Transcriptional repression of the transforming growth factor β (TGF-β) Pseudoreceptor BMP and activin membrane-bound inhibitor (BAMBI) by Nuclear Factor κB (NF-κB) p50 enhances TGF-β signaling in hepatic stellate cells

Abstract

TLR4 signaling induces down-regulation of the bone morphogenic protein (BMP) and activin membrane-bound inhibitor (BAMBI), which enhances TGF-β signaling during hepatic stellate cell (HSC) activation. We investigated the mechanism by which TLR4 signaling down-regulates BAMBI expression in HSCs and found that TLR4- and TNF-α-mediated BAMBI down-regulation is dependent on regulation of BAMBI promoter activity through the interaction with NF-κBp50 and HDAC1 in HSCs. Bambi was predominantly expressed in HSCs, at high levels in quiescent HSCs but at low levels in in vivo-activated and LPS-stimulated HSCs. In human HSCs, BAMBI expression was down-regulated in response to LPS and TNF-α. A BAMBI reporter assay demonstrated that the regulatory element to repress BAMBI transcription is located between 3384 and 1560 bp upstream from the transcription start site. LPS stimulation down-regulated BAMBI expression in cells with NF-κBp65 knockdown. However, it failed to down-regulate BAMBI in cells with inactivation of NF-κB or NF-κBp50 silencing, indicating that NF-κBp50 is a factor for BAMBI down-regulation. ChIP analysis revealed that LPS and TNF-α induced binding of the NF-κBp50/p50 homodimer to the BAMBI promoter region. We also found that HDAC1 is bound to this region as part of the NF-κBp50-HDAC1 complex, repressing transcriptional activity of the BAMBI promoter. Finally, we confirmed that LPS does not repress BAMBI reporter activity using a BAMBI reporter construct with a mutation at 3166 bp upstream of the coding region. In summary, our study demonstrates that LPS- and TNF-α-induced NF-κBp50-HDAC1 interaction represses BAMBI transcriptional activity, which contributes to TLR4-mediated enhancement of TGF-β signaling in HSCs during liver fibrosis.

Keywords: Hepatic Stellate Cells; Lipopolysaccharide (LPS); NF-κB; Toll-like Receptors (TLR); Transcription Promoter; Transforming Growth Factor β (TGF-β).

FIGURE 1.

BAMBI regulation by TLR4 signaling. A, Bambi mRNA expression in primary mouse HSCs, Kupffer cells (KC), and hepatocytes was determined by RT-PCR. B, Bambi mRNA levels in quiescent and 5-day culture-activated HSCs, HSCs isolated from bile duct ligation (BDL) and CCl₄-treated mice, and quiescent HSCs stimulated with LPS (100 ng/ml) for 4 h were determined by qPCR. C and D, BAMBI mRNA levels in LX2 cells (C) and hHSCs (D) 4 h after LPS (100 ng/ml) or TNF-α (10 ng/ml) stimulation were determined by qPCR. Un, untreated. Data represent mean ± S.D. *, p < 0.05; **, p < 0.01.

FIGURE 2.

Identification of the element in the promoter region responsible for repression of BAMBI by LPS and TNF-α. Nucleotide sequence positions are indicated relative to the transcriptional start site. LX2 cells were transfected with the indicated hBAMBI reporter constructs. 6 h after transfection, cells were treated with LPS (100 ng/ml) (A) or TNF-α (10 ng/ml) (B) for 16 h, followed by measurement for luciferase activities. The Renilla luciferase reporter was cotransfected to normalize transfection efficiency. Un, untreated. Experiments were performed three times and are shown as mean ± S.D. **, p < 0.01.

FIGURE 3.

NF-κBp50 mediates BAMBI repression by LPS and TNF-α. A and B, BAMBI mRNA levels in LX2 cells after 4 h of stimulation with LPS (100 ng/ml) (A) or TNF-α (10 ng/ml) (B) were determined by qPCR. Before treatment, LX2 cells were transfected with the Ad-GFP or Ad-IκB superrepressor overnight. Un, untreated. Data represent mean ± S.D. *, p < 0.05; **, p < 0.01. C and D, BAMBI mRNA levels in hHSCs after 4 h of LPS (100 ng/ml) (C) or TNF-α (10 ng/ml) (D) treatment were determined by qPCR. Before treatment, hHSCs were transfected with control Ad-GFP or the Ad-IκB superrepressor overnight. Un, untreated. Data represent mean ± S.D. **, p < 0.01. E, after transfection with siRNA for p50 or a control for 72 h, LX2 cells were stimulated with LPS (100 ng/ml) or TNF-α (10 ng/ml) for 4 h. The knockdown efficiency of siRNA p50 was evaluated by Western blot analysis for p50. BAMBI mRNA was measured by qPCR. Un, untreated. Data represent mean ± S.D. *, p < 0.05; **, p < 0.01. F, LX2 cells were transfected with siRNA for p65 or a control for 72 h, followed by stimulation with LPS (100 ng/ml) or TNF-α (10 ng/ml) for 4 h. p65 expression was evaluated by Western blot analysis. BAMBI mRNA was measured by qPCR. Un, untreated. Data represent mean ± S.D. *, p < 0.05; **, p < 0.01. G, BAMBI luciferase reporter activity was measured. LX2 cells were cotransfected with −3384/+82-luc and siRNA p50. The Renilla luciferase reporter was used to normalize transfection efficiency. Data represent mean ± S.D. **, p < 0.01. H, two κB binding sites within −3384 to −1560 of the hBAMBI promoter region were identified using bioinformatics analysis. I, effects of LPS and TNF-α on the recruitment of p50 to the κB binding site in the hBAMBI promoter were assessed by ChIP analysis. LX2 cells (top panel) and hHSCs (bottom panel) were treated with LPS (100 ng/ml) or TNF-α for 2 h. After fixation, soluble chromatin was immunoprecipitated using anti-p50 or control Ab (normal mouse IgG). BAMBI promoter fragments containing the −3166 binding site were amplified by PCR. J, effects of Ad-IκBsr on the recruitments of p50 to the κB binding site in the hBAMBI promoter were assessed. LX2 cells were treated with control Ad-GFP or Ad-IκBsr overnight and then treated with LPS (100 ng/ml) or TNF-α for 2 h. A ChIP analysis using anti-p50 or control Ab (normal mouse IgG) was performed. BAMBI promoter fragments containing the −3166 binding site were amplified by PCR. Un, untreated.

FIGURE 4.

Mechanism of BAMBI repression by HDAC1. A, the knockdown efficiencies of siRNA HDAC1 were estimated by Western blot analysis. LX2 cells were transected with siRNA HDAC1 or a control for 72 h, followed by treatment with LPS (100 ng/ml) or TNF-α (10 ng/ml) for 4 h. B, LX2 cells were transfected with siRNA HDAC1 for 72 h. Then, hBAMBI mRNA was measured by qPCR. 18 S was used as an internal control for normalization. Un, untreated. Data represent mean ± S.D. *, p < 0.05. C, effects of LPS and TNF-α on the recruitment of HDAC1 to the κB binding site in the hBAMBI promoter were assessed. LX2 (left panel) and hHSCs (right panel) were treated with LPS (100 ng/ml) or TNF-α (10 ng/ml) for 2 h. A ChIP analysis using anti-HDAC1 or control Ab (normal rabbit polyclonal IgG) was performed. BAMBI promoter fragments containing the −3166 binding site were amplified by PCR. D, effects of TSA on the recruitment of HDAC1 to the κB binding site in the hBAMBI promoter were analyzed. LX2 cells were pretreated with TSA (300 nm/ml) for 8 h and then treated with LPS or TNF-α for 2 h. A ChIP analysis using anti-HDAC1 or control Ab (normal rabbit polyclonal IgG) was employed. BAMBI promoter fragments containing the −3166 binding site were amplified by PCR. E, effects of NF-κB inhibition on the binding of HDAC1 to the κB binding site in the hBAMBI promoter. LX2 cells were treated with control Ad-GFP or Ad-IκBsr overnight and then treated with LPS or TNF-α for 2 h. A ChIP analysis on the BAMBI promoter using anti-HDAC1 or control Ab (normal rabbit polyclonal IgG) was performed. F, ChIP analysis for the assessment of the effects of TSA on the binding of p50 to the κB binding site in the hBAMBI promoter. G, direct interaction of p50 with HDAC1 after LPS or TNF-α stimulation was assessed. LX2 cells were treated with control Ad-GFP or Ad-IκBsr overnight and then treated with LPS and TNF-α for 2 h. After immunoprecipitation (IP) with anti-p50 antibody, Western blotting (WB) for HDAC1 was performed.

FIGURE 5.

Effect of mutation of the −3166 κB-binding site in the BAMBI promoter. A, the predicted p50 binding sequence (top panel) and designed mutant sequence (bottom panel). B, hBAMBI p-3384+82-luc, which contains an NF-κB binding site, was used to assess the functional significance of the −3166 binding site. The p-3384+82-luc construct was responsive to an LPS or TNF-α stimulus, whereas the promoter constructs harboring a mutation of the predicted −3166 κB binding site (p-3384+82-mutant-luc) abrogated the LPS- or TNF-α-induced suppression of luciferase expression. Un, untreated. Data represent mean ± S.D. **, p < 0.01.

FIGURE 6.

LPS enhances TGF-β signaling in hHSCs through down-regulation of BAMBI. A, LX2 cells were pretreated with LPS (100 ng/ml) or TNF-α (10 ng/ml) for 12 h and subsequently treated with TGF-β1 (5 ng/ml) for 30 min. A Western blot analysis for phospho-Smad2/3 was performed. B, LX2 cells were infected with Ad-IκBsr or Ad-GFP overnight, followed by treatment with LPS or TNF-α for 12 h. Subsequently, cells were treated with TGF-β1 (5 ng/ml) for 24 h. COL1A1 mRNA was measured by qPCR, and 18 S was used as an internal control. Data represent mean ± S.D. *, p < 0.05; **, p < 0.01. n.s., not significant. C, LX2 cells were transfected with siRNA BAMBI for 48 h and then treated with TGF-β1 (5 ng/ml) for an additional 24 h. BAMBI protein expression was analyzed by immunoblotting (left panel), and COL1A1 mRNA expression was measured by qPCR (right panel). Data represent mean ± S.D. *, p < 0.05.