HHM inhibits TGF-β signalling in a cell response-specific manner. (A) HHM attenuated TGF-β-induced growth inhibition. NMuMG cells infected with Ad-LacZ, Ad-Smad7, or Ad-HHM were seeded and treated with or without TGF-β (1 ng/ml). Cell numbers were counted 6 days after stimulation. Error bars represent s.d. (B) HHM inhibited TGF-β-induced cell migration in chamber assay. Error bars represent s.d. (C) HHM inhibited TGF-β-promoted wound closure in wound healing assay. The result was quantified as shown in the graph on the right side. Migration of wound edges was measured at three random points on the photograph. (D) HHM failed to affect TGF-β-induced actin reorganization. Cells were stimulated with TGF-β (1 ng/ml) for 24 h, and TRITC-phalloidin staining was then performed. (E) Effects of HHM on the expressions of E-cadherin and fibronectin. Cells were infected with Ad-LacZ, Ad-Smad7, or Ad-HHM, and cultured with or without TGF-β for 24 h. Proteins were extracted for immunoblotting. The bottom panel shows protein expression of Smad7 and HHM determined by anti-Flag antibody. (F) Effects of overexpression of HHM on the expressions of target genes of TGF-β, PAI-1, p21^WAF, p15^INK4b, c-myc, Snail, and E-cadherin. NMuMG cells were infected with Ad-LacZ or Ad-HHM, and then stimulated with 1 ng/ml TGF-β for 1 h (except for E-cadherin; 24 h). Expression of each gene was determined by quantitative real-time PCR analyses. Values were normalized to the amount of GAPDH mRNA. Error bars represent s.d.

Olig1 is associated with Smad2/3, and their cooperative action on chromosome is abrogated by HHM. (A) TGF-β signalling-dependent association of Olig1 with Smad2 and Smad3. Physical interactions of Flag–Smads with 6Myc–Olig1 were examined by immunoprecipitation with anti-Flag antibody followed by immunoblotting with anti-Myc antibody in transfected HEK293T cells. TGF-β signalling was induced by ALK5-TD. (B) Physical interaction of endogenous Olig1 with endogenous Smad2 and Smad3 in U373MG cells. U373MG cells were treated with or without TGF-β (2.5 ng/ml) for 3 h before harvest. Cell lysates were subjected to immunoprecipitation with anti-Smad2/3 antibody, or normal mouse IgG as a negative control, followed by immunoblotting with anti-Olig1 antibody. (C) Stronger binding between endogenous Olig1 and Smad2/3 when HHM was knocked down. U373MG cells were transfected with control or HHM siRNA duplex and treated with or without TGF-β (2.5 ng/ml) for 3 h before harvest. Cell lysates were subjected to immunoprecipitation with anti-Olig1 antibody, or normal mouse IgG as a negative control, followed by immunoblotting with anti-Smad2/3 antibody. Smad3 is dominantly detected by this antibody in U373MG cells. (D) Interaction of Olig1 with the activated Smad complex bound to DNA is abrogated by HHM. HEK293T cells were transfected as indicated. Cell lysates were subjected to DNA-affinity precipitation assay using biotinylated 3 × CAGA as a probe. (E) Association of Olig1–Smad complex with the PAI-1 and Smad7 promoter regions is abrogated by HHM. ChIP analysis was performed using U373MG cells infected with Ad-LacZ or Ad-HHM. Cells were treated with TGF-β (2.5 ng/ml) for 1 h and harvested. Eluted DNAs were subjected to quantitative real-time PCR analysis. Values were normalized to the amount of the first intron of hypoxanthine phosphoribosyltransferase 1. Error bars represent s.d. Primer sequences are listed on the Supplementary Table S2.

Effects of endogenous Olig1 and HHM on the growth of U373MG glioma cells in vitro and in vivo. (A) Knockdown of Olig1 and HHM using microRNA. U373MG cells were infected with lentivirus-encoding control microRNA, Olig1 microRNA, or HHM microRNA. Expression of Olig1 and HHM proteins were determined by immunoblot. Expression of α-tubulin was determined as a loading control. NC, negative-control microRNA. (B) In vitro proliferation assay of U373MG glioma cells in which Olig1 or HHM was knocked down. Results were expressed as the change from control (untreated cells). Error bars represent s.e. (C) TGF-β-induced expression of PDGF-B in U373MG glioma cells in which Olig1 or HHM was knocked down. mRNAs were prepared from cells treated with TGF-β (1 ng/ml) for 3 h. Expression of PDGF-B, Olig1, or HHM was determined by quantitative real-time PCR analyses. Values were normalized to the amount of GAPDH mRNA. Error bars represent s.d. (D) Association of Olig1 and Smad2/3 with the PDGF-B promoter region. ChIP analysis was performed using U373MG cells infected with lentivirus-encoding control microRNA, Olig1 microRNA, or HHM microRNA. Cells were treated with TGF-β (2.5 ng/ml) for 1 h and harvested. Eluted DNAs were subjected to quantitative real-time PCR analysis. Values were normalized to the amount of the first intron of hypoxanthine phosphoribosyltransferase 1. Error bars represent s.d. Primer sequences are listed on the Supplementary Table S2. (E) In vivo growth of U373MG cells in which Olig1 or HHM was knocked down by infection with lentivirus-encoding control microRNA, Olig1 microRNA, or HHM microRNA. Values are plotted as the mean tumour volume±s.e. of nine tumours per condition. Panels on the right side show the results of histological examination of the samples. Tumour tissues dissected at 3 weeks after transplantation appeared encapsulated and exhibited no significant variation histologically. Tissue sections were stained with haematoxylin–eosin or Azan. Scale bars: 60 μm.

HHM attenuates TGF-β signalling through suppression of Smad-dependent transcriptional activity. (A) Effects of HHM on the transactivation of p3TP-Luc induced by TGF-β ligand (1 ng/ml) or constitutively active TβR-I (ALK5-TD) in NIH3T3 cells. Error bars represent s.d. Smad7 was used as a positive control. (B) Effects of HHM knockdown by shRNA on the transactivation of p3TP-Luc induced by constitutively active TβR-I (ALK5-TD) in HeLa cells (top panel). Error bars represent s.d. Expression of HHM protein was determined by anti-HHM antibody (middle panel). The lower panel shows expression level of tubulin protein as a loading control. (C) Effects of HHM on the transcriptional activity of p800-Luc in NMuMG cells (upper panel) and (CAGA)₁₂-MLP-Luc in NIH3T3 cells (lower panel) induced by ALK5-TD. Error bars represent s.d. Smad7 was used as a positive control.

Olig1 upregulates expression of PAI-1, but not Snail, in concert with Smad2/3, and their synergistic effects are antagonized by HHM. (A) Physical interaction of endogenous HHM with endogenous Olig1 in NMuMG cells. Cell lysates were subjected to immunoprecipitation with anti-Olig1 antibody, anti-HHM antibody as a positive control, or normal mouse IgG as a negative control, followed by immunoblotting with anti-HHM. (B) Knockdown of Olig1 in NMuMG cells. NMuMG cells were transfected with control or Olig1 siRNA duplex, and the expression of Olig1 protein was determined by immunoblotting. Expression of endogenous α-tubulin is also shown as a loading control. NC, negative control siRNA. (C) Effects of Olig1 knockdown on the expression of target genes of TGF-β, PAI-1, and Snail. NMuMG cells were transfected with control or Olig1 siRNA and then stimulated with 1 ng/ml TGF-β for 1 h. Expressions of PAI-1 (upper panel) and Snail (lower panel) were examined by quantitative real-time PCR analyses. Values were normalized to the amount of GAPDH mRNA. Error bars represent s.d. (D) Cooperation of Smad3 with Olig1 in transactivation of p800-Luc. Cells were co-transfected with the p800-Luc reporter construct and various combinations of indicated cDNAs, and luciferase activities in cell lysates were determined. Error bars represent s.d. (E) Knockdown of HHM in A549 cells. A549 cells were transfected with control or HHM siRNA duplex, and expression of HHM protein was determined by immunoblotting. Expression of endogenous α-tubulin is also shown as a loading control. (F) A549 cells were transfected with control or HHM siRNA duplex. Twenty-four hours after transfection, cells were infected with Ad-LacZ or Ad-HHM and cultured for another 24 h. Cells were then treated with or without TGF-β (1 ng/ml) for 1 h before harvest, and then expression of PAI-1 and Snail was examined by quantitative real-time PCR analyses. Error bars represent s.d.

HHM antagonizes the TGF-β-induced proliferation of glioma cells accelerated by Olig1. (A) Model of promotion of the growth of glioma by TGF-β through induction of PDGF-B exerting an effect through an autocrine mechanism (Bruna et al, 2007). (B) Physical interaction of endogenous Olig1 and HHM in U373MG cells. U373MG cells were treated with or without TGF-β (1 ng/ml) for 3 h before harvest. Cell lysates were subjected to immunoprecipitation with anti-Olig1 antibody, anti-HHM antibody as a positive control, or normal mouse IgG as a negative control, followed by immunoblotting with anti-HHM. (C) Olig1 promotes TGF-β-induced proliferation of glioma cells, whereas HHM suppresses it. U373MG cells were infected with Ad-LacZ, Ad-Olig1, or Ad-HHM and seeded. After 24 h, the cells were treated with TGF-β (1 ng/ml) and/or STI571 (5 μM), and cell number was counted 72 h after treatment. Results were expressed as the change from control (untreated cells). Error bars represent s.e. (D) TGF-β-induced expression of PDGF-B in Ad-Olig1- or Ad-HHM-infected cells. Expression of PDGF-B was examined by quantitative real-time PCR analyses after infected U373MG cells were treated with TGF-β (1 ng/ml) for 3 h. Values were normalized to the amount of GAPDH mRNA. Error bars represent s.d.

(A) Model of synexpression group-restricted inhibition of TGF-β signalling by HHM. On activation of TGF-β signalling, Smads form complexes with transcription factors, including Olig1 in the nucleus, and interact with the promoter region of target genes. HHM binds to a subset of the transcription factors including Olig1 (i) and Y (ii), but not Z (iii) and inhibits their physical interaction with Smads, which results in the dissociation of both the Smad complex and the partner transcription factors from the promoter regions. Y, unidentified Smad-binding transcription factors that interact with HHM; Z, Smad-binding transcription factors that do not interact with HHM. (B) Diagram representing data obtained from the microarray analysis of gene expression profiles of NMuMG cells transfected with negative-control siRNA, HHM siRNA, or Olig1 siRNA and treated for 1 h with TGF-β (1 ng/ml). Black square, TGF-β-up regulated genes; green square, HHM-regulated genes; red square, Olig1-regulated genes. Raw data are shown in Supplementary Table S1.