATG GGG CCC ACC AGC GTC CCG-3′ and 5′-GCT CTA GAC TAG GAA GAA GAG TTG ACG TTG CC-3′; S1P2: 5′-CAT TGC CAA GGT CAA GCT GT-3′ and 5′-ACG ATG GTG ACC GTC TTG AG-3′; S1P3: 5-CCC AAG CTT ATG GAC ACT GCC CTC CCG-3′ and 5′-CGG GAT CCT CAG TTG CAG AAG ATC CC-3′; S1P4: 5′-ACG GGA GGG CCT GCT CTT CA-3′ and 5′-AAG GCC AGC AGG ATC ATC AG-3′; S1P5: 5′-GTG GAC TTG AGC TTC AAG AC-3′ and 5′-CAC TTT GGG GAG GAT TTG GA-3′. Polymerase chain reaction amplification was carried out in a Thermocycler (T Gradient, Whatman Biometra, Göttingen, Germany) using the Thermoprime Plus polymerase (Advanced Biotechnologies, Columbia, MD) under the following cycling conditions: (1) 94 C for 1 min; (2) 94°C for 30 s; (3) 55°C for 30 s; (4) 72°C for 1 min; (5) repeat of steps 2–4 for 30 cycles; (6) 72°C for 2 min; and (7) 4°C for 1 s. Polymerase chain reaction products were size-fractionated in a 2% agarose gel, and visualized by ethidium bromide staining. Keratinocytes were washed with ice-cold PBS, scraped in buffer A containing 20 mM Tris (pH 7.4), 500 μM PMSF, 1 μg per ml each leupeptin and aprotinin, and 0.5 μg pepstatin per ml and homogenized by passing through a 28 gauge needle 10 times. The homogenate was centrifuged for 5 min at 5000×g and the pellet was discarded. The supernatant was spun for 40 min at 43,000×g, the resulting pellet was resuspended in buffer A and frozen at -80°C until use. Ten micrograms of protein per assay were incubated for 45 min in buffer B containing 20 mM Tris (pH 7.4), 5 mM MgCl2, 1 mM ethylenediamine tetraacetic acid, 1 mM ethyleneglycol-bis-(β-aminoethylether)-N,N,N′,N′-tetraacetic acid, 100 μM PMSF, 1 μg per ml each leupeptin and aprotinin, 0.5 μg pepstatin per ml, 3 μM guanosine diphosphate (GDP), 50 pM [35S]-GTPγS, and indicated concentrations of S1P. The samples were rapidly filtered on GF/B glass microfiber filters (Whatman, Kent, U.K.) presoaked in buffer C (20 mM Tris, pH 7.4, 10 mM MgCl2, 100 mM NaCl, 1 mMβ-mercaptoethanol). The filters were washed three times with buffer C and radioactivity was determined in a scintillation counter. Keratinocytes (approximately 60% confluent) were cultured on gridded glass coverslips. Then cells were microinjected using an Eppendorf Micromanipulator 5171 and Microinjector 5242 (Eppendorf, Hamburg, Germany) at a pressure of 210 hPa for 0.3 s. Approximately 400 cells were microinjected cytoplasmatically with S1P diluted in injection buffer (27 mM K2HPO4, 8 mM Na2HPO4, 26 mM KH2PO4, and 1 mg per ml BSA pH 7.2). Texas red-conjugated dextran (5 mg per ml) was coinjected for identification of microinjected cells. Cells injected only with Texas red-conjugated dextran and injection buffer served as a control. After microinjection of 400 cells, they were cultured in KGM supplemented with bromodesoxyuridine (BrdU, 10 mM) for 24 h at 37°C. Then keratinocytes were fixed in acidic ethanol (70% in 50 mM glycine buffer, pH 2.0) and stained with mouse monoclonal anti-BrdU antibody according to the manufacturer's instructions (Boehringer Mannheim GmbH, Mannheim, Germany). Coverslips were mounted on slides and fixed with 20% glycerol/PBS. Cell counts of nuclear BrdU labeling were determined by a Zeiss Axiovert 100 fluorescence microscope (Zeiss, Jena, Germany) using a triple bandpass filter. As S1P is released from degranulating platelets at wound sites, we have investigated its effects on different parameters of dermal and epidermal cells involved in wound healing of human skin. In particular these parameters include migration and proliferation of keratinocytes responsible for the reconstruction of the cutaneous barrier as well as proliferation of and matrix formation by fibroblasts. Exogenous S1P possessed no mitogenic effect in primary human quiescent keratinocytes, isolated from skin samples and cultured in KBM. But intriguingly, S1P decreased thymidine incorporation of proliferating keratinocytes, cultured in KGM, in a concentration-dependent manner indicating an anti-proliferative action in these epidermal cells. An inhibition of DNA synthesis of more than 60% was observed at a concentration of 10 μM S1P with an IC50 of 1 μM (Fig 1A). Direct assessment of cell growth confirmed the anti-proliferative property of 10 μM S1P with a 40% inhibition of cell growth after 3 d. In additio
Referência(s)