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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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Characterization of Smad7liver-KO mice.(A) Liver-specific deletion of Smad7. RT-PCR analysis was performed with total RNA isolated from multiple tissues in Alb-Cre heterozygous or wild type mice (all mice having Smad7loxP/loxP) with specific primers that amplify mRNA regions corresponding to exons 1–3 or exons 3–4 of Smad7 respectively. (B) Analysis of the Smad7 by immunohistochemistry staining. Representative liver sections (400 X) from wild type and Smad7liver-KO mice were used in immunohistochemistry staining with an anti-Smad7 antibody. The nuclei were stained with haematoxylin. (C) Smad2 phosphorylation is elevated in the liver of Smad7liver-KO mice. Smad2 phosphorylation was analyzed by immunohistochemistry using liver sections from either wild type or Smad7liver-KO mice. The nuclei were stained with hematoxylin. Note that nuclear phospho-Smad2 staining is increased in the liver of Smad7liver-KO mice. (D) TGF-β-induced Smad2 and Smad3 phosphorylation is enhanced by Smad7 deletion in primary hepatocytes. Immunoblotting was performed using total protein lysate extracted from primary hepatocytes using antibodies as indicated. The cells were treated with or without 5 ng/ml of TGF-β1 for 24 hours as indicated after overnight serum starvation.
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pone-0017415-g001: Characterization of Smad7liver-KO mice.(A) Liver-specific deletion of Smad7. RT-PCR analysis was performed with total RNA isolated from multiple tissues in Alb-Cre heterozygous or wild type mice (all mice having Smad7loxP/loxP) with specific primers that amplify mRNA regions corresponding to exons 1–3 or exons 3–4 of Smad7 respectively. (B) Analysis of the Smad7 by immunohistochemistry staining. Representative liver sections (400 X) from wild type and Smad7liver-KO mice were used in immunohistochemistry staining with an anti-Smad7 antibody. The nuclei were stained with haematoxylin. (C) Smad2 phosphorylation is elevated in the liver of Smad7liver-KO mice. Smad2 phosphorylation was analyzed by immunohistochemistry using liver sections from either wild type or Smad7liver-KO mice. The nuclei were stained with hematoxylin. Note that nuclear phospho-Smad2 staining is increased in the liver of Smad7liver-KO mice. (D) TGF-β-induced Smad2 and Smad3 phosphorylation is enhanced by Smad7 deletion in primary hepatocytes. Immunoblotting was performed using total protein lysate extracted from primary hepatocytes using antibodies as indicated. The cells were treated with or without 5 ng/ml of TGF-β1 for 24 hours as indicated after overnight serum starvation.

Mentions: To investigate the potential function of Smad7 in the liver, we crossed Albumin-Cre transgenic mice with Smad7loxP/loxP mice that contain two loxP fragments flanking the 5′ half of exon 4 of Smad7 gene [17]. The Albumin-Cre transgenic mice specifically express Cre recombinase in hepatocytes under control of a rat albumin promoter/enhancer. Specific deletion of the MH2 domain (encoded by the 5′ half of exon 4) of Smad7 in the mouse liver was confirmed by RT-PCR (Figure 1A). As expected, we found that the mRNA region corresponding to exon 1–3 was not deleted in the liver-specific Smad7-deleted mouse (Smad7liver-KO mouse). However, only the mRNA region corresponding to exon 3–4 was lost in the liver of Smad7liver-KO mouse, but not changed in the other tissues such as brain, lung, heart, and kidney, indicating liver-specific deletion of Smad7 MH2 domain. To verify Smad7 deletion, the protein level of Smad7 was also examined by immunohistochemistry staining in the liver sections. The result revealed that the protein level of Smad7 was markedly decreased in the liver of Smad7liver-KO mouse in comparison with wide type animals (Figure 1B).


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)

Characterization of Smad7liver-KO mice.(A) Liver-specific deletion of Smad7. RT-PCR analysis was performed with total RNA isolated from multiple tissues in Alb-Cre heterozygous or wild type mice (all mice having Smad7loxP/loxP) with specific primers that amplify mRNA regions corresponding to exons 1–3 or exons 3–4 of Smad7 respectively. (B) Analysis of the Smad7 by immunohistochemistry staining. Representative liver sections (400 X) from wild type and Smad7liver-KO mice were used in immunohistochemistry staining with an anti-Smad7 antibody. The nuclei were stained with haematoxylin. (C) Smad2 phosphorylation is elevated in the liver of Smad7liver-KO mice. Smad2 phosphorylation was analyzed by immunohistochemistry using liver sections from either wild type or Smad7liver-KO mice. The nuclei were stained with hematoxylin. Note that nuclear phospho-Smad2 staining is increased in the liver of Smad7liver-KO mice. (D) TGF-β-induced Smad2 and Smad3 phosphorylation is enhanced by Smad7 deletion in primary hepatocytes. Immunoblotting was performed using total protein lysate extracted from primary hepatocytes using antibodies as indicated. The cells were treated with or without 5 ng/ml of TGF-β1 for 24 hours as indicated after overnight serum starvation.
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Related In: Results  -  Collection

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pone-0017415-g001: Characterization of Smad7liver-KO mice.(A) Liver-specific deletion of Smad7. RT-PCR analysis was performed with total RNA isolated from multiple tissues in Alb-Cre heterozygous or wild type mice (all mice having Smad7loxP/loxP) with specific primers that amplify mRNA regions corresponding to exons 1–3 or exons 3–4 of Smad7 respectively. (B) Analysis of the Smad7 by immunohistochemistry staining. Representative liver sections (400 X) from wild type and Smad7liver-KO mice were used in immunohistochemistry staining with an anti-Smad7 antibody. The nuclei were stained with haematoxylin. (C) Smad2 phosphorylation is elevated in the liver of Smad7liver-KO mice. Smad2 phosphorylation was analyzed by immunohistochemistry using liver sections from either wild type or Smad7liver-KO mice. The nuclei were stained with hematoxylin. Note that nuclear phospho-Smad2 staining is increased in the liver of Smad7liver-KO mice. (D) TGF-β-induced Smad2 and Smad3 phosphorylation is enhanced by Smad7 deletion in primary hepatocytes. Immunoblotting was performed using total protein lysate extracted from primary hepatocytes using antibodies as indicated. The cells were treated with or without 5 ng/ml of TGF-β1 for 24 hours as indicated after overnight serum starvation.
Mentions: To investigate the potential function of Smad7 in the liver, we crossed Albumin-Cre transgenic mice with Smad7loxP/loxP mice that contain two loxP fragments flanking the 5′ half of exon 4 of Smad7 gene [17]. The Albumin-Cre transgenic mice specifically express Cre recombinase in hepatocytes under control of a rat albumin promoter/enhancer. Specific deletion of the MH2 domain (encoded by the 5′ half of exon 4) of Smad7 in the mouse liver was confirmed by RT-PCR (Figure 1A). As expected, we found that the mRNA region corresponding to exon 1–3 was not deleted in the liver-specific Smad7-deleted mouse (Smad7liver-KO mouse). However, only the mRNA region corresponding to exon 3–4 was lost in the liver of Smad7liver-KO mouse, but not changed in the other tissues such as brain, lung, heart, and kidney, indicating liver-specific deletion of Smad7 MH2 domain. To verify Smad7 deletion, the protein level of Smad7 was also examined by immunohistochemistry staining in the liver sections. The result revealed that the protein level of Smad7 was markedly decreased in the liver of Smad7liver-KO mouse in comparison with wide type animals (Figure 1B).

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