TGF-β downstream of Smad3 and MAPK signaling antagonistically regulate the viability and partial epithelial–mesenchymal transition of liver progenitor cells

Results

TGF-β induced growth inhibition and partial epithelial–mesenchymal transition in liver progenitor cells

To investigate the role of TGF-β and its downstream signaling in liver progenitor cells, we first treated liver progenitor cell lines (WB-F344 and LE/6 cells) with TGF-β (10 ng/ml) for 3 days and then analyzed cell proliferation (CCK-8) assays (repeated three times). After treatment with TGF-β, the proliferation of WB-F344 and LE/6 cells was reduced (Figure 1A), although the inhibitory effect on cell growth was limited to both WB-F344 (39.24% inhibition, P < 0.001 for the TGF-β-treated vs. the PBS-treated WB-F344 cell line) and LE/6 (31.67% inhibition, P < 0.0001 for the TGF-β-treated vs. the PBS-treated LE/6 cell line) cells. Considering that TGF-β is a classic inducer of epithelial–mesenchymal transition (EMT) in cells and that EMT and partial EMT of LPCs contribute to liver fibrosis and the initiation of liver cancer [12–14], we subsequently performed EMT-related experiments to confirm whether EMT occurs in LPCs after treatment with TGF-β. Intriguingly, after exposure to a high concentration of TGF-β (10 ng/ml) for 3 days, phase contrast microscopic scanning and fluorescent F-actin staining revealed morphological changes (Figure 1B) and rearrangement of actin fibers (Figure 1C) in both WB-F344 and LE/6 cells, although this trend was not obvious. Western blot analyses of WB-F344 and LE/6 cells after treatment with TGF-β (10 ng/ml) for three days revealed that the expression of an epithelial marker (E-cadherin) was reduced, while the expression of a mesenchymal marker (vimentin) was slightly increased (Figure 1D and Supplementary Figure 1). In vitro motility assays showed that the migration of WB-F344 and LE/6 cells was enhanced by stimulation with TGF-β (Figure 1E), which suggests that the mesenchymal state of LPCs was enhanced after stimulation with TGF-β. Taken together, these results suggested that despite TGF-β-induced cytostasis and EMT in LPCs, these effects were not obvious.

Figure 1. TGF-β inhibited the growth and promoted epithelial–mesenchymal transition in liver progenitor cells. (A) Liver progenitor cell lines (WB-F344 and LE/6) were treated with TGF-β (10 ng/ml) for 3 days and then subjected to CCK-8 analyses. OD values were normalized to those of the control groups. (B) Phase contrast images of TGF-β (10 ng/ml, 3 days)-treated WB-F344 and LE/6 cells. Scale bar, 100 μm. (C) Phalloidin staining for F-actin (red) in TGF-β (10 ng/ml, 3 days)-induced WB-F344 and LE/6 cells. DAPI was used to show the location of the nucleus (blue). The white arrows indicate rearrangements of F-actin. Scale bar, 25 μm. (D) Western blot analyses of the indicated cell lines with antibodies against E-cadherin and vimentin. GAPDH was used as a loading control. (E) Transwell analyses of the motility of WB-F344 and LE/6 cells treated with or without TGF-β (10 ng/ml, 7 hr and 24 hr). Representative images of migrated cells are shown (left panel); migrated cells were counted, and the average number of cells in three independent experiments is shown (right panel). Two-tailed Student’s t-tests were used for statistical analysis. The data are presented as the mean ± S.E.M. ^***p < 0.001.

Both Smad and MAPK signaling were activated in liver progenitor cells under TGF-β stimulation

We subsequently investigated the signaling cascades that contributed to the TGF-β-induced growth inhibition and EMT in LPCs. Previous studies have reported that Smad signaling is the classic downstream signaling cascade of TGF-β. We then carried out Western blot analyses, and the results showed that under stimulation with TGF-β, both Smad2 and Smad3 were phosphorylated in a time-dependent manner in WB-F344 and LE/6 cells, which suggested that canonical TGF-β downstream of Smad signaling was activated in LPCs (Figure 2A). In addition, Western blot analyses revealed that in both WB-F344 and LE/6 cells, Erk, JNK and p38 MAPK were phosphorylated in a time-dependent manner, which suggested that in LPCs, the major components of MAPK signaling (Erk, JNK and p38 MAPK signaling) are also activated by TGF-β. In detail, we found that the induced phosphorylation of Smad2 and 3 at the C-terminus peaked at 0.5 h to 1 h after TGF-β induction, whereas the TGF-β-induced phosphorylation of p-Erk, JNK and p38 MAPK peaked at 1–3 h after induction, accompanied by a reduction in phosphorylated Smad2 and 3 (Figure 2B).

Figure 2. Both Smad and MAPK signaling are activated by TGF-β in liver progenitor cells. (A, B) LPCs (LE/6 and WB-F344 cells) were treated with TGF-β (10 ng/ml) for the indicated times, and the lysates were subjected to Western blot analyses with antibodies against the indicated proteins. Representative blot images of three independent experiments are shown, and GAPDH was used as a loading control.

Suppression of TGF-β downstream of Erk, JNK or p38 MAPK signaling sensitizes LPCs to TGF-β-induced cytostatic effects

We found that TGF-β induced limited cytostatic effects in LPCs, and our previous studies demonstrated that Smad signaling contributed to these inhibitory effects on the growth of LPCs. To explore the role of TGFβ-MAPK signaling in LPCs, we treated LPCs with both TGF-β and MAPK inhibitors, including U0126 (an Erk inhibitor, 10 μM), SP600125 (a JNK inhibitor, 10 μM) and SB203580 (a p38 MAPK inhibitor, 5 μM), and then carried out Western blot and CCK-8 analyses. In LPCs, TGF-β activated Erk, JNK, and p38 MAPK signaling, which was indicated by the phosphorylation of Erk, c-jun and ATF-2, which was inhibited by U0126 (Figure 3A and Supplementary Figure 2), SP600125 (Figure 3B), and SB201580 (Figure 3C), respectively. The results of the CCK-8 assays revealed that in both WB-F344 and LE/6 cells, the inhibition of Erk signaling by U0126 augmented the suppressive effect of TGF-β on cell growth (Figure 3D). Similar effects were also found for the SP600125- or SB203580-treated LPCs (Figure 3E, 3F). Taken together, these results suggested that suppressing TGF-β downstream of MAPK signaling sensitizes LPCs to TGF-β-induced cytostatic effects.

Figure 3. Suppression of TGF-β downstream of Erk, JNK or p38 MAPK signaling strengthens the TGF-β-induced cytostatic effects in LPCs. (A–C) WB-F344 and LE/6 cells were treated with TGF-β, U0126 (an Erk inhibitor), SP600125 (a JNK inhibitor), and/or SB203580 (a p38 MAPK inhibitor) as indicated, and Western blot analyses were carried out with antibodies against phospho-ERK and ERK (A), phospho-c-jun and c-jun (B), and phospho-ATF2 and ATF2 (C). GAPDH was used as a loading control. (D–F) WB-F344 and LE/6 cells were treated with TGF-β and/or the inhibitors as indicated for 3 days, after which CCK-8 analyses were performed. The normalized OD values of each group were compared, and the average OD values of three independent experiments are shown. One-way ANOVA was used for statistical analysis. The data are presented as the mean ± S.E.M. ^***p < 0.001; ^****p < 0.0001.

Inhibition of TGF-β-induced Erk, JNK or p38 MAPK signaling augmented TGF-β-mediated EMT in LPCs

Our previous studies demonstrated that TGFβ induces EMT in LPCs through activating Smad signaling. To further investigate the role of TGF-β in activating Erk, JNK and p38 MAPK signaling in LPCs, we suppressed the TGF-β downstream effectors Erk, JNK and p38 MAPK via specific inhibitors in LPCs. The results showed that TGF-β induced obvious EMT after Erk, JNK or p38 MAPK signaling was inhibited in LPCs, which was shown by the elevated rearrangement of F-actin (Figure 4A–4C), decreased expression of the epithelial marker E-cadherin, increased expression of the mesenchymal marker vimentin (Figure 4D–4F and Supplementary Figure 3), and enhanced capacity for cell migration (Figure 4G and Supplementary Figure 4). These results suggested that TGF-β-induced Erk, JNK and p38 MAPK signaling plays an inhibitory role in the TGF-β-induced EMT of LPCs.

Figure 4. Inhibition of TGF-β-induced Erk, JNK or p38 MAPK signaling augmented TGF-β-mediated EMT and motility in LPCs. (A–C) Liver progenitor cell lines (WB-F344 and LE/6) were treated with TGF-β and/or kinase inhibitors as indicated for 3 days and then subjected to phalloidin staining for F-actin (red). DAPI (blue) was used to stain the cell nuclei. The white arrows indicate F-actin rearrangements. Scale bar, 25 μm. (D–F) WB-F344 and LE/6 cells were treated with TGF-β and/or kinase inhibitors as indicated for three days, and Western blot analyses were carried out with antibodies against E-cadherin and vimentin. Representative bands of three independent experiments are shown, and GAPDH was used as a loading control. (G) Cell motility analyses of LPCs treated with TGF-β and/or kinase inhibitors as indicated (for WB-F344 cells, TGF-β and/or kinase inhibitors were added for seven hrs, and for LE/6 cells, TGF-β and/or kinase inhibitors were added for 24 hrs). The average number of migrated cells per field in three independent experiments is shown. One-way ANOVA was used for statistical analysis. The data are presented as the mean ± S.E.M. ^*p < 0.05; ^**p < 0.01; and ^***p < 0.001.

TGF-β activated Erk, JNK and p38 MAPK signaling and inhibited TGFβ-Smad3 signaling in LPCs

We found that TGF-β downstream of MAPK cascades suppressed TGFβ-induced cytostatic and EMT effects in LPCs. To explore whether TGFβ-MAPK signaling inhibits TGFβ-Smad signaling in LPCs, we treated LPCs with TGF-β and specific MAPK inhibitors, including U0126, SP600125 and SB203580, and carried out Western blotting to analyze the activation of Smad signaling in LPCs. Treatment with U0126, SP600125 or SB203580 had little effect on the phosphorylation of Smad2 in LPCs stimulated with TGF-β, whereas the TGF-β-induced phosphorylation of Smad3 in LPCs was elevated after treatment with MAPK inhibitors, including U0126 (Figure 5A), SP600125 (Figure 5B) or SB203580 (Figure 5C). These results suggested that in LPCs, TGFβ-MAPK signaling inhibited TGFβ-Smad3 signaling but did not alter the status of Smad2. To further confirm these findings, we performed luciferase reporter analyses, and the results showed that TGF-β activated SBE4-luc (a Smad3 binding element) in both WB-F344 and LE/6 cells, and this activation was further strengthened after the activation of Erk, JNK, or p38 MAPK was inhibited (Figure 5D–5F). Taken together, these results suggested that in LPCs, the signaling of the TGF-β downstream effectors Erk, JNK and p38 MAPK inhibited the activation of Smad signaling. In particular, TGF-β, which is downstream of Smad3 but not Smad2 signaling, was inhibited by TGFβ-MAPK signaling in LPCs.

Figure 5. TGF-β activated Erk, JNK and p38 MAPK signaling and inhibited TGFβ-Smad3 signaling in LPCs. (A–C) LPCs were treated with TGF-β and/or kinase inhibitors as indicated, and the levels of phospho-Smad2 and Smad3 at the C-terminus and related total Smad2 and 3 proteins in the cells were analyzed via Western blotting. GAPDH was used as a loading control. (D–F) LPCs were cotransfected with pRL-TK and SBE4-luc and treated with TGF-β and/or kinase inhibitors as indicated. Luciferase activity was normalized to Renilla luciferase activity and is expressed as the means ± SEMs of triplicate measurements. The following comparisons of the bars were made: a, P < 0.01, second with first; b, P < 0.001, third with first; c, P < 0.01, third with second; d, P < 0.01, fourth with second; e, P < 0. 001, fourth with third; f, P < 0.05, third with second. The experiments were repeated three times. One-way ANOVA was used for statistical analysis. The data are presented as the mean ± S.E.M.

Inhibition of Smad signaling downstream of TGF-β abrogated TGF-β-induced cytostasis and EMT and strengthened MAPK signaling in LPCs

We then suppressed Smad signaling in LPCs through shRNA-mediated interference of Smad4 expression (Figure 6A and Supplementary Figure 5) and carried out CCK-8 analyses. The results showed that TGF-β-induced cytostasis was abrogated after Smad4 was knocked down in LPCs (Figure 6B). The results of Western blot analyses of EMT-related markers (Figure 6C), F-actin fluorescence staining (Figure 6D) and cell motility (Figure 6E and Supplementary Figure 6) in LPCs revealed that TGF-β-induced EMT and motility in LPCs were mitigated after Smad4 knockdown. In addition, the results of Western blot analyses of LPCs showed that knockdown of Smad4 strengthened TGF-β-induced MAPK signaling, as indicated by the phosphorylation of Erk, c-jun and p38 MAPK (Figure 6F). These results suggested that TGF-β-induced Smad signaling suppressed downstream MAPK signaling in LPCs.

Figure 6. Inhibition of Smad signaling downstream of TGF-β abrogated TGF-β-induced cytostasis and EMT and strengthened MAPK signaling in LPCs. (A) Western blotting was used to measure the expression of Smad4 in WB-shSmad4, LE-shSmad4, and control cells (WB-shScramble or LE-shcramble cells). GAPDH was used as a loading control. (B) CCK-8 assays of WB-shSmad4, LE-shSmad4 and control cells before and after treatment with TGF-β (10 ng/ml, 3d). OD values were normalized to those of the control groups. (C) WB-shSmad4, LE-shSmad4, and control cells were treated with TGF-β (10 ng/ml, 3d) as indicated, and Western blot analyses were carried out with antibodies against E-cadherin and vimentin. GAPDH was used as a loading control. (D) WB-shSmad4, LE-shSmad4 and control cells were treated with TGF-β (10 ng/ml) for 3 days and then subjected to phalloidin staining for F-actin (red). DAPI (blue) was used to stain the cell nuclei. The white arrows indicate F-actin rearrangements. Scale bar, 25 μm. (E) Cell motility analyses of WB-shSmad4, LE-shSmad4 and control cells treated with TGF-β. The average number of migrated cells per field is shown. (F) Western blot analyses of Smad4, p-ERK, ERK, p-p38 MAPK, p38 MAPK, p-c-Jun and c-Jun expression in WB-shSmad4, LE-shSmad4, and control cells. GAPDH was used as a loading control. WB-shSmad4, LE-shSmad4 and control cells were treated with TGF-β (10 ng/ml) for 1 h or 3 h, respectively. These experiments were repeated three times. One-way ANOVA was used for statistical analysis. The data are presented as the mean ± S.E.M. ^*p < 0.05; ^**p < 0.01; and ^***p < 0.001.

TGF-β downstream MAPK signaling promotes linker phosphorylation of Smad3 in liver progenitor cells

Previous studies have reported that in mammalian cells, both Smad2 and Smad3 can be phosphorylated at their linker and C-terminal regions through different upstream signals, and Smad isoform signals play distinct roles in the proliferation, EMT, motility, and carcinogenesis of cells [15] (Figure 7A and Supplementary Figure 7). To explore the occurrence of Smad phosphoisoform signaling in LPCs, we treated LPCs with TGF-β and carried out Western blot analysis. The results showed that TGF-β induced linker phosphorylation of Smad2 and Smad3 in LPCs (Figure 7B). In addition, blocking the activity of Erk, JNK or p38 MAPK with specific inhibitors (U0126, SP600125, and SB203580, respectively) reduced the TGF-β-induced linker phosphorylation of Smad3, whereas the TGF-β-induced linker phosphorylation of Smad2 was not influenced by the inhibition of Erk, JNK or p38 MAPK signaling in LPCs (Figure 7C–7E). Taken together, these results suggested that TGF-β-MAPK signaling contributed to the linker phosphorylation of Smad3 in LPCs.

Figure 7. TGF-β downstream of MAPK signaling promotes linker phosphorylation of Smad3 in liver progenitor cells. (A) Schematic representation of the phosphorylation sites in Smad2 and Smad3. (B) WB-F344 and LE/6 cells were treated with TGF-β (10 ng/ml) for the indicated times, and lysates were subjected to Western blot analyses with antibodies against the indicated phospho- and total Smad proteins. GAPDH was used as a loading control. (C–E) WB-F344 and LE/6 cells were treated with TGF-β (10 ng/ml, 1 h) and/or kinase inhibitors as indicated for 1 h or 3 h, and Western blot analyses were carried out with antibodies against the indicated phospho- and total Smad proteins. GAPDH was used as a loading control. The experiments were repeated three times, and representative images are shown.

Smad3 phosphorylation affects TGF-β-induced cytostasis and EMT in LPCs

To explore the roles of the linker and C-terminal phosphorylation of Smad3 in LPCs, we used recombinant adenoviruses harboring constitutively expressed wild-type Smad3 (Ad-Smad3) or its mutant at the C-terminal (Ad-3SA) or linker region (Ad-EPSM), in which all serine/threonine phosphorylation sites were replaced with alanine (A) or valine (V, Figure 8A and Supplementary Figures 8 and 9). An adenovirus carrying green fluorescent protein (Ad-GFP) was used as a control in the present study. We first infected Ad-Smad3, Ad-S3A, Ad-EPSM, and Ad-GFP into WB-F344 and LE-6 cells, and the Western blotting results showed that after infection of LPCs with Ad-Smad3, the phosphorylation of Smad3 at both the C-tail and linker region was elevated in a multiplicity of infection (MOI)-dependent manner (Figure 8B). In addition, infection of LPCs with Ad-3SA or Ad-EPSM caused a reduction in the C-terminus (Figure 8C) and linker phosphorylation of Smad3 (Figure 8D), respectively, in the presence or absence of TGF-β. Moreover, infection of LPCs with Ad-3SA promoted TGF-β-induced linker phosphorylation of Smad3 (Figure 8C), and infection of LPCs with Ad-EPSMs increased the C-terminal phosphorylation of Smad3, as indicated by stimulation with TGF-β (Figure 8D). We then carried out cell proliferation analyses, and the results showed that infection with Ad-Smad3 in LPCs augmented TGF-β-induced cytostasis, and infection with Ad-EPSMs markedly enhanced the cytostatic effect of TGF-β. In contrast, LPCs infected with Ad-3SA were resistant to TGF-β-induced cytostasis, and TGF-β even promoted the proliferation of these cells (Figure 8E). The results of EMT-related analyses revealed that infection of LPCs with Ad-3SA abrogated EMT and cell migration in the presence and absence of TGF-β, whereas infection with Ad-EPSMs enhanced the effects of TGF-β-induced EMT in LPCs (Figure 8F–8H and Supplementary Figure 10). Collectively, these results suggested that overexpression of mutated Smad3 at linker phosphorylation sites intensifies TGF-β-induced cytostasis and EMT, mimicking the effects of MAPK inhibition in LPCs, whereas mutation of Smad3 at the C-terminus caused LPCs to blunt TGF-β-induced cytostasis and EMT, similar to the effects of Smad4 knockdown (Figure 9).

Figure 8. Smad3 phosphorylation affects TGF-β-induced cytostasis and EMT in LPCs. (A) Schematic representation of the phosphorylation sites in Smad3, the corresponding C-terminal region (3SA) and the corresponding linker region (EPSM). (B–D) WB-F344 and LE/6 cells were infected with adenoviruses carrying GFP (Ad-GFP), Smad3 (Ad-Smad3), or Smad3 (Ad-3SA or Ad-EPSM) at the indicated MOIs and were treated with TGF-β (10 ng/ml) for 3 1 h. Cell lysates were subjected to Western blot analyses with antibodies against the indicated phospho- and total Smad3 proteins. GAPDH was used as a loading control. (E) WB-F344 (upper panel) and LE/6 (lower panel) cells were infected with adenoviruses carrying Smad3 or its mutants, treated with TGF-β (10 ng/ml, 3d), and subjected to CCK-8 analyses. OD values were measured and compared between groups as indicated. (F) WB-F344 and LE/6 cells infected with adenoviruses carrying Smad3 or its mutants as indicated were treated with TGF-β (10 ng/ml, 3d) and then subjected to phalloidin staining for F-actin (red). DAPI (blue) was used to stain the cell nuclei. The white arrows indicate F-actin rearrangements. Scale bar, 25 μm. (G) WB-F344 and LE/6 cells infected with adenoviruses carrying Smad3 or its mutants as indicated were treated with TGF-β (10 ng/ml) for 3 days, and Western blot analyses were performed with antibodies against E-cadherin and vimentin. GAPDH was used as a loading control. (H) Cell motility analyses of WB-F344 and LE/6 cells infected with adenovirus carrying Smad3 or its mutants as indicated and control cells treated with TGF-β. The average number of migrated cells per field is shown. The experiments were repeated three times. Two-tailed Student’s t-tests were used for statistical analysis. The data are presented as the mean ± S.E.M. ^*p < 0.05; ^**p < 0.01; and ^***p < 0.001.

Figure 9. Schematic illustration of this study. In LPCs, TGF-β activated Smad and MAPK signaling, and activated MAPK signaling contributed to linker phosphorylation of Smad3 at T179 and S213. TGF-β-activated Smad signaling contributes to the cytostasis and EMT of LPCs, whereas TGF-β-activated MAPK signaling and phosphorylation of Smad3 at T179 and S213 have opposite effects.

Abbreviations

LPCs: liver progenitor cells; ECM: extracellular matrix; EMT: epithelial–mesenchymal transition; TGF-β: transforming growth factor-β; MAPK: mitogen-activated protein kinase; SBE: Smad3 binding elements; GSK3β: glycogen synthetase kinase 3β; CDKs: cyclin-dependent kinases; HBx: HBV antigen X; pSmad3C: C-terminal phosphorylated Smad3; pSmad3L: linker phosphorylated Smad3.

Author Contributions

YS and GJ performed the experiments, analyzed the data, designed the figures, and drafted the manuscript. YW, GL, and GJ were also involved in performing the experiments. HL, BZ, TY, ZL and XC assisted with analyzing and interpreting the data and provided technical support. ZD designed the research, critically revised the manuscript, and obtained funding. All the authors read and approved the final manuscript.

Acknowledgments

We thank Dr. Xiaofeng Hu (HanBio, Shanghai, China) for his technical support in the production of the recombinant adenovirus.

Conflicts of Interest

ZD served as a speaker and consultant for Bayer, Eisai, Roche, MSD, AstraZeneca, Innovent, Hengrui, and BeiGene. The remaining authors have nothing to disclose.

Funding

This work was supported by the National Nature Science Foundation of China (No. 82273441, 81874065 and 81401997); the Knowledge Innovation Program of Wuhan-Shuguang Project (No. 2022020801020456); the Tongji Hospital (HUST) Foundation for Excellent Young Scientists (No. 2020YQ05); the National Basic Research Program of China (2020YFA0710700); and the First Level of the Public Health Youth Top Talent Project of Hubei Province (No. 2022SCZ051).

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