Hepatic Knockdown of Splicing Regulator Slu7 Ameliorates Inflammation and Attenuates Liver Injury in Ethanol-Fed Mice
2018; Elsevier BV; Volume: 188; Issue: 8 Linguagem: Inglês
10.1016/j.ajpath.2018.05.004
ISSN1525-2191
AutoresJiayou Wang, Noah Kainrad, Hong Shen, Zhou Zhou, Paula Rote, Yanqiao Zhang, Laura E. Nagy, Jiashin Wu, Min You,
Tópico(s)Lipid metabolism and biosynthesis
ResumoAberrant precursor mRNA splicing plays a pivotal role in liver diseases. However, roles of splicing regulators in alcoholic liver disease are unknown. Herein, we investigated a splicing regulator, Slu7, in the development of alcoholic steatohepatitis. Adenovirus-mediated alteration of hepatic Slu7 expression in mice pair fed either with or without (as control) ethanol in their diet was used. Knockdown of hepatic Slu7 by adenovirus-Slu7shRNA treatment ameliorated inflammation and attenuated liver injury in mice after ethanol administration. Mechanistically, reducing liver Slu7 expression increased the expression of sirtuin 1 (SIRT1) full-length and repressed the splicing of SIRT1 into SIRT1-ΔExon8 isoform in ethanol-fed mice. Knockdown of hepatic Slu7 in the ethanol-fed mice also ameliorated splicing of lipin-1 and serine/arginine-rich splicing factor 3 (Srsf3). In concordance with ameliorated splicing of SIRT1, lipin-1, and Srsf3, knockdown of hepatic Slu7 inhibited the activity of NF-κB, normalized iron and zinc homeostasis, reduced oxidative stress, and attenuated liver damage in ethanol-fed mice. In addition, hepatic Slu7 was significantly elevated in patients with alcoholic steatohepatitis. Our present study illustrates a novel role of Slu7 in alcoholic liver injury and suggests that dysregulated Slu7 may contribute to the pathogenesis of human alcoholic steatohepatitis. Aberrant precursor mRNA splicing plays a pivotal role in liver diseases. However, roles of splicing regulators in alcoholic liver disease are unknown. Herein, we investigated a splicing regulator, Slu7, in the development of alcoholic steatohepatitis. Adenovirus-mediated alteration of hepatic Slu7 expression in mice pair fed either with or without (as control) ethanol in their diet was used. Knockdown of hepatic Slu7 by adenovirus-Slu7shRNA treatment ameliorated inflammation and attenuated liver injury in mice after ethanol administration. Mechanistically, reducing liver Slu7 expression increased the expression of sirtuin 1 (SIRT1) full-length and repressed the splicing of SIRT1 into SIRT1-ΔExon8 isoform in ethanol-fed mice. Knockdown of hepatic Slu7 in the ethanol-fed mice also ameliorated splicing of lipin-1 and serine/arginine-rich splicing factor 3 (Srsf3). In concordance with ameliorated splicing of SIRT1, lipin-1, and Srsf3, knockdown of hepatic Slu7 inhibited the activity of NF-κB, normalized iron and zinc homeostasis, reduced oxidative stress, and attenuated liver damage in ethanol-fed mice. In addition, hepatic Slu7 was significantly elevated in patients with alcoholic steatohepatitis. Our present study illustrates a novel role of Slu7 in alcoholic liver injury and suggests that dysregulated Slu7 may contribute to the pathogenesis of human alcoholic steatohepatitis. Alcoholic steatohepatitis is a prevalent early form of alcoholic liver disease (ALD), which can progress to fibrosis, cirrhosis, hepatocellular carcinoma, and liver failure in clinical patients.1Farooq M.O. Bataller R. Pathogenesis and management of alcoholic liver disease.Dig Dis. 2016; 34: 347-355Crossref PubMed Scopus (35) Google Scholar Despite intensive studies over the past decades, the pathogenic mechanisms of alcoholic steatohepatitis remain incompletely understood, and patients with alcoholic steatohepatitis lack effective treatments. Alternative splicing of precursor mRNA (pre-mRNA) is a vital step in gene expression that eliminates the introns and ligates the exons to form mature mRNAs that can be translated into proteins.2Gallego-Paez L.M. Bordone M.C. Leote A.C. Saraiva-Agostinho N. Ascensão-Ferreira M. Barbosa-Morais N.L. Alternative splicing: the pledge, the turn, and the prestige: the key role of alternative splicing in human biological systems.Hum Genet. 2017; 136: 1015-1042Crossref PubMed Scopus (66) Google Scholar, 3Berasain C. Goñi S. Castillo J. Latasa M.U. Prieto J. Avila M.A. Impairment of pre mRNA splicing in liver disease: mechanisms and consequences.World J Gastroenterol. 2010; 16: 3091-3102Crossref PubMed Scopus (35) Google Scholar Defects in pre-mRNA splicing have been implicated in various human diseases, including liver diseases.3Berasain C. Goñi S. Castillo J. Latasa M.U. Prieto J. Avila M.A. Impairment of pre mRNA splicing in liver disease: mechanisms and consequences.World J Gastroenterol. 2010; 16: 3091-3102Crossref PubMed Scopus (35) Google Scholar In recent years, aberrant mRNAs and dysregulated splicing regulators in liver diseases are emerging as targets for new therapeutic interventions.3Berasain C. Goñi S. Castillo J. Latasa M.U. Prieto J. Avila M.A. Impairment of pre mRNA splicing in liver disease: mechanisms and consequences.World J Gastroenterol. 2010; 16: 3091-3102Crossref PubMed Scopus (35) Google Scholar However, alternative pre-mRNA splicing is an underappreciated mechanism in the pathogenesis of ALD. The pre-mRNA splicing is catalyzed in two steps by spliceosome, a large ribonucleoprotein complex.4Chua K. Reed R. The RNA splicing factor hSlu7 is required for correct 3' splice-site choice.Nature. 1999; 402: 207-210Crossref PubMed Scopus (77) Google Scholar, 5James S.A. Turner W. Schwer B. How Slu7 and Prp18 cooperate in the second step of yeast pre-mRNA splicing.RNA. 2011; 8: 1068-1077Crossref Scopus (70) Google Scholar, 6Chua K. Reed R. Human step II splicing factor hSlu7 functions in restructuring the spliceosome between the catalytic steps of splicing.Genes Dev. 1999; 13: 841-850Crossref PubMed Scopus (47) Google Scholar, 7Shomron N. Reznik M. Ast G. Splicing factor hSlu7 contains a unique functional domain required to retain the protein within the nucleus.Mol Biol Cell. 2004; 15: 3782-3795Crossref PubMed Google Scholar The splicing regulator Slu7 (human homolog hSlu7) participates in the selection of the 3′ site during the second step of splicing.4Chua K. Reed R. The RNA splicing factor hSlu7 is required for correct 3' splice-site choice.Nature. 1999; 402: 207-210Crossref PubMed Scopus (77) Google Scholar, 5James S.A. Turner W. Schwer B. How Slu7 and Prp18 cooperate in the second step of yeast pre-mRNA splicing.RNA. 2011; 8: 1068-1077Crossref Scopus (70) Google Scholar, 6Chua K. Reed R. Human step II splicing factor hSlu7 functions in restructuring the spliceosome between the catalytic steps of splicing.Genes Dev. 1999; 13: 841-850Crossref PubMed Scopus (47) Google Scholar, 7Shomron N. Reznik M. Ast G. Splicing factor hSlu7 contains a unique functional domain required to retain the protein within the nucleus.Mol Biol Cell. 2004; 15: 3782-3795Crossref PubMed Google Scholar, 8Shomron N. Alberstein M. Reznik M. Ast G. Stress alters the subcellular distribution of hSlu7 and thus modulates alternative splicing.J Cell Sci. 2005; 118: 1151-1159Crossref PubMed Scopus (50) Google Scholar, 9Castillo J. Goñi S. Latasa M.U. Perugorría M.J. Calvo A. Muntané J. Bioulac-Sage P. Balabaud C. Prieto J. Avila M.A. Berasain C. Amphiregulin induces the alternative splicing of p73 into its oncogenic isoform DeltaEx2p73 in human hepatocellular tumors.Gastroenterology. 2009; 137: 1805-1815Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar Human homolog hSlu7 protein has three functional domains, a zinc-knuckle motif, a lysine-rich region, and a nuclear localization signal, that play distinctive roles in determining its subcellular localization.8Shomron N. Alberstein M. Reznik M. Ast G. Stress alters the subcellular distribution of hSlu7 and thus modulates alternative splicing.J Cell Sci. 2005; 118: 1151-1159Crossref PubMed Scopus (50) Google Scholar, 9Castillo J. Goñi S. Latasa M.U. Perugorría M.J. Calvo A. Muntané J. Bioulac-Sage P. Balabaud C. Prieto J. Avila M.A. Berasain C. Amphiregulin induces the alternative splicing of p73 into its oncogenic isoform DeltaEx2p73 in human hepatocellular tumors.Gastroenterology. 2009; 137: 1805-1815Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar Slu7 is the only second step splicing protein with a potential zinc-binding domain in the form of a zinc-knuckle motif, suggesting the potential involvement of Slu7 in regulation of metabolism of metals, such as zinc and iron.4Chua K. Reed R. The RNA splicing factor hSlu7 is required for correct 3' splice-site choice.Nature. 1999; 402: 207-210Crossref PubMed Scopus (77) Google Scholar, 5James S.A. Turner W. Schwer B. How Slu7 and Prp18 cooperate in the second step of yeast pre-mRNA splicing.RNA. 2011; 8: 1068-1077Crossref Scopus (70) Google Scholar, 6Chua K. Reed R. Human step II splicing factor hSlu7 functions in restructuring the spliceosome between the catalytic steps of splicing.Genes Dev. 1999; 13: 841-850Crossref PubMed Scopus (47) Google Scholar, 7Shomron N. Reznik M. Ast G. Splicing factor hSlu7 contains a unique functional domain required to retain the protein within the nucleus.Mol Biol Cell. 2004; 15: 3782-3795Crossref PubMed Google Scholar, 8Shomron N. Alberstein M. Reznik M. Ast G. Stress alters the subcellular distribution of hSlu7 and thus modulates alternative splicing.J Cell Sci. 2005; 118: 1151-1159Crossref PubMed Scopus (50) Google Scholar, 9Castillo J. Goñi S. Latasa M.U. Perugorría M.J. Calvo A. Muntané J. Bioulac-Sage P. Balabaud C. Prieto J. Avila M.A. Berasain C. Amphiregulin induces the alternative splicing of p73 into its oncogenic isoform DeltaEx2p73 in human hepatocellular tumors.Gastroenterology. 2009; 137: 1805-1815Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 10Urtasun R. Elizalde M. Azkona M. Latasa M.U. García-Irigoyen O. Uriarte I. Fernández-Barrena M.G. Vicent S. Alonso M.M. Muntané J. Prieto J. Ávila M.A. Berasain C. Splicing regulator SLU7 preserves survival of hepatocellular carcinoma cells and other solid tumors via oncogenic miR-17-92 cluster expression.Oncogene. 2016; 35: 4719-4729Crossref PubMed Scopus (22) Google Scholar, 11Elizalde M. Urtasun R. Azkona M. Latasa M.U. Goñi S. García-Irigoyen O. Uriarte I. Segura V. Collantes M. Di Scala M. Lujambio A. Prieto J. Ávila M.A. Berasain C. Splicing regulator SLU7 is essential for maintaining liver homeostasis.J Clin Invest. 2014; 124: 2909-2920Crossref PubMed Scopus (42) Google Scholar Slu7 not only functions as a splicing regulator but also plays an essential coregulatory role for maintaining liver functions.8Shomron N. Alberstein M. Reznik M. Ast G. Stress alters the subcellular distribution of hSlu7 and thus modulates alternative splicing.J Cell Sci. 2005; 118: 1151-1159Crossref PubMed Scopus (50) Google Scholar, 9Castillo J. Goñi S. Latasa M.U. Perugorría M.J. Calvo A. Muntané J. Bioulac-Sage P. Balabaud C. Prieto J. Avila M.A. Berasain C. Amphiregulin induces the alternative splicing of p73 into its oncogenic isoform DeltaEx2p73 in human hepatocellular tumors.Gastroenterology. 2009; 137: 1805-1815Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 10Urtasun R. Elizalde M. Azkona M. Latasa M.U. García-Irigoyen O. Uriarte I. Fernández-Barrena M.G. Vicent S. Alonso M.M. Muntané J. Prieto J. Ávila M.A. Berasain C. Splicing regulator SLU7 preserves survival of hepatocellular carcinoma cells and other solid tumors via oncogenic miR-17-92 cluster expression.Oncogene. 2016; 35: 4719-4729Crossref PubMed Scopus (22) Google Scholar, 11Elizalde M. Urtasun R. Azkona M. Latasa M.U. Goñi S. García-Irigoyen O. Uriarte I. Segura V. Collantes M. Di Scala M. Lujambio A. Prieto J. Ávila M.A. Berasain C. Splicing regulator SLU7 is essential for maintaining liver homeostasis.J Clin Invest. 2014; 124: 2909-2920Crossref PubMed Scopus (42) Google Scholar In liver, Slu7 participates in the regulation of splicing and expression of multiple genes implicated in lipid metabolism, inflammation, development, and progression of fibrosis and cirrhosis.11Elizalde M. Urtasun R. Azkona M. Latasa M.U. Goñi S. García-Irigoyen O. Uriarte I. Segura V. Collantes M. Di Scala M. Lujambio A. Prieto J. Ávila M.A. Berasain C. Splicing regulator SLU7 is essential for maintaining liver homeostasis.J Clin Invest. 2014; 124: 2909-2920Crossref PubMed Scopus (42) Google Scholar Defective Slu7 is likely to contribute to metabolic diseases, such as nonalcoholic fatty liver diseases, because experimental knockdown of Slu7 disrupted the gene expression involved in lipogenesis and impaired physiological metabolic responses in mouse liver.11Elizalde M. Urtasun R. Azkona M. Latasa M.U. Goñi S. García-Irigoyen O. Uriarte I. Segura V. Collantes M. Di Scala M. Lujambio A. Prieto J. Ávila M.A. Berasain C. Splicing regulator SLU7 is essential for maintaining liver homeostasis.J Clin Invest. 2014; 124: 2909-2920Crossref PubMed Scopus (42) Google Scholar Sirtuin (SIRT) 1, an NAD+-dependent class III protein deacetylase, is an essential player in controlling the pathways of inflammatory responses and lipid metabolism.12Chang H.C. Guarente L. 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HuR and TIA1/TIAL1 are involved in regulation of alternative splicing of SIRT1 pre-mRNA.Int J Mol Sci. 2014; 15: 2946-2958Crossref PubMed Scopus (22) Google Scholar Because exon 8 encodes the deacetylase domain of SIRT1, the exclusion of exon 8 markedly attenuates SIRT1-ΔExon8 deacetylase activity.13Lynch C.J. Shah Z.H. Allison S.J. Ahmed S.U. Ford J. Warnock L.J. Li H. Serrano M. Milner J. SIRT1 undergoes alternative splicing in a novel auto-regulatory loop with p53.PLoS One. 2010; 5: e13502Crossref PubMed Scopus (43) Google Scholar, 14Zhao W. Zhao J. Hou M. Wang Y. Zhang Y. Zhao X. Zhang C. Guo D. HuR and TIA1/TIAL1 are involved in regulation of alternative splicing of SIRT1 pre-mRNA.Int J Mol Sci. 2014; 15: 2946-2958Crossref PubMed Scopus (22) Google Scholar Abnormality of SIRT1 and its signaling has been implicated in the pathogenesis of alcoholic steatosis/steatohepatitis.15You M. Jogasuria A. Taylor C. Wu J. Sirtuin 1 signaling and alcoholic fatty liver disease.Hepatobiliary Surg Nutr. 2015; 4: 88-100PubMed Google Scholar However, whether and how ethanol exposure influences alternative splicing of SIRT1 pre-mRNA and contributes to liver damage are currently unknown. Interestingly, SIRT1 is a target gene of Slu7 in cultured human hepatic cells, suggesting that Slu7 may regulate SIRT1 splicing.11Elizalde M. Urtasun R. Azkona M. Latasa M.U. Goñi S. García-Irigoyen O. Uriarte I. Segura V. Collantes M. Di Scala M. Lujambio A. Prieto J. Ávila M.A. Berasain C. Splicing regulator SLU7 is essential for maintaining liver homeostasis.J Clin Invest. 2014; 124: 2909-2920Crossref PubMed Scopus (42) Google Scholar Lipin-1 regulates lipid metabolism and inflammation process via functioning as a phosphatidic acid phosphohydrolase enzyme and as a transcriptional coregulator.16Han G.S. Carman G.M. Characterization of the human LPIN1-encoded phosphatidate phosphatase isoforms.J Biol Chem. 2010; 285: 14628-14638Crossref PubMed Scopus (107) Google Scholar Lipin-1 pre-mRNA splicing produces two isoforms, lipin-1α and lipin-1β.16Han G.S. Carman G.M. Characterization of the human LPIN1-encoded phosphatidate phosphatase isoforms.J Biol Chem. 2010; 285: 14628-14638Crossref PubMed Scopus (107) Google Scholar, 17Péterfy M. Phan J. Reue K. Alternatively spliced lipin isoforms exhibit distinct expression pattern, subcellular localization, and role in adipogenesis.J Biol Chem. 2005; 280: 32883-32889Crossref PubMed Scopus (174) Google Scholar Lipin-1α is generated through skipping exon 7 from lipin-1β.17Péterfy M. Phan J. Reue K. Alternatively spliced lipin isoforms exhibit distinct expression pattern, subcellular localization, and role in adipogenesis.J Biol Chem. 2005; 280: 32883-32889Crossref PubMed Scopus (174) Google Scholar Dysregulated lipin-1 and its pre-mRNA splicing have been implicated in metabolic diseases, including obesity.17Péterfy M. Phan J. Reue K. Alternatively spliced lipin isoforms exhibit distinct expression pattern, subcellular localization, and role in adipogenesis.J Biol Chem. 2005; 280: 32883-32889Crossref PubMed Scopus (174) Google Scholar, 18Phan J. Reue K. Lipin, a lipodystrophy and obesity gene.Cell Metab. 2005; 1: 73-83Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar Ethanol exposure inhibits lipin-1 pre-mRNA splicing by inhibiting lipin-1α generation, inducing the ratio of Lpin1 β/α in the livers of mice and of patients with alcoholic hepatitis.19Yin H. Hu M. Liang X. Ajmo J.M. Li X. Bataller R. Odena G. Stevens Jr., S.M. You M. Deletion of SIRT1 from hepatocytes in mice disrupts lipin-1 signaling and aggravates alcoholic fatty liver.Gastroenterology. 2014; 146: 801-811Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 20Hu M. Yin H. Mitra M.S. Liang X. Ajmo J.M. Nadra K. Chrast R. Finck B.N. You M. Hepatic-specific lipin-1 deficiency exacerbates experimental alcohol-induced steatohepatitis in mice.Hepatology. 2013; 58: 1953-1963Crossref PubMed Scopus (56) Google Scholar More important, aberrant SIRT1 signaling is associated with disruption of lipin-1 pre-mRNA splicing by ethanol in mice.19Yin H. Hu M. Liang X. Ajmo J.M. Li X. Bataller R. Odena G. Stevens Jr., S.M. You M. Deletion of SIRT1 from hepatocytes in mice disrupts lipin-1 signaling and aggravates alcoholic fatty liver.Gastroenterology. 2014; 146: 801-811Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar Serine/arginine-rich splicing factor 3 (Srsf3; alias SRp20) belongs to the highly conserved family of SR proteins.21Sen S. Jumaa H. Webster N.J. Splicing factor SRSF3 is crucial for hepatocyte differentiation and metabolic function.Nat Commun. 2013; 4: 1336Crossref PubMed Scopus (89) Google Scholar Srsf3 pre-mRNA alternative splicing generates a full-length isoform lacking exon 4 (Iso1) and an alternative isoform including exon 4 (Iso2).21Sen S. Jumaa H. Webster N.J. Splicing factor SRSF3 is crucial for hepatocyte differentiation and metabolic function.Nat Commun. 2013; 4: 1336Crossref PubMed Scopus (89) Google Scholar, 22Lareau L.F. Inada M. Green R.E. Wengrod J.C. Brenner S.E. Unproductive splicing of SR genes associated with highly conserved and ultraconserved DNA elements.Nature. 2007; 446: 926-929Crossref PubMed Scopus (441) Google Scholar, 23Sen S. Langiewicz M. Jumaa H. Webster N.J.G. Deletion of serine/arginine-rich splicing factor 3 in hepatocytes predisposes to hepatocellular carcinoma in mice.Hepatology. 2015; 61: 171-183Crossref PubMed Scopus (62) Google Scholar Slu7 regulates Srsf3 pre-mRNA splicing.11Elizalde M. Urtasun R. Azkona M. Latasa M.U. Goñi S. García-Irigoyen O. Uriarte I. Segura V. Collantes M. Di Scala M. Lujambio A. Prieto J. Ávila M.A. Berasain C. Splicing regulator SLU7 is essential for maintaining liver homeostasis.J Clin Invest. 2014; 124: 2909-2920Crossref PubMed Scopus (42) Google Scholar Furthermore, errors in Slu7-Srsf3 axis contribute to abnormality of liver functions in rodents and humans.11Elizalde M. Urtasun R. Azkona M. Latasa M.U. Goñi S. García-Irigoyen O. Uriarte I. Segura V. Collantes M. Di Scala M. Lujambio A. Prieto J. Ávila M.A. Berasain C. Splicing regulator SLU7 is essential for maintaining liver homeostasis.J Clin Invest. 2014; 124: 2909-2920Crossref PubMed Scopus (42) Google Scholar, 21Sen S. Jumaa H. Webster N.J. Splicing factor SRSF3 is crucial for hepatocyte differentiation and metabolic function.Nat Commun. 2013; 4: 1336Crossref PubMed Scopus (89) Google Scholar, 22Lareau L.F. Inada M. Green R.E. Wengrod J.C. Brenner S.E. Unproductive splicing of SR genes associated with highly conserved and ultraconserved DNA elements.Nature. 2007; 446: 926-929Crossref PubMed Scopus (441) Google Scholar, 23Sen S. Langiewicz M. Jumaa H. Webster N.J.G. Deletion of serine/arginine-rich splicing factor 3 in hepatocytes predisposes to hepatocellular carcinoma in mice.Hepatology. 2015; 61: 171-183Crossref PubMed Scopus (62) Google Scholar We tested a hypothesis in the present study that dysregulated expression of liver Slu7 might be pivotal in the development of alcoholic steatohepatitis. Using adenovirus (Ad)–mediated gene alteration of hepatic Slu7 expression in a chronic-plus-binge ethanol feeding mouse model,24Bertola A. Mathews S. Ki S.H. Wang H. Gao B. Mouse model of chronic and binge ethanol feeding (the NIAAA model).Nat Protoc. 2013; 8: 627-637Crossref PubMed Scopus (601) Google Scholar we demonstrate, for the first time, that knockdown of Slu7 ameliorates the ethanol-induced inflammation and attenuates alcoholic liver injury in mice via modifying pre-mRNA splicing of SIRT1, lipin-1, and Srsf3. Most supplies and chemicals were purchased from Sigma Chemical (St. Louis, MO), Schleicher and Schuell (Keene, NH), Gibco-BRL (Grand Island, NY), and DuPont NEN Research Products (Boston, MA). Murine vectors expressing Ad–green fluorescent protein (GFP; control), Ad-Slu7 [adenoviral type 5 (dE1/E3); promoter, cytomegalovirus], Ad-shRNA (control), and Ad-shSlu7 [adenoviral type 5 (dE1/E3); promoter, U6] were custom designed and obtained from Vector BioLabs, Inc. (Malvern, PA). Antibodies against Slu7, Srsf3, nuclear factor of activated T cells 4 (NFATc4), and acetylated forkhead box protein O 1 were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX). Acetylated NF-κB, NF-κB, and SIRT1 antibodies were purchased from Cell Signaling (Danvers, MA). Hepatocyte-specific SIRT1 knockout mice were kindly gifted by Dr. Xiaoling Li (National Institute of Environmental Health Sciences/NIH, Research Triangle Park, NC).19Yin H. Hu M. Liang X. Ajmo J.M. Li X. Bataller R. Odena G. Stevens Jr., S.M. You M. Deletion of SIRT1 from hepatocytes in mice disrupts lipin-1 signaling and aggravates alcoholic fatty liver.Gastroenterology. 2014; 146: 801-811Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar A chronic-plus-binge mouse model (a single binge after chronic ethanol consumption) was used.24Bertola A. Mathews S. Ki S.H. Wang H. Gao B. Mouse model of chronic and binge ethanol feeding (the NIAAA model).Nat Protoc. 2013; 8: 627-637Crossref PubMed Scopus (601) Google Scholar Mice were fed with liquid diets providing 1 kcal/mL, which were freshly prepared daily from powder with Lieber-DeCarli formulation (Frenchtown, NJ). Female SIRT1 knockout and wild-type mice (10 to 12 weeks old) were divided into pair-fed dietary groups: ethanol and control (without ethanol). During the first 10 days, diet in the ethanol groups contained 5% ethanol, whereas the control mice were pair fed with same calories of dextrin maltose. At day 11, mice were gavaged either a single dose of ethanol [5 g/kg body weight, 31.5% ethanol (ethanol group)] or an isocaloric dose of dextrin maltose (control group). The mice were euthanized, and blood and tissue samples were collected 9 hours after gavage. In vivo Ad-mediated gene transfer experiments were performed, as described previously.25Yin H. Liang X. Jogasuria A. Davidson N.O. You M. miR-217 regulates ethanol-induced hepatic inflammation by disrupting sirtuin 1-lipin-1 signaling.Am J Pathol. 2015; 185: 1286-1296Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 26Li Y. Xu S. Giles A. Nakamura K. Lee J.W. Hou X. Donmez G. Li J. Luo Z. Walsh K. Guarente L. Zang M. Hepatic overexpression of SIRT1 in mice attenuates endoplasmic reticulum stress and insulin resistance in the liver.FASEB J. 2011; 25: 1664-1679Crossref PubMed Scopus (238) Google Scholar During the 10 days of the chronic-plus-binge ethanol feeding period, overexpression of Slu7 or knockdown of Slu7 in female C57BL/6J mice was accomplished via retro-orbital injection of Ad-GFP, Ad-Slu7, Ad-shRNA, and Ad-shSlu7 (0.5 to 1.0 × 109 active viral particles in 200 μL phosphate-buffered saline) to mice twice on days 1 and 5. On the final day, mice were sacrificed, and tissue (liver and/or serum) samples were harvested. All of the animal experiments were approved by the Institutional Animal Care and Use Committee at Northeast Ohio Medical University. Mouse AML-12 hepatocytes (ATCC, Manassas, VA) were cultured in Dulbecco's modified Eagle's medium/F12 medium supplemented with 100 μg/mL streptomycin, 10% fetal bovine serum, 0.1 μmol/L dexamethasone, 63 μg/mL penicillin G, and insulin-transferrin-selenium (Gibco-BRL). They were seeded in 6-well plates for 24 hours. On the day of transfection, cells were washed and switched to reduced serum medium (Invitrogen, Carlsbad, CA) and then infected with Ad-GFP, Ad-Slu7, Ad-shRNA, and Ad-shSlu7 for 36 to 48 hours.27Cai Y. Jogasuria A. Yin H. Xu M.J. Hu X. Wang J. Kim C. Wu J. Lee K. Gao B. You M. The detrimental role played by lipocalin-2 in alcoholic fatty liver in mice.Am J Pathol. 2016; 186: 2417-2428Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar Western blot analyses were performed on liver tissue extract with 10% Bio-Rad MiniPROTEIN Tetra Cell system and then transferred to polyvinylidene difluoride membranes with Bio-Rad Blotting Module (Bio-Rad Laboratories, Hercules, CA).27Cai Y. Jogasuria A. Yin H. Xu M.J. Hu X. Wang J. Kim C. Wu J. Lee K. Gao B. You M. The detrimental role played by lipocalin-2 in alcoholic fatty liver in mice.Am J Pathol. 2016; 186: 2417-2428Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 28Hu X. Jogasuria A. Wang J. Kim C. Han Y. Shen H. Wu J. You M. MitoNEET deficiency alleviates experimental alcoholic steatohepatitis in mice by stimulating endocrine adiponectin-Fgf15 axis.J Biol Chem. 2016; 291: 22482-22495Crossref PubMed Scopus (24) Google Scholar The signals obtained for liver protein extracts were normalized by using polyclonal rabbit anti-actin β antibody (Sigma, Ronkonkoma, NY). Quantification and analysis of multiple protein bands were performed using AlphaView software version 3.4 (ProteinSimple, San Jose, CA). The lipid peroxidation end product, malondialdehyde (MDA), in liver was measured on the basis of the formation of thiobarbituric reactive substances and expressed as the extent of MDA production.28Hu X. Jogasuria A. Wang J. Kim C. Han Y. Shen H. Wu J. You M. MitoNEET deficiency alleviates experimental alcoholic steatohepatitis in mice by stimulating endocrine adiponectin-Fgf15 axis.J Biol Chem. 2016; 291: 22482-22495Crossref PubMed Scopus (24) Google Scholar Liver tissues were homogenized in ice-cold phosphate-buffered saline. Total lipids were extracted with chloroform/methanol. Liver triglyceride and cholesterol contents were measured with BioVision assay kits (BioVision, Milpitas, CA), as described previously.27Cai Y. Jogasuria A. Yin H. Xu M.J. Hu X. Wang J. Kim C. Wu J. Lee K. Gao B. You M. The detrimental role played by lipocalin-2 in alcoholic fatty liver in mice.Am J Pathol. 2016; 186: 2417-2428Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 28Hu X. Jogasuria A. Wang J. Kim C. Han Y. Shen H. Wu J. You M. MitoNEET deficiency alleviates experimental alcoholic steatohepatitis in mice by stimulating endocrine adiponectin-Fgf15 axis.J Biol Chem. 2016; 291: 22482-22495Crossref PubMed Scopus (24) Google Scholar Liver myeloperoxidase (MPO) activity was measured using assay kits (Abcam, Cambridge, MA), as previously reported.28Hu X. Jogasuria A. Wang J. Kim C. Han Y. Shen H. Wu J. You M. MitoNEET deficiency alleviates experimental alcoholic steatohepatitis in mice by stimulating endocrine adiponectin-Fgf15 axis.J Biol Chem. 2016; 291: 22482-22495Crossref PubMed Scopus (24) Google Scholar Liver tissues were fixed in 10% formalin and embedded in paraffin.27Cai Y. Jogasuria A. Yin H. Xu M.J. Hu X. Wang J. Kim C. Wu J. Lee K. Gao B. You M. The detrimental role played by lipocalin-2 in alcoholic fatty liver in mice.Am J Pathol. 2016; 186: 2417-2428Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 28Hu X. Jogasuria A. Wang J. Kim C. Han Y. Shen H. Wu J. You M. MitoNEET deficiency alleviates experimental alcoholic steatohepatitis in mice by stimulating endocrine adiponectin-Fgf15 axis.J Biol Chem. 2016; 291: 22482-22495Crossref PubMed Scopus (24) Google Scholar Liver sections were stained with hematoxylin and eosin. Serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured by using the MaxDiscovery ALT and AST Assay Kits (Bio Scientific, Austin, TX). Serum triglyceride and cholesterol were measured by using the Infinity Cholesterol and Triglyceride Reagents (Thermo Fisher Scientific, Waltham, MA). All analyses were performed using a SpectraMax i3× microplate reader (Molecular De
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