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Hepatic deletion of Smad7 in mouse leads to spontaneous liver dysfunction and aggravates alcoholic liver injury.

Zhu L, Wang L, Wang X, Luo X, Yang L, Zhang R, Yin H, Xie D, Pan Y, Chen Y - PLoS ONE (2011)

Bottom Line: It is currently unclear whether endogenous Smad7 has an effect on liver function and alcoholic liver damage.Furthermore, alcohol-induced ADH1 expression was significantly abrogated by Smad7 deletion in hepatocytes.In this study, we provided in vivo evidence revealing that endogenous Smad7 plays an important role in liver function and alcohol-induced liver injury.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Graduate School of the Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, China.

ABSTRACT

Background: TGF-β has been known to play an important role in various liver diseases including fibrosis and alcohol-induced fatty liver. Smad7 is an intracellular negative regulator of TGF-β signaling. It is currently unclear whether endogenous Smad7 has an effect on liver function and alcoholic liver damage.

Methodology/principal findings: We used Cre/loxP system by crossing Alb-Cre mice with Smad7(loxP/loxP) mice to generate liver-specific deletion of Smad7 with loss of the indispensable MH2 domain. Alcoholic liver injury was achieved by feeding mice with a liquid diet containing 5% ethanol for 6 weeks, followed by a single dose of ethanol gavage. Deletion of Smad7 in the liver was associated with increased Smad2/3 phosphorylation in the liver or upon TGF-β treatment in primary hepatocytes. The majority of mice with liver specific deletion of Smad7 (Smad7(liver-KO)) were viable and phenotypically normal, accompanied by only slight or no reduction of Smad7 expression in the liver. However, about 30% of Smad7(liver-KO) mice with high efficiency of Smad7 deletion had spontaneous liver dysfunction, demonstrated as low body weight, overall deterioration, and increased serum levels of AST and ALT. Degeneration and elevated apoptosis of liver cells were observed with these mice. TGF-β-induced epithelial to mesenchymal transition (EMT) was accelerated in Smad7-deleted primary hepatocytes. In addition, alcohol-induced liver injury and steatosis were profoundly aggravated in Smad7 deficient mice, associated with upregulation of critical genes involved in lipogenesis and inflammation. Furthermore, alcohol-induced ADH1 expression was significantly abrogated by Smad7 deletion in hepatocytes.

Conclusion/significance: In this study, we provided in vivo evidence revealing that endogenous Smad7 plays an important role in liver function and alcohol-induced liver injury.

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Deletion of Smad7 enhances TGF-β-induced EMT.(A) Confirmation of Smad7 deletion in primary hepatocytes isolated from Smad7liver-KO mouse. Real time RT-PCR was performed with total RNA isolated from wild type or Smad7liver-KO mice with primers to detect the mRNA region corresponding to exon4 of Smad7 gene. The data are shown as mean ± SD and ** indicates p<0.01 as comparison between the groups as indicated by Student's t-test. (B) TGF-β-induced EMT-like morphology changes. Immunofluorescence labeling were performed with wild type or Smad7liver-KO hepatocytes treated with or without TGF-β1 (5 ng/ml) for 48 h. F-actin was stained with fluorescein isothiocyanate-labeled phalloidin (Red) and the nuclei were labeled by Hoechst 33342 (Blue). (C) Analysis of cell motility by a wound-healing assay. Cultured primary hepatocytes were analyzed by phase contrast microscopy. The cells were treated with or without TGF-β1 (5 ng/ml) for 48 h. Quantitation of the cell motility is shown in lower right panel as mean ± SD and ** indicates p<0.01 by Student's t-test. (D) Analysis of EMT markers E-cadherin and vimentin. Primary hepatocytes were treated with or without TGF-β1 (5 ng/ml) for 48 h and the cell lysate was used in immunoblotting with the antibodies as indicated.
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pone-0017415-g004: Deletion of Smad7 enhances TGF-β-induced EMT.(A) Confirmation of Smad7 deletion in primary hepatocytes isolated from Smad7liver-KO mouse. Real time RT-PCR was performed with total RNA isolated from wild type or Smad7liver-KO mice with primers to detect the mRNA region corresponding to exon4 of Smad7 gene. The data are shown as mean ± SD and ** indicates p<0.01 as comparison between the groups as indicated by Student's t-test. (B) TGF-β-induced EMT-like morphology changes. Immunofluorescence labeling were performed with wild type or Smad7liver-KO hepatocytes treated with or without TGF-β1 (5 ng/ml) for 48 h. F-actin was stained with fluorescein isothiocyanate-labeled phalloidin (Red) and the nuclei were labeled by Hoechst 33342 (Blue). (C) Analysis of cell motility by a wound-healing assay. Cultured primary hepatocytes were analyzed by phase contrast microscopy. The cells were treated with or without TGF-β1 (5 ng/ml) for 48 h. Quantitation of the cell motility is shown in lower right panel as mean ± SD and ** indicates p<0.01 by Student's t-test. (D) Analysis of EMT markers E-cadherin and vimentin. Primary hepatocytes were treated with or without TGF-β1 (5 ng/ml) for 48 h and the cell lysate was used in immunoblotting with the antibodies as indicated.

Mentions: We next analyzed the cellular function of Smad7 on epithelial to mesenchymal transition (EMT) in hepatocytes as TGF-β plays a pivotal role in EMT [22]. We isolated primary hepatocytes from wild type and Smad7liver-KO mice. The cultured hepatocytes isolated from Smad7liver-KO mice did have significant reduction of the mRNA encoded by Smad7 exon 4 (Figure 4A), confirming that Smad7 was successfully deleted in these cells. When the cultured primary hepatocytes were treated with TGF-β1, the cells underwent morphological changes characteristic of EMT (Figure 4B). Untreated hepatocytes exhibited a cuboidal phenotype, while TGF-β1 treatment induced a fibroblastic transition resulting in elongated and spindle-like cell morphology. Interestingly, the TGF-β1-induced EMT morphology was robustly enhanced by Smad7 deletion (Figure 4B, right panel). We also analyzed the cell motility using standard scratch-wound assays as previously described [23]. At 48 h after wounding, the untreated cells from both wild type and Smad7-deleted mice were unable to migrate into the wound area (Figure 4C). TGF-β1 treatment was able to induce migration of the cells and such effect was significantly accelerated when Smad7 was deleted (Figure 4C). TGF-β-induced EMT was further analyzed by immunoblotting to detect expression of E-cadherin and vimentin, two well-recognized markers for EMT [22]. We found that TGF-β1-induced reduction of E-cadherin and increase of vimentin was profoundly enhanced by Smad7 deletion (Figure 4D). These data, therefore, reveal that Smad7 deficiency is able to enhance TGF-β-induced EMT in hepatocytes.


Hepatic deletion of Smad7 in mouse leads to spontaneous liver dysfunction and aggravates alcoholic liver injury.

Zhu L, Wang L, Wang X, Luo X, Yang L, Zhang R, Yin H, Xie D, Pan Y, Chen Y - PLoS ONE (2011)

Deletion of Smad7 enhances TGF-β-induced EMT.(A) Confirmation of Smad7 deletion in primary hepatocytes isolated from Smad7liver-KO mouse. Real time RT-PCR was performed with total RNA isolated from wild type or Smad7liver-KO mice with primers to detect the mRNA region corresponding to exon4 of Smad7 gene. The data are shown as mean ± SD and ** indicates p<0.01 as comparison between the groups as indicated by Student's t-test. (B) TGF-β-induced EMT-like morphology changes. Immunofluorescence labeling were performed with wild type or Smad7liver-KO hepatocytes treated with or without TGF-β1 (5 ng/ml) for 48 h. F-actin was stained with fluorescein isothiocyanate-labeled phalloidin (Red) and the nuclei were labeled by Hoechst 33342 (Blue). (C) Analysis of cell motility by a wound-healing assay. Cultured primary hepatocytes were analyzed by phase contrast microscopy. The cells were treated with or without TGF-β1 (5 ng/ml) for 48 h. Quantitation of the cell motility is shown in lower right panel as mean ± SD and ** indicates p<0.01 by Student's t-test. (D) Analysis of EMT markers E-cadherin and vimentin. Primary hepatocytes were treated with or without TGF-β1 (5 ng/ml) for 48 h and the cell lysate was used in immunoblotting with the antibodies as indicated.
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pone-0017415-g004: Deletion of Smad7 enhances TGF-β-induced EMT.(A) Confirmation of Smad7 deletion in primary hepatocytes isolated from Smad7liver-KO mouse. Real time RT-PCR was performed with total RNA isolated from wild type or Smad7liver-KO mice with primers to detect the mRNA region corresponding to exon4 of Smad7 gene. The data are shown as mean ± SD and ** indicates p<0.01 as comparison between the groups as indicated by Student's t-test. (B) TGF-β-induced EMT-like morphology changes. Immunofluorescence labeling were performed with wild type or Smad7liver-KO hepatocytes treated with or without TGF-β1 (5 ng/ml) for 48 h. F-actin was stained with fluorescein isothiocyanate-labeled phalloidin (Red) and the nuclei were labeled by Hoechst 33342 (Blue). (C) Analysis of cell motility by a wound-healing assay. Cultured primary hepatocytes were analyzed by phase contrast microscopy. The cells were treated with or without TGF-β1 (5 ng/ml) for 48 h. Quantitation of the cell motility is shown in lower right panel as mean ± SD and ** indicates p<0.01 by Student's t-test. (D) Analysis of EMT markers E-cadherin and vimentin. Primary hepatocytes were treated with or without TGF-β1 (5 ng/ml) for 48 h and the cell lysate was used in immunoblotting with the antibodies as indicated.
Mentions: We next analyzed the cellular function of Smad7 on epithelial to mesenchymal transition (EMT) in hepatocytes as TGF-β plays a pivotal role in EMT [22]. We isolated primary hepatocytes from wild type and Smad7liver-KO mice. The cultured hepatocytes isolated from Smad7liver-KO mice did have significant reduction of the mRNA encoded by Smad7 exon 4 (Figure 4A), confirming that Smad7 was successfully deleted in these cells. When the cultured primary hepatocytes were treated with TGF-β1, the cells underwent morphological changes characteristic of EMT (Figure 4B). Untreated hepatocytes exhibited a cuboidal phenotype, while TGF-β1 treatment induced a fibroblastic transition resulting in elongated and spindle-like cell morphology. Interestingly, the TGF-β1-induced EMT morphology was robustly enhanced by Smad7 deletion (Figure 4B, right panel). We also analyzed the cell motility using standard scratch-wound assays as previously described [23]. At 48 h after wounding, the untreated cells from both wild type and Smad7-deleted mice were unable to migrate into the wound area (Figure 4C). TGF-β1 treatment was able to induce migration of the cells and such effect was significantly accelerated when Smad7 was deleted (Figure 4C). TGF-β-induced EMT was further analyzed by immunoblotting to detect expression of E-cadherin and vimentin, two well-recognized markers for EMT [22]. We found that TGF-β1-induced reduction of E-cadherin and increase of vimentin was profoundly enhanced by Smad7 deletion (Figure 4D). These data, therefore, reveal that Smad7 deficiency is able to enhance TGF-β-induced EMT in hepatocytes.

Bottom Line: It is currently unclear whether endogenous Smad7 has an effect on liver function and alcoholic liver damage.Furthermore, alcohol-induced ADH1 expression was significantly abrogated by Smad7 deletion in hepatocytes.In this study, we provided in vivo evidence revealing that endogenous Smad7 plays an important role in liver function and alcohol-induced liver injury.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Graduate School of the Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, China.

ABSTRACT

Background: TGF-β has been known to play an important role in various liver diseases including fibrosis and alcohol-induced fatty liver. Smad7 is an intracellular negative regulator of TGF-β signaling. It is currently unclear whether endogenous Smad7 has an effect on liver function and alcoholic liver damage.

Methodology/principal findings: We used Cre/loxP system by crossing Alb-Cre mice with Smad7(loxP/loxP) mice to generate liver-specific deletion of Smad7 with loss of the indispensable MH2 domain. Alcoholic liver injury was achieved by feeding mice with a liquid diet containing 5% ethanol for 6 weeks, followed by a single dose of ethanol gavage. Deletion of Smad7 in the liver was associated with increased Smad2/3 phosphorylation in the liver or upon TGF-β treatment in primary hepatocytes. The majority of mice with liver specific deletion of Smad7 (Smad7(liver-KO)) were viable and phenotypically normal, accompanied by only slight or no reduction of Smad7 expression in the liver. However, about 30% of Smad7(liver-KO) mice with high efficiency of Smad7 deletion had spontaneous liver dysfunction, demonstrated as low body weight, overall deterioration, and increased serum levels of AST and ALT. Degeneration and elevated apoptosis of liver cells were observed with these mice. TGF-β-induced epithelial to mesenchymal transition (EMT) was accelerated in Smad7-deleted primary hepatocytes. In addition, alcohol-induced liver injury and steatosis were profoundly aggravated in Smad7 deficient mice, associated with upregulation of critical genes involved in lipogenesis and inflammation. Furthermore, alcohol-induced ADH1 expression was significantly abrogated by Smad7 deletion in hepatocytes.

Conclusion/significance: In this study, we provided in vivo evidence revealing that endogenous Smad7 plays an important role in liver function and alcohol-induced liver injury.

Show MeSH
Related in: MedlinePlus