Stimulation of inducible nitric oxide by hepatitis B virus transactivator protein HBx requires MTA1 coregulator

Abstract

Nitric oxide has been implicated in the pathogenesis of inflammatory disorders, including hepatitis B virus-associated hepatocellular carcinoma. Transactivator protein HBx, a major regulator of cellular responses of hepatitis B virus, is known to induce the expression of MTA1 (metastasis-associated protein 1) coregulator via NF-kappaB signaling in hepatic cells. However, the underlying mechanism of HBx regulation of the inducible nitric-oxide synthase (iNOS) pathway remains unknown. Here we provide evidence that MTA1 is a positive regulator of iNOS transcription and plays a mechanistic role in HBx stimulation of iNOS expression and activity. We found that the HBx-MTA1 complex is recruited onto the human iNOS promoter in an NF-kappaB-dependent manner. Pharmacological inhibition of the NF-kappaB signaling prevented the ability of HBx to stimulate the transcription, the expression, and the activity of iNOS; nevertheless, these effects could be substantially rescued by MTA1 dysregulation. We further discovered that HBx-mediated stimulation of MTA1 is paralleled by the suppression of miR-661, a member of the small noncoding RNAs, recently shown to target MTA1. We observed that miR-661 controls of MTA1 expression contributed to the expression and activity of iNOS in HBx-expressing HepG2 cells. Accordingly, depletion of MTA1 by either miR-661 or siRNA in HBx-expressing cells severely impaired the ability of HBx to modulate the endogenous levels of iNOS and nitrite production. Together, these findings reveal an inherent role of MTA1 in HBx regulation of iNOS expression and consequently its function in the liver cancer cells.

FIGURE 1.

HBx targets miR-661. A, transcriptional levels of miR-661 and MTA1 in HepG2 cells expressing HBx and control were quantified by real-time PCR. U6 RNA was used as an internal control for miR-661 quantification. The levels of mRNA for MTA1 were normalized to that of β-actin mRNA. B, HBx regulates the MTA1 3′-UTR reporter. HepG2 cells were co-transfected with HBx expression plasmid and MTA1 3′-UTR reporter construct. Luciferase reporter assay was performed after 48 h of transfection. All experiments were repeated three times, and data are shown as mean ± S.D. in -fold change compared with control.

FIGURE 2.

miR-661 plays an instrumental role in HBx regulating iNOS. Effects of miR-661 on HepG2 cells expressing HBx. HepG2 cells were transfected with 100 nmol of either miR-661 or negative control mimic (Dharmacon, Lafayette, CO) using Oligofectamine (Invitrogen). After 24 h, transfected HepG2 cells were again subjected to co-transfection with either control vector or HBx (250 ng/reaction in a 6-well plate) and iNOS promoter reporter construct. A and B, cell lysates were subjected to Western blot analysis for iNOS, HBx, and MTA1 expression. Vinculin was used as a loading control. Transfection efficiency of miR-661 in HepG2 cells was evaluated by quantitative real time PCR. U6 RNA was used as an internal control for miR-661 quantification. C, MTA1 ectopic expression rescued miR-661 effects. iNOS promoter luciferase assay was performed 48 h after HBx transfection. MicroRNA-transfected HepG2 cells were subsequently co-transfected with HBx and pcDNA-MTA1-T7-tagged or control expression vector (500 ng/reaction in a 6-well plate) (n = 3). Results are presented in -fold change compared with control, mean ± S.E., n = 3. D and E, cells were subjected to Western blot analysis for iNOS and MTA1 expression. T7 tag was analyzed for transfection efficiency. β-Actin was used as an internal control. E, transfection efficiency of miR-661 in HepG2 cells being transfected with miRNA-661, HBx, and MTA1 expression vector was evaluated by quantitative real-time PCR. U6 RNA was used as an internal control for miR-661 quantification. F, conditioned medium at the time of harvesting was collected and assayed for nitrite levels as described under “Experimental Procedures.” The levels of nitrite accumulation/10⁵ cells are presented as mean ± S.E. All experiments were repeated at least three times.

FIGURE 3.

MTA1 is required for HBx-induced iNOS activity. A, quantitative PCR analysis of iNOS and MTA1 mRNAs in HBx-expressing HepG2 cells or control-transfected HepG2 cells with or without MTA1 knockdown by siRNA-MTA1. Control siRNA was used as indicated in the experiments. Expression levels of iNOS were normalized using iNOS amplicon, and MTA1 levels were normalized with β-actin. Results are presented as mean ± S.E., n = 3, with p < 0.001 considered to be statistically significant. B, representative Western blot analysis of iNOS, HBx, and MTA1 in HepG2 cells transfected with increased amounts (100, 250, and 500 ng/reaction) of either control vector (lanes 1–3) or HBx expression vector (lanes 4–6). HepG2 cells with MTA1 knockdown by siRNA-MTA1 (lanes 7 and 8) were transfected with (250 ng/reaction) of either control (lane 8) or HBx expression vector (lane 7). β-Actin was used as an internal control.

FIGURE 4.

Selectively knocking down MTA1 compromises iNOS expression. A, HepG2 cells with or without MTA1 knockdown by siRNA-MTA1 were co-transfected with HBx and iNOS promoter luciferase reporter construct. Cell lysates were analyzed for iNOS and MTA1 protein expression. Vinculin was used as an internal control. B, promoter activity was assessed 48 h after transfection. Results are presented as -fold change compared with control (mean ± S.E., n = 3). C, wild type MEFs (■) or MTA1^−/− MEFs (□) were co-transfected with HBx and iNOS luciferase reporter construct. iNOS promoter activity was assessed after 48 h. Results are presented as -fold change compared with control (mean ± S.E., n = 3). D, cell lysates were analyzed for iNOS protein expression by Western blot. β-Actin was used as a loading control.

FIGURE 5.

MTA1-HBx protein complex interacts with NF-κB Sequence of the iNOS gene promoter. A, recruitment of HBx or MTA1 to iNOS chromatin (−1 to −246, −5344 to −5593, and −5707 to −5941) by ChIP assay in HepG2 cells after being transfected with either pCMV vector control or pCMV-HBx. B, recruitment of HBx followed by MTA1 to iNOS chromatin after HBx-expressing cells were treated with parthenolide (5 μm). Recruitment of HBx followed by MTA1 to iNOS chromatin (−1 to −246 and −5344 to −5593) was analyzed by sequential double ChIP assay in HepG2 cells. C, nucleus extracts of HBx and control vector transient transfected cells (2000 ng/reaction) were subjected to EMSA. A PCR product of the iNOS promoter region encompassing the functional NF-κB consensus sequence. Probe control (0.3 ng/lane), MTA1 antibody (MTA1 Ab.), and IgG control (1000 ng/lane) were used. Reactions were also carried out in the presence or absence of parthernolide. IP, immunoprecipitation.

FIGURE 6.

Ectopically expressed MTA1 rescued inhibitory effects of parthernolide. A, HBx-expressing HepG2 cells were treated with parthernolide and subsequently transfected with pcDNA-MTA1 control expression vector (500 ng/reaction in a 6-well plate). Cells were then subjected to an iNOS promoter assay. B, subsequently, cell lysates were subjected to Western blot analysis for iNOS expression. Western blot analysis for HBx and T7 tag was carried out for transfection efficiency. Vinculin was used as a loading control. C, the levels of nitrite accumulation/10⁵ cells are presented as mean ± S.E., n = 3.