Hepatic Knockdown of Splicing Regulator Slu7 Ameliorates Inflammation and Attenuates Liver Injury in Ethanol-Fed Mice

Abstract

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.

Figure 1

Ad-mediated hepatic Slu7 knockdown attenuates liver injury but slightly aggravates steatosis in mice after ethanol administration. Female C57BL/6J mice were pair fed either a control diet or an ethanol (E)–containing diet for 10 days, followed by single gavage of ethanol. During the 10-day chronic-plus-binge ethanol feeding period, Ad-shSlu7 or Ad-shRNA control (0.5 to 1.0 × 10⁹ active viral particles in 200 μL of phosphate-buffered saline) was given to mice twice on days 1 and 5. A: Serum alanine aminotransferase (ALT). B: Serum aspartate aminotransferase (AST). C: Hepatic triglyceride (TG) content. D: Hematoxylin and eosin (H&E) staining of liver sections. E: Liver myeloperoxidase (MPO) activity. F: Relative liver mRNA levels of Ly6G. G: Relative liver mRNA levels of tumor necrosis factor-α (TNF-α), IL-1β, IL-6, inducible nitric oxide synthase (iNOS), monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1α, MIP-1β, E-selectin, intercellular adhesion molecule (ICAM), and vascular cell adhesion molecule (VCAM). Data are expressed as means ± SEM (A–C and E–G). n = 5 to 12 mice. ^∗P < 0.05 versus pair-fed Ad-shRNA controls; ^†P < 0.05 versus ethanol-fed Ad-shRNA. Original magnification, ×40 (D).

Figure 2

Ad-mediated knockdown of hepatic Slu7 regulates hepatic sirtuin (SIRT) 1 pre-mRNA splicing in mice after ethanol administration. A: Mouse AML-12 hepatocytes were infected with Ad-GFP, Ad-Slu7, and Ad-shSlu7. Relative abundance of SIRT1-ΔExon8 or SIRT1 full length (SIRT1-FL). B: Female C57BL/6J mice were pair fed either a control diet or an ethanol (E)–containing diet for 10 days, followed by single gavage of ethanol. During the 10-day chronic-plus-binge ethanol feeding period, Ad-shSlu7 or Ad-shRNA control (0.5 to 1.0 × 10⁹ active viral particles in 200 μL of phosphate-buffered saline) was given to mice twice on days 1 and 5. Relative abundance of SIRT1-ΔExon8 or SIRT1-FL. C: Relative liver mRNA levels of SIRT1-FL and SIRT1-ΔExon8 and ratio of SIRT1-ΔExon8/SIRT1-FL. D: Western blot analysis of liver SIRT1 and acetylated (Ac) forkhead box protein O (FoxO) 1 (on separate gels). Data are expressed as means ± SEM. n = 3 replications (A and B); n = 5 to 12 mice (C and D). ^∗P < 0.05 versus Ad-GFP controls; ^†P < 0.05 versus pair-fed Ad-shRNA controls; ^‡P < 0.05 versus ethanol-fed Ad-shRNA. M, markers.

Figure 3

Ad-mediated knockdown of hepatic Slu7 regulates hepatic lipin-1 and serine/arginine-rich splicing factor (Srsf) 3 splicing in ethanol-fed mice. Female C57BL/6J mice were pair fed either a control diet or an ethanol (E)–containing diet for 10 days, followed by single gavage of ethanol. During the 10-day chronic-plus-binge ethanol feeding period, Ad-shSlu7 or Ad-shRNA control (0.5 to 1.0 × 10⁹ active viral particles in 200 μL of phosphate-buffered saline) was given to mice twice on days 1 and 5. A: Relative hepatic mRNA levels of lipin-1, lipin-1α, lipin-1β, and lipin-1α/β. B: Relative liver mRNA levels of Srsf3, Srsf3–full-length isoform lacking exon 4 (Iso1), Srsf3–alternative isoform including exon 4 (Iso2), and ratio of Srsf3-Iso2/Iso1. C: Real-time PCR analyses of liver tissues from normal livers and patients with alcoholic steatohepatitis (ASH). Data are expressed as means ± SEM. n = 5 to 12 mice (A and B); n = 7 (C, normal liver); n = 5 (C, ASH). ^∗P < 0.05 versus pair-fed Ad-shRNA controls; ^†P < 0.05 versus ethanol-fed Ad-shRNA; ^‡P < 0.05 versus pair-fed Ad-shSlu7; ^§P < 0.05, ^§§§P < 0.001 versus normal livers.

Figure 4

Slu7 knockdown modulates hepatic iron and zinc homeostasis and reduces oxidative stress in mice. Female C57BL/6J mice were pair fed either a control diet or an ethanol (E)–containing diet for 10 days, followed by single gavage of ethanol. During the 10-day chronic-plus-binge ethanol feeding period, Ad-shSlu7 or Ad-shRNA control (0.5 to 1.0 × 10⁹ active viral particles in 200 μL of phosphate-buffered saline) was given to mice twice on days 1 and 5. A: Liver total iron (Fe), ferrous (Fe²⁺), ferric (Fe³⁺) concentrations. B: Serum ferritin concentrations. C: Liver zinc (Zn), magnesium (Mg), manganese (Mn), and calcium (Ca). D: Liver malondialdehyde (MDA) contents. Data are expressed as means ± SEM. n = 5 to 12 mice. ^∗P < 0.05 versus pair-fed Ad-shRNA controls.

Figure 5

Slu7 knockdown attenuates hepatic NF-κB and nuclear factor of activated T cells 4 (NFATc4) activities in the ethanol administrated mice. Female C57BL/6J mice were pair fed either a control diet or an ethanol (E)–containing diet for 10 days, followed by single gavage of ethanol. During the 10-day chronic-plus-binge ethanol feeding period, Ad-shSlu7 or Ad-shRNA control (0.5 to 1.0 × 10⁹ active viral particles in 200 μL of phosphate-buffered saline) was given to mice twice on days 1 and 5. A: Representative Western blot analysis of liver nuclear NFATc4, acetylated (Ac) NF-κB, and NF-κB (on separate gels). The β-actin blot is the same as shown in Figure 2D. B: Relative liver protein levels of liver nNFATc4, Ac-NF-κB, and NF-κB. Data are expressed as means ± SEM (B). n = 5 to 12 mice. ^∗P < 0.05 versus pair-fed Ad-shRNA controls.

Figure 6

Ethanol (E) up-regulates hepatic Slu7 in mice and in patients with alcoholic steatohepatitis (ASH). A: Mice were pair fed either a control (Con) diet or an ethanol-containing diet for 10 days, followed by single gavage of ethanol. Western blot analysis of hepatic Slu7. B and C: Hepatic levels of SLU7 were determined in liver tissues from normal livers and patients with ASH. B: Relative hepatic mRNA levels of SLU7. C: Western blot analysis of hepatic SLU7. Data are expressed as means ± SEM (A–C). n = 7 (normal liver); n = 5 (ASH); n = 4 to 6 mice (A). ^∗P < 0.05 versus pair-fed controls; ^†††P < 0.001 versus normal livers. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 7

Proposed role of Slu7 in alcoholic liver injury. Chronic-plus-binge ethanol feeding induces hepatic Slu7 expression. Elevated Slu7 subsequently disrupts sirtuin (SIRT) 1, lipin-1, and serine/arginine-rich splicing factor (Srsf) 3 pre-mRNA splicing and/or expression, which, in turn, activates NF-κB and nuclear factor of activated T cells 4 (NFATc4), disturbs metal homeostasis, enhances reactive oxygen species (ROS), increases hepatic inflammation, and promotes alcoholic liver injury.