Digoxin Suppresses Pyruvate Kinase M2-Promoted HIF-1α Transactivation in Steatohepatitis

Abstract

Sterile inflammation after tissue damage is a ubiquitous response, yet it has the highest amplitude in the liver. This has major clinical consequences, for alcoholic and non-alcoholic steatohepatitis (ASH and NASH) account for the majority of liver disease in industrialized countries and both lack therapy. Requirements for sustained sterile inflammation include increased oxidative stress and activation of the HIF-1α signaling pathway. We demonstrate the ability of digoxin, a cardiac glycoside, to protect from liver inflammation and damage in ASH and NASH. Digoxin was effective in maintaining cellular redox homeostasis and suppressing HIF-1α pathway activation. A proteomic screen revealed that digoxin binds pyruvate kinase M2 (PKM2), and independently of PKM2 kinase activity results in chromatin remodeling and downregulation of HIF-1α transactivation. These data identify PKM2 as a mediator and therapeutic target for regulating liver sterile inflammation, and demonstrate a novel role for digoxin that can effectively protect the liver from ASH and NASH.

Keywords: HIF-1α; NASH; ROS; alcohol; digoxin; liver; pyruvate kinase M2; steatohepatitis; sterile inflammation; therapy.

Copyright © 2018 Elsevier Inc. All rights reserved.

Figures

Figure 1. Digoxin reduces endotoxin-induced hepatic damage

(A–G) Mice were injected with indicated dosages of digoxin or vehicle, followed by LPS/D-GalN 1h later and examination after a further 6 h. (A–B) Dose-dependent reduction of hemorrhage, cellular necrosis, neutrophilic infiltrate and serum alanine transaminase in digoxin treated animals. (C–E) Reduced mRNA levels of inflammatory genes in liver tissues by digoxin as measured by qRT-PCR. (F) Reduced serum levels of IL-1β protein by digoxin as measured by ELISA. (G) Reduced protein expression level of HIF-1α, caspase-1 (P10), and pro IL-1β genes by digoxin in liver as measured by western blot Data represent mean ± SD (n=5). *p

Figure 2. Digoxin prevents chronic hepatic damage and inflammation in high fat diet model of non-alcoholic steatohepatitis

(A–E) Mice were injected Intraperitoneally with indicated dosages of digoxin or vehicle twice a week with concurrent HFD feeding for 12 weeks. (A) Digoxin dose-dependently reduces HFD-induced steatosis by histological analysis of H & E stained sections. (B) Digoxin reduces HFD-induced hepatic steatosis and inflammation as scored quantitatively from H & E stained sections. (C) Digoxin dose-dependently prevents HFD-induced hepatocellular damage as measured by the serum level of ALT. Reduced liver neutrophil (D) and monocyte (E) infiltration by digoxin in HFD as measured by Ly6G/CD11b and Ly6C/CD11b positive cells in non-parenchymal cell populations using flow cytometry analysis. Data represent mean ± SD (n=4–5). *p

Figure 3. Digoxin reduces hepatitis steatosis and inflammation when given therapeutically

(A–H) Mice were fed with HFD for 5 weeks, and then given intraperitoneal injection of digoxin (1 mg/kg) or vehicle twice a week with continued HFD feeding for a total of 12 weeks. (A) Digoxin reduces HFD-induced hepatic steatosis from histological analysis by H & E stained sections. (B) Digoxin reduces HFD-induced hepatic steatosis and inflammation as scored quantitatively from H & E stained sections. (C) Reduced liver neutrophil, and (D) monocyte infiltration by digoxin in HFD as measured by Ly6G/CD11b and Ly6C/CD11b positive cells in non-parenchymal cell populations using flow cytometry. (E) Digoxin reduces HFD-induced hepatocellular damage as measured by the serum ALT. (F–G) Digoxin attenuates hepatic steatosis as measured by oil red staining with quantification. (H) Digoxin reduces liver TG content as measured by chemical analysis. All data throughout the figure are shown as the mean ± SD from 4–5 mice in each group. *p

Figure 4. Digoxin improves cellular redox status from a wide range of stimuli

(A) Mice were injected with digoxin (1 mg/kg) or vehicle for 1 h, and then injected with LPS/D-GalN for 6 h. DHE was given i.v.15 min before sacrifice. Reduction in liver oxidative stress monitored by fluorescence microscopy is shown (left) and quantification in accounting multiple views of positive areas using Image J (right). (B) Mice were intraperitoneally injected with digoxin (1 mg/kg) or vehicle twice a week with concurrent HFD feeding for 12 weeks. DHE was given i.v. 90 min before analysis. Reduction in liver oxidative stress was monitored by fluorescence microscopy (left) and quantified using Image J (right). (C) Mice were treated as Figure 4A, except without DHE injection. Peritoneal neutrophils were harvested by lavage, and stained with CM-H2DCFDA followed by kinetic reading of ROS with fluorescent plate reader. (D–E) THP-1 cells were treated with digoxin (10 nM) or vehicle for 3 h and then LPS (1μg/ml) (D), or H2O2 (10 mM) (E), for additional 3 h. MitoSOX ™ Red mitochondrial superoxide indicator was added 30 min before analysis to monitor mitochondrial ROS. The cells were collected and washed following flow cytometry analysis. (F) THP-1 cells were treated with indicated dosages of digoxin or vehicle for 3 h and then H2O2 (10 mM) for additional 3 h. CM-H2DCFDA was added in last 30 min to monitor intracellular ROS. Cells were collected and washed following flow cytometry. (G) Human primary neutrophils were isolated and treated with dosages of digoxin or vehicle for 3 h. The cells were then incubated with CM-H2DCFDA for 30 min, and washed following kinetic analysis of intracellular ROS with fluorescent plate reader. (H) Human primary neutrophils were isolated and treated with dosages of digoxin, or vehicle for 3 h. Cells were then incubated with JC-1 for 30 min to monitor mitochondrial membrane potential. The kinetic analysis of mitochondrial membrane potential was performed using a fluorescent plate reader.

Figure 5. Digoxin inhibits the transcription of HIF-1α signature genes in inflamed liver

(A–D) Mice were intraperitoneally injected with digoxin (1 mg/kg) or vehicle twice a week with concurrent HFD feeding for 12 weeks. (A) Microarray analysis of liver tissue was performed and digoxin regulated genes further narrowed down into multiple categories based on PANTHER analysis. (B) Significance of biological functions refers to the –log (p-value) obtained by the Ingenuity Pathway Analysis (IPA). Biological functions with a greater than 2-fold reduction are shown. (C) Heat map showing the fold changes of digoxin down-regulated genes based microarray data (digoxin treated group versus vehicle group in HFD model). (D) Validation of gene expression for HIF-1α signatures from liver tissue was performed by RT-PCR. (E) HEK293 T cells were transfected with HRE-luciferase construct for 12 h, and then cobalt chloride treatment in the presence of digoxin (10 nM) for additional 12 h. HRE luciferase activity was measured by dual-luciferase reporter assay. The relevant luciferase activity was based on renilla control. (F) Primary neutrophils were collected from HIF-1αf/f-LysM (HIF-1α-cKO), and wild type controls. The cells were treated with digoxin as indicated dosages for 3 h, and then CM-H2DCFDA staining. The fluorescence density was measured kinetically by plate reader and the data showed at 6 h point. (G) Bone marrow derived macrophages were collected from HIF-1α-cKO and wild type controls. The cells were stimulated with LPS for 6 h and challenged with ATP for 30 min. The FACS analysis was performed by Mito-Tracker Green and Mito-Tracker Deep Red. All data throughout the figure are shown as the mean ± SD from 5 mice in each group. *p

Figure 6. Digoxin directly binds to PKM2 and inhibits PKM2-HIF-1α axis activation

(A) Whole cell lysates from LPS stimulated Raw264.7 cells were applied on digoxin-immobilized affinity resin (upper). LC-Mass spectrum (LC-MS/MS) of digoxin bound proteins identified PKM2 (middle), and the detected peptide sequence of PKM2 was identical and shown as red (bottom). (B) Cell lysates from LPS stimulated Raw264.7 cells were applied on digoxin immobilized affinity resin and eluted proteins examined by western blot using anti-PKM2 antibody. (C) Cell lysates from LPS stimulated Raw264.7 cells were incubated with or without digoxin (10 mM) for overnight, and then applied on digoxin immobilized affinity resin and eluted proteins examined by western blot using anti-PKM2 antibody. (D) PKM2 enzyme activity was measured in the presence of digoxin (50 nM) and PKM2 activator (Pka 550602) in pancreatic β cells (INS). (E–G) HEK293T cells were co-transfected with HRE-luciferase (E, F), or NFκB-luciferase (G) construct for 12 h, treated with digoxin for additional 12 h. The luciferase activity was measured by dual-luciferase reporter assay and normalized to renilla control. (H–P) Raw264.7 cells were treated with digoxin (50 nM) for 3 h, and the stimulated with LPS for 6 h, and whole cell lysates were assayed by ChIP-PCR using anti-PKM2 antibody (H–L), and anti-histone H3 (M–P), and the DNA binding was determined by qPCR.

Figure 7. Digoxin disrupts the protein interaction of PKM2 to histones in the nucleus

(A) Nuclear protein lysates from LPS stimulated Raw264.7 cells were immune-precipitated using anti-PKM2 antibody. PKM2 bound proteins were identified by LC-MS/MS. The PKM2 bound histones were extracted as A. (B) The nuclear cell lysates from (A) were applied for PKM2 immune-precipitation and blotted with indicated antibodies. (C) PKM2, HIF-1α stable knockout Raw264.7 macrophages were stimulated with LPS in the presence or absence of digoxin (50 nM) for 6 h. Reduced mRNA levels of inflammatory genes as indicated by digoxin were measured using qRT-PCR. The data are representative as the mean ± SD from quadruplicate wells of three independent experiments in one colony and two more independent experiments in additional colony of stable cells. (D) Proposed mechanism of digoxin on PKM2 triggered transactivation.