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Yang J, Gong Y, Xu W, Li L, Shi Z, Wang Q, He Y, Zhang C, Luo C, Fang Z, Yang Y. Smad3 gene C-terminal phosphorylation site mutation exacerbates CCl 4-induced hepatic fibrogenesis by promoting pSmad2L/C-mediated signaling transduction. Naunyn Schmiedebergs Arch Pharmacol 2021; 394:1779-1786. [PMID: 34191114 DOI: 10.1007/s00210-021-02114-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2021] [Accepted: 06/15/2021] [Indexed: 11/26/2022]
Abstract
Current researches have confirmed that Smads, mediators of TGF-β signaling, are strictly controlled by domain-specific site phosphorylation in the process of hepatic disease. Usually, Smad3 phospho-isoform pSmad3L and pSmad3C are reversible and antagonistic; pSmad2L/C could act together with pSmad3L by stimulating PAI-1 expression and ECM synthesis to transmit fibrogenic signals. Our recent study found that pSmad3C mutation is supposed to perform a vigorous role on the early phase of liver injury and abates salvianolic acid B's anti-hepatic fibrotic-carcinogenesis. However, whether pSmad3C mutation expedites pSmad2L/C-mediated signaling transduction during hepatic fibrogenesis remains vague. Presently, Smad3 gene C-terminal phosphorylation site mutation heterozygote (pSmad3C+/-) mice were constructed to probe if and how pSmad3C retards CCl4-induced hepatic fibrogenesis by inhibiting pSmad2L/C-mediated signaling transduction. Twelve 6-week-old pSmad3C+/- C57BL/6J mice were intraperitoneally injection with CCl4 for 6 weeks to induce liver fibrogenesis. Results showed that pSmad3C mutation aggravates the relative liver weight, biochemical parameters, collagenous fibers and fibrotic septa formation, contributes to fibrogenesis in HT-CCl4 mice. Furthermore, fibrotic-related proteins TGF-β1, pSmad2C, pSmad2L, and PAI-1 were also increased in CCl4-induced pSmad3C+/- mice. These results suggest that pSmad3C mutation exacerbates hepatic fibrogenesis which relates to intensifying pSmad2L/C-mediated signaling transduction.
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Affiliation(s)
- Juan Yang
- Department of Pharmacology, Key Laboratory of Anti-Inflammatory and Immunopharmacology, Ministry of Education, Anhui Medical University, 81 Meishan Road, Hefei, 230032, China
| | - Yongfang Gong
- Department of Pharmacology, Key Laboratory of Anti-Inflammatory and Immunopharmacology, Ministry of Education, Anhui Medical University, 81 Meishan Road, Hefei, 230032, China
| | - Wenjing Xu
- Department of Pharmacology, Key Laboratory of Anti-Inflammatory and Immunopharmacology, Ministry of Education, Anhui Medical University, 81 Meishan Road, Hefei, 230032, China
| | - Lili Li
- Department of Pharmacology, Key Laboratory of Anti-Inflammatory and Immunopharmacology, Ministry of Education, Anhui Medical University, 81 Meishan Road, Hefei, 230032, China
| | - Zhenghao Shi
- Department of Pharmacology, Key Laboratory of Anti-Inflammatory and Immunopharmacology, Ministry of Education, Anhui Medical University, 81 Meishan Road, Hefei, 230032, China
| | - Qin Wang
- Department of Pharmacology, Key Laboratory of Anti-Inflammatory and Immunopharmacology, Ministry of Education, Anhui Medical University, 81 Meishan Road, Hefei, 230032, China
| | - Yinghao He
- Department of Pharmacology, Key Laboratory of Anti-Inflammatory and Immunopharmacology, Ministry of Education, Anhui Medical University, 81 Meishan Road, Hefei, 230032, China
| | - Chong Zhang
- Department of Pharmacology, Key Laboratory of Anti-Inflammatory and Immunopharmacology, Ministry of Education, Anhui Medical University, 81 Meishan Road, Hefei, 230032, China
| | - Chenchen Luo
- Department of Pharmacology, Key Laboratory of Anti-Inflammatory and Immunopharmacology, Ministry of Education, Anhui Medical University, 81 Meishan Road, Hefei, 230032, China
| | - Zhirui Fang
- Department of Pharmacology, Key Laboratory of Anti-Inflammatory and Immunopharmacology, Ministry of Education, Anhui Medical University, 81 Meishan Road, Hefei, 230032, China
| | - Yan Yang
- Department of Pharmacology, Key Laboratory of Anti-Inflammatory and Immunopharmacology, Ministry of Education, Anhui Medical University, 81 Meishan Road, Hefei, 230032, China.
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Ding H, Fang M, Gong Y, Li D, Zhang C, Wen G, Wu C, Yang J, Yang Y. Smad3 gene C-terminal phosphorylation site mutation aggravates CCl 4 -induced inflammation in mice. J Cell Mol Med 2020; 24:7044-7054. [PMID: 32406200 PMCID: PMC7299733 DOI: 10.1111/jcmm.15385] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/16/2019] [Revised: 02/17/2020] [Accepted: 04/27/2020] [Indexed: 12/13/2022] Open
Abstract
The expression of C‐terminal phosphorylated Smad3 (pSmad3C) is down‐regulated with the progression of liver disease. Thus, we hypothesized that pSmad3C expression may be negatively related to liver disease. To develop novel therapeutic strategies, a suitable animal model is required that will allow researchers to study the effect of Smad3 domain‐specific phosphorylation on liver disease progression. The current study aimed to construct a new mouse model with the Smad3 C‐terminal phosphorylation site mutation and to explore the effects of this mutation on CCl4‐induced inflammation. Smad3 C‐terminal phosphorylation site mutant mice were generated using TetraOne™ gene fixed‐point knock‐in technology and embryonic stem cell microinjection. Resulting mice were identified by genotyping, and the effects on inflammation were explored in the presence or absence of CCl4. No homozygous mice were born, indicating that the mutation is embryonic lethal. There was no significant difference in liver phenotype and growth between the wild‐type (WT) and heterozygous (HT) mice in the absence of reagent stimulation. After CCl4‐induced acute and chronic liver damage, liver pathology, serum transaminase (ALT/AST) expression and levels of inflammatory factors (IL‐6/TNF‐α) were more severely altered in HT mice than in WT mice. Furthermore, pSmad3C protein levels were lower in liver tissue from HT mice. These results suggest that Smad3 C‐terminal phosphorylation may have a protective effect during the early stages of liver injury. In summary, we have generated a new animal model that will be a novel tool for future research on the effects of Smad3 domain‐specific phosphorylation on liver disease progression.
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Affiliation(s)
- Hanyan Ding
- Department of Pharmacology, Key Laboratory of Anti-inflammatory and Immunopharmacology, Ministry of Education, Anhui Medical University, Hefei, China
| | - Meng Fang
- Department of Anatomy, Anhui Medical University, Hefei, China
| | - Yongfang Gong
- Department of Pharmacology, Key Laboratory of Anti-inflammatory and Immunopharmacology, Ministry of Education, Anhui Medical University, Hefei, China
| | - Dong Li
- Department of Pharmacology, Key Laboratory of Anti-inflammatory and Immunopharmacology, Ministry of Education, Anhui Medical University, Hefei, China
| | - Chong Zhang
- Department of Pharmacology, Key Laboratory of Anti-inflammatory and Immunopharmacology, Ministry of Education, Anhui Medical University, Hefei, China
| | - Guanghua Wen
- Department of Pharmacology, Key Laboratory of Anti-inflammatory and Immunopharmacology, Ministry of Education, Anhui Medical University, Hefei, China
| | - Chao Wu
- Department of Pharmacology, Key Laboratory of Anti-inflammatory and Immunopharmacology, Ministry of Education, Anhui Medical University, Hefei, China
| | - Jingjing Yang
- Department of Pharmacology, Key Laboratory of Anti-inflammatory and Immunopharmacology, Ministry of Education, Anhui Medical University, Hefei, China
| | - Yan Yang
- Department of Pharmacology, Key Laboratory of Anti-inflammatory and Immunopharmacology, Ministry of Education, Anhui Medical University, Hefei, China
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Ikeda A, Fujii W, Sugiura K, Naito K. High-fidelity endonuclease variant HypaCas9 facilitates accurate allele-specific gene modification in mouse zygotes. Commun Biol 2019; 2:371. [PMID: 31633062 PMCID: PMC6787007 DOI: 10.1038/s42003-019-0627-8] [Citation(s) in RCA: 34] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/01/2019] [Accepted: 09/23/2019] [Indexed: 02/04/2023] Open
Abstract
CRISPR/Cas9 has been widely used for the efficient generation of genetically modified animals; however, this system could have unexpected off-target effects. In the present study, we confirmed the validity of a high-fidelity Cas9 variant, HypaCas9, for accurate genome editing in mouse zygotes. HypaCas9 efficiently modified the target locus while minimizing off-target effects even in a single-nucleotide mismatched sequence. Furthermore, by applying HypaCas9 to the discrimination of SNP in hybrid strain-derived zygotes, we accomplished allele-specific gene modifications and successfully generated mice with a monoallelic mutation in an essential gene. These results suggest that the improved accuracy of HypaCas9 facilitates the generation of genetically modified animals.
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Affiliation(s)
- Arisa Ikeda
- Department of Animal Resource Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 Japan
| | - Wataru Fujii
- Department of Animal Resource Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 Japan
| | - Koji Sugiura
- Department of Animal Resource Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 Japan
| | - Kunihiko Naito
- Department of Animal Resource Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 Japan
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Generation of genetically modified mice using SpCas9-NG engineered nuclease. Sci Rep 2019; 9:12878. [PMID: 31501500 PMCID: PMC6733909 DOI: 10.1038/s41598-019-49394-5] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2019] [Accepted: 08/24/2019] [Indexed: 11/23/2022] Open
Abstract
Although genetically modified mice can be generated with high efficiency by using CRISPR/Cas9-mediated genome editing in mouse zygotes, only the loci with a protospacer-adjacent motif (PAM) sequence are targetable. The present study investigated the usability of engineered Streptococcus pyogenes Cas9 (SpCas9-NG) in mouse zygotes. In addition to the 5′-NGG sequence, SpCas9-NG recognized the 5′-NGA, 5′-NGC and 5′-NGT sequences in mouse zygotes as PAMs that were appropriate for the generation of knockout mice. Moreover, SpCas9-NG-mediated genome editing enabled the generation of knock-in mice untargetable by the conventional SpCas9 in mouse zygotes. These results suggest that SpCas9-NG-mediated genome editing in zygotes is available for the generation of knockout and knock-in mice at the locus corresponding to NGN-PAM.
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Fujii W, Ikeda A, Sugiura K, Naito K. Efficient Generation of Genome-Modified Mice Using Campylobacter jejuni-Derived CRISPR/Cas. Int J Mol Sci 2017; 18:ijms18112286. [PMID: 29088065 PMCID: PMC5713256 DOI: 10.3390/ijms18112286] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2017] [Revised: 10/26/2017] [Accepted: 10/26/2017] [Indexed: 12/31/2022] Open
Abstract
Mammalian zygote-mediated genome-engineering by Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas is currently used for the generation of genome-modified animals. Here, we report that a Campylobacter jejuni-derived orthologous CRISPR/Cas system recognizes a 5′-NNNVRYAC sequence as a protospacer-adjacent motif in mouse zygotes, and is applicable for efficient generation of knockout mice. Moreover, this novel CRISPR/Cas can be used for zygote-mediated knock-in at a unique locus, suggesting that this system could help to expand the feasibility of the zygote-mediated generation of genome-modified animals.
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Affiliation(s)
- Wataru Fujii
- Department of Animal Resource Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1, Yayoi, Tokyo 113-8657, Japan.
| | - Arisa Ikeda
- Department of Animal Resource Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1, Yayoi, Tokyo 113-8657, Japan.
| | - Koji Sugiura
- Department of Animal Resource Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1, Yayoi, Tokyo 113-8657, Japan.
| | - Kunihiko Naito
- Department of Animal Resource Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1, Yayoi, Tokyo 113-8657, Japan.
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