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E3 ubiquitin ligase synoviolin is involved in liver fibrogenesis.

Hasegawa D, Fujii R, Yagishita N, Matsumoto N, Aratani S, Izumi T, Azakami K, Nakazawa M, Fujita H, Sato T, Araya N, Koike J, Tadokoro M, Suzuki N, Nagata K, Senoo H, Friedman SL, Nishioka K, Yamano Y, Itoh F, Nakajima T - PLoS ONE (2010)

Bottom Line: We also analyzed the effect of synoviolin on collagen synthesis in the cell line from HSCs (LX-2) using siRNA-synoviolin and a mutant synoviolin in which E3 ligase activity was abolished.Furthermore, collagen expression in LX-2 cells was upregulated by synoviolin overexpression, while synoviolin knockdown led to reduced collagen expression.Moreover, in Syno(-/-) MEF cells, the amounts of intracellular and secreted mature collagen were significantly decreased, and procollagen was abnormally accumulated in the endoplasmic reticulum.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Science, St Marianna University School of Medicine, Kawasaki, Japan.

ABSTRACT

Background and aim: Chronic hepatic damage leads to liver fibrosis, which is characterized by the accumulation of collagen-rich extracellular matrix. However, the mechanism by which E3 ubiquitin ligase is involved in collagen synthesis in liver fibrosis is incompletely understood. This study aimed to explore the involvement of the E3 ubiquitin ligase synoviolin (Syno) in liver fibrosis.

Methods: The expression and localization of synoviolin in the liver were analyzed in CCl(4)-induced hepatic injury models and human cirrhosis tissues. The degree of liver fibrosis and the number of activated hepatic stellate cells (HSCs) was compared between wild type (wt) and Syno(+/-) mice in the chronic hepatic injury model. We compared the ratio of apoptosis in activated HSCs between wt and Syno(+/-) mice. We also analyzed the effect of synoviolin on collagen synthesis in the cell line from HSCs (LX-2) using siRNA-synoviolin and a mutant synoviolin in which E3 ligase activity was abolished. Furthermore, we compared collagen synthesis between wt and Syno(-/-) mice embryonic fibroblasts (MEF) using quantitative RT-PCR, western blotting, and collagen assay; then, we immunohistochemically analyzed the localization of collagen in Syno(-/-) MEF cells.

Results: In the hepatic injury model as well as in cirrhosis, synoviolin was upregulated in the activated HSCs, while Syno(+/-) mice developed significantly less liver fibrosis than in wt mice. The number of activated HSCs was decreased in Syno(+/-) mice, and some of these cells showed apoptosis. Furthermore, collagen expression in LX-2 cells was upregulated by synoviolin overexpression, while synoviolin knockdown led to reduced collagen expression. Moreover, in Syno(-/-) MEF cells, the amounts of intracellular and secreted mature collagen were significantly decreased, and procollagen was abnormally accumulated in the endoplasmic reticulum.

Conclusion: Our findings demonstrate the importance of the E3 ubiquitin ligase synoviolin in liver fibrosis.

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Related in: MedlinePlus

Involvement of synoviolin in collagen synthesis in the maturation of collagen I.(A) Amount of soluble secreted collagen in culture supernatants of wt and Syno−/− MEFs as quantified by the sircol collagen assay method. The amount of collagen was expressed as a ratio by normalization to the protein concentration in each sample. Data are represented as mean ± SD (n = 3 per group). Unpaired Student's t-test was used to evaluate statistical significance. * P<0.01. Immunoblotting analysis of whole-cell lysates of wt and Syno−/− MEFs by using anti-synoviolin antibody. (B) Effect of synoviolin on COL1A1 mRNA expression in wt and Syno−/− MEFs. The expression level of COL1A1 mRNA was quantified by real-time RT-PCR. The mRNA level was normalized relative to the amount of the transcript of β-actin, a housekeeping gene. Data are represented as mean ± SD (n = 4 per group). Unpaired Student's t-test was used for statistical analysis. n.s.  =  not significant. (C) Effect of synoviolin on the expression of intracellular collagen I. The amount of intracellular collagen I in wt and Syno−/− MEFs was analyzed by immunoblot analysis using anti-collagen I, synoviolin, and β-actin antibodies. The results were derived from 5 independent experiments. (D) The amount of procollagen I and mature collagen I in wt and Syno−/− MEFs semiquantified by NIH Image software. Data are represented as mean ± SEM (n = 5 per group). Unpaired Student's t-test was used for statistical analysis. * P<0.05, ** P<0.001. (E) Double immunofluorescence staining was performed using antibodies against type I collagen (AB765P) and GM130 (a marker of cis-Golgi compartment) or protein disulfide isomerase (PDI; a marker of ER). The results were derived from 3 independent experiments. Scale bar  = 20 µm.
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pone-0013590-g005: Involvement of synoviolin in collagen synthesis in the maturation of collagen I.(A) Amount of soluble secreted collagen in culture supernatants of wt and Syno−/− MEFs as quantified by the sircol collagen assay method. The amount of collagen was expressed as a ratio by normalization to the protein concentration in each sample. Data are represented as mean ± SD (n = 3 per group). Unpaired Student's t-test was used to evaluate statistical significance. * P<0.01. Immunoblotting analysis of whole-cell lysates of wt and Syno−/− MEFs by using anti-synoviolin antibody. (B) Effect of synoviolin on COL1A1 mRNA expression in wt and Syno−/− MEFs. The expression level of COL1A1 mRNA was quantified by real-time RT-PCR. The mRNA level was normalized relative to the amount of the transcript of β-actin, a housekeeping gene. Data are represented as mean ± SD (n = 4 per group). Unpaired Student's t-test was used for statistical analysis. n.s.  =  not significant. (C) Effect of synoviolin on the expression of intracellular collagen I. The amount of intracellular collagen I in wt and Syno−/− MEFs was analyzed by immunoblot analysis using anti-collagen I, synoviolin, and β-actin antibodies. The results were derived from 5 independent experiments. (D) The amount of procollagen I and mature collagen I in wt and Syno−/− MEFs semiquantified by NIH Image software. Data are represented as mean ± SEM (n = 5 per group). Unpaired Student's t-test was used for statistical analysis. * P<0.05, ** P<0.001. (E) Double immunofluorescence staining was performed using antibodies against type I collagen (AB765P) and GM130 (a marker of cis-Golgi compartment) or protein disulfide isomerase (PDI; a marker of ER). The results were derived from 3 independent experiments. Scale bar  = 20 µm.

Mentions: To investigate the underlying mechanisms, we further analyzed the effect of synoviolin in the process of collagen synthesis using primary Syno−/−MEFs (E12.5; Fig. 5). The amount of soluble secreted collagen protein in the supernatants of cultured Syno−/− MEFs was significantly lower than in that of wt MEFs (Fig. 5A), while the mRNA expression levels of the type I collagen gene (COL1A1), that is 80% of all types of collagen in HSCs [30], were equivalent between Syno−/− and wt MEFs (Fig. 5B). We further analyzed the amounts of soluble collagen I protein in the whole cell lysates of Syno−/− MEFs using anti-collagen I antibody (Fig. 5C). Interestingly, the amount of cleaved mature collagen I (70 kDa) was significantly decreased, while the amounts of procollagen I (148 kDa) were slightly decreased (Fig. 5C and D). These results suggest that synoviolin is involved in the maturation of collagen I. Since it is known that the procollagen molecules are modified and folded in the ER where synoviolin is located [17] (Fig. S4) and then the mature collagen is transported to the Golgi apparatus [8], we next analyzed the intracellular localization of collagen I in wt and Syno−/− by immunofluorescence staining (Fig. 5E). In wt MEFs, a major portion of the intracellular collagen I was co-localized with GM130, a marker of cis-Golgi compartment, while a minor portion was co-localized with PDI—a marker of ER—consistent with previous observations [7], [31]–[33] (Fig. 5E). In contrast, in Syno−/− MEFs, the majority of collagen I was co-localized with PDI, suggesting that the lack of synoviolin caused accumulation of collagen I in the ER. Notably, in Syno−/− MEFs, this abnormal transport was not observed for laminin, which is the one of the proteins secreted via the ER-Golgi apparatus pathway, similar to collagen I (Fig. S5).


E3 ubiquitin ligase synoviolin is involved in liver fibrogenesis.

Hasegawa D, Fujii R, Yagishita N, Matsumoto N, Aratani S, Izumi T, Azakami K, Nakazawa M, Fujita H, Sato T, Araya N, Koike J, Tadokoro M, Suzuki N, Nagata K, Senoo H, Friedman SL, Nishioka K, Yamano Y, Itoh F, Nakajima T - PLoS ONE (2010)

Involvement of synoviolin in collagen synthesis in the maturation of collagen I.(A) Amount of soluble secreted collagen in culture supernatants of wt and Syno−/− MEFs as quantified by the sircol collagen assay method. The amount of collagen was expressed as a ratio by normalization to the protein concentration in each sample. Data are represented as mean ± SD (n = 3 per group). Unpaired Student's t-test was used to evaluate statistical significance. * P<0.01. Immunoblotting analysis of whole-cell lysates of wt and Syno−/− MEFs by using anti-synoviolin antibody. (B) Effect of synoviolin on COL1A1 mRNA expression in wt and Syno−/− MEFs. The expression level of COL1A1 mRNA was quantified by real-time RT-PCR. The mRNA level was normalized relative to the amount of the transcript of β-actin, a housekeeping gene. Data are represented as mean ± SD (n = 4 per group). Unpaired Student's t-test was used for statistical analysis. n.s.  =  not significant. (C) Effect of synoviolin on the expression of intracellular collagen I. The amount of intracellular collagen I in wt and Syno−/− MEFs was analyzed by immunoblot analysis using anti-collagen I, synoviolin, and β-actin antibodies. The results were derived from 5 independent experiments. (D) The amount of procollagen I and mature collagen I in wt and Syno−/− MEFs semiquantified by NIH Image software. Data are represented as mean ± SEM (n = 5 per group). Unpaired Student's t-test was used for statistical analysis. * P<0.05, ** P<0.001. (E) Double immunofluorescence staining was performed using antibodies against type I collagen (AB765P) and GM130 (a marker of cis-Golgi compartment) or protein disulfide isomerase (PDI; a marker of ER). The results were derived from 3 independent experiments. Scale bar  = 20 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2963597&req=5

pone-0013590-g005: Involvement of synoviolin in collagen synthesis in the maturation of collagen I.(A) Amount of soluble secreted collagen in culture supernatants of wt and Syno−/− MEFs as quantified by the sircol collagen assay method. The amount of collagen was expressed as a ratio by normalization to the protein concentration in each sample. Data are represented as mean ± SD (n = 3 per group). Unpaired Student's t-test was used to evaluate statistical significance. * P<0.01. Immunoblotting analysis of whole-cell lysates of wt and Syno−/− MEFs by using anti-synoviolin antibody. (B) Effect of synoviolin on COL1A1 mRNA expression in wt and Syno−/− MEFs. The expression level of COL1A1 mRNA was quantified by real-time RT-PCR. The mRNA level was normalized relative to the amount of the transcript of β-actin, a housekeeping gene. Data are represented as mean ± SD (n = 4 per group). Unpaired Student's t-test was used for statistical analysis. n.s.  =  not significant. (C) Effect of synoviolin on the expression of intracellular collagen I. The amount of intracellular collagen I in wt and Syno−/− MEFs was analyzed by immunoblot analysis using anti-collagen I, synoviolin, and β-actin antibodies. The results were derived from 5 independent experiments. (D) The amount of procollagen I and mature collagen I in wt and Syno−/− MEFs semiquantified by NIH Image software. Data are represented as mean ± SEM (n = 5 per group). Unpaired Student's t-test was used for statistical analysis. * P<0.05, ** P<0.001. (E) Double immunofluorescence staining was performed using antibodies against type I collagen (AB765P) and GM130 (a marker of cis-Golgi compartment) or protein disulfide isomerase (PDI; a marker of ER). The results were derived from 3 independent experiments. Scale bar  = 20 µm.
Mentions: To investigate the underlying mechanisms, we further analyzed the effect of synoviolin in the process of collagen synthesis using primary Syno−/−MEFs (E12.5; Fig. 5). The amount of soluble secreted collagen protein in the supernatants of cultured Syno−/− MEFs was significantly lower than in that of wt MEFs (Fig. 5A), while the mRNA expression levels of the type I collagen gene (COL1A1), that is 80% of all types of collagen in HSCs [30], were equivalent between Syno−/− and wt MEFs (Fig. 5B). We further analyzed the amounts of soluble collagen I protein in the whole cell lysates of Syno−/− MEFs using anti-collagen I antibody (Fig. 5C). Interestingly, the amount of cleaved mature collagen I (70 kDa) was significantly decreased, while the amounts of procollagen I (148 kDa) were slightly decreased (Fig. 5C and D). These results suggest that synoviolin is involved in the maturation of collagen I. Since it is known that the procollagen molecules are modified and folded in the ER where synoviolin is located [17] (Fig. S4) and then the mature collagen is transported to the Golgi apparatus [8], we next analyzed the intracellular localization of collagen I in wt and Syno−/− by immunofluorescence staining (Fig. 5E). In wt MEFs, a major portion of the intracellular collagen I was co-localized with GM130, a marker of cis-Golgi compartment, while a minor portion was co-localized with PDI—a marker of ER—consistent with previous observations [7], [31]–[33] (Fig. 5E). In contrast, in Syno−/− MEFs, the majority of collagen I was co-localized with PDI, suggesting that the lack of synoviolin caused accumulation of collagen I in the ER. Notably, in Syno−/− MEFs, this abnormal transport was not observed for laminin, which is the one of the proteins secreted via the ER-Golgi apparatus pathway, similar to collagen I (Fig. S5).

Bottom Line: We also analyzed the effect of synoviolin on collagen synthesis in the cell line from HSCs (LX-2) using siRNA-synoviolin and a mutant synoviolin in which E3 ligase activity was abolished.Furthermore, collagen expression in LX-2 cells was upregulated by synoviolin overexpression, while synoviolin knockdown led to reduced collagen expression.Moreover, in Syno(-/-) MEF cells, the amounts of intracellular and secreted mature collagen were significantly decreased, and procollagen was abnormally accumulated in the endoplasmic reticulum.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Science, St Marianna University School of Medicine, Kawasaki, Japan.

ABSTRACT

Background and aim: Chronic hepatic damage leads to liver fibrosis, which is characterized by the accumulation of collagen-rich extracellular matrix. However, the mechanism by which E3 ubiquitin ligase is involved in collagen synthesis in liver fibrosis is incompletely understood. This study aimed to explore the involvement of the E3 ubiquitin ligase synoviolin (Syno) in liver fibrosis.

Methods: The expression and localization of synoviolin in the liver were analyzed in CCl(4)-induced hepatic injury models and human cirrhosis tissues. The degree of liver fibrosis and the number of activated hepatic stellate cells (HSCs) was compared between wild type (wt) and Syno(+/-) mice in the chronic hepatic injury model. We compared the ratio of apoptosis in activated HSCs between wt and Syno(+/-) mice. We also analyzed the effect of synoviolin on collagen synthesis in the cell line from HSCs (LX-2) using siRNA-synoviolin and a mutant synoviolin in which E3 ligase activity was abolished. Furthermore, we compared collagen synthesis between wt and Syno(-/-) mice embryonic fibroblasts (MEF) using quantitative RT-PCR, western blotting, and collagen assay; then, we immunohistochemically analyzed the localization of collagen in Syno(-/-) MEF cells.

Results: In the hepatic injury model as well as in cirrhosis, synoviolin was upregulated in the activated HSCs, while Syno(+/-) mice developed significantly less liver fibrosis than in wt mice. The number of activated HSCs was decreased in Syno(+/-) mice, and some of these cells showed apoptosis. Furthermore, collagen expression in LX-2 cells was upregulated by synoviolin overexpression, while synoviolin knockdown led to reduced collagen expression. Moreover, in Syno(-/-) MEF cells, the amounts of intracellular and secreted mature collagen were significantly decreased, and procollagen was abnormally accumulated in the endoplasmic reticulum.

Conclusion: Our findings demonstrate the importance of the E3 ubiquitin ligase synoviolin in liver fibrosis.

Show MeSH
Related in: MedlinePlus