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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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Related in: MedlinePlus

Alcohol-induced liver injury and steatosis were aggravated in Smad7liver-KO mice.(A) Confirmation of Smad7 deletion. Wild type and Smad7liver-KO mice were fed with control or ethanol-containing diet for 6 weeks, followed by a single gavage of 10% ethanol or maltose respectively (8 mice per group). Real time RT-PCR was performed with total RNA isolated from the mice with primers to detect the mRNA region corresponding to exon4 of Smad7 gene. (B) and (C) Measurement of serum ALT and AST. The data are shown as mean ± SD and ** indicates p<0.01 by Student's t-test. (D) Histological analysis of the liver. Representative images of H&E staining are shown for each group of mice. Please note that fatty liver degeneration induced by alcohol administration is enhanced in Smad7liver-KO mice. (E) Oil-Red-O staining of the liver. (F) Triglyceride level of the liver. The data are shown as mean ± SD. * and ** indicates p<0.05 and p<0.01 respectively.
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pone-0017415-g005: Alcohol-induced liver injury and steatosis were aggravated in Smad7liver-KO mice.(A) Confirmation of Smad7 deletion. Wild type and Smad7liver-KO mice were fed with control or ethanol-containing diet for 6 weeks, followed by a single gavage of 10% ethanol or maltose respectively (8 mice per group). Real time RT-PCR was performed with total RNA isolated from the mice with primers to detect the mRNA region corresponding to exon4 of Smad7 gene. (B) and (C) Measurement of serum ALT and AST. The data are shown as mean ± SD and ** indicates p<0.01 by Student's t-test. (D) Histological analysis of the liver. Representative images of H&E staining are shown for each group of mice. Please note that fatty liver degeneration induced by alcohol administration is enhanced in Smad7liver-KO mice. (E) Oil-Red-O staining of the liver. (F) Triglyceride level of the liver. The data are shown as mean ± SD. * and ** indicates p<0.05 and p<0.01 respectively.

Mentions: We further investigated the potential function of Smad7 deletion on alcohol-induced liver damage. Both the wild type and Smad7liver-KO mice at 10 to 12 weeks old were fed with a liquid diet containing 5% ethanol or a control diet for up to 6 weeks. On the day of animal sacrifice, a single dose of gavage with 10% ethanol or isocaloric maltose dextrin was administered. We first analyzed the expression level of mRNA encoded by exon 4 of Smad7 gene to confirm that Smad7 expression was indeed significantly reduced in the Smad7liver-KO mice (Figure 5A). We tracked the alteration of food intake and body weight for the entire 6-week period and found that there were no significant differences among the four groups in food intake (data not shown), except for a slightly reduced body weight gain in Smad7liver-KO mice (Table 1). Alcohol exposure significantly increased the liver/body weight ratio in both wild type and Smad7liver-KO mice (Table 1). Alcohol exposure decreased the levels of serum triglyceride and cholesterol in wild type mice, while Smad7-deleted mice only had a significant reduction of serum triglyceride but not cholesterol after alcohol administration (Table 1). As expected, alcohol exposure could increase serum ALT and AST activities in the mice (Figure 5B and 5C). However, the alcohol-induced raise of these enzymes was more significant in Smad7liver-KO mice than the wild type animals (Figure 5B and 5C).


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)

Alcohol-induced liver injury and steatosis were aggravated in Smad7liver-KO mice.(A) Confirmation of Smad7 deletion. Wild type and Smad7liver-KO mice were fed with control or ethanol-containing diet for 6 weeks, followed by a single gavage of 10% ethanol or maltose respectively (8 mice per group). Real time RT-PCR was performed with total RNA isolated from the mice with primers to detect the mRNA region corresponding to exon4 of Smad7 gene. (B) and (C) Measurement of serum ALT and AST. The data are shown as mean ± SD and ** indicates p<0.01 by Student's t-test. (D) Histological analysis of the liver. Representative images of H&E staining are shown for each group of mice. Please note that fatty liver degeneration induced by alcohol administration is enhanced in Smad7liver-KO mice. (E) Oil-Red-O staining of the liver. (F) Triglyceride level of the liver. The data are shown as mean ± SD. * and ** indicates p<0.05 and p<0.01 respectively.
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Related In: Results  -  Collection

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pone-0017415-g005: Alcohol-induced liver injury and steatosis were aggravated in Smad7liver-KO mice.(A) Confirmation of Smad7 deletion. Wild type and Smad7liver-KO mice were fed with control or ethanol-containing diet for 6 weeks, followed by a single gavage of 10% ethanol or maltose respectively (8 mice per group). Real time RT-PCR was performed with total RNA isolated from the mice with primers to detect the mRNA region corresponding to exon4 of Smad7 gene. (B) and (C) Measurement of serum ALT and AST. The data are shown as mean ± SD and ** indicates p<0.01 by Student's t-test. (D) Histological analysis of the liver. Representative images of H&E staining are shown for each group of mice. Please note that fatty liver degeneration induced by alcohol administration is enhanced in Smad7liver-KO mice. (E) Oil-Red-O staining of the liver. (F) Triglyceride level of the liver. The data are shown as mean ± SD. * and ** indicates p<0.05 and p<0.01 respectively.
Mentions: We further investigated the potential function of Smad7 deletion on alcohol-induced liver damage. Both the wild type and Smad7liver-KO mice at 10 to 12 weeks old were fed with a liquid diet containing 5% ethanol or a control diet for up to 6 weeks. On the day of animal sacrifice, a single dose of gavage with 10% ethanol or isocaloric maltose dextrin was administered. We first analyzed the expression level of mRNA encoded by exon 4 of Smad7 gene to confirm that Smad7 expression was indeed significantly reduced in the Smad7liver-KO mice (Figure 5A). We tracked the alteration of food intake and body weight for the entire 6-week period and found that there were no significant differences among the four groups in food intake (data not shown), except for a slightly reduced body weight gain in Smad7liver-KO mice (Table 1). Alcohol exposure significantly increased the liver/body weight ratio in both wild type and Smad7liver-KO mice (Table 1). Alcohol exposure decreased the levels of serum triglyceride and cholesterol in wild type mice, while Smad7-deleted mice only had a significant reduction of serum triglyceride but not cholesterol after alcohol administration (Table 1). As expected, alcohol exposure could increase serum ALT and AST activities in the mice (Figure 5B and 5C). However, the alcohol-induced raise of these enzymes was more significant in Smad7liver-KO mice than the wild type animals (Figure 5B and 5C).

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