A disintegrin and metalloprotease 10 (ADAM10) is a central regulator of murine liver tissue homeostasis

A Disintegrin And Metalloprotease (ADAM) 10 exerts essential roles during organ development and tissue integrity in different organs, mainly through activation of the Notch pathway. However, only little is known about its implication in liver tissue physiology. Here we show that in contrast to its role in other tissues, ADAM10 is dispensable for the Notch2-dependent biliary tree formation. However, we demonstrate that expression of bile acid transporters is dependent on ADAM10. Consequently, mice deficient for Adam10 in hepatocytes, cholangiocytes and liver progenitor cells develop spontaneous hepatocyte necrosis and concomitant liver fibrosis. We furthermore observed a strongly augmented ductular reaction in 15-week old ADAM10Δhep/Δch mice and demonstrate that c-Met dependent liver progenitor cell activation is enhanced. Additionally, liver progenitor cells are primed to hepatocyte differentiation in the absence of ADAM10. These findings show that ADAM10 is a novel central node controlling liver tissue homeostasis. Highlights: Loss of ADAM10 in murine liver results in hepatocyte necrosis and concomitant liver fibrosis. ADAM10 directly regulates expression of bile acid transporters but is dispensable for Notch2-dependent formation of the biliary system. Activation of liver progenitor cells is enhanced through increased c-Met signalling, in the absence of ADAM10. Differentiation of liver progenitor cells to hepatocytes is augmented in the absence of ADAM10.


Histological analysis
Liver tissues were fixed in phosphate buffered saline (PBS) containing 4% formaldehyde, dehydrated and embedded in paraffin, and cut into 4 µm-thick sections. Sections were stained with haematoxylin-eosin or Sirius Red using standard protocols. Additional sections were stained immunohistochemically. Primary antibodies were detected by biotinylated secondary antibodies and subsequently stained using a peroxidase DAB kit (Dako, Hamburg, Germany). Antibody specifications are listed in Supplementary Table 2. Haematoxylin was used for counterstaining. Necrotic, collagen containing and Ki67 + areas were quantified by morphometry (ImageJ, NIH). For morphometric assessment, 10 nonoverlapping fields at 10x magnification (necrosis) or 20x magnification (collagen, Ki67) per section were evaluated for each mouse. For immunofluorescence analyses, sections were deparaffinised, rehydrated and subjected to Proteinase K-mediated enzymatic or heatinduced antigen retrieval in either citrate buffer (pH6) or EDTA buffer (pH9). Sections were incubated with primary antibodies (Supplementary

Determination of total biliary acids
Total biliary acids from serum were determined using the total bile acids assay kit (Diazyme, Dresden, Germany) according to manufacturer's instructions. Absorption was measured at 405 nm in a microplate reader (Tecan, Mainz, Germany) or an AU480 (Beckman-Coulter, Germany) biochemical analyser.

BMOL tube formation assay
A 24-well plate was coated with 300 µl of slowly thawed Matrigel (Corning, Bedford (MA), USA) per well. Matrigel had a protein concentration of > 10 mg/ml and was left for at least 3h at 37°C, 5% CO 2 , and 95% RH to solidify. 300 µl of a 2x10 5 cells/ml cell suspension plus the respective inhibitors were seeded on top of the Matrigel in each pre-coated well.
Cells were incubated for 24h to form tubes. Pictures of formed tubes were taken with a AZ100 microscope with a DS-Fi2 camera (Nikon, Düsseldorf, Germany) and tubelength and branching was evaluated with the ImageJ plugin 2D Skeleton.
Transfection was performed at 0 h and again at 48 h after seeding using Interferin as transfection reagent (Hiss Diagnostics, Freiburg, Germany). Cells were used for stimulation experiments 72 h after seeding.

BMOL cell differentiation
2.5 x 10 5 BMOL cells were seeded either in six well plates or on cover slips and transfected with 50 pmol control or mADAM10 siRNA (Life Technologies, Darmstadt, Germany), respectively. Transfection was performed on day -4 and -2 and day 5 of differentiation ( Figure S5C) using Interferin as transfection reagent (Hiss Diagnostics, Freiburg, Germany). Four days after intital cell seeding, medium was changed to differentiation medium as described previously (Tirnitz-Parker et al., 2007), which was replaced again on day 3, 5 and 7 ( Figure S5C). Cells were harvested on day 10 of differentiation and either subjected to lysis, RNA isolation or immunofluorescence staining.

Protein isolation and immunoblotting
Samples were lysed in RIPA buffer supplied with 50 mM NaF, protease and phosphatase inhibitors. Proteins were separated by electrophoresis on 10 % SDS gels and transferred to PVDF or nitrocellulose membranes. Membranes were incubated with primary antibodies (Supplementary Table 2) overnight at 4°C and with horseradish peroxidase-conjugated secondary antibodies at room temperature for 1h. An ECL substrate kit was used for detection (Thermo Scientific, St. Leon-Rot, Germany).

Gene expression analysis
Total RNA was isolated from cryopreserved whole liver tissue using TRIzol

Hydroxyproline assay
The assay was performed as described previously (Uchinami et al., 2006).

Multiplex ELISA
Levels of HGF were measured by multiplex assay with a Bio-Plex mouse array (Bio-Rad Laboratories, Prague, Czech Republic) using the Bio-Plex 200 System (Bio-Rad Laboratories, Prague, Czech Republic). Mouse HGF multiplex beads were produced by amine coupling reaction with primary antibody from R&D (Minneapolis, USA) and validated according to Luminex validation procedures and recommendations.