ERRγ suppression by Sirt6 alleviates cholestatic liver injury and fibrosis

Orphan nuclear receptor estrogen-related receptor γ (ERRγ) stimulates bile acid production; however, the role and the regulatory mechanism of ERRγ in cholestatic liver disease are largely unknown. This study identifies that Sirt6 is a deacetylase of ERRγ and suggests a potentially novel mechanism by which Sirt6 activation alleviates cholestatic liver damage and fibrosis through regulating ERRγ. We observed that hepatic expression of Sirt6 is repressed, whereas hepatic expression of ERRγ is upregulated in murine cholestasis models. Hepatocyte-specific Sirt6-KO mice were more severely injured after a bile duct ligation (BDL) than WT mice, and adenoviral reexpression of Sirt6 reversed liver damage and fibrosis as demonstrated by biochemical and histological analyses. Mechanistically, Sirt6 deacetylated ERRγ, thereby destabilizing ERRγ and inhibiting its transcriptional activity. Elimination of hepatic ERRγ using Ad-shERRγ abolished the deleterious effects of Sirt6 deficiency, whereas ERRγ overexpression aggravated cholestatic liver injury. Administration of a Sirt6 deacetylase activator prevented BDL-induced liver damage and fibrosis. In patients with cholestasis, Sirt6 expression was decreased, whereas total ERRγ and acetylated ERRγ levels were increased, confirming negative regulation of ERRγ by Sirt6. Thus, Sirt6 activation represents a potentially novel therapeutic strategy for treating cholestatic liver injury.


Animals
Male Sirt6 KO mice and wild-type littermates were subjected to bile duct ligation at 8-10 weeks of age. After midline laparotomy, the common bile duct was exposed and ligated with 6-0 silk sutures twice. In the sham operation, the bile duct was touched and the abdomen was closed with 6-0 suture and mice were allowed to wake up on a heating pad. On day 3 after bile duct ligation, some mice were sacrificed to check neutrophil infiltration, and on day 10, serum was collected and the liver was harvested to isolate primary hepatocytes or processed for qPCR, western blotting, and histology. Adenoviruses were administered 2 days before BDL surgery. MDL801 (Chemscene, Monmouth Junction, NJ, USA) dissolved in 4% DMSO + 48% PBS + 48% PEG400 was administered to mice intraperitoneally at a dose of 100 mg/kg body weight 1 day before BDL surgery and every other day after BDL surgery (five times).

Isolation of primary hepatocytes and Kupffer cells
Primary hepatocytes and Kupffer cells were isolated from 8-to 10-week-old male Sirt6 KO mice and wild type (WT) littermates. Anesthetized mice were intubated in the inferior vena cava, and the liver was perfused at a flow rate of 0.35 ml/min with calcium-free 10 mM HEPES buffer (pH 7.4) for 2 min, followed by perfusion with 15 ml of HEPES buffer containing 5 μg of collagenase IV (Sigma-Aldrich) and 5 mM calcium chloride. Hepatocytes were resuspended in Medium199 supplemented with 10% FBS, 10 units/ml penicillin, 10 μg/ml streptomycin, and 10 nM dexamethasone mixed with 42% Percoll and then centrifuged for 5 min at 1,300 rpm to remove dead cells. Live hepatocytes were plated at 1 × 10 6 cells/well in 6-well culture dishes. After 5 h, culture medium was changed.
To isolate Kupffer cells, hepatocytes were removed by centrifugation at 500 rpm for 3 min and washed 3 times. After the last wash, the supernatant was transferred to a new 50 ml tube and centrifuged for 7 min at 1,600 rpm. The cell pellet was then resuspended in 4 ml 17.5% OptiPrep (Sigma-Aldrich, St. Louis, MO, USA) and loaded carefully into the bottom of a new tube containing 2 ml 11.5% OptiPrep and 2 ml HBSS (Sigma-Aldrich). After centrifugation at 2,700 rpm for 17 min, the Kupffer cells-enriched layer was harvested from the 11.5% and 17.5% OptiPrep interphase. After washing with MACS buffer (Miltenyi Biotech, Paris, France) and blocking with anti-mouse CD16/32 (eBioscience, San Diego, CA, USA) for 20 min, cells were incubated with anti-mouse CD11b biotin (eBioscience) for 30 min at 4°C, then washed and incubated with anti-biotin microbeads (Miltenyi Biotech) for 20 min at 4°C. Cells were washed twice more using MACS buffer to remove excess microbeads. The supernatant was removed and the cell pellet was re-suspended in MACS buffer, and the cell suspension was placed in a magnetic column (Miltenyi Biotech) for separation. After washing with MACS buffer, the column was removed from the magnetic field, and the fraction containing the CD11b positive cells was eluted with MACS buffer. Cells were centrifuged at 1,800 rpm for 5 min, and the pellet was then resuspended in DMEM supplemented with 10% FBS and antibiotics and cells were plated at a density of 5 × 10 5 cells/well in a 12-well plate.

Western blots and co-immunoprecipitation
Cell lysates (10 μg) were separated using 7-14% SDS-PAGE and transferred to PVDF membranes. After blocking with 5% skim milk for 1 h, blots were probed with primary antibodies overnight at 4 ○ C. For co-immunoprecipitations, 600 μg protein was incubated with anti-ERRγ, anti-Ac-Lys, or anti-Sirt6 antibodies overnight at 4 ○ C followed by protein Gagarose for 2 h at 4 ○ C. Blots were probed with primary antibodies against Ac-Lys, Sirt6, or ERRγ, and antibody signals were detected using a Las-4000 imager (GE Healthcare Life Science, Pittsburgh, PA, USA).

RNA isolation and qPCR
Total RNA was extracted from frozen liver tissues or primary hepatocytes using TRIzol reagent (Invitrogen). RNA was precipitated with isopropanol, dried using 70% ETOH, and dissolved in diethyl pyrocarbonate-treated distilled water. First-strand cDNA was generated using the random hexamer primer provided in the first-strand cDNA synthesis kit (Applied Biosystems, Foster City, CA, USA). Specific primers were designed using PrimerBank (https://pga.mgh.harvard.edu/primerbank, Table S2). qPCR reactions were performed in a final volume of 10 µl containing 10 ng reverse-transcribed total RNA, 200 nM forward and reverse primers, and PCR master mixture. qPCR was performed in 384-well plates using an ABI Prism 7900HT Sequence Detection System (Applied Biosystems).

Histology
Liver tissues were removed and placed immediately in fixative (10% formalin solution in 0.1 M PBS). Histological sections (5 μm) were cut from formalin-fixed paraffin-embedded tissue blocks. Tissue sections were stained with hematoxylin-eosin (H&E) under standard conditions. Immunohistochemical staining was performed using the DAKO Envision system (DAKO, Carpinteria, CA, USA). After deparaffinization and hydration, tissue sections were subjected to a microwave antigen-retrieval procedure in 0.01 M sodium citrate buffer. After blocking endogenous peroxidase, sections were incubated with protein blocking buffer to block nonspecific staining. Sections were incubated with primary antibodies against F4/80, Gr-1, and CK19 at 4 ○ C overnight and secondary antibody for 30 min at room temperature. Peroxidase activity was detected with 3-amino-9-ethyl carbazole. The number of positive staining cells was counted in five microscopic fields (magnification, 100 or 200×) from each sample. TUNEL staining was performed following the manufacturer's instructions.

LC-MS/MS
After staining with colloidal Coomassie blue, protein gels were subjected to in-gel tryptic digestion as described by Shevchenko and co-workers (3). The resulting tryptic peptides were analyzed by LC-MS/MS. All mass analyses were performed on an LTQ-orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) equipped with a nanoelectrospray ion source. To separate the peptide mixture, we used a C18 reverse-phase HPLC column (150 mm × 75 μm ID) and an acetonitrile/0.1% formic acid gradient from 13 to 30% for 60 min at a flow rate of 300 nl/min. For MS/MS analysis, the precursor ion scan MS spectra (m/z 400~2,000) were acquired in the Orbitrap at a resolution of 60,000 at m/z 400 with an internal lock mass. The 20 most intensive ions were isolated and fragmented by collision-induced dissociation.

LC-MS/MS data processing for protein identification
All MS/MS samples were analyzed using the Sequest Sorcerer platform (Sagen-N Research, San Jose, CA, USA). Sequest was set up to search the mouse protein sequence database (51552 entries, UniProt, http://www.uniprot.org/), which includes frequently observed contaminants assuming digestion with trypsin. Sequest was searched with a fragment ion mass tolerance of 0.60 Da and a parent ion tolerance of 10.0 ppm. Carbamidomethyl of cysteine was specified in Sequest and X! Tandem as a fixed modification. Deamidation of asparagine and glutamine; methylation of glutamic acid, lysine, glutamine, and arginine; oxidation of methionine; acetylation of lysine, serine, and threonine, and the N-terminus; and phosphorylation of serine, threonine, and tyrosine were specified in Sequest and X! Tandem as variable modifications. Additionally, Glupyro-Glu at the N-terminus, ammonia loss of the N-terminus, and Glnpyro-Glu at the N-terminus were also specified in X! Tandem as variable modifications. Scaffold (Version 4.9.0, Proteome Software, Portland, OR, USA) was used to validate MS/MS-based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 99.0% probability to achieve a false discovery rate less than 1.0% by the Scaffold Local FDR algorithm. Protein identifications were accepted if they could be established at greater than 99.0% probability to achieve an FDR less than 1.0% and contained at least two identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm (4). Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. Table S1. Biochemical parameters of study subjects AST, aspartate aminotransferase; ALT, alanine aminotransferase; TB, total bilirubin; CB, conjugated bilirubin; ALP, alkaline phosphatase; GGT, γ-glutamyltransferase; Dx, diagnosis; NA, not applicable; IHD St, intrahepatic duct stone; IHCC, intrahepatic cholangiocarcinoma; * These patients underwent preoperative biliary drainage procedures (endoscopic retrograde cholangio-pancreatography for patient 6 and percutaneous transhepatic biliary drainage for patient 8) before segmental hepatectomy. # Metavir score was determined by histopathological evaluation in H&E stained liver sections (F0, no fibrosis; F1, portal fibrosis without septa; F2, portal fibrosis with few septa; F3, portal fibrosis with numerous septa; F4, cirrhosis).  Values are means ± SEM. Comparisons were made using paired, two-tailed Student's t test. ** , p<0.01 versus control.