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. 2019 Mar 1;294(9):3137-3151.
doi: 10.1074/jbc.RA118.005761. Epub 2019 Jan 4.

Transforming growth factor β (TGFβ) cross-talk with the unfolded protein response is critical for hepatic stellate cell activation

Zhikui Liu  1 , Chao Li  1 , Ningling Kang  2 , Harmeet Malhi  1 , Vijay H Shah  1 , Jessica L Maiers  3
Affiliations

Transforming growth factor β (TGFβ) cross-talk with the unfolded protein response is critical for hepatic stellate cell activation

Zhikui Liu et al. J Biol Chem. .

Abstract

Transforming growth factor β (TGFβ) potently activates hepatic stellate cells (HSCs), which promotes production and secretion of extracellular matrix (ECM) proteins and hepatic fibrogenesis. Increased ECM synthesis and secretion in response to TGFβ is associated with endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). TGFβ and UPR signaling pathways are tightly intertwined during HSC activation, but the regulatory mechanism that connects these two pathways is poorly understood. Here, we found that TGFβ treatment of immortalized HSCs (i.e. LX-2 cells) induces phosphorylation of the UPR sensor inositol-requiring enzyme 1α (IRE1α) in a SMAD2/3-procollagen I-dependent manner. We further show that IRE1α mediates HSC activation downstream of TGFβ and that its role depends on activation of a signaling cascade involving apoptosis signaling kinase 1 (ASK1) and c-Jun N-terminal kinase (JNK). ASK1-JNK signaling promoted phosphorylation of the UPR-associated transcription factor CCAAT/enhancer binding protein β (C/EBPβ), which is crucial for TGFβ- or IRE1α-mediated LX-2 activation. Pharmacological inhibition of C/EBPβ expression with the antiviral drug adefovir dipivoxil attenuated TGFβ-mediated activation of LX-2 or primary rat HSCs in vitro and hepatic fibrogenesis in vivo Finally, we identified a critical relationship between C/EBPβ and the transcriptional regulator p300 during HSC activation. p300 knockdown disrupted TGFβ- or UPR-induced HSC activation, and pharmacological inhibition of the C/EBPβ-p300 complex decreased TGFβ-induced HSC activation. These results indicate that TGFβ-induced IRE1α signaling is critical for HSC activation through a C/EBPβ-p300-dependent mechanism and suggest C/EBPβ as a druggable target for managing fibrosis.

Keywords: C/EBPβ; IRE1α; adefovir dipivoxil; apoptosis signal-regulating kinase 1 (ASK1); c-Jun N-terminal kinase (JNK); cell signaling; collagen; endoplasmic reticulum stress (ER stress); fibronectin; fibrosis; p300.

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Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
TGFβ induction of IRE1α is critical for HSC activation. A, LX-2 cells were stably infected with an shRNA targeting procollagen 1α1 (shCollagen 1α1) or an NT control. Cells were treated with 5 ng/ml TGFβ for 0, 1, 2, 4, or 24 h, followed by assessment of phosphorylation and protein levels of IRE1α, fibronectin (FN), αSMA, and collagen I. HSC70 served as a loading control. Quantification is shown adjacent. B, LX-2 cells were transfected with an siRNA targeting SMAD2 (siSMAD2) or a nontargeting siRNA (siControl). 24 h post-transfection, cells were serum-starved and treated with TGFβ for 0, 1, 2, 4, or 24 h. Cell lysates were harvested, and levels of phosphorylated and total IRE1α, SMAD2/3, and HSC70 (loading control) were assessed by immunoblotting. Quantification is shown adjacent. C, primary HSCs were isolated from IRE1αfl/fl mice and infected with adenovirus expressing Cre recombinase (AdCre) or LacZ as a control. 48 h postinfection, cells were treated with TGFβ or vehicle. Cell lysates were harvested and analyzed by immunoblotting for FN, collagen I, αSMA, and HSC70 (loading control). Quantification is shown adjacent. D and E, LX-2 cells were pretreated with the IRE1α inhibitor 4μ8C (15 μm) for 1 h, followed by TGFβ or vehicle for 24 h. Cell lysates or mRNA were harvested and analyzed by either immunoblotting (D) for FN, collagen I, and HSC70 (loading control) or qPCR (E) to assess fibronectin, procollagen 1α1 and 1α2, αSMA, or PPARγ expression. Quantification for D is shown below the blots. Statistics were performed using ANOVA followed by Tukey post hoc analysis (*, p < 0.05; **, p < 0.01; ***, p < 0.001). Error bars, S.E.
Figure 2.
Figure 2.
IRE1α kinase activity promotes HSC activation. A, LX-2 cells were stably infected with doxycycline-inducible IRE1α constructs encoding for WT IRE1α (doxIRE1α WT), a kinase-dead mutant (K599A), or an endonuclease-dead mutant (K907A). Cells were treated with doxycycline (5 μg/ml) or vehicle for 24 h, cells were lysed, and protein levels of IRE1α, fibronectin, and collagen I levels were assessed. HSC70 served as a loading control. Quantification is shown adjacent. B, doxIRE1α cells were treated with GS-444217 (2 μm), SP600125 (10 μm), or SB203580 (0.5 μm) for 1 h to inhibit ASK1, JNK, or p38, respectively, followed by doxycycline treatment for 24 h. Cell lysates were harvested, and IRE1α, fibronectin, collagen I, phosphorylated and total JNK, and phosphorylated and total p38 were assessed by immunoblotting. HSC70 served as a loading control. Quantification is shown adjacent. C, doxIRE1α cells were pretreated with U0126 (5 μm) to inhibit ERK1/2 phosphorylation, followed by doxycycline treatment for 24 h. Cells were lysed and assessed by immunoblotting for IRE1α, fibronectin, collagen I, phosphorylated and total ERK1/2, and HSC70 as a loading control. Quantification is shown adjacent. Statistics were performed using ANOVA followed by Tukey post hoc analysis (*, p < 0.05; **, p < 0.01). n ≥ 3 biological replicates for each experiment. Error bars, S.E.
Figure 3.
Figure 3.
TGFβ promotes C/EBPβ expression. A, LX-2 cells were treated with 5 ng/ml TGFβ for 0, 1, 2, 4, or 24 h, harvested, and assessed by immunoblotting for expression of C/EBPβ isoforms (LAP1/2 and LIP). HSC70 served as a loading control. Quantification is shown below. B, shCollagen 1α1 or NT cells were treated with TGFβ for 0, 1, 2, 4, or 24 h, and cell lysates were analyzed by immunoblotting for collagen I or C/EBPβ isoforms. HSC70 served as a loading control. Quantification is shown below. C, LX-2 cells were treated with 5 ng/ml TGFβ for 4 h, fixed, permeabilized, and stained for C/EBPβ (green) and DAPI (blue). Representative images are shown. Statistics were performed using ANOVA followed by Tukey post hoc analysis (*, p < 0.05; **, p < 0.01). n ≥ 3 biological replicates. Error bars, S.E.
Figure 4.
Figure 4.
C/EBPβ expression and phosphorylation is increased by TGFβ in an IRE1α-dependent manner. A, LX-2 cells were pretreated for 1 h with 15 μm 4μ8C, followed by TGFβ (5 ng/ml) treatment for 0, 1, 2, 4, or 24 h. Cell lysates were harvested, and protein expression or phosphorylation of C/EBPβ isoforms on Thr-235 (corresponding to Thr-223 on LAP2 and Thr-37 on LIP) was examined by immunoblotting. HSC70 served as a loading control. Quantification is shown in adjacent graphs. B, doxIREα WT, K599A, or K907A cells were treated with doxycycline (5 μg/ml) or vehicle for 24 h. Cells were lysed, and C/EBPβ phosphorylation, total C/EBPβ, and IRE1α expression were analyzed by immunoblotting. HSC70 served as a loading control. Quantification is shown in adjacent graphs. C, doxIRE1α cells were treated with GS-444217 (2 μm) or SP600125 (10 μm) for 1 h to inhibit ASK1 or JNK, respectively, followed by doxycycline treatment for 24 h. Cells were lysed, and phosphorylation of C/EBPβ isoforms and total C/EBPβ expression, as well as IRE1α expression, was analyzed. HSC70 served as a loading control. Quantification is shown in adjacent graphs. Statistics for A and C were performed using ANOVA followed by Tukey post hoc analysis (*, p < 0.05; **, p < 0.01; ***, p < 0.001). For B, paired t tests were performed. n ≥ 3 biological replicates for each experiment. Error bars, S.E.
Figure 5.
Figure 5.
C/EBPβ is critical for HSC activation in response to TGFβ or IRE1α signaling. A, LX-2 cells were infected with a lentivirus expressing an shRNA against C/EBPβ (sh-C/EBPβ) or an NT control. Two clonal cell lines were engineered and were assessed by immunoblotting for expression of C/EBPβ isoforms (LAP1/2 and LIP). HSC70 served as a loading control. Quantification is shown below. B and C, sh-C/EBPβ cells (clones 1 and 2) or NT cells were treated with TGFβ (5 ng/ml) for 24 h. Cell lysates were harvested and assessed by immunoblotting for collagen I, FN, C/EBPβ, and HSC70 (loading control). Quantification is shown below. D, sh-C/EBPβ or NT cells were treated with TGFβ (5 ng/ml), after which mRNA was harvested and qPCR was performed to assess fibronectin, procollagen 1α1 and 1α2, or αSMA expression. E, DoxIRE1α cells were infected with lentivirus expressing shRNA targeting C/EBPβ (sh-C/EBPβ) or an NT control, and clonal cell populations were selected. Cells were treated with doxycycline for 24 h, after which cell lysates were harvested and assessed by immunoblotting for IRE1α, fibronectin, collagen I, αSMA, C/EBPβ, and HSC70 (loading control). Quantification is shown adjacent. Statistics were performed using ANOVA followed by Tukey post hoc analysis (*, p < 0.05; **, p < 0.01; ***, p < 0.001). n ≥ 3 biological replicates for each experiment. Error bars, S.E.
Figure 6.
Figure 6.
Adefovir dipivoxil limits HSC activation through decreasing C/EBPβ expression. A and B, LX-2 cells were pretreated with 10 μm adefovir dipivoxil (ADV) for 1 h, followed by TGFβ (5 ng/ml) for 24 h. Cell lysates or mRNA were harvested and assessed by immunoblotting (A) for FN, collagen I, C/EBPβ isoforms LAP1/2 and LIP, and HSC70 (loading control) or qPCR (B) for fibronectin, procollagen 1α1 and 1α2, or PPARγ expression. Quantification for A is shown in adjacent graphs. C, cells were treated with TGFβ in the presence of adefovir dipivoxil or vehicle for 0, 1, 2, 4, or 24 h. Cell lysates were harvested and assessed for phosphorylated SMAD3 or total SMAD2/3. HSC70 served as a loading control. D, primary HSCs were harvested from rats and pretreated with adefovir dipivoxil for 1 h, followed by either vehicle or TGFβ for 24 h. Lysates were harvested and analyzed by immunoblotting for fibronectin or collagen I. HSC70 served as a loading control. Quantification is shown in the graphs below. E, primary HSCs were harvested from rats, followed by adefovir dipivoxil treatment on days 2, 4, and 6 post-isolation. Cell lysates were harvested on day 7 and analyzed by immunoblotting for fibronectin, collagen I expression, or HSC70 (loading control). Quantification is shown in adjacent graphs. Statistics were performed using ANOVA followed by Tukey post hoc analysis for A–D and paired t test for E (*, p < 0.05; **, p < 0.01; ***, p < 0.001). n ≥ 3 biological replicates for each experiment. Error bars, S.E.
Figure 7.
Figure 7.
Adefovir dipivoxil–treated mice display reduced fibrogenesis. Age- and sex-matched C57Bl/6 mice (n = 7–8 mice/group) were treated with CCl4 or olive oil twice a week for 6 weeks, in conjunction with injections of 10 mg/kg adefovir dipivoxil or 0.05 m citric acid (vehicle) 5 times a week during the same time period. Whole liver was harvested and analyzed for fibrosis. A, liver sections underwent sirius red staining, and quantification was performed using ImageJ (shown below). B, hydroxyproline analysis for collagen content. C, whole liver lysates were harvested and immunoblotted for fibronectin, αSMA, and HSC70 (loading control). Quantification is shown adjacent. D, qPCR was performed on mRNA harvested from whole liver to analyze expression of procollagen 1α1, TIMP1, αSMA, and PDGFRα. E, immunofluorescence was performed on frozen liver sections to assess αSMA, desmin (a marker of HSCs), and collagen I (green). DAPI was used as a nuclear stain (blue). Representative images are shown. Staining was quantified using ImageJ and is shown in the adjacent graphs. F, whole-liver lysates were harvested and immunoblotted for phospho-IRE1α, total IRE1α, C/EBPβ, and HSC70 (loading control). Statistics were performed using ANOVA followed by Tukey post hoc analysis (*, p < 0.05; **, p < 0.01; ***, p < 0.001). Error bars, S.E.
Figure 8.
Figure 8.
p300 is critical for TGFβ- or UPR-mediated HSC activation and may involve a C/EBPβ-dependent mechanism. A, whole-liver lysates harvested from mice treated with olive oil, CCl4, adefovir dipivoxil, or CCl4 + adefovir dipivoxil and assessed by immunoblotting for protein levels of p300. HSC70 served as a loading control. B and C, LX-2 cells were infected with a lentivirus encoding an shRNA targeting p300 (sh-p300) or NT control and were selected to yield a clonal cell population. NT or sh-p300 cells were treated with 5 ng/ml TGFβ (B) or 1 μg/ml Tm (C) for 24 h, and cell lysates were harvested and analyzed by immunoblotting for p300, fibronectin, collagen I, αSMA, and HSC70 (loading control). Quantification is adjacent. D, doxIRE1α cells were stably infected with a lentivirus encoding an shRNA targeting p300 (doxIRE1α sh-p300) or NT, treated with doxycycline, and assessed by immunoblotting for IRE1α, p300, fibronectin, collagen I, αSMA, and HSC70 (loading control). Quantification is adjacent. Statistics were performed using ANOVA followed by Tukey post hoc analysis (*, p < 0.05; **, p < 0.01; ***, p < 0.001). n ≥ 3 biological replicates for each experiment. Error bars, S.E.
Figure 9.
Figure 9.
Helenalin acetate limits TGFβ-induced HSC activation. LX-2 cells were pretreated with 1 μm helenalin acetate (HA) for 1 h, followed by TGFβ treatment for 24 h. mRNA (A) or cell lysates (B and C) were harvested and analyzed by qPCR for fibronectin, procollagen 1α1 and 1α2, αSMA, or PPARγ or by immunoblotting for fibronectin, collagen I, p-SMAD3, total SMAD2/3, and HSC70 (loading control). Quantification for B and C is shown below the blots. D, primary rat HSCs (rHSCs) were pretreated with helenalin acetate for 1 h, followed by TGFβ for 48 h. Cells were lysed and assessed for protein expression of fibronectin, collagen I, and αSMA. HSC70 served as a loading control. Quantification is shown in adjacent graphs. Statistics were performed using ANOVA followed by Tukey post hoc analysis (*, p < 0.05; **, p < 0.01; ***, p < 0.001). n ≥ 3 biological replicates for each experiment. Error bars, S.E.
Figure 10.
Figure 10.
Cross-talk between TGFβ and IRE1α promotes HSC activation. 1, TGFβ-mediated phosphorylation of SMAD2/3 leads to increased expression of ECM proteins leading to ER stress. 2, ER stress leads to IRE1α autotransphosphorylation. 3, IRE1α signaling leads to phosphorylation of C/EBPβ downstream of ASK1 and JNK. 4, activation of C/EBPβ facilitates ECM production through a mechanism that involves p300. 5, together, this signaling cascade is critical for fibrogenesis.
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