Functional interaction of E1AF and Sp1 in glioma invasion

Abstract

Transcription factor E1AF is widely known to play critical roles in tumor metastasis via directly binding to the promoters of genes involved in tumor migration and invasion. Here, we report for the first time E1AF as a novel binding partner for ubiquitously expressed Sp1 transcription factor. E1AF forms a complex with Sp1, contributes to Sp1 phosphorylation and transcriptional activity, and functions as a mediator between epidermal growth factor and Sp1 phosphorylation and activity. Sp1 functions as a carrier bringing E1AF to the promoter region, thus activating transcription of glioma-related gene for beta1,4-galactosyltransferase V (GalT V; EC 2.4.1.38). Biologically, E1AF functions as a positive invasion regulator in glioma in cooperation with Sp1 partly via up-regulation of GalT V. This report describes a new mechanism of glioma invasion involving a cooperative effort between E1AF and Sp1 transcription factors.

FIG. 1.

Identification E1AF as a Sp1 binding protein. (A) In vivo association of E1AF with Sp1 determined using cells of the glioma SHG44 cell line and a coimmunoprecipitation assay. Lysates from SHG44 cells were immunoprecipitated (IP) with anti-Sp1 antibody (Ab) or control IgG in the absence or presence of EtBr (50 μg/ml, 200 μg/mln or 400 μg/ml) and sequentially immunoblotted with anti-E1AF or anti-Sp1 antibody. (B) Sp1 IP of glioma tissue (T) and normal brain tissue (N) lysates in the absence or presence of EtBr (50 μg/ml) probed with anti-E1AF, anti-Sp1, anti-EGFR, or anti-GAPDH antibodies. Expression of GAPDH served as a loading control. (C) Schematic representations of E1AF and myc-tagged E1AF mutants used in a coimmunoprecipitation assay. The ETS domain and acidic domain (AD) are shown as gray boxes. The Gln-rich domain is shown as a black box. Amino acid numbers mark the N and C termini and the deletion breakpoints. (D) SHG44 cells were transfected with constructs for expression of control or myc-tagged E1AF mutants and harvested 48 h after transfection. The results of Sp1 IP of these cell lysates in the absence or presence of EtBr (50 μg/ml) blotted with anti-myc or anti-Sp1 antibodies are shown. (E) The structural domains of Sp1 and HA-tagged Sp1 mutants in this work are diagrammed. (F) SHG44 cells were transfected with expression constructs for control or HA-tagged Sp1 or its mutants and harvested 48 h after transfection. The results of E1AF IP of these cell lysates in the absence or presence of EtBr (50 μg/ml) blotted with anti-HA or anti-E1AF antibodies are shown.

FIG. 2.

Activation of Sp1 transcription potential by E1AF. (A to D) PcDNA3.0 and/or E1AF and/or Sp1 expression vector was transiently cotransfected into SHG44 cells with a pSp1-Luc, pmSp1-Luc, CDK2-Luc, mCDK2-Luc, hSR-Luc, mhSR-Luc, AP2γ-Luc, or mAP2γ-Luc construct. The luciferase activity was determined as described in Materials and Methods. (E) PcDNA3.0 and/or E1AF and/or Sp1 expression vectors were transiently cotransfected into SHG44 cells with GalT V-Luc, M(Sp1), or M(EBS). The luciferase activity was determined as described in Materials and Methods. (F) PcDNA3.0 and/or E1AF(R^397/400K) and/or Sp1 expression vector was transiently cotransfected into SHG44 cells with a pSp1-Luc, CDK2-Luc, hSR-Luc, AP2γ-Luc, or GalT V-Luc construct. The luciferase activity was determined as previously described.

FIG. 3.

Analysis of E1AF/Sp1 complex binding to the GalT V promoter in glioma cell and glioma tissue in vitro. (A) Oligonucleotides used in an electrophoretic mobility shift assay. The putative Sp1 and Ets binding sites are indicated with boxes. The mutated nucleotides are underlined. (B) An electrophoretic mobility shift assay was performed using nuclear proteins of SHG44 cells and a human GalT V promoter sequence (−82 to −57) double-stranded radiolabeled probe. Competition assays were carried out with a 10- to 20-fold excess of GalT V promoter sequence (−82 to −57) oligonucleotides with or without the Ets-binding site or Sp1-binding site mutated or Sp1 consensus oligonucleotides. The protein-DNA complexes (arrows a to c) and free DNA are indicated. (C) E1AF/Sp1 bound to a GC box site within a human GalT V promoter. Nuclear extracts from SHG44 cells were incubated with ³²P-labeled double-stranded oligonucleotides spanning the GC box and an Ets-binding site within the GalT V promoter in the presence or absence of control IgG or an antibody specific to Sp1 or E1AF. The unlabeled arrow indicates the protein-DNA-antibody complex. (D) The same amounts of nuclear extracts from glioma tissues or normal brain tissues were incubated with biotin-labeled oligonucleotides as described in Materials and Methods. Proteins bound to these nucleotides were isolated with streptavidin-agarose, and E1AF or Sp1 was detected by immunoblotting. PARP expression served as a loading control. (E) The same nuclear extracts from SHG44 cells transiently transfected with control or E1AF-myc plasmids incubated with biotin-labeled oligonucleotides as described in Materials and Methods. Proteins bound to these nucleotides were isolated with streptavidin-agarose, and E1AF, Sp1, or myc was detected using immunoblotting. PARP expression served as a loading control.

FIG. 4.

Analysis of E1AF/Sp1 complex binding to the GalT V promoter in glioma cell in vivo. (A) ChIP assay of endogenous E1AF or Sp1 on GalT V-Luc (upper panel) or M(Sp1) (lower panel) constructs in SHG44 cells. Immunoprecipitations were carried out with rabbit IgG (R.IgG), mouse IgG (M.IgG), anti-E1AF antibody (E1AF-Ab), or anti-Sp1 antibody (Sp1-Ab). Coprecipitating DNA was revealed by PCR with the indicated primers. Input DNA was diluted 10-fold before amplification. (B) A ChIP assay was performed using SHG44 cells transfected with control or E1AF expression vector and control IgG or an antibody against Sp1. PCR primers for the GalT V promoter or the GAPDH promoter were used to detect promoter fragments in immunoprecipitates (left panel). The presence of transfected E1AF is indicated (right panel). (C) A ChIP assay was performed using SHG44 cells transfected with control, C^658/688S, control RNAi, or Sp1 RNAi vector and control IgG or an antibody against E1AF. PCR primers for the GalT V promoter or the GAPDH promoter were used to detect promoter fragments in immunoprecipitates (upper panel). The presence of endogenous Sp1 expression or C^658/688S is indicated (lower panel).

FIG. 5.

E1AF was associated with phosphorylation of Sp1. (A, WCL panels) Whole-cell lysates from SHG44 cells transfected with control or E1AF expression vectors in the absence or presence of EtBr (50 μg/ml) were loaded onto a 8% denatured polyacrylamide gel, and E1AF and Sp1 protein levels were determined by Western blotting using anti-E1AF or anti-Sp1 antibody (Sp1-Ab). (IP panels) The results of Sp1 immunoprecipitation of the lysates of SHG44 cells transfected with control or E1AF expression vectors in the absence of EtBr or in the presence of EtBr (50 μg/ml) blotted with the indicated antibodies are shown. (B) Whole-cell lysates from SHG44 cells transfected with control or E1AF expression vectors labeled with ³²PO4 for 2 h prior to harvesting and the levels of ³²P labeling of Sp1 were determined as described in Materials and Methods. (C, WCL panels) Whole-cell lysates from SHG44 cells transfected with control or myc-E1AF expression vector and pcDNA3.0-HA, HA-Sp1, S59A, or S131A were loaded onto an 8% denatured polyacrylamide gel, and E1AF and HA-Sp1 protein levels were determined by Western blotting using anti-myc or anti-HA antibody. (IP panels) The results of Sp1 IP of the lysates of SHG44 cells transfected with control or myc-E1AF expression vector and pcDNA3.0-HA, HA-Sp1, S59A, or S131A blotted with the indicated antibodies are shown. (D, WCL panels) Whole-cell lysates from SHG44 cells transfected with the indicated constructs were loaded onto an 8% denatured polyacrylamide gel, and E1AF and HA-Sp1 protein levels were determined by Western blotting using anti-myc or anti-HA antibody. (IP panels) The results of Sp1 IP of the lysates of SHG44 cells transfected with the indicated constructs blotted with the indicated antibodies are shown. (E) PcDNA3.0 and/or E1AF and/or Sp1 and/or Sp1 (S131A/T453A) expression vectors and GalT V-Luc were transiently cotransfected into SHG44 cells. The luciferase activity was determined as described in Materials and Methods.

FIG. 6.

The contribution of E1AF to EGF-induced GalT V promoter activity. (A) SHG44 cells transiently cotransfected with GalT V-Luc and control RNAi or E1AF RNAi construct were treated with EGF (50 ng/ml) for 24 h or left untreated, and the luciferase activity was assayed as described in Materials and Methods. (B) EGF increased E1AF expression and enhanced the interaction between E1AF and Sp1. (WCL panels) Expression of E1AF and Sp1 in SHG44 cells left untreated or treated with EGF (50 ng/ml) for 24 h was studied by immunoblot analysis using the indicated antibodies. (IP panels) The results of Sp1 immunoprecipitation of the lysates of these cells blotted with the indicated antibodies are shown. (C) The role of E1AF in the phosphorylation of Sp1 induced by EGF. (WCL panels) Whole-cell lysates from SHG44 cells transfected with control or E1AF RNAi construct in the presence of EGF (50 ng/ml) for 24 h were determined by Western blotting using anti-E1AF or anti-Sp1 antibody. (IP panels) The results of Sp1 immunoprecipitation of the lysates of these cells blotted with the indicated antibodies are shown. (D, WCL panels) Whole-cell lysates from SHG44 cells transfected with control or E1AF RNAi construct in presence of EGF (50 ng/ml) for 24 h and after being labeled with ³²PO4 for 2 h were investigated by Western blotting using anti-E1AF or anti-Sp1 antibody. (IP panels) Sp1 immunoprecipitation of the lysates of these cells transfected with control or E1AF RNAi construct in the presence of EGF (50 ng/ml) for 24 h were labeled with ³²PO4 for 2 h prior to harvesting, and the levels of ³²P labeling of Sp1 were determined as described in Materials and Methods. (E, WCL panels) Whole-cell lysates from SHG44 cells cotransfected with control or E1AF RNAi and HA-Sp1 or HA-Sp1(S131A/T453A) construct in presence of EGF (50 ng/ml) for 24 h were subjected to Western blot analysis using anti-E1AF, anti-HA, and anti-GAPDH antibody. (IP panels) Results of HA IP of the lysates of these cells blotted with the indicated antibodies are shown.

FIG. 7.

Positive correlation between levels of E1AF and GalT V in glioma. (A) RT-PCR analysis of Sp1, E1AF, and GalT V mRNA expression in normal brain tissues (n = 3) and glioma tissues (n = 5). The relative quantities of mRNA expression compared to that of the first sample are indicated. (B) The correlation of expression of E1AF and GalT V mRNA in normal brain tissues (n = 6) and glioma tissues (n = 14). The values are presented as upregulation compared to the results seen with the first sample. (C) Immunohistochemical analysis of Sp1 and E1AF protein expression was performed with normal brain tissues (n = 2) and glioma tissues (n = 4). Scale bar, 10 μm. (D) RT-PCR analysis of GalT V and E1AF mRNA expression levels in SHG44 cells transfected with control or E1AF RNAi construct. GAPDH mRNA expression served as a loading control. (E) Control RNAi or increasing amounts of E1AF RNAi construct were transiently cotransfected into SHG44 cells with GalT V-Luc. The luciferase activity was measured as described in Materials and Methods.

FIG. 8.

E1AF promotes glioma migration and invasion in cooperation with Sp1. (A) Western blot assay demonstrating E1AF-GFP expression using U251 cells stably transfected with EGFPN3 or E1AF-GFP construct and anti-GFP antibody. (B) Control cells and E1AF-GFP-transfected U251 cells were subjected to actin staining by fluorescein isothiocyanate-phalloidin. Scale bar, 10 μm. (C) E1AF overexpression in U251 cells increased invasive ability as assayed in a modified Boyden chamber (P < 0.05; n = 3). Photomicrographs of the bottom of a Transwell filter (8-μm pores) are shown (upper panel). Scale bar, 100 μm. For quantification of invasion assays, the numbers of invading cells in 10 photographic fields from three separate experiments were counted. The values represent n-fold activation compared to control cell results. Data represent the means ± standard deviations of the results of three independent experiments (lower panel). (D) Cell migration assay of control or E1AF-GFP-transfected U251 cells. A wound-healing assay was prepared as described in Material and Methods. Scale bar, 50 μm. For quantification of migration assays, the wound-induced migration of cells was measured after 24 h. (E) U251 cells stably transfected with EGFPN3 or E1AF-GFP construct were plated on top of gels. Mithramycin A (0.1 μM) was added to the medium 1 day later. Quantification of the invasion assay was performed as described in Materials and Methods. (F) Control and/or Sp1 RNAi and/or myc-E1AF expression vectors were cotransfected into U251 cells. After 24 h, these cells were subjected to invasion assays. The values represent activation compared to control cell results (upper panel). Expression of myc-E1AF and Sp1 is indicated; expression of GAPDH served as a loading control (lower panel). (G) PcDNA3.0 and/or Sp1 and/or E1AF expression vector was cotransfected into U251 cells. After 24 h, these cells were subjected to wound-healing assays (upper panel). Expression of E1AF and Sp1 is indicated; expression of GAPDH served as a loading control (lower panel). (H) PcDNA3.0 and/or Sp1 and/or E1AF expression vector was cotransfected into U251 cells. After 24 h, these cells were subjected to invasion assays. The values represent activation compared to control cell results (upper panel). Expression of E1AF and Sp1 is indicated; expression of GAPDH served as a loading control (lower panel). (I) Control Sp1 or HA-Sp1 or its mutant was transiently cotransfected into U251 cells with control or myc-tagged E1AF expression vector. After 24 h, these cells were subjected to invasion assays. The values represent activation compared to control cell results (upper panel). Expression of myc-E1AF and HA is indicated; expression of GAPDH served as a loading control (lower panel). (J) Control, Myc-E1AF, R^397/400K, or 148-244 expression vector was transiently cotransfected into U251 cells with control or HA-Sp1 expression vector. After 24 h, these cells were subjected to invasion assays. The values represent activation compared to control cell results (upper panel). Expression of myc and HA-Sp1 is indicated; expression of GAPDH served as a loading control (lower panel).

FIG. 9.

Downregulation of E1AF expression decreases the invasion of glioma left untreated or treated with EGF. (A) A Western blot assay demonstrated E1AF expression by use of SHG44 cells stably transfected with control RNAi or E1AF RNAi construct and anti-E1AF antibody. (B) Cell migration assay of control or E1AF RNAi-transfected SHG44 cells (left panel). A wound-healing assay was prepared as described in Material and Methods. Scale bar, 50 μm. The wound-induced migration of cells was measured after 24 h (right panel). (C) Decreasing E1AF expression in SHG44 cells inhibited invasive ability as assayed in a modified Boyden chamber (P < 0.05, n = 3) (left panel). Scale bar, 100 μm. Quantification of invasion assays is shown in the right panel. (D) SHG44 cells stably transfected with control or E1AF RNAi vector was plated on the top of gels, and EGF (50 ng/ml) was added to the medium 1 day later. The numbers of invading cells in 10 photographic fields from three separate experiments were counted. The values represent activation compared to levels observed in untreated cells. (E) Control and/or E1AF RNAi and/or HA-GalT V expression vector was cotransfected into SHG44 cells. After 24 h, these cells were subjected to invasion assays. The values represent activation compared to control cell results (upper panel). Expression of E1AF and HA-GalT V is indicated; expression of GAPDH served as a loading control (lower panel). (F) Control and/or E1AF RNAi and/or HA-GalT V expression vector was cotransfected into SHG44 cells. After 24 h, these cells were subjected to wound-healing assays (upper panel). Expression of E1AF and HA-GalT V is indicated; expression of GAPDH served as a loading control (lower panel).