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, 9 (1), 10673

A Critical Role of Autophagy in Regulating the Mesenchymal Transition of Ductular Cells in Liver Cirrhosis

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A Critical Role of Autophagy in Regulating the Mesenchymal Transition of Ductular Cells in Liver Cirrhosis

Tzu-Min Hung et al. Sci Rep.

Abstract

Our previous studies have shown that autophagy mediates the link between ductular reaction (DR) and liver cirrhosis. Whether the subsequent fibrogenic response is regulated by increased autophagy in DR remains unclear. Here, using both human liver specimens and a rat model of liver cirrhosis induced by 2-acetylaminofluorene (AAF) and carbon tetrachloride (CCL4), we explored the involvement of autophagy in regulating mesenchymal transition of ductular cells. Ductular cells from AAF/CCL4 livers exhibited increased autophagy compared to those of normal livers. These cells showed morphological and functional characteristics of mesenchymal cells. Blocking autophagy using bafilomycin A1 or siRNA targeting ATG7 reduced the expression of mesenchymal markers in these ductular cells from AAF/CCL4 livers, indicating a role for autophagy in regulating the mesenchymal phenotype of ductular cells. Furthermore, we show that the mesenchymal transition in DR requires the activation of transforming growth factor-β (TGF-β) signaling in an autophagy-dependent manner. Importantly, in cirrhotic human livers, ductular cells that are positive for LC3B also showed increased expression of TGF-β and fibroblast-specific protein-1. Our data suggest activation of autophagy in ductular cells, and also demonstrate that it is required for the mesenchymal transition during the DR, processes that are critically involved in the pathogenesis of cirrhosis.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Immunohistochemical detection of autophagy markers in liver tissues from patients with cirrhosis. (A,B) Paraffin-embedded three consecutive liver sections were stained with antibodies against LC3B, p62 or normal IgG. Ductular reaction in cirrhotic liver (B) showed strong expression of LC3B but were negative for p62, whereas hepatocytes in cirrhotic liver were positive for both LC3B and p62. Rabbit normal IgG was used as a negative control counterstain. Asterisks indicate the ductular reaction in cirrhotic livers. Scale bar, 50 μm. (C) Higher magnifications of the ductular reaction in corresponding cirrhotic liver sections are enlarged micrographs from the respective boxed area. The dotted lines indicated the same region among three consecutive sections. Scale bar, 20 μm.
Figure 2
Figure 2
Characterization of ductular cells from AAF/CCL4-induced cirrhosis rat model. (A,B) Flow cytometry analysis of freshly isolated ductular cells from normal (A) and AAF/CCL4 (B) livers. The y-axis indicates EpCAM expression (PE fluorescence) and the x-axis represents side scatter (SSC). The positive and negative fractions of each liver were collected as described in the Materials and Methods. Percentage of cells expressing EpCAM is shown. (C) Identity of EpCAM-expressing cells in the positive fraction was further confirmed by staining for SOX9 and CK19.
Figure 3
Figure 3
Autophagy is increased in ductular cells from AAF/CCL4 livers. (A) Immunoblots and (B) quantification depicting expression of ATG7, ATG5, LC3B-II and p62 in ductular cells from normal and AAF/CCL4 livers. β-actin served as a loading control. Full-length blots are presented in Supplementary Fig. S4. (C) The presence of LC3B punctae was analyzed using immunofluorescence. (D) Transmission electron microscopy illustrating a larger number and size of autophagic vacuoles in ductular cells from AAF/CCL4 livers than in those from normal livers. Arrows indicate autophagic vacuoles. (Right) Quantification data are expressed in relative surface of autophagic vacuole to cytoplasm. (E) Quantitative PCR analysis of mRNA for LC3B and p62 in ductular cells from normal and AAF/CCL4 livers. Data were normalized by the amount of β-actin mRNA, expressed relative to the corresponding value for normal cells. (F,G) Autophagic flux assay of ductular cells from normal and AAF/CCL4 livers. Immunoblots (F) and immunofluorescence images (G) depicting expression of LC3B and p62 in ductular cell. Ductular cells from AAF/CCL4 liver were treated with bafilomycin A1 (Baf, 100 nM) for 3 hours further increased the levels of both LC3B and p62, indicating increased autophagy. Full-length blots are presented in Supplementary Fig. S5. The graph underneath the blot shows the relative intensities of the LC3B-II and p62 bands normalized to β-actin from three independent experiments (mean ± SD). Quantification of immunofluorescence images was performed using Zen-Pro software (Zeiss) and normalized to the number of nuclei. Statistical analysis was analyzed by Student’s unpaired t-test. *P < 0.05 compared with normal group; #P < 0.05 compared with AAF/CCL4 groups.
Figure 4
Figure 4
Ductular cells from AAF/CCL4 livers undergo mesenchymal transition. (A) Phase-contrast micrographs of ductular cells isolated from normal and AAF/CCL4 livers on day 2. Normal cells form colonies with epithelial morphology. AAF/CCL4 cells have a spindle-like morphology. (B) Ultrastructural analysis of ductular cells from AAF/CCL4 livers revealing accumulation of microfilaments (red arrows) and vesicles (asterisks) in the cytoplasm. The magnified image is of the area indicated by the square. (C) Immunoblot depicting expression of mesenchymal markers in ductular cells from normal and AAF/CCL4 livers. Full-length blots are presented in Supplementary Fig. S6. Bar graph shows the relative intensities of the type I collagen, vimentin and α-SMA bands normalized to β-actin from three independent experiments (mean ± SD) (D) Transwell migration assay of ductular cells isolated from normal and AAF/CCL4 livers. A statistical analysis of relative migration rate was shown on the right. Statistical analysis was analyzed by Student’s unpaired t-test. *P < 0.05 compared with normal group.
Figure 5
Figure 5
Effect of autophagy on the mesenchymal phenotype of ductular cells from AAF/CCL4 livers. (A,B) Immunofluorescence images of isolated ductular cells demonstrating LC3B was associated with the mesenchymal markers vimentin (A) and α-SMA (B). (Right) Quantification of images was performed using Zen-Pro software (Zeiss) and normalized to the number of nuclei. (C) Ductular cells were cultured for 3 days and then treated with or without bafilomycin A1 (Baf, 100 nM) for another 24 hours. Representative Western blot analysis confirmed the increased expression of mesenchymal markers in AAF/CCL4 cells compared to normal cells. Treatment of AAF/CCL4 cells with Baf suppressed the expression of mesenchymal markers. Full-length blots are presented in Supplementary Fig. S7. Quantitative analysis of mesenchymal markers (left panel), and autophagic markers (right panel) was shown below. (D) Inhibition of autophagy by ATG7 small interfering RNA (siATG7) reduced the protein levels of mesenchymal markers. Full-length blots are presented in Supplementary Fig. S8. Quantitative analysis of mesenchymal markers (left panel), and autophagic markers (right panel) was shown below. Statistical analysis was analyzed by Student’s unpaired t-test. *P < 0.05 compared with normal group; s#P < 0.05 compared with AAF/CCL4 groups.
Figure 6
Figure 6
Autophagy-induced TGF-β/Smad2/3 signaling drives mesenchymal transition of ductular cells from AAF/CCL4 livers. (A) Immunofluorescence images of Smad2/3 in ductular cells from normal and AAF/CCL4 livers. Nuclear localization was confirmed with DAPI staining. The percentage of Smad2/3 localized in the nucleus was determined by counting 50 immunofluorescence‐positive cells for each sample. (B) Cell culture supernatant of ductular cells transfected with control (Ctrl) or siRNA against ATG7 (siATG7) was subjected to ELISA detection for TGF-β1 levels. Data are expressed as mean ± SD from three independent experiments. (C) Representative Western blot analysis of phosphorylated (p)- and total Smad2/3 in ductular cells transfected with control or siATG7. The graph below shows the relative intensities of the p-Smad2/3 bands normalized to those for total Smad2/3 from three independent experiments (mean ± SD, n = 3). Full-length blots are presented in Supplementary Fig. S9. Statistical analysis was analyzed by Student’s unpaired t-test. *P < 0.05 compared with normal group; #P < 0.05 compared with AAF/CCL4 groups; +P < 0.05 compared with AAF/CCL4 with siATG7 group. (D) Immunoblots and quantification depicting expression of type I collagen, vimentin, α-SMA and ATG7 in ductular cells, which transfected with control or siATG7 for 72 hr, and subsequently treated with or without 20 ng/ml TGF-β1 for another 24 hr. Relative intensity (RI) shown was calculated by normalization of the intensities of each marker to the β-actin and to the value of normal cell. Full-length blots are presented in Supplementary Fig. S10.
Figure 7
Figure 7
Co-expression of autophagy and mesenchymal markers in the ductular reaction of human cirrhotic livers. A representative series of immunofluorescence results from cirrhotic liver tissues showing expression of LC3B, TGF‐β, FSP-1 and CK19. (A) Immunostaining for LC3B and TGF‐β. Merged images show co-expression of LC3B (green) and TGF‐β (red) in ductule structures. (B) Panels I and II are consecutive sections from the same cirrhotic tissues. The images in panel I show co-expression of the mesenchymal marker FSP-1 (green) and CK19 (red), and those in panel II show co-expression of LC3B (green) and CK19 (red). The insets in panel I show ductular cells with low expression of CK19 (red) and high expression of FSP-1 (green) that appear to be acquiring a fibroblast morphology. Arrows indicate LC3B-positive ductular cells potentially undergoing the mesenchymal transition during cirrhosis. (C) Consecutive sections are from non‐cirrhotic tissue with a bile duct showing regular morphology. The bile duct expressed LC3B (green, panel I) and CK19 (red, panel III) but not TGF‐β (panel II) or FSP-1 (panel IV).

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References

    1. Gouw ASH, Clouston AD, Theise ND. Ductular reactions in human liver: diversity at the interface. Hepatology. 2011;54:1853–1863. doi: 10.1002/hep.24613. - DOI - PubMed
    1. Williams MJ, Clouston AD, Forbes SJ. Links between hepatic fibrosis, ductular reaction, and progenitor cell expansion. Gastroenterology. 2014;146:349–356. doi: 10.1053/j.gastro.2013.11.034. - DOI - PubMed
    1. Clouston AD, et al. Fibrosis correlates with a ductular reaction in hepatitis C: Roles of impaired replication, progenitor cells and steatosis. Hepatology. 2005;41:809–818. doi: 10.1002/hep.20650. - DOI - PubMed
    1. Richardson MM, et al. Progressive fibrosis in nonalcoholic steatohepatitis: association with altered regeneration and a ductular reaction. Gastroenterology. 2007;133:80–90. doi: 10.1053/j.gastro.2007.05.012. - DOI - PubMed
    1. Wood MJ, Gadd VL, Powell LW, Ramm GA, Clouston AD. Ductular reaction in hereditary hemochromatosis: the link between hepatocyte senescence and fibrosis progression. Hepatology. 2014;59:848–857. doi: 10.1002/hep.26706. - DOI - PubMed
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