Intracellular Ca2+ and Zn2+ Levels Regulate the Alternative Cell Density-dependent Secretion of S100B in Human Glioblastoma Cells
2001; Elsevier BV; Volume: 276; Issue: 33 Linguagem: Inglês
10.1074/jbc.m103541200
ISSN1083-351X
AutoresGabriela E. Davey, Petra Murmann, Claus W. Heizmann,
Tópico(s)Antimicrobial Peptides and Activities
ResumoIn recent years, protein translocation has been implicated as the mechanism that controls assembly of signaling complexes and induction of signaling cascades. Several members of the multifunctional Ca2+- (Zn2+- and Cu2+)-binding S100 proteins appear to translocate upon cellular stimulation, and some are even secreted from cells, exerting extracellular functions. We transfected cells with S100B-green fluorescent fusion proteins and followed the relocation in real time. A small number of cells underwent translocation spontaneously. However, the addition of thapsigargin, which increases Ca2+ levels, intensified ongoing translocation and secretion or induced these processes in resting cells. On the other hand, EGTA or BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid), the Ca2+-chelating agents, inhibited these processes. In contrast, relocation of S100B seemed to be negatively dependent on Zn2+ levels. Treatment of cells with TPEN (N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine), a Zn2+-binding drug, resulted in a dramatic redistribution and translocation of S100B. Secretion of S100B, when measured by ELISA, was dependent on cell density. As cells reached confluence the secretion drastically declined. However, an increase in Ca2+ levels, and even more so, a decrease in Zn2+ concentration, reactivated secretion of S100B. On the other hand, secretion did not decrease by treatment with brefeldin A, supporting the view that this process is independent of the endoplasmic reticulum-Golgi classical secretion pathway. In recent years, protein translocation has been implicated as the mechanism that controls assembly of signaling complexes and induction of signaling cascades. Several members of the multifunctional Ca2+- (Zn2+- and Cu2+)-binding S100 proteins appear to translocate upon cellular stimulation, and some are even secreted from cells, exerting extracellular functions. We transfected cells with S100B-green fluorescent fusion proteins and followed the relocation in real time. A small number of cells underwent translocation spontaneously. However, the addition of thapsigargin, which increases Ca2+ levels, intensified ongoing translocation and secretion or induced these processes in resting cells. On the other hand, EGTA or BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid), the Ca2+-chelating agents, inhibited these processes. In contrast, relocation of S100B seemed to be negatively dependent on Zn2+ levels. Treatment of cells with TPEN (N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine), a Zn2+-binding drug, resulted in a dramatic redistribution and translocation of S100B. Secretion of S100B, when measured by ELISA, was dependent on cell density. As cells reached confluence the secretion drastically declined. However, an increase in Ca2+ levels, and even more so, a decrease in Zn2+ concentration, reactivated secretion of S100B. On the other hand, secretion did not decrease by treatment with brefeldin A, supporting the view that this process is independent of the endoplasmic reticulum-Golgi classical secretion pathway. green fluorescent protein Dulbecco's modified Eagle's medium N,N,N′,N′-tetrakis(2-pyridylmethyl)-ethylenediamine 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid endoplasmic reticulum N-acetyl-Asp-Glu-Val-Asp enzyme-linked immunosorbent assay 1,4-piperazinediethanesulfonic acid phosphate-buffered saline interleukin S100B is a small acidic protein containing two distinct EF-hands, predominantly expressed in astrocytes, oligodendrocytes, and Schwann cells. Intracellularly, S100B regulates the cytoskeletal dynamics through disassembly of tubulin filaments and binding to fibrillary proteins such as CapZ (1Kligman D. Marshak D.R. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 7136-7139Crossref PubMed Scopus (334) Google Scholar, 2Van Eldik L.J. Christie-Pope B. Bolin L.M. Shooter E.M. 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Chem. 2000; 275: 40096-40105Abstract Full Text Full Text PDF PubMed Scopus (513) Google Scholar). However, the extracellular concentration of S100B plays a crucial role in the physiological response. Although nanomolar quantities have trophic effects on cells, high levels of this protein have been implicated in glial activation (a prominent feature in Down syndrome and Alzheimer's disease), up-regulation of nitric-oxide synthase, and apoptosis (11Huttunen H.J. Kuja-Panula J. Sorci G. Agneletti A.L. Donato R. Rauvala H. J. Biol. Chem. 2000; 275: 40096-40105Abstract Full Text Full Text PDF PubMed Scopus (513) Google Scholar, 12Hu J. Van Eldik L.J. Biochim. Biophys. Acta. 1996; 1313: 239-245Crossref PubMed Scopus (123) Google Scholar, 13Hu J. Van Eldik L.J. Brain Res. 1999; 842: 46-54Crossref PubMed Scopus (91) Google Scholar). Recently, Schmidt and colleagues (14Hofmann M.A. Drury S. Fu C. Qu W. Taguchi A. Lu Y. Avila C. Kambham N. Bierhaus A. Nawroth P. Neurath M.F. Slattery T. Beach D. McClary J. Nagashima M. Morser J. Stern D. Schmidt A.M. Cell. 1999; 97: 889-901Abstract Full Text Full Text PDF PubMed Scopus (1609) Google Scholar,15Schmidt A.M. Yan S.D. Yan S.F. Stern D.M. Biochim. Biophys. Acta. 2000; 1498: 99-111Crossref PubMed Scopus (607) Google Scholar) identified the surface receptor RAGE (receptor for advanced glycation endproducts) for S100B, shedding more light on its extracellular function. Others also demonstrated that the binding of extracellular S100B to RAGE promotes cell survival by induction of NF-κB and Bcl-2 (11Huttunen H.J. Kuja-Panula J. Sorci G. Agneletti A.L. Donato R. Rauvala H. J. Biol. Chem. 2000; 275: 40096-40105Abstract Full Text Full Text PDF PubMed Scopus (513) Google Scholar). On the other hand, the antioxidant-dependent apoptosis induced by high levels of S100B requires expression of the full-length receptor. In fact, the deletion mutant of RAGE was unable to transduce trophic or toxic stimuli of S100B. This indicates that extracellular S100B can induce cell survival or apoptosis only when the functional receptor is expressed on the cell surface. However, the mechanisms of regulation of S100B secretion still remain unclear. In resting cells, S100 proteins are localized in specific cellular compartments from which they relocate upon Ca2+ activation (16Mandinova A. Atar D. Schäfer B.W. Spiess M. Aebi U. Heizmann C.W. J. Cell Sci. 1998; 111: 2043-2054Crossref PubMed Google Scholar, 17Mueller A. Bachi T. Hochli M. Schafer B.W. Heizmann C.W. Histochem. Cell Biol. 1999; 111: 453-459Crossref PubMed Scopus (63) Google Scholar, 18Davey G.E. Murmann P. Hoechli M. Tanaka T. Heizmann C.W. Biochim. Biophys. Acta. 2000; 1498: 220-232Crossref PubMed Scopus (30) Google Scholar). This suggests that translocation might be a temporal and spatial determinant of their interactions with different partner proteins. Most of the S100B cascades, whether induced through interaction with intracellular partners or binding to extracellular receptor domains, are most likely preceded by translocation and/or secretion of S100B. Therefore, we investigated factors that might regulate these mechanisms. Because S100B binds Ca2+ and Zn2+ (19Heizmann C.W. Cox J.A. Biometals. 1998; 11: 383-397Crossref PubMed Scopus (260) Google Scholar, 20Barber K.R. McClintock K.A. Jamieson Jr., G.A. Dimlich R.V. Shaw G.S. J. Biol. Chem. 1999; 274: 1502-1508Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar) we focused on the effects of intracellular Ca2+/Zn2+ concentration on translocation/secretion. Astrocytes (glial cells), which secrete S100B, have evolved a complex machinery that enables them to change intracellular calcium concentration. They sense and respond to external signals produced by neurons and communicate with each other through calcium waves (21Giaume C. Venance L. Glia. 1998; 24: 50-64Crossref PubMed Scopus (221) Google Scholar). Xiong et al. (22Xiong Z. O'Hanlon D. Becker L.E. Roder J. MacDonald J.F. Marks A. Exp. Cell Res. 2000; 257: 281-289Crossref PubMed Scopus (100) Google Scholar) used glial cells derived from S100B null mice and demonstrated that S100B modulates Ca2+ homeostasis in these cells, confirming the earlier results of Barger and Van Eldik (23Barger S.W. Van Eldik L.J. J. Biol. Chem. 1992; 267: 9689-9694Abstract Full Text PDF PubMed Google Scholar), who showed that S100B stimulates calcium fluxes in glial and neuronal cells. Therefore, we selected two human glioblastoma cell lines, one expressing and the other not expressing endogenous S100B, to examine the translocation/secretion pathway using S100B-green fluorescent protein (GFP).1 To fuse S100B to GFP (enhanced form, modified for brighter fluorescence,CLONTECH), we designed primers, including a linker of 5 glycines, 20 bases of the S100B-coding sequence, and restriction sites compatible with the GFP-containing vectors (XhoI/BamHI for pEGFP-N1 orBglII/SalI for pEGFP-C1) followed by three additional bases. The primers were incorporated into the S100B sequence by polymerase chain reaction. Oligonucleotides used for construction of pEGFP-N1 were 5′-TAACTCGAGATGTCTGAG CTGGAGAAGGC-3′ and 5′-ATTACCTAGGGC(CCA)5GAGTACAAGTTTCTTGAGCA; of pEGFP-C1, 5′-AATAGATCT(GGT)5TCTGAGCTGGAGAAGGCCAt-3′ and 5′-ATTCAGCTGAGTGAGTACAAGTTTTCTTGA. The polymerase chain reaction conditions were as follows: pEGFP-N1, 58 °C, 30 cycles; pEGFP-C1, 56 °C, 30 cycles. The resulting products were digested, purified, and ligated in the correct reading frame to the corresponding vectors. All constructs were verified by restriction digestion and DNA sequencing. The human glioblastoma cell lines U-373MG and U-87MG (HTB-17 and HTB-14, respectively, available from ATCC) were maintained in DMEM containing 10% FBC and penicillin-streptomycin. GFP constructs were transfected using LipofectAMINE (Life Technologies, Inc.). Cells were seeded at 2–2.5 × 105 cells/35-mm dish, plated, and incubated overnight. Plasmid DNA (1 µg) in DMEM (100 µl total volume) and LipofectAMINE (5 µl) reagent in DMEM (95 µl) were combined and incubated for 15–30 min before being diluted to 1 ml with DMEM and added to the cells. After incubating for 5 h, a solution of 10% fetal bovine serum in DMEM was added to the cells and incubated overnight. The following day, the transfection medium was removed and fresh medium was added. Stable transfectants were selected and maintained in medium containing G418. To examine localization and translocation of endogenous S100B and recombinant fusion proteins by confocal microscopy, 2–5 × 104 cells were plated on coverslips in 24-well dishes 24 h prior to the experiment. Cells were rinsed with DMEM and incubated for 10 min at 37 °C with TPEN (Sigma, 5 µm) or thapsigargin (Sigma, 100 nm-1 µm) (24Thastrup O. Cullen P.J. Drobak B.K. Hanley M.R. Dawson A.P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2466-2470Crossref PubMed Scopus (3001) Google Scholar, 25Diarra A. Sauve R. Pflugers Arch. Eur. J. Physiol. 1992; 422: 40-47Crossref PubMed Scopus (11) Google Scholar, 26Gromada J. Jorgensen T.D. Dissing S. FEBS Lett. 1995; 360: 303-306Crossref PubMed Scopus (45) Google Scholar). For treatment with thapsigargin, cells were incubated in a stimulation buffer (140 mm NaCl, 5 mm KCl, 1 mmMgCl2, 10 mm glucose, and 10 mmHEPES supplemented with 1 mm CaCl2 or 10 mm EGTA). The viability of cells was assessed after prolonged treatment with thapsigargin, TPEN, or EGTA (2.5 × 105). The cells were seeded in 35-mm dishes (in triplicates), incubated with drugs for up to 90 min, trypsinized, stained with trypan blue, and counted. To observe the translocation of S100B in live cells, 5–10 × 105 cells expressing fusion proteins were seeded in 35-mm glass-bottom dishes (Matek) and treated with different drugs on the microscope stage in a temperature-controlled CO2 chamber. The time course for S100B translocation after thapsigargin (100 nm-1 µm) treatment was monitored by microscope time lapse photography. The effects of Ca2+ and Zn2+ on translocation were monitored by incubating the cells with Ca2+-chelators such as 10 mm EGTA or membrane-permeable 100 µm BAPTA-AM (Molecular Probes (27Zhang Z. Hernandez-Lagunas L. Horne W.C. Baron R. J. Biol. Chem. 1999; 274: 25093-25098Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 28Wang H.J. Guay G. Pogan L. Sauve R. Nabi I.R. J. Cell Biol. 2000; 150: 1489-1498Crossref PubMed Scopus (143) Google Scholar)) or with a Zn2+ chelator, i.e. 5 µm TPEN. Cells were treated with 0.5–5 µg/ml brefeldin A, which reversibly blocks the ER-Golgi-dependent pathway (Calbiochem (29Rammes A. Roth J. Goebeler M. Klempt M. Hartmann M. Sorg C. J. Biol. Chem. 1997; 272: 9496-9502Abstract Full Text Full Text PDF PubMed Scopus (478) Google Scholar)). To disrupt actin or tubulin filaments, cells were treated with 1 µm amlexanox (Takeda Chemical Industries Ltd. (30Carreira C.M. LaVallee T.M. Tarantini F. Jackson A. Lathrop J.T. Hampton B. Burgess W.H. Maciag T. J. Biol. Chem. 1998; 273: 22224-22231Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 31Landriscina M. Prudovsky I. Carreira C.M. Soldi R. Tarantini F. Maciag T. J. Biol. Chem. 2000; 275: 32753-32762Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar)) or 1 µm demecolcine (Sigma (32Caron J.M. Jones A.L. Rall L.B. Kirschner M.W. Nature. 1985; 317: 648-651Crossref PubMed Scopus (62) Google Scholar)), respectively. For ELISAs, a medium (1 ml) from each sample was collected, and the cells were extracted in 1 ml of buffer B (50 mm Tris-HCl, pH 8, 250 mm NaCl, 2 mm EDTA, 1% Nonidet P-40, supplemented with protease inhibitors: 10 µg/ml phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin). S100B was quantified by ELISA using an immunoradiometric assay (Sangtec) according to the manufacturer's protocol. The amount of S100B in the sample was calculated using a standard curve with known concentrations of human recombinant S100B. To assess potential apoptotic effects, cells were resuspended in 75 µl of cell extraction buffer (50 mm Pipes, pH 7.0, 50 mm KCl, 5 mm EGTA, 2 mm MgCl2, 1 mm dithiothreitol, and 1 µg/ml CLAP (chymostatin, leupeptin, aprotinin, pepstatin A, 10 µg/ml phenylmethylsulfonyl fluoride)) and then frozen and thawed twice. After centrifugation at 14,000 × g, 4 °C for 15 min, the supernatant was transferred to a new tube, and the protein concentration was determined by the Coomassie Blue assay. Protein extracts from each sample were diluted to a final concentration of 40 µg/100 µl. Then, 100 µl-aliquots from each sample were added to two wells of a 96-well plate. One of the wells was left untreated, while the other was treated with 10 µm DEVD-Chinese hamster ovary inhibitor (Sigma) at 37 °C for 30 min. Then 10 µl of 0.8 mmDEVD-pNa substrate was added to each well, and the plate was analyzed after 2, 6, and 24 h at 405 nm. As a positive control, cells were treated with 100 nm staurosporine. Cell proteins (5–6 µg/ml) were extracted in 1 ml of buffer B (see above) and immunoprecipitated with 20 µl of rabbit anti-human recombinant S100B (F 3802) (33Ilg E.C. Schäfer B.W. Heizmann C.W. Int. J. Cancer. 1996; 68: 325-332Crossref PubMed Scopus (218) Google Scholar) bound to 15 mg of protein A-Sepharose. The samples were analyzed by SDS-polyacrylamide gel electrophoresis. After blotting onto nitrocellulose membranes and blocking in nonfat milk (5% in washing buffer: 25 mm Tris-HCl, pH 7.4, 0.15 m NaCl, 0.5% Triton X-100), membranes were incubated with anti-S100B serum (dilution, 1:1500), washed for 30 min, and incubated with horseradish peroxidase-conjugated goat anti-rabbit serum (dilution 1:500, Bio-Rad). Visualization of immunoreactive proteins was achieved by enhanced chemiluminescence (Amersham Pharmacia Biotech). Cells were rinsed with DMEM, fixed with 3.7% formaldehyde in DMEM for 15 min at 37 °C, and permeabilized with methanol for 10 min at room temperature. After washing in 5% horse serum-DMEM, the cells were incubated with anti-S100B (dilution 1:500) for 1 h at 37 °C followed by staining with Cy3-conjugated anti-rabbit IgG (H+L) (goat, Jackson ImmunoResearch Laboratories) for 45 min at 37 °C. As a control, cells were incubated with the antibodies preabsorbed with human S100B antigen (1 µg/1 µl). The cells to be stained with the monoclonal Cy3-conjugated anti-actin (mouse IgG2a isotype; Sigma) or anti-β-tubulin (mouse IgG1 isotype; Sigma) were fixed with formaldehyde (3.7% in PBS with Ca2+/Mg2+) at room temperature for 20–30 min, washed twice with PBS, treated with NH4Cl (50 mm in PBS), washed with PBS, and permeabilized with Triton X-100 (0.1% in PBS) as described (34Zhu X. Roovers K. Davey G. Assoian R.K. Guan J.-L. Signaling through Cell Adhesion Molecules. CRC Press, Boca Raton, FL1999: 129-140Google Scholar). Cells were incubated with antibodies against actin (dilution 1:50) or tubulin (dilution 1:100) for 30 min and washed four times with PBS and once with water. To mount the slides, a small drop of mounting media (Molwiol, Hoechst) containing 0.75% n-propyl gallate as an anti-bleaching agent, was placed on the slide. Mounted slides were left to dry for 24 h at room temperature and stored in the dark at 4 °C until viewed. Fixed cells were analyzed using a Zeiss Axioplan fluorescence microscope (under a 63×-oil objective lens) equipped with a confocal scanning unit MRC-600 (Bio-Rad) and an argon-krypton laser with an excitation wavelength of 568 nm for Cy3 and 488 nm for enhanced GFP. Subsequently, the images were processed using Graphic Converter (version 3.8) and Photoshop (Adobe System, version 5.5) software on a Macintosh computer. Live cells were analyzed by time lapse photography after plating in 35-mm glass bottom dishes (Matek), using a wide-field Leica DM IRBE microscope equipped with a Hamamatsu camera controller (C4742–95) and connected to a Macintosh computer. The imaging and recording was operated by Open Lab software, version 2.1.1. The 25–50 images (depending on the rapidity of the cellular response) were recorded, one every 4 s, with each exposure set at 50–300 ms (depending on the intensity of the signal, but constant in each series) using a set filter for GFP (Leica), and neutral density filters to minimize bleaching. The images obtained were processed using Photoshop (Adobe System, version 5.5) or converted into quick-time files using a public domain NIH Image program (developed at the National Institutes of Health, rsb.info.nih.gov/nih.-image/). Before examining the localization, mechanisms of translocation, and secretion of S100B, we assessed the endogenous levels of S100B in two glioblastoma cell lines, U373 and U87, both described to be similar to primary astrocytic tumors (35Kiss R. Camby I. Salmon I. Van Ham P. Brotchi J. Pasteels J.L. Cytometry. 1995; 20: 118-126Crossref PubMed Scopus (11) Google Scholar, 36Camby I. Salmon I. Oiry C. Galleyrand J.C. Nagy N. Danguy A. Brotchi J. Pasteels J.L. Martinez J. Kiss R. Neuropeptides. 1996; 30: 433-437Crossref PubMed Scopus (9) Google Scholar, 37De Hauwer C. Camby I. Darro F. Decaestecker C. Gras T. Salmon I. Kiss R. Van Ham P. Biochem. Biophys. Res. Commun. 1997; 232: 267-272Crossref PubMed Scopus (32) Google Scholar, 38De Hauwer C. Camby I. Darro F. Migeotte I. Decaestecker C. Verbeek C. Danguy A. Pasteels J.L. Brotchi J. Salmon I. Van Ham P. Kiss R. J. Neurobiol. 1998; 37: 373-382Crossref PubMed Scopus (55) Google Scholar). Proteins in cellular extracts were immunoprecipitated with anti-human S100B and analyzed by Western blotting (Fig.1 a) and ELISA (Fig.1 b). In U373 cells, endogenous S100B levels are highest at confluence (∼5 ng/100,000 cells), whereas in U87 cells they are undetectable. Next we prepared S100B-GFP fusion proteins connected by a 5 glycine-linker, allowing for rotational freedom between the S100B protein and GFP. S100B fused to the N terminus of GFP (through ligation to pEGFP-N1, Fig. 2 a) was named GnB, and that fused to the C terminus of GFP (through ligation to pEGFP-C1, Fig. 2 b) was named GcB. To examine the localization of GnB and GcB in comparison to the endogenous S100B, U373 (Fig. 3 a) and U87 (Fig.3 b) cells were either stained with anti-S100B and examined by indirect immunofluorescence or U87 cells were transfected with either construct (named GnB-U87 or GcB-U87) and directly analyzed (3c-d).Figure 2Construction of S100B-GFP vectors. S100B cDNA was amplified by polymerase chain reaction using specific primers/adaptors and then ligated into the corresponding vector.a, S100B was fused to the N terminus of GFP (GnB), and thus its stop codon was eliminated and a 5-glycine linker was added at the 3′-end. b, S100B was fused to the C terminus of the GFP (GcB), and thus its start codon was removed and the 5-glycine linker was added at the 5′-end. The indicated restriction sites were added at the 5′- and 3′-ends of the primers to clone into multiple cloning sites (MCS). ext, extension; CMV, cytomegalovirus.View Large Image Figure ViewerDownload (PPT)Figure 3Localization of endogenous S100B in U373 cells is comparable with that of S100B-GFP fusion proteins in U87 cells. U373 (a) and U87 cells (b) were fixed and stained with anti-S100B and Cy3-conjugated goat anti-rabbit IgG. U87 cells were transfected with either GcB (c) or GnB (d) and analyzed directly. All samples were analyzed by laser-scanning confocal microscopy (scale bar,15 µm).View Large Image Figure ViewerDownload (PPT) The localization of the fusion proteins in U87 cells was comparable with that of endogenous S100B in U373. Some of the differences might be because of the variation between the appearance of GFP fluorescence, as opposed to the immuno staining, which might yield a more punctate pattern. Nonetheless, the endogenous and the fusion proteins were both absent from the nucleus, as assessed by confocal microscopy, in contrast to S100A11, which was found in the nucleus (18Davey G.E. Murmann P. Hoechli M. Tanaka T. Heizmann C.W. Biochim. Biophys. Acta. 2000; 1498: 220-232Crossref PubMed Scopus (30) Google Scholar), and they were both predominantly localized to the perinuclear area. However, the staining of the S100B-GFP extended further into the cytoplasm, potentially caused by slightly higher concentration of the fusion protein in the cell. Similar results were obtained with transient and stable transfectants. To determine the effects of intracellular Ca2+/Zn2+ levels on the localization of endogenous S100B, we either left U373 cells untreated (Fig.4 a) or we treated them for 10 min with thapsigargin (data not shown) or TPEN (5 µm, Fig. 4 b) and compared the localization of S100B by indirect immunofluorescence. Similarly, we analyzed untreated (Fig.4 c) and TPEN-treated GcB-U87 cells (Fig. 4 d). In both cell types we observed vesicle-like structures located toward the periphery of the cell, indicating that both the endogenous protein and the S100B-GFP fusion protein behave similarly and translocate in response to increasing Ca2+ or decreasing Zn2+concentration. To study these processes in real time, U87-GnB or U87-GcB cells were studied by wide-field microscopy, using either transiently or stably transfected cells. Initial observation revealed that a small number of the S100B-GFP-positive cells were undergoing spontaneous translocation. The S100B seemed to be moving toward the newly formed cytoplasmic extensions. These extensions were dynamically forming and disappearing, however, they were not always occupied by S100B when observed under phase-contrast. Although these glioblastoma cell lines are in general quite motile (35Kiss R. Camby I. Salmon I. Van Ham P. Brotchi J. Pasteels J.L. Cytometry. 1995; 20: 118-126Crossref PubMed Scopus (11) Google Scholar, 36Camby I. Salmon I. Oiry C. Galleyrand J.C. Nagy N. Danguy A. Brotchi J. Pasteels J.L. Martinez J. Kiss R. Neuropeptides. 1996; 30: 433-437Crossref PubMed Scopus (9) Google Scholar, 37De Hauwer C. Camby I. Darro F. Decaestecker C. Gras T. Salmon I. Kiss R. Van Ham P. Biochem. Biophys. Res. Commun. 1997; 232: 267-272Crossref PubMed Scopus (32) Google Scholar, 38De Hauwer C. Camby I. Darro F. Migeotte I. Decaestecker C. Verbeek C. Danguy A. Pasteels J.L. Brotchi J. Salmon I. Van Ham P. Kiss R. J. Neurobiol. 1998; 37: 373-382Crossref PubMed Scopus (55) Google Scholar), the process of translocation did not appear to be dependent on this characteristic. Translocation would subside in one cell (Fig. 5 a) and begin in another in 10–20% of cells in the population at any time point. Addition of 0.5 µm thapsigargin (Fig. 5 b) caused relocation of S100B in 70–80% of the cells within 1–2 min, supporting the idea that this process depends on calcium fluxes that normally occur in glial cells and can be intensified by a sudden increase in the intracellular Ca2+ concentration through drug stimulation. These results did not depend on the expression levels of S100B-GFP. The experiments were repeated with different stable and transient transfectants that display a wide range of expression levels of S100B-GFP, yielding the same results. As a control, cells undergoing S100B translocation were treated with 10 mm EGTA (Fig. 6,a–c) or 100 µm BAPTA-AM (data not shown) to chelate endogenous Ca2+, resulting in an inhibition of the translocation process within 2–5 min. Similarly, pretreatment of unstimulated cells with either Ca2+-chelator blocked or significantly reduced the response of cells to thapsigargin. In fact, the vesicles would get smaller as they were moving about in the cell, to the point when they were no longer detectable. The cell membrane retracted only after prolonged incubation with EGTA, which removes Ca2+ from the interactions between integrins and extracellular matrix, resulting in a slow cell detachment. However, when EGTA-containing medium was replaced by normal medium, cells fully reattached and resumed the basal level of translocation (data not shown). Therefore, induction and inhibition of S100B translocation are both Ca2+-dependent and reversible. On the other hand, chelating endogenous Zn2+ by addition of 5–10 µm TPEN (a membrane-permeant Zn2+-chelator) produced a result opposite to that of Ca2+ removal by EGTA/BAPTA-AM. Within seconds after treatment, a dramatic change in fluorescence intensity of S100B-GFP occurred (Fig. 6 e), followed by an onset of translocation (Fig. 6 f). If TPEN was removed by incubation in fresh media, cells would return to normal conditions. Viability of cells after 0, 15, 45, and 90 min of treatment with 1 µm thapsigargin, 20 µm TPEN, or 10 mm EGTA was assessed by staining triplicate samples with trypan blue, showing no increase in cell death even when drug concentration and exposure time were higher than those used in the time lapse experiments. We also analyzed and compared relocation of S100B-GFP between the two cell lines, showing that the translocation of the fusion proteins is unaffected by the presence or absence of endogenous S100B. Here, we present analysis mostly done in the U87 cells, which do not express endogenous S100B. To determine whether S100B translocation upon [Ca2+] increase depends on the ER-Golgi pathway, U87 cells expressing fusion proteins were seeded onto glass-bottom plates, incubated with 5 µg/ml of brefeldin A (29Rammes A. Roth J. Goebeler M. Klempt M. Hartmann M. Sorg C. J. Biol. Chem. 1997; 272: 9496-9502Abstract Full Text Full Text PDF PubMed Scopus (478) Google Scholar,39Jareb M. Banker G. J. Neurosci. 1997; 17: 8955-8963Crossref PubMed Google Scholar), which blocks the classical secretion pathway. In an effort to identify cellular components involved in the process of translocation, the cells were treated with 1 µm amlexanox (30Carreira C.M. LaVallee T.M. Tarantini F. Jackson A. Lathrop J.T. Hampton B. Burgess W.H. Maciag T. J. Biol. Chem. 1998; 273: 22224-22231Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 31Landriscina M. Prudovsky I. Carreira C.M. Soldi R. Tarantini F. Maciag T. J. Biol. Chem. 2000;
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