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• W2019679148 abstract "Bcl-xl and the hepatocyte growth factor (HGF) receptor c-Met are both highly expressed in mesotheliomas, where they protect cells from apoptosis and can confer resistance to conventional therapeutic agents. In our current study, we investigate a model for the transcriptional control of Bcl-xl that involves ETS transcription factors and the HGF/Met axis. In addition, the effects of activated c-Met on the phosphorylation of the ETS family transcriptional factors were examined. The transient expression of ETS-2 and PU.1 cDNAs in mesothelioma cell lines resulted in an increase in the promoter activity of Bcl-xl and consequently in its mRNA and protein expression levels, whereas the transcriptional repressor Tel suppressed Bcl-xl transcription. The activation of the HGF/Met axis led to rapid phosphorylation of ETS family transcription factors in mesothelioma cells through the mitogen-activated protein kinase pathway and via nuclear accumulation of ETS-2 and PU.1. A chromatin immunoprecipitation assay further demonstrated that the activation of c-Met enhanced the binding of ETS transcriptional factors to the Bcl-x promoter. Finally, we determined the Bcl-xl and phosphorylated c-Met expression levels in mesothelioma patient samples; these data suggest a strong correlation between Bcl-xl and phosphorylated c-Met levels. Taken together, these findings support a role for c-Met as an inhibitor of apoptosis and an activator of Bcl-xl. Bcl-xl and the hepatocyte growth factor (HGF) receptor c-Met are both highly expressed in mesotheliomas, where they protect cells from apoptosis and can confer resistance to conventional therapeutic agents. In our current study, we investigate a model for the transcriptional control of Bcl-xl that involves ETS transcription factors and the HGF/Met axis. In addition, the effects of activated c-Met on the phosphorylation of the ETS family transcriptional factors were examined. The transient expression of ETS-2 and PU.1 cDNAs in mesothelioma cell lines resulted in an increase in the promoter activity of Bcl-xl and consequently in its mRNA and protein expression levels, whereas the transcriptional repressor Tel suppressed Bcl-xl transcription. The activation of the HGF/Met axis led to rapid phosphorylation of ETS family transcription factors in mesothelioma cells through the mitogen-activated protein kinase pathway and via nuclear accumulation of ETS-2 and PU.1. A chromatin immunoprecipitation assay further demonstrated that the activation of c-Met enhanced the binding of ETS transcriptional factors to the Bcl-x promoter. Finally, we determined the Bcl-xl and phosphorylated c-Met expression levels in mesothelioma patient samples; these data suggest a strong correlation between Bcl-xl and phosphorylated c-Met levels. Taken together, these findings support a role for c-Met as an inhibitor of apoptosis and an activator of Bcl-xl. Malignant mesotheliomas are aggressive tumors commonly associated with asbestos exposure.1Greillier L Astoul P Mesothelioma and asbestos-related pleural diseases.Respiration. 2008; 76: 1-15Crossref PubMed Scopus (41) Google Scholar Although there has been some progress in the treatment of these cancers, the overall prognosis remains very poor. Bcl-xl is a key antiapoptotic protein expressed in many tumor types and its overexpression is believed to contribute to chemotherapeutic resistance in mesotheliomas. In previous studies, Bcl-xl expression was found to be influenced by a variety of transcription factors and signal transduction pathways. In addition to nuclear factor-κB (NF-κB) and signal transducers and activators of transcription (STATs), analysis of human Bcl-xl promoter has revealed nine potential ETS-binding sites.2Chen C Edelstein LC Gelinas C The Rel/NF-κB family directly activates expression of the apoptosis inhibitor Bcl-xL.Mol Cell Biol. 2000; 20: 2687-2695Crossref PubMed Scopus (689) Google Scholar, 3Kirito K Watanabe T Sawada Ki Endo H Ozawa K Komatsu N Thrombopoietin regulates Bcl-xL gene expression through Stat5 and phosphatidylinositol 3-kinase activation pathways.J Biol Chem. 2002; 277: 8329-8337Crossref PubMed Scopus (84) Google Scholar, 4Sevilla L Aperlo C Dulic V Chambard JC Boutonnet C Pasquier O Pognonec P Boulukos KE The Ets2 transcription factor inhibits apoptosis induced by colony-stimulating factor 1 deprivation of macrophages through a Bcl-xL-dependent mechanism.Mol Cell Biol. 1999; 19: 2624-2634Crossref PubMed Scopus (90) Google Scholar Bcl-xl overexpression in various tumors is known to contribute to tumorigenesis and resistance to therapeutic agents. By decreasing Bcl-xl expression through antisense or small interfering (si) RNAs and inhibiting the Bcl-xl protein using BH3 mimetics, an apoptotic response is induced, and the tumor cells are rendered sensitive to chemotherapy.5Cao X Rodarte C Zhang L Morgan CD Littlejohn J Smythe WR Bcl2/bcl-xL inhibitor engenders apoptosis and increases chemosensitivity in mesothelioma.Cancer Biol Ther. 2007; 6: 246-252Crossref PubMed Scopus (36) Google Scholar, 6Littlejohn JE Cao X Miller SD Ozvaran MK Jupiter D Zhang L Rodarte C Smythe WR Bcl-xL antisense oligonucleotide and cisplatin combination therapy extends survival in SCID mice with established mesothelioma xenografts.Int J Cancer. 2008; 123: 202-208Crossref PubMed Scopus (26) Google Scholar, 7Ozvaran MK Cao XX Miller SD Monia BA Hong WK Smythe WR Antisense oligonucleotides directed at the bcl-xl gene product augment chemotherapy response in mesothelioma.Mol Cancer Ther. 2004; 3: 545-550PubMed Google Scholar, 8Smythe WR Mohuiddin I Ozveran M Cao XX Antisense therapy for malignant mesothelioma with oligonucleotides targeting the bcl-xl gene product.J Thorac Cardiovasc Surg. 2002; 123: 1191-1198Abstract Full Text Full Text PDF PubMed Scopus (44) Google ScholarThe ETS family of transcription factors consists of more than 30 members, which are conserved from sea urchin to human beings. Each ETS family member contains a conserved DNA-binding domain of 85 amino acids, the ETS domain, which binds to a purine-rich GGAA/T core sequence.9Oikawa T Yamada T Molecular biology of the Ets family of transcription factors.Gene. 2003; 303: 11-34Crossref PubMed Scopus (520) Google Scholar ETS proteins bind to DNA as monomers and can activate transcription alone or in conjunction with other transcription factors. Most ETS proteins are nuclear targets of diverse signaling pathways such as the mitogen-activated protein (MAP) kinase signaling pathway and undergo post-translational modifications including phosphorylation, glycosylation, acetylation, ubiquitination, and sumoylation.10Tootle TL Rebay I Post-translational modifications influence transcription factor activity: a view from the ETS superfamily.Bioessays. 2005; 27: 285-298Crossref PubMed Scopus (146) Google Scholar These modifications have a profound impact on the activity and subcellular localization of the ETS proteins.It has been reported that several receptor tyrosine kinases (RTKs) are activated in mesothelioma, including epidermal growth factor receptor, platelet-derived growth factor receptor, and hepatocyte growth factor (HGF) receptor (c-Met). Clinical trials of imatinib (a platelet-derived growth factor receptor inhibitor) and gefitinib (epidermal growth factor receptor inhibitor) in mesothelioma tumors have shown limited success. Thus, there is great interest in identifying an alternative receptor tyrosine kinase target in these cancers. c-Met is overexpressed and activated in most cases of mesothelioma in comparison with normal adjacent tissue.11Arai H Maki K Waga K Sasaki K Nakamura Y Imai Y Kurokawa M Hirai H Mitani K Functional regulation of TEL by p38-induced phosphorylation.Biochem Biophys Res Commun. 2002; 299: 116-125Crossref PubMed Scopus (22) Google Scholar, 12Ramos-Nino ME Blumen SR Sabo-Attwood T Pass H Carbone M Testa JR Altomare DA Mossman BT HGF mediates cell proliferation of human mesothelioma cells through a PI3K/MEK5/Fra-1 pathway.Am J Respir Cell Mol Biol. 2008; 38: 209-217Crossref PubMed Scopus (60) Google Scholar In addition, the circulating serum levels of HGF are twofold greater in patients with mesothelioma compared with the healthy population.13Jagadeeswaran R Ma PC Seiwert TY Jagadeeswaran S Zumba O Nallasura V Ahmed S Filiberti R Paganuzzi M Puntoni R Kratzke RA Gordon GJ Sugarbaker DJ Bueno R Janamanchi V Bindokas VP Kindler HL Salgia R Functional analysis of c-Met/hepatocyte growth factor pathway in malignant pleural mesothelioma.Cancer Res. 2006; 66: 352-361Crossref PubMed Scopus (169) Google Scholar There are numerous signal transduction cascade mechanisms that are activated on HGF stimulation, c-Met phosphorylation, MAP kinase activation, and phosphatidylinositol 3-kinase kinase activation.12Ramos-Nino ME Blumen SR Sabo-Attwood T Pass H Carbone M Testa JR Altomare DA Mossman BT HGF mediates cell proliferation of human mesothelioma cells through a PI3K/MEK5/Fra-1 pathway.Am J Respir Cell Mol Biol. 2008; 38: 209-217Crossref PubMed Scopus (60) Google Scholar, 13Jagadeeswaran R Ma PC Seiwert TY Jagadeeswaran S Zumba O Nallasura V Ahmed S Filiberti R Paganuzzi M Puntoni R Kratzke RA Gordon GJ Sugarbaker DJ Bueno R Janamanchi V Bindokas VP Kindler HL Salgia R Functional analysis of c-Met/hepatocyte growth factor pathway in malignant pleural mesothelioma.Cancer Res. 2006; 66: 352-361Crossref PubMed Scopus (169) Google Scholar Attenuation of c-Met through siRNA and the small molecular inhibitor SU11274 has been found to inhibit both tumor cell growth and migration.14Ma PC Jagadeeswaran R Jagadeesh S Tretiakova MS Nallasura V Fox EA Hansen M Schaefer E Naoki K Lader A Richards W Sugarbaker D Husain AN Christensen JG Salgia R Functional expression and mutations of c-Met and its therapeutic inhibition with SU11274 and small interfering RNA in non-small cell lung cancer.Cancer Res. 2005; 65: 1479-1488Crossref PubMed Scopus (478) Google ScholarThe association between Bcl-xl and c-Met expression levels was well established in a number of previous studies.15Fornoni A Li H Foschi A Striker GE Striker LJ Hepatocyte growth factor, but not insulin-like growth factor I protects podocytes against cyclosporin A-induced apoptosis.Am J Pathol. 2001; 158: 275-280Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 16Grad JM Zeng XR Boise LH Regulation of Bcl-xL: a little bit of this and a little bit of STAT.Curr Opin Oncol. 2000; 12: 543-549Crossref PubMed Scopus (205) Google Scholar The elevation of Bcl-xl in both tumor and normal cells on HGF exposure indicates a role for activated c-Met in Bcl-xl transcriptional regulation.17Fan S Ma YX Wang JA Yuan RQ Meng Q Cao Y Laterra JJ Goldberg ID Rosen EM The cytokine hepatocyte growth factor/scatter factor inhibits apoptosis and enhances DNA repair by a common mechanism involving signaling through phosphatidyl inositol 3′ kinase.Oncogene. 2000; 19: 2212-2223Crossref PubMed Scopus (141) Google Scholar, 18Mitchell C Mallet VO Guidotti JE Goulenok C Kahn A Gilgenkrantz H Liver repopulation by Bcl-xL transgenic hepatocytes.Am J Pathol. 2002; 160: 31-35Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 19Zhang J Yang J Liu Y Role of Bcl-xL induction in HGF-mediated renal epithelial cell survival after oxidant stress.Int J Clin Exp Pathol. 2008; 1: 242-253PubMed Google Scholar The anti-apoptotic survival role of activated c-Met has also been partly explained by its activation of the phosphatidylinositol 3-kinase-AKT kinase pathway12Ramos-Nino ME Blumen SR Sabo-Attwood T Pass H Carbone M Testa JR Altomare DA Mossman BT HGF mediates cell proliferation of human mesothelioma cells through a PI3K/MEK5/Fra-1 pathway.Am J Respir Cell Mol Biol. 2008; 38: 209-217Crossref PubMed Scopus (60) Google Scholar and its angiogenic properties.20Gupta A Karakiewicz PI Roehrborn CG Lotan Y Zlotta AR Shariat SF Predictive value of plasma hepatocyte growth factor/scatter factor levels in patients with clinically localized prostate cancer.Clin Cancer Res. 2008; 14: 7385-7390Crossref PubMed Scopus (37) Google Scholar There has been no report to date, however, that has addressed the mechanism underlying the up-regulation of Bcl-xl after c-Met activation. Given also that Bcl-xl and Akt are independent guardians of the mitochondria, which provide the gateway to the intrinsic apoptosis pathways, it is of some importance to elucidate the mechanism by which HGF up-regulates Bcl-xl expression.We have examined the role of HGF signaling in controlling apoptosis in a human mesothelioma model. The role of c-Met activation in the regulation of Bcl-xl expression via the ETS family of transcription factors has been further clarified.Materials and MethodsCell Lines and ReagentsThe human mesothelioma cell lines H28, HAY, I45, MSTO, REN, and ROB were maintained in RPMI 1640 medium (Invitrogen, Grand Island, NY) containing 10% fetal bovine serum (Invitrogen). The mesothelioma cell lines SF.HAT and SF.ORT were maintained in Dulbecco’s modified Eagle’s medium (Invitrogen) also containing 10% fetal bovine serum. The human lung cancer cell line, H1299, was maintained in RPMI 1640 medium containing 10% fetal bovine serum. I45 cells are sarcomatous subtypes of mesothelioma. REN and H28 are epithelial subtypes. MSTO is a biphasic subtype. The subtypes of HAY, ROB, SF.HAT, and SF.ORT are unknown. Hepatocyte growth factor was obtained from R&D Systems (Minneapolis, MN). Anti-Bcl-xl antibody and all anti-MAP kinase antibodies were purchased from Cell Signaling Technology (Beverly, MA). All antibodies used to detect ETS family transcriptional factors were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-actin monoclonal antibody was purchased from Sigma-Aldrich (St. Louis, MO). Mitogen-activated protein kinase (MEK) inhibitor (MEK inhibitor I) and c-Jun NH2-terminal kinase (JNK) inhibitor (SB203580) were obtained from Calbiochem (San Diego, CA). P38 kinase inhibitor (SB202190) was purchased from LC Laboratories (Woburn, MA).Plasmids and DNA TransfectionA 1.2-kb fragment of the human Bcl-xl promoter was cloned into the luciferase reporter plasmid pGL2 (Promega, Madison, WI) to generate pXLPR. All deletion mutant constructs were generated by PCR and fully sequenced for verification. The cytomegalovirus (CMV) promoter-based ETS-1, ETS-2, PU.1, and Tel cDNA expression vectors were purchased from Origene (Rockville, MD). Each of these plasmids was purified using a Qiafilter Maxi kit (Qiagen, Valencia, CA). I45 cells were transfected in 24-well plates using FuGENE 6 (Roche, Indianapolis, IN) with 200 ng of pXLPR, and each of the serial deletion plasmid constructs was supplemented with 20 ng of pCMV-βGal as an internal control for transfection efficiency. Several independent experiments using I45 cells were performed in triplicate. At 48 hours after transfection, cell lysates were prepared in 25 mmol/L Tris (pH 7.5)-10% glycerol-1% Triton X-100–2 mmol/L dithiothreitol and analyzed for luciferase and β-galactosidase activities as described by the manufacturer (Applied Biosystems, Foster City, CA). All luciferase activities were normalized to the β-galactosidase internal control.Immunoprecipitation and Western BlottingWestern blot analyses were performed using a standard method. In brief, cells were lysed in Laemmli buffer, and equal amounts of total protein were electrophoresed on 4 to 20% polyacrylamide/bisacrylamide gels. The proteins resolved were then transferred to a nitrocellulose membrane and incubated with Bcl-xl, actin, MAP kinase, and ETS antibodies. Signals were visualized using the ECL system (GE Amersham, Little Chalfont, Buckinghamshire, UK). For immunoprecipitation experiments, I45 cells were transfected with Tel, ETS-2, and PU.1 expression plasmids using FuGENE 6 and then cultured for 24 hours. These cells were then either untreated or treated with 100 ng/ml HGF for 30 minutes and harvested in 750 μl of lysis buffer (50 mmol/L Tris-HCl [pH 7.4], 150 mmol/L NaCl, 0.05% SDS, 1% deoxycholic acid, 1% Triton X-100, 500 U/ml ulinastatin, and 2 mmol/L phenylmethylsulfonyl fluoride) per 100-mm diameter culture dish. Immunoprecipitations were performed using Tel, ETS-2, and PU.1 antibodies and the Catch and Release V.20 kit (Upstate, Charlottesville, VA). The signals were detected by electrophoresis and autoradiography.Immunohistochemistry and Immunofluorescent MicroscopyThe expression of Bcl-xl and c-Met was determined by immunohistochemical analysis on formalin-fixed and paraffin-embedded mesothelioma tissues arrays. This study was approved by the Scott & White Memorial Hospital Texas Health Science Center Institutional Review Board. Five-micrometer-thick sections of these mesothelioma tissue arrays were deparaffinized in xylene substitute and rehydrated in PBS. Antigen retrieval was performed with citrate buffer (pH 6.5) for 20 minutes at 99°C, followed by the block of endogenous peroxidase activity (3% hydrogen peroxide for 10 minutes). Sections were incubated with blocking serum in PBS containing 5% bovine serum albumin, followed by incubation with rabbit anti-human Bcl-xl polyclonal antibody (Cell Signaling Technology) or with rabbit anti-phosphorylated human c-Met polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 hour, followed by incubation with a biotinylated goat secondary anti-rabbit antibody (1:200 dilution, Vector Laboratories, Burlingame, CA). Immunoreactive signals were detected using a streptavidin-biotin-peroxidase complex from Vector Laboratories, according to the manufacturer’s recommended procedures. All of the slides were counterstained with hematoxylin (Sigma-Aldrich). For the negative control, slides were subjected to the same procedures, including antigen retrieval, except for treatment of samples with control rabbit IgG. This negative control clearly demonstrated the specificity of the immunostaining that we observed.Subcellular localization of ETS proteins was detected by indirect immunofluorescence. In brief, ETS-2-, PU.1- or Tel-transfected I45 cells were plated on coverslips in RMPI 1640 medium containing 10% fetal bovine serum. The cells were then serum-starved or grown in 10% fetal bovine serum for 24 hours. The serum-starved cells were exposed to 100 ng/ml HGF for 20 minutes, fixed, and then stained with ETS-2, PU.1, or Tel antibodies. Positive immunostaining was detected by incubation with a fluorescein isothiocyanate-conjugated secondary antibody plus a 5 ng/ml concentration of Hoechst dye and visualized using epifluorescence microscopy (Olympus BX70).Quantitative Measurements of Bcl-xl mRNABcl-xl mRNA levels in both patient samples and cell lines were measured using real-time PCR. Total RNAs were extracted using TRIzol from Sigma-Aldrich, and 1-μg aliquots of total RNA from each sample were reverse-transcribed using a TaqMan reverse transcription kit (Applied Biosystems). Primers and probes to detect Bcl-xl and glyceraldehyde-3-phosphate dehydrogenase were obtained from Applied Biosystems. Human total RNA was used as a related standard and human glyceraldehyde-3-phosphate dehydrogenase was used as the internal PCR control. Real-time PCR was performed using an MX4000 Multiplex quantitative PCR system (Stratagene, La Jolla, CA). All reactions were performed in triplicate.Chromatin Immunoprecipitation AssayThe chromatin immunoprecipitation (ChIP) assay was performed essentially as described by Saccani et al21Saccani S Pantano S Natoli G Two waves of nuclear factor κB recruitment to target promoters.J Exp Med. 2001; 193: 1351-1360Crossref PubMed Scopus (336) Google Scholar with minor modifications. In brief, I45 cells were treated with 1% formaldehyde for 15 minutes. Cross-linked chromatin was then prepared and sonicated to an average size of 1000 bp before being immunoprecipitated with antibodies against Tel, PU.1, or ETS-2 or with control rabbit IgG at 4°C overnight. After reversal of the cross-linking, the immunoprecipitated chromatin was PCR-amplified with primers specific for the Bcl-xl promoter as follows: forward 5′-GCCTAAGGCGGATTTGAATGTAG-3′); reverse 5′-GAAGGGAGAGAAAGAGATTCAGGAA-3′.Statistical analysisRelationships between Bcl-xl and phosphorylated c-Met were analyzed statistically using χ2 analysis.ResultsBcl-xl Is Highly Elevated in Human MesotheliomasBcl-xl expression levels in mesothelioma cell lines and in normal lung and pleural tissue (NL1 and NL2) were evaluated by Western blotting with an anti-human Bcl-xl polyclonal antibody. The robust expression of Bcl-xl was evident in all mesothelioma cell lines in contrast with the two normal tissues examined (Figure 1A). Differential Bcl-xl expression in human tumor samples was demonstrated by immunohistochemical analysis in which a strong Bcl-xl signal was detected in the tumor area, whereas the adjacent normal tissue showed no expression of this protein (Figure 1B). The differences in the Bcl-xl RNA levels between the mesotheliomas and normal tissue were further confirmed using real-time PCR analysis of the same human samples used for immunohistochemical staining (Figure 1C).Functional Analysis of the Bcl-xl PromoterTo identify the potential regulatory elements of the human Bcl-xl gene, we performed a transient luciferase assay using a series of 5′ deletions of the Bcl-xl promoter linked to the luciferase reporter gene. pCMV-β-gal cDNA was cotransfected as an internal control (Figure 2A). The data indicate that the Bcl-xl regulatory elements are spread along the entire promoter region. Similar results were obtained in other mesothelioma cell lines. We used the TESS (Transcription Element Search Software) package from the Department of Computational Biology and Informatics Laboratory at the University of Pennsylvania to analyze the putative transcription factor binding sites within the Bcl-xl promoter. Nine ETS-binding sites were identified in the promoter region (Figure 2B) in addition to two NF-κB binding sites and one STAT binding site. Several transcription factors have been reported previously to be involved in the regulation of Bcl-xl expression in a variety of tissues, including ETS-1,2 PU.1, TEL, C-REL, REL A, and STATs.16Grad JM Zeng XR Boise LH Regulation of Bcl-xL: a little bit of this and a little bit of STAT.Curr Opin Oncol. 2000; 12: 543-549Crossref PubMed Scopus (205) Google Scholar, 22Sevilla L Zaldumbide A Pognonec P Boulukos KE Transcriptional regulation of the bcl-x gene encoding the anti-apoptotic Bcl-xL protein by Ets, Rel/NFκB, STAT and AP1 transcription factor families.Histol Histopathol. 2001; 16: 595-601PubMed Google Scholar To evaluate the possible roles of NF-κB and STATs in regulating the Bcl-xl promoter, NF-κB activity was inhibited by the proteasome inhibitor MG132 in the I45 and REN mesothelioma cell lines. Bcl-xl expression was then analyzed by Western blotting but was unaffected at 24 hours after exposure, although the tumor cells had already undergone apoptosis (Figure 2C). The Jak kinase inhibitor, tyrphostin AG490 was used to block the activity of the JAK-STAT pathway in the same mesothelioma cell lines (Figure 2C) but there were no detectable effects on Bcl-xl expression after 24 hours of exposure.Figure 2A: Analysis of Bcl-xl promoter activity by serial deletions of the promoter region. I45 cells were seeded in 96-well plates and transfected with a Bcl-xl promoter reporter, pxl-luc, or a series of 5′ deletions of the Bcl-xl promoter along with a p-CMV-β-galactosidase cDNA control vector using FuGENE 6. A p-GL2 luciferase vector was also transfected as a negative control. At 24 hours after transfection, cells were lysed and luciferase and β-galactosidase activities were measured by luminometry using a dual-light luciferase kit (Applied Biosystems). The luciferase activity was normalized to β-galactosidase activity, and the data shown are the averages of triplicate determinations. This promoter analysis was repeated three times. B: Schematic representation of the location of transcription factor binding sites on the Bcl-xl promoter: triangles, NF-κB; oval, STAT; rectangles, ETS. C: Inhibition of NF-κB or JAK kinase does not suppress Bcl-xl protein expression. The mesothelioma cell lines, I45 and REN, were exposed to proteasome inhibitor MG-132 or JAK kinase inhibitor AG490. These cells were then harvested and Bcl-xl expression was determined by Western blot. RLU, relative light units.View Large Image Figure ViewerDownload Hi-res image Download (PPT)HGF Induces Bcl-xl Expression through the ETS Family of Transcription FactorsTo next determine whether the ETS family of transcription factors regulates Bcl-xl expression, different ETS transcription factor cDNAs or a green fluorescent protein (GFP) cDNA control were cotransfected into I45 cells with the Bcl-xl promoter construct. Cells transfected with the ETS-1, ETS-2, and PU.1 constructs showed much higher luciferase activities than the controls (Figure 3A). We then cotransfected I45 cells with a TEL expression or GFP control vector and the Bcl-xl promoter construct and found from the luciferase activity measurements that the Bcl-xl promoter was much inhibited (Figure 3A).Figure 3A: ETS family transcription factors up-regulate Bcl-xl promoter activity. I45 cells seeded in 96-well plates were cotransfected with Bcl-xl promoter p-XL and either GFP, ETS-1,−2, PU.1, Tel, or STAT expression vectors along with p-CMV-β-galactosidase. A p-GL2 luciferase vector was transfected as a negative control. Twenty-four hours after transfection, the cells were lysed, and the luciferase and β-galactosidase activities were measured with a luminometer. The luciferase activities were normalized to those of β-galactosidase, and the data shown are the average of triplicate determinations. This experiment was repeated twice. B: HGF stimulates Bcl-xl protein expression after ETS-2 and PU.1 transfection. I45 cells were seeded into six-well plates (106 cells) and were transfected with ETS-2, PU.1 and GFP control expression vectors. The cells were then cultured under normal conditions, serum starvation, or serum starvation plus HGF (100 ng/ml). Bcl-xl expression was then determined by Western blot. Protein expression was quantified using an UN-SCAN-IT automated digitalized system (Silk Scientific Corporation, Orem, UT). RLU, relative light units.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We next investigated whether a connection existed between the HGF receptor, c-Met, and Bcl-xl expression in mesothelioma cells and whether overexpressed ETS transcriptional factors could increase the Bcl-xl expression levels. We expressed ETS-2, PU.1, and GFP control cDNA in I45 cells under normal growth conditions or under serum starvation conditions and then exposed the cells to HGF. Compared with the serum-starved samples, Bcl-xl expression was found to be significantly elevated in the untreated I45 cells expressing ETS-2 and the same cells exposed to HGF, respectively (Figure 3B). These results indicate that ETS transcription factors and exposure to HGF activate Bcl-xl gene expression.HGF Induces the Phosphorylation of ETS Transcription FactorsPost-translational modifications are known to influence transcription factor activities. In this regard, the ETS proteins have been reported to be regulated by phosphorylation, glycosylation, acetylation, ubiquitination, and sumoylation.10Tootle TL Rebay I Post-translational modifications influence transcription factor activity: a view from the ETS superfamily.Bioessays. 2005; 27: 285-298Crossref PubMed Scopus (146) Google Scholar To further examine how HGF may affect ETS functions, we analyzed the levels of phosphorylation of the ETS-2 and PU.1 proteins in I45 cells under conditions of serum starvation or HGF stimulation by immunoprecipitation and Western blot analysis. Cell lysates was immunoprecipitated using ETS-2 and PU.1 antibodies, and the phosphor-serine and -threonine levels were detected using phosphor-serine-specific antibodies. Whereas the total ETS levels were observed to be equivalent in the cells, the levels of phosphorylated ETS-2 and PU.1 were clearly elevated (Figure 4A). We next determined whether physical binding occurs between extracellular signal-regulated kinase, ETS-2, and PU.1. ETS-2 and PU.1 proteins were immunoprecipitated from I45 cell lysates that had been treated with PBS or HGF for 30 minutes and subjected to Western blotting. The signals on these blots demonstrated that extracellular signal-regulated kinase is indeed associated with these ETS proteins (Figure 4B).Figure 4HGF induces the phosphorylation of ETS-2 and PU.1 in human mesothelioma cells. The mesothelioma cell line, I45, was grown under conditions of serum starvation or serum starvation plus 20 minutes of HGF (100 ng/ml) stimulation. The endogenous ETS-2 and PU.1 proteins were precipitated from the cell lysates using respective antibodies. The immunoprecipitates were then analyzed by Western blot using phosphor-Ser/Thr antibodies (A) and an ERK antibody (B). The loading was normalized by measuring the ETS-2 and PU.1 expression levels in the cell lysates by Western blotting. Protein expression was quantified using an UN-SCAN-IT automated digitalized system.View Large Image Figure ViewerDownload Hi-res image Download (PPT)HGF Stimulates Bcl-xl Expression by Enhancing Bcl-xl Promoter Transcriptional ActivityWe analyzed the subcellular distribution of ETS-2 and PU" @default.
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• W2019679148 title "Up-Regulation of Bcl-xl by Hepatocyte Growth Factor in Human Mesothelioma Cells Involves ETS Transcription Factors" @default.
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• W2019679148 doi "https://doi.org/10.2353/ajpath.2009.090070" @default.
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