S100P Stimulates Cell Proliferation and Survival via Receptor for Activated Glycation End Products (RAGE)
2004; Elsevier BV; Volume: 279; Issue: 7 Linguagem: Inglês
10.1074/jbc.m310124200
ISSN1083-351X
AutoresThiruvengadam Arumugam, Diane M. Simeone, Ann Marie Schmidt, Craig D. Logsdon,
Tópico(s)Alzheimer's disease research and treatments
ResumoS100P is a member of the S100 protein family that is expressed in several malignant neoplasms. Currently the effects of this molecule on cell function are unknown. In the present study we investigated the biological effects and mechanisms of action of S100P using NIH3T3 cells. Expression of S100P in NIH3T3 cells led to the presence of S100P in the culture medium, increased cellular proliferation, and enhanced survival after detachment from the culture substrate or after exposure to the chemotherapeutic agent 5-flurouracil. The proliferation and survival effects of S100P expression were duplicated in a time- and concentration-dependent manner by the extracellular addition of purified S100P to wild-type NIH3T3 cells and correlated with the activation of extracellular-regulated kinases (Erks) and NF-κB. To determine the mechanisms involved in these effects, we tested the hypothesis that S100P activated RAGE (receptor for activated glycation end products). We found that S100P co-immunoprecipitated with RAGE. Furthermore, the effects of S100P on cell signaling, proliferation, and survival were blocked by agents that interfere with RAGE including administration of an amphoterin-derived peptide known to antagonize RAGE activation, anti-RAGE antibodies, and by expression of a dominant negative RAGE. These data suggest that S100P can act in an autocrine manner via RAGE to stimulate cell proliferation and survival. S100P is a member of the S100 protein family that is expressed in several malignant neoplasms. Currently the effects of this molecule on cell function are unknown. In the present study we investigated the biological effects and mechanisms of action of S100P using NIH3T3 cells. Expression of S100P in NIH3T3 cells led to the presence of S100P in the culture medium, increased cellular proliferation, and enhanced survival after detachment from the culture substrate or after exposure to the chemotherapeutic agent 5-flurouracil. The proliferation and survival effects of S100P expression were duplicated in a time- and concentration-dependent manner by the extracellular addition of purified S100P to wild-type NIH3T3 cells and correlated with the activation of extracellular-regulated kinases (Erks) and NF-κB. To determine the mechanisms involved in these effects, we tested the hypothesis that S100P activated RAGE (receptor for activated glycation end products). We found that S100P co-immunoprecipitated with RAGE. Furthermore, the effects of S100P on cell signaling, proliferation, and survival were blocked by agents that interfere with RAGE including administration of an amphoterin-derived peptide known to antagonize RAGE activation, anti-RAGE antibodies, and by expression of a dominant negative RAGE. These data suggest that S100P can act in an autocrine manner via RAGE to stimulate cell proliferation and survival. The S100 family consists of Ca2+-binding proteins of the EF-hand type with at least 20 members (1Donato R. Int. J. Biochem. Cell Biol. 2001; 33: 637-668Crossref PubMed Scopus (1308) Google Scholar, 2Heizmann C.W. Fritz G. Schafer B.W. Front. Biosci. 2002; 7: 1356-1368Crossref PubMed Google Scholar). Although these molecules are widely expressed, none appear to be ubiquitous, and several have highly restricted distributions (1Donato R. Int. J. Biochem. Cell Biol. 2001; 33: 637-668Crossref PubMed Scopus (1308) Google Scholar). The functions of these proteins vary widely between individual members. S100 proteins can function as both intracellular and extracellular signaling molecules. Intracellular actions of S100 proteins are isoform-specific and include Ca2+-dependent regulation of a variety of intracellular activities including protein phosphorylation, enzyme activities, cytoskeletal function, intracellular Ca2+ homeostasis, and protection from oxidative cell damage (1Donato R. Int. J. Biochem. Cell Biol. 2001; 33: 637-668Crossref PubMed Scopus (1308) Google Scholar, 3McNutt N.S. J. Cutan. Pathol. 1998; 25: 521-529Crossref PubMed Scopus (54) Google Scholar, 4Schafer B.W. Heizmann C.W. Trends Biochem. Sci. 1996; 21: 134-140Abstract Full Text PDF PubMed Scopus (1035) Google Scholar, 5Zimmer D.B. Cornwall E.H. Landar A. Song W. Brain Res. Bull. 1995; 37: 417-429Crossref PubMed Scopus (806) Google Scholar). Several S100s are also known to be released from cells and to act extracellularly. Although the mechanisms for the extracellular effects of many S100 proteins are not known and there may be differences between isoforms, both S100A12 and S100B proteins have previously been shown to act extracellularly through their abilities to activate the receptor for activated glycation end products (RAGE), 1The abbreviations used are: RAGEreceptor for activated glycation end productsErkextracellular-regulated kinaseELISAenzyme-linked immunosorbent assay5-FU5-fluorouracilpoly-HEMApolyhydroxyethyl methacrylateDndominant negativeMTS[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfphenyl)-2-H-tetrazolium, inner salt).1The abbreviations used are: RAGEreceptor for activated glycation end productsErkextracellular-regulated kinaseELISAenzyme-linked immunosorbent assay5-FU5-fluorouracilpoly-HEMApolyhydroxyethyl methacrylateDndominant negativeMTS[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfphenyl)-2-H-tetrazolium, inner salt). leading to the suggestion that this may be an important mechanism for extracellular effects of S100 proteins (6Hofmann 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 (1582) Google Scholar).One of the least studied members of the S100 family is S100P, a 95-amino acid protein first purified from placenta with a restricted cellular distribution (7Becker T. Gerke V. Kube E. Weber K. Eur. J. Biochem. 1992; 207: 541-547Crossref PubMed Scopus (154) Google Scholar, 8Emoto Y. Kobayashi R. Akatsuka H. Hidaka H. Biochem. Biophys. Res. Commun. 1992; 182: 1246-1253Crossref PubMed Scopus (71) Google Scholar). The molecular structure of S100P has been well described and supports its classification in the S100 family of proteins (9Zhang H. Wang G. Ding Y. Wang Z. Barraclough R. Rudland P.S. Fernig D.G. Rao Z. J. Mol. Biol. 2003; 325: 785-794Crossref PubMed Scopus (48) Google Scholar). Expression of S100P has been noted in esophageal epithelial cells during their differentiation, indicating that it may play a role in normal development (10Sato N. Hitomi J. Anat. Rec. 2002; 267: 60-69Crossref PubMed Scopus (33) Google Scholar). There is also considerable evidence that S100P plays a role in cancer. S100P expression has been noted in various cancer cell lines including breast cancer, where it was associated with cellular immortalization (11Guerreiro I, D.S. Hu Y.F. Russo I.H. Ao X. Salicioni A.M. Yang X. Russo J. Int. J. Oncol. 2000; 16: 231-240PubMed Google Scholar), and colon cancer, where its expression was elevated in doxorubicin-resistant cells (12Bertram J. Palfner K. Hiddemann W. Kneba M. Anticancer Drugs. 1998; 9: 311-317Crossref PubMed Scopus (76) Google Scholar). S100P has also been shown to be expressed in tumors, including prostate cancer, where its expression is androgen-sensitive (13Averboukh L. Liang P. Kantoff P.W. Pardee A.B. Prostate. 1996; 29: 350-355Crossref PubMed Google Scholar), and pancreatic adenocarcinoma, where expression has been localized to the neoplastic epithelium of pancreatic (14Logsdon C.D. Simeone D.M. Binkley C. Arumugam T. Greenson J.K. Giordano T.J. Misek D.E. Hanash S. Cancer Res. 2003; 63: 2649-2657PubMed Google Scholar). Furthermore, S100P expression has been shown to be correlated with decreased survival in patients with lung cancer (15Beer D.G. Kardia S.L. Huang C.C. Giordano T.J. Levin A.M. Misek D.E. Lin L. Chen G. Gharib T.G. Thomas D.G. Lizyness M.L. Kuick R. Hayasaka S. Taylor J.M. Iannettoni M.D. Orringer M.B. Hanash S. Nat. Med. 2002; 8: 816-824Crossref PubMed Scopus (1645) Google Scholar). Despite the evidence indicating the potential importance of this molecule in normal and transformed cell function, its effects on cell function are unknown. Previous studies have utilized affinity columns to identify molecules that may interact with S100P. It was observed that S100P can interact with the cytoskeletal protein ezrin in a Ca2+-dependent manner and influence its ability to bind actin (16Koltzscher M. Neumann C. Konig S. Gerke V. Mol. Biol. Cell. 2003; 14: 2372-2384Crossref PubMed Scopus (88) Google Scholar). S100P has also been reported to be able to interact with CacyBP/SIP, a component of a novel ubiquitinylation pathway, leading to β catenin degradation (17Filipek A. Jastrzebska B. Nowotny M. Kuznicki J. J. Biol. Chem. 2002; 277: 28848-28852Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). However, it is not known whether S100P interacts with these molecules under in vivo conditions or what effects these interactions might have on cell function.RAGE is a member of the immunoglobulin superfamily of cell surface molecules that is activated by multiple ligands (6Hofmann 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 (1582) Google Scholar, 18Bucciarelli L.G. Wendt T. Rong L. Lalla E. Hofmann M.A. Goova M.T. Taguchi A. Yan S.F. Yan S.D. Stern D.M. Schmidt A.M. Cell. Mol. Life Sci. 2002; 59: 1117-1128Crossref PubMed Scopus (261) Google Scholar, 19Schmidt A.M. Stern D.M. Front. Biosci. 2001; 6: 1151-1160Crossref PubMed Google Scholar). RAGE was originally identified based on its ability to bind advanced glycation end products, which are adducts formed by glycoxidation (20Cerami A. Vlassara H. Brownlee M. Diabetes Care. 1988; 11: 73-79PubMed Google Scholar). Subsequently, it has been observed that RAGE can be activated by a number of specific ligands including amphoterin, amyloid-β peptide, and members of the S100 family and play important roles in a variety of disease states including inflammation, diabetes, Alzheimer's disease, and cancer (21Schmidt A.M. Yan S.D. Yan S.F. Stern D.M. J. Clin. Invest. 2001; 108: 949-955Crossref PubMed Scopus (1046) Google Scholar). In the context of cancer cells, activation of RAGE has been found to stimulate cell proliferation, survival, and motility (22Taguchi A. Blood D.C. del Toro G. Canet A. Lee D.C. Qu W. Tanji N. Lu Y. Lalla E. Fu C. Hofmann M.A. Kislinger T. Ingram M. Lu A. Tanaka H. Hori O. Ogawa S. Stern D.M. Schmidt A.M. Nature. 2000; 405: 354-360Crossref PubMed Scopus (1094) Google Scholar). The intracellular signaling mechanisms activated by RAGE are not completely understood, but occupation of RAGE by its ligands leads to stimulation of extracellular-regulated kinase (Erk), Rho, and Jak/Stat signaling as well as activation of the transcription factor NF-κB (23Stern D.M. Yan S.D. Yan S.F. Schmidt A.M. Ageing Res. Rev. 2002; 1: 1-15Crossref PubMed Scopus (232) Google Scholar). The role of RAGE activation in S100P signaling has not been previously examined.In the current study, we observed that expression of S100P led to the release of this molecule into the culture media and conferred proliferation and survival benefits to NIH3T3 cells. We then tested the hypothesis that extracellular S100P could influence cell function through the activation of RAGE. NIH3T3 cells were treated with purified S100P, which led to dose- and time-dependent increases in cell proliferation and survival. The effects of S100P on cell function correlated with its ability to activate Erks and the transcription factor NF-κB. Evidence that these biological effects were mediated by S100P activation of RAGE included the observation that these two molecules could be co-immunoprecipitated. Furthermore, blocking the interaction between S100P and RAGE using anti-RAGE antibodies, administration of a peptide antagonist derived from amphoterin, or expression of a dominant negative truncated RAGE inhibited the biological and signaling effects of S100P. Taken together, these data suggest that S100P can act in an autocrine manner to stimulate cell growth and survival through activation of RAGE.MATERIALS AND METHODSDevelopment of Stable Cell Lines—NIH3T3 cells obtained from the American Type Culture collection (Manassas, VA) were transfected using LipofectAMINE reagent (Invitrogen) with plasmids encoding human S100P or a dominant negative RAGE (22Taguchi A. Blood D.C. del Toro G. Canet A. Lee D.C. Qu W. Tanji N. Lu Y. Lalla E. Fu C. Hofmann M.A. Kislinger T. Ingram M. Lu A. Tanaka H. Hori O. Ogawa S. Stern D.M. Schmidt A.M. Nature. 2000; 405: 354-360Crossref PubMed Scopus (1094) Google Scholar) cloned into pcDNA3.1 vector and selected for resistance to G418 (0.5 mg/ml). Wild-type and stably transfected NIH3T3 cells were routinely cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum at 37 °C in a humidified atmosphere of 5% CO2. Data are shown for a single representative stable cell line, but all experiments were reproduced with at least two other monoclonally selected cell lines (data not shown).Expression and Purification of S100P—Full-length human S100P cDNA was cloned into the pTrcHis2 vector and transformed into one-shot TOP10 competent Escherichia coli (Invitrogen). The bacterial culture was incubated at 37 °C to an A600 = 0.6, then isopropyl-1-thio-β-d-galactopyranoside (1 mm) was added, and the bacteria were cultured for another 3 h. His-S100P was purified using a Probond resin column as described by the manufacturer (Invitrogen). The fraction was further dialyzed against 10 mm Tris, pH 8.0, containing 0.1% Triton X 100 overnight at 4 °C using a Slide-A-Lyzer 10K (Pierce). The dialyzed protein was further concentrated by a Centricon centrifugal filter device YM10 (Millipore, Bedford, MA). The purity of the S100P protein was confirmed by SDS-PAGE and Western blotting, and ELISA was used for in vitro experiments. To monitor for nonspecific effects, proteins from non-induced bacteria were utilized as a control.SDS-PAGE, Western Blot Analysis, and Co-immunoprecipitation— Western blot analysis was utilized for the detection of S100P, RAGE, and Erks by minor modifications of previously published methods (24Ji B. Chen X.Q. Misek D.E. Kuick R. Hanash S. Ernst S. Najarian R. Logsdon C.D. Physiol. Genomics. 2003; 14: 59-72Crossref PubMed Scopus (99) Google Scholar). Cell lysates were prepared and separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose. Membranes were blocked for 1 h at room temperature or overnight at 4 °C in 5% milk solution. S100P was detected by incubating the transferred membrane overnight at 4 °C with anti-human monoclonal antibody (Transduction Laboratories, San Diego, CA) at 1:1000 dilution in 5% milk solution. RAGE was detected by incubating the transferred membrane for 1 h at room temperature with anti-human goat polyclonal antibody (Santa Cruz, Santa Cruz, CA) at 1:100 dilution in 5% milk solution. Erk activation was estimated by detection of phosphorylated forms of Erk 1 and 2 using phospho-p44/42 mitogen-activated protein kinase (Thr-202/Tyr-204) antibody (Cell Signaling, Beverly, MA) and as a loading control a rabbit polyclonal antibody for total Erk 1 and 2 (Santa Cruz, Santa Cruz, CA) by incubating the membrane at 4 °C for overnight with antibody diluted 1:100 in 5% milk solution. Secondary antibody anti-mouse, anti-rabbit, or anti-goat IgG plus horseradish peroxidase antibody was incubated for 1 h at room temperature, and the signal was detected by the ECL detection system (Amersham) as per the manufacturer's protocol.For co-immunoprecipitation experiments cell lysates were incubated in the absence or presence of purified S100P (1 μg) at 4 °C overnight. S100P was immunoprecipitated using a monoclonal antibody against S100P for 6 h at 4 °C and IgG-immobilized beads (Pierce). Antibody-associated proteins were electrophoresed on 10% polyacrylamide gel and transferred to a nitrocellulose membrane. The transferred membrane was blocked by 5% milk solution overnight at 4 °C. RAGE was detected by Western blotting as described.Cell Growth Studies—Cell growth was analyzed using the MTS reagent (Promega, Madison, WI) according to the manufacturer's directions. For studies on the effects of exogenously applied S100P, purified S100P was added at the indicated concentrations for the specified times. Control cells were treated with non-induced bacterial protein. For studies on cell survival cells were treated with 5-fluorouracil (5-FU) at the concentrations indicated for the specified times, or cells were plated on dishes previously coated with polyhydroxyethyl methacrylate (poly-HEMA; Sigma-Aldrich). Poly-HEMA dissolved at 10 mg/ml in ethanol and 3 ml of solution was added to each well, and plates were kept at 37 °C for 5 days to evaporate solvent completely. Cell numbers were estimated using MTS, which was added to the wells 1 h before taking the photometric reading.ELISA for S100P—S100P was quantified in the media collected from S100P-transfected NIH3T3 cells. S100P was captured between a anti-bovine S100B rabbit polyclonal antibody (Abcam Ltd., Cambridge, UK) and the S100P monoclonal antibody using an ELISA kit (KPL, Gaithersburg, MD). Cells were plated at 1 × 105 cells per well for 3 days, and medium was collected and concentrated using YM10 Centricon concentrating filters. Concentrated samples (200 μl) were incubated for 2 h at room temperature in antibody-coated plates and washed three times with wash buffer. Bound S100P was detected with horseradish peroxidase-labeled anti-mouse secondary antibody and TMB (3,3′,5,5′-tetramethylbenzidene) substrate. Color development was blocked with 1 m phosphoric acid and read at 450 nm. Purified S100P was used as a standard, and placental lysate was used as a positive control.NF-κB Electrophoretic Mobility Shift Assay—Nuclear extracts were prepared and used for electrophoretic mobility shift assays as previously described (25Han B. Logsdon C.D. Am. J. Physiol. 1999; 277: 74-82Crossref PubMed Google Scholar). For NF-κB DNA binding the reaction was started by the addition of 10,000 cpm of the 22-base pair oligonucleotide 5′-AGT TGA GGG GAC TTT CCC AGG C-3′ containing the NF-κB consensus sequence that had been labeled with [γ-32P]ATP (10 mCi/mmol) by T4 polynucleotide kinase. The reaction was allowed to proceed for 30 min at room temperature. For cold competition experiments unlabeled NF-κB oligonucleotide or OCT1 oligonucleotide as a nonspecific competitor (300×) was added to the binding reaction 5 min before the addition of the radiolabeled probe. For antibody supershift assays 2 μl of specific antibodies to NF-κB protein subunits p65, p50, and c-Rel were incubated with nuclear extracts for 1 h at room temperature before the addition of labeled probe. All reaction mixtures were subjected to PAGE on 4.5% gel in 0.5× Tris borate-EDTA buffer at 200 V. Gels were dried and directly exposed to a B-1 phosphorimaging screen and visualized with a GS-250 Molecular Imaging System (Bio-Rad).Fluorescence-activated Cell Sorter Analysis—Standard propidium iodide staining by the hypotonic lysis method was used for cell cycle and apoptosis studies. Wild-type and S100P-transfected NIH-3T3 cells were seeded in 100-mm plates for 48 h. Apoptosis was induced by treating with 150 μg/ml 5-FU. After 48 h cells were collected by trypsinization, washed once with cold phosphate-buffered saline, mixed with 500 μl of hypotonic solution (0.1% sodium citrate, 0.1% Triton X-100, 100 μg/ml RNase, 50μg/ml propidium iodide), and analyzed by flow cytometry after 30 min incubation. Cells undergoing apoptosis that had lost part of their DNA (due to the DNA fragmentation) were detected as the population of cells with sub-G1 DNA contents.RESULTSExpression of S100P Stimulates NIH3T3 Cell Growth and Survival—To evaluate the influence of S100P on cell function, NIH3T3 cells stably expressing this molecule were produced using standard transfection techniques. S100P was detected in cell lysates after but not before transfection (Fig. 1A). S100P expression did not stimulate these cells to form colonies in soft agar, suggesting a lack of transforming ability (data not shown). However, S100P expression increased the proliferation rate of NIH3T3 cells to 251 ± 42% that of control (n = 3, p < 0.05) within 72 h (Fig. 1B). This increase in proliferation rate correlated with an increased proportion of the cell population in S phase (Fig. 1C). Similar results were observed in two other independently derived stable NIH cell lines (data not shown).S100P expression also influenced NIH3T3 cell survival after two apoptotic insults, detachment from the growth substrate (causing anoikis) or treatment with the cytotoxic agent 5-FU. When wild-type NIH3T3 cells were plated on dishes coated with poly-HEMA, which prevents cell attachment, the cells underwent rapid induction of anoikis, indicated by a reduction in cell numbers (Fig. 2A). In contrast, S100P-expressing NIH3T3 cells were remarkably resistant to this treatment. Furthermore, the chemotherapeutic agent 5-FU (150 μg/ml) was able to efficiently kill wild-type NIH3T3 cells, but S100P-expressing cells were resistant to this treatment (Fig. 2B). The survival benefits of S100P expression were due to an inhibition of apoptosis, as indicated by a reduced proportion of cells with sub-G1 levels of DNA content after 72 h of treatment with 5-FU (Fig. 2C) and by a reduction in the appearance of active caspase 3 after plating on poly-HEMA for 3 h (data not shown).Fig. 2Expression of S100P protects NIH3T3 cells against cell death induced by detachment or 5-FU.A, effects of detachment on wild-type and S100P-expressing NIH3T3 cells. Cells were plated on poly-HEMA-coated dishes to prevent cell attachment and incubated for indicated times, and cell viability was analyzed using the MTS method. Data shown are the means ± S.E. for three experiments (*, p < 0.05). B, effects of 5-FU treatment on wild-type (WT) and S100P-expressing NIH-3T3 cell numbers. Cells were cultured in the presence of 5-FU (150 μg/ml) for the indicated times, and cell viability was analyzed using the MTS method. Data shown are the means ± S.E. for three experiments (*, p < 0.05). C, effects of 5-FU treatment on apoptosis in wild-type and S100P-expressing NIH-3T3 cells. Cells were cultured in the presence of 5-FU (150 μg/ml) for 48 h, and the number of apoptotic cells was estimated as the percentage of cells with sub-G1 DNA contents as quantitated using fluorescence-activated cell sorter analysis. Data shown are the means ± S.E. for three experiments (*, p < 0.05).View Large Image Figure ViewerDownload Hi-res image Download (PPT)We hypothesized that the effects of S100P expression might be due to secretion of S100P and activation of autocrine signaling mechanisms. Therefore, we examined the level of S100P in conditioned media from NIH3T3 cells using an ELISA assay. S100P was detected in media bathing cells stably transfected with S100P (22 ± 4 ng/ml, n = 5) but not media from wild-type NIH3T3 cells (undetectable). Therefore, to avoid the complications of potential intracellular actions of S100P, we subsequently focused on the effects of extracellularly added S100P.Extracellular S100P Stimulates NIH3T3 Cell Growth and Survival—To test the effects of extracellular S100P on cell function we produced purified S100P as a histidine-tagged fusion protein in bacteria. Purity of the protein was analyzed by SDS-PAGE (Fig. 3A), and specificity was confirmed by Western blot (Fig. 3B). Non-induced bacterial proteins were used as a control. The addition of purified S100P (0.01–1000 nm) to NIH3T3 cells stimulated cell proliferation in a concentration-dependent manner. Effects were noted with 0.01 nm, and maximal effects (188 ± 23% of control, p < 0.05, n = 3) were observed with 100 nm S100P (Fig. 3C). The effects of S100P on cell proliferation were also time-dependent, with a significant increase in cell proliferation noted within 48 h after the addition of 100 nm S100P to the culture medium (Fig. 3D).Fig. 3Exogenous S100P stimulates NIH3T3 cell proliferation and survival.A, S100P was synthesized in E. coli, and His-S100P protein was purified using a nickel column. Expression and purity of protein was confirmed by SDS-PAGE. M. Wt. kD, molecular mass in kDa. B, specificity of S100P was confirmed by Western blot by using a monoclonal S100P antibody. BXPc3 cell lysate was used as a positive control (+), and non-induced bacterial protein was used as a negative control (Bacterial Protein). C, S100P stimulated proliferation was concentration-dependent. Wild-type NIH3T3 cells were incubated with the indicated concentrations of S100P for 48 h. D, S100P stimulated proliferation was time-dependent. Cells were incubated with 100 nm S100P for the indicated times. E, S100P increased cell survival after treatment with 5-FU in a concentration-dependent manner. Wild-type NIH3T3 cells were incubated in the presence of 5-FU (150 μg/ml) and the indicated concentrations of S100P for 48 h. F, S100P increased cell survival after cell detachment was time-dependent. Wild-type NIH3T3 cells were plated onto poly-HEMA-coated dishes for the indicated times in the presence and absence of S100P (100 nm). Protein from non-induced bacteria was used as a control. Cell numbers were estimated using MTS, and data shown are the means ± S.E. (n = 3; *, p < 0.05).View Large Image Figure ViewerDownload Hi-res image Download (PPT)The addition of S100P to the culture medium also increased the survival of NIH3T3 cells. Pretreatment of wild-type NIH3T3 cells with S100P (100 nm) reduced the loss of cells due to treatment with 5-FU (150 μg/ml) in a concentration-dependent manner with significant protection (p < 0.05, n = 3) observed at 10 nm (Fig. 3E) and nearly complete protection at 100 nm S100P. The effects of S100P on cell survival were also time-dependent, with protection from the effects of 5-FU treatment noted within 36 h and persisting for at least 48 h (Fig. 3F).S100P Activates Erks and NF-κB—We examined the effects of extracellular S100P on cell growth and survival signaling pathways. Erk activation is commonly associated with stimulation of cell proliferation. Treatment of NIH3T3 cells with purified S100P induced Erk 1 and 2 phosphorylation in a time-dependent manner, with significant effects noted within 10 min, and a maximal increase over control levels (458 ± 30% of control, n = 3) was observed after 30 min (Fig. 4A). Beyond 30 min Erk phosphorylation levels returned toward base line but remained significantly elevated for at least 2 h. The effects of S100P on Erk phosphorylation were also concentration-dependent, with significant effects noted at 0.01 nm and maximal effects noted with 100 nm S100P (Fig. 4B).Fig. 4Exogenous S100P stimulates Erk activation in NIH3T3 cells in a concentration- and time-dependent manner. Purified S100P was added to medium bathing wild-type NIH3T3 cells, and activation of Erks 1 and 2 was visualized by Western blotting with anti-phospho-Erk (P-Erk), then the membranes were stripped and reprobed with antibodies for total Erks (T-Erk). A, induction of Erks by S100P was time-dependent. Wild-type NIH3T3 cells were treated with S100P (100 nm) for the indicated times (0–120 min). B, induction of Erks by S100P was concentration-dependent. Wild-type NIH3T3 cells were treated with purified S100P at the indicated concentrations or with non-induced bacterial protein (BP) for 30 min. Data are representative of three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)NF-κB activation is often associated with increased cell survival. Therefore, we investigated whether extracellular S100P activated this transcription factor. S100P caused a time-dependent increase in NF-κB DNA binding in NIH3T3 cells that was significant within 10 min and was maintained for at least 1 h (Fig. 5A). The specificity of the NF-κB band observed in these assays was indicated by competition with unlabeled κB site oligonucleotides. Furthermore, super-shift analysis using antibodies specific for individual NF-κB subunits indicated the presence of p50, p65, and to a lesser extent c-Rel in the induced complexes (Fig. 5A). These effects on NF-κB activation were also concentration-dependent, with significant effects noted at 0.1 nm, and a maximal effect was observed with 100 nm S100P (494 ± 38% of control, n = 3) (Fig. 5B).Fig. 5Exogenous S100P stimulates NF-κB activation in NIH3T3 cells. Purified S100P was added to the medium bathing wild-type NIH3T3 cells, and NF-κB activation was assessed by electrophoretic mobility shift assays. A, S100P effects on NF-κB were time-dependent. Nuclear extracts from wild-type NIH3T3 cells treated with S100P at 100 nm for the indicated times (0–60 min) were subjected to electrophoretic mobility shift assays with equal amounts of nuclear protein per sample. To assess specificity, competition with 300× excess of unlabeled κB oligonucleotide (cold oligo) was added to the reaction before the addition of labeled κB oligonucleotide. For supershift analysis nuclear extracts were incubated for 1
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