Hepatic Deletion of Janus Kinase 2 Counteracts Oxidative Stress in Mice

Genetic deletion of the tyrosine kinase JAK2 or the downstream transcription factor STAT5 in liver impairs growth hormone (GH) signalling and thereby promotes fatty liver disease. Hepatic STAT5 deficiency accelerates liver tumourigenesis in presence of high GH levels. To determine whether the upstream kinase JAK2 exerts similar functions, we crossed mice harbouring a hepatocyte-specific deletion of JAK2 (JAK2Δhep) to GH transgenic mice (GHtg) and compared them to GHtgSTAT5Δhep mice. Similar to GHtgSTAT5Δhep mice, JAK2 deficiency resulted in severe steatosis in the GHtg background. However, in contrast to STAT5 deficiency, loss of JAK2 significantly delayed liver tumourigenesis. This was attributed to: (i) activation of STAT3 in STAT5-deficient mice, which was prevented by JAK2 deficiency and (ii) increased detoxification capacity of JAK2-deficient livers, which diminished oxidative damage as compared to GHtgSTAT5Δhep mice, despite equally severe steatosis and reactive oxygen species (ROS) production. The reduced oxidative damage in JAK2-deficient livers was linked to increased expression and activity of glutathione S-transferases (GSTs). Consistent with genetic deletion of Jak2, pharmacological inhibition and siRNA-mediated knockdown of Jak2 led to significant upregulation of Gst isoforms and to reduced hepatic oxidative DNA damage. Therefore, blocking JAK2 function increases detoxifying GSTs in hepatocytes and protects against oxidative liver damage.

2 incubated in Coon's F-12 medium (Sigma) for 10 minutes at 37°C. For laser scanning microscopy liver slices were stained simultaneously with fluorescent dyes sensitive to mitochondrial membrane potential (MitoTracker Deep Red 633, ex/em 630/662 nm), mitochondrial ROS (CM-H2XROS, ex /em 550/599 nm), and cytoplasmic ROS (DCF-DA ex /em: 492/517-527 nm). Staining was performed according to the manufacturer's protocol (Invitrogen). Thereafter, liver slices were transferred on cover glasses (Carlroth) and fixed to the optical table. Imaging was performed with an inverted confocal microscope (LSM 510, Zeiss, Germany) and x630 oil immersion objective. Image analysis was carried out with the histogram toolbar (LSM 510,Zeiss,Germany). Analysis was performed on hepatocytes excluding hepatic infiltrates. Absolute fluorescence intensity was defined as Mean x Area + Area x Threshold.
Transmission electron microscopy. For electron microscopy mouse livers were cut into 2 mm pieces and fixed overnight in 1.6% glutaraldehyde. Pictures were made with a magnification at 8,000x using a transmission electron microscope.
Histology and immunohistochemistry and quantification. Sections prepared from paraffinembedded formalin-fixed organ specimens were stained with hematoxylin-eosin (H&E).
All quantifications were performed with 10 fields/liver from 5-10 mice/genotype.  1 μg of total RNA from control and mutant animals (n≥5/genotype) was reverse-transcribed into complementary DNA using the Revert Aid cDNA synthesis kit (Thermo Fisher Scientific).
qRT-PCR was performed using the SYBR green method. mRNA levels were normalised for glyceraldehyde 3-phosphate dehydrogenase (Gapdh), and relative abundance was calculated using the ΔCt method (gene-specific expression level relative to that of an endogenous housekeeping gene). Each reaction condition was performed in triplicate. Primer sequences are listed in Supplementary Table 1. Cell Culture. Hepatocytes of p19 ARF-/mice were isolated and propagated as described 1 .
p19 ARF-/hepatocytes (we used passage 10-30) are proficient to lower growth-suppressive functions of p53 and due to p19 deficiency primary cells can bypass cellular senescence.

GH and ruxolitinib treatment
Hepatocytes were treated for 20 minutes with 0.5 µg/ml human growth hormone (hGH) (ImmunoTools, Friesoythe, Germany) or with 3 µM ruxolitinib (CaymanChemical, Michigan, 5 USA) at indicated time points. Ruxolitinib was freshly dissolved in DMSO at a concentration of 5 mM for individual kinetics. In all controls, equal amounts of DMSO were added to media. After the indicated treatments, cells were washed twice with PBS, fixed with 4% (vol/vol) formaldehyde for 15 minutes at room temperature followed by three washes with PBS (pH 8,5 min each). Slides were blocked for 1 hour at room temperature in blocking buffer (PBS pH 8 / 5% donkey serum / 0.3% Triton X-100) before incubated with pH2AX antibodies (1:400, Cell Signalling #9718) overnight at 4°C temperature in antibody dilution buffer (PBS pH 8 / 1% BSA / 0.3% Triton X-100). Slides were washed three times in PBS before incubation in the dark with Alexa Fluor 488-conjugated secondary antibodies (1:400 in antibody dilution buffer, Invitrogen, A21206) for 2 hours at room temperature. After three washes in PBS, slides were mounted in Vectashield media containing DAPI (4′,6-diamidino-2-phenylindole; Vector Laboratories). Cell imaging and data collection (20x magnification) was performed with an EVOS FL Cell Imaging System (ThermoFisher). The exposure length and gain were maintained at a constant level for all samples. Quantification of pH2AX positive cells was performed using ImageJ.

Mice. All mice were kept under standardised conditions at the Decentralised Biomedical
Facilities, Medical University of Vienna (Vienna, Austria). Mice were kept on a 12 hours lightdark cycle and fed a standard diet (#V1126-000, ssniff Spezialdiäten GmbH, Soest, Germany).