NF-kappaB signaling mediates the induction of MTA1 by hepatitis B virus transactivator protein HBx

Abstract

Metastasis-associated protein 1 (MTA1), a master chromatin modifier, has been shown to regulate cancer progression and is widely upregulated in human cancer, including hepatitis B virus-associated hepatocellular carcinomas (HCCs). Here we provide evidence that hepatitis B virus transactivator protein HBx stimulates the expression of MTA1 but not of MTA2 or MTA3. The underlying mechanism of HBx stimulation of MTA1 involves HBx targeting of transcription factor nuclear factor (NF)-kappaB and the recruitment of HBx/p65 complex to the NF-kappaB consensus motif on the relaxed MTA1 gene chromatin. We also discovered that MTA1 depletion in HBx-expressing cells severely impairs the ability of HBx to stimulate NF-kappaB signaling and the expression of target proinflammatory molecules. Furthermore, the presence of HBx in HBx-infected HCCs correlated well with increased MTA1 and NF-kappaB-p65. Collectively, these findings revealed a previously unrecognized integral role of MTA1 in HBx stimulation of NF-kappaB signaling and consequently, the expression of NF-kappaB targets gene products with functions in inflammation and tumorigenesis.

Figure 1

HBx induces MTA1 expression at the transcriptional level. (A) Western blot analysis of HBx and MTA1 in HepG2 cells transfected with either control vector (250 ng/reaction) or HBx expression vector (50, 100, and 250 ng/reaction). (B) Western blot analysis of MTA2 and MTA3 proteins in HepG2 cells transfected with control vector (250 ng/reaction) or HBx expression vector (50 and 250 ng/reaction). (C) Western blot analysis of MTA1 protein in HepG2X, a HBx-stable transfectant of HepG2, and HEK 293 cells transfected with either control vector (250 ng/reaction) or HBx (50 and 250 ng/reaction). (D) Western blot analysis of MTA1 protein in HepG2 cells transfected with either control vector or HCV-core (250 ng/reaction). (E) q-PCR analysis of MTA1 in HepG2 cells treated with actinomycin D (Act D) (5 μg/ml) after being transfected with either control vector or HBx (50–200 ng/reaction). Expression levels of MTA1 were normalized with β-Actin. F, MTA1 promoter activity in HepG2 cells and HEK 293 cells transfected with either control vector (250 ng/reaction) or HBx (100–250 ng/reaction)(*P<0.05,**P<0.001). G, MTA1 promoter activity in HepG2X.

Figure 2

HBx activates MTA1 transcription through NF-κB. (A) qPCR analysis of MTA1 in HepG2 cells treated with parthenolide (5 μM) after being transfected with either control vector or HBx expression vector. Expression levels of MTA1 were normalized with β-Actin. (B) Effect of HBx or NF-κB-p65 on MTA1 promoter activity in HepG2 cells. (C) Recruitment of HBx or NF-κB-p65 or Acetyl H4 to MTA1-chromatin (−3814 to −4152 and −2874 to −3207) by ChIP assay in NIH 3T3 cells using Flag-tagged antibody (Flag Ab.), NF-κB-p65 antibody (p65 Ab.) or Acetyl H4 antibody (Acetyl H4 Ab.) after cotransfecting with pCMV vector control or pCMV-HBx. Row 3: Sequential ChIP assay showing the recruitment of HBx followed by NF-κB-p65 to MTA1-chromatin (−3814 to −4152 and −2874 to −3207). (D) Recruitment of HBx followed by NF-κB-p65 to MTA1-chromatin (−3814 to −4152 and −2874 to −3207) by sequential double ChIP assay in NIH 3T3 cells treated with parthenolide after being transfected with either pCMV vector control or pCMV-HBx.

Figure 3

Role of YFKD motif of HBX in regulation of MTA1 transcription. (A) MTA1 promoter activity in HepG2 cells transfected with either control vector or HBx expression vector or HBx-deletion constructs. Inset: Western blot analysis of MTA1 protein in HepG2 cells after transfecting with vector or HBx or HBx- deletion constructs. (B) MTA1 promoter activity in HepG2 or NIH 3T3 or MEF cells after being transfected with either control vector or HBx expression vector or mut-HBx (YFKD was mutated to FAEN) expression vector. Lower panels are Western blot analysis of MTA1 protein in HepG2 or NIH 3T3 or MEF cells after being transfected with either control vector or HBx expression vector or mut-HBx expression vector. (C) GST pulldown assays with ³⁵S-labeled in vitro-translated HBx and mut-HBx and GST- NF-κB-p65 protein. (D) Recruitment of HBx to MTA1-chromatin (−3814 to −4152 and −2874 to −3207) by ChIP assay in the NIH 3T3 cells. (E) EMSA analysis of NF-κB-p65 binding to the mouse MTA1 promoter using PCR product encompassing functional NF-κB consensus sequence in transfected cells and controls. Nuclear extract of HepG2 cell transfected with either Flag-tagged HBx, or Flag-tagged mut-HBx, or Flag-tagged ΔD-deletion HBx (2000 ng/lane), probe control (0.3 ng/lane), NF-κB-p65 antibody (Ab.), anti-Flag antibody, and IgG control (1000 ng/lane), cold probe (15 ng/lane) were used.

Figure 4

MTA1 is required for HBx transactivation function. (A) MTA1 promoter activity in wild type (WT) and MTA1−/ − MEFs after being transfected with either either control vector or HBx. (B) MTA1-promoter activity in HepG2 cells with or without MTA1 knockdown by siRNA after being transfected with either control vector or HBx.

Figure 5

MTA1 is needed for HBx stimulated NF-κB signalling. (A) Effect of selective knockdown of MTA1 on the activation status of the NF-κB signaling components in HepG2 cells by Western blot analysis after being transfected with either control vector or HBx (250 ng per reaction in a 6-well plate). HepG2 cells transfected with increased amount of control vector and HBx (50 ng, 250 ng, and 600 ng per reaction) were used as controls. (B) NF-κB-promoter activity in HepG2 cells with or without MTA1 knockdown by siRNA-MTA1 after being transfected with either vector or HBx. Lower panel is the control Western blot analysis for the aforementioned experiments. Vinculin was used as a control. (C) NF-κB-promoter activity in MEF cells after being transfected with either vector or HBx. Lower panel is the control Western blot analysis for the aforementioned experiments. Vinculin was used as a control. (D) Nucleus extracts from HepG2 cells transfected with either vector control or HBx expression vector after MTA1 knockdown by siRNA-MTA1 were subjected to EMSA analysis using a NF-κB-consensus sequence. Extracts from wild type HepG2 transiently transfected with HBx were used as controls. (E) qPCR analysis of COX2, TNF-α, MTA1, and HBx mRNAs in HepG2 cells with or without MTA1 knockdown by siRNA-MTA1 after being transfected with control vector or vector expressing HBx. Control siRNA was used in indicated experiments. Expression levels of COX2, TNF-α, MTA1, and HBx were normalized with β-Actin. Inset: Western blot analysis for MTA1 in HepG2 cells after being co-transfected with siRNA-MTA1 and HBx or control vector. Vinculin was used as a control.

Figure 6

Prevalence of HBx, MTA1, and NF-κB-p65 in hepatocellular carcinoma. (A) Western blot analysis for MTA1, NF-κB-p65, and HBx proteins in human HCC specimens and matched non-tumor tissue from the same patient (n=44). Statistical analysis of the data was performed using Fisher’s Exact test. (B) Representative images of immunohistochemical analysis for HBx, MTA1 NF-κB-p65, and NF-κB-phospho-p65 in HCC and non-HCC samples in microtissue array. Circular marks denoted positive staining regions. (C) A multiple linear mixed model for the MTA1 intensity (arbitrary unit, AU) with those of HBx or NF-κB-p65 and the interaction between these markers and disease group fitted as covariates was assembled. Positive association between MTA1 and HBx and MTA1 and NF-κB-p65 intensity were observed in the HCC patients but non-HCC patients. Particularly, in HBx positive HCC patients, there was a significant positive association between NF-κB-p65 intensity and MTA1 intensity (P< 0.0001).