Interleukin-6 is required for cell cycle arrest and activation of DNA repair enzymes after partial hepatectomy in mice
- Shingo Tachibana†1, 2,
- Xiuying Zhang†1, 3,
- Kazushige Ito†1, 2,
- Yoshihiro Ota1,
- Andrew M Cameron1,
- George Melville Williams1 and
- Zhaoli Sun1Email author
© Tachibana et al.; licensee BioMed Central Ltd. 2014
Received: 15 October 2013
Accepted: 5 November 2013
Published: 3 February 2014
Interleukin-6 (IL-6) has been shown to be vital for liver regeneration, however the specific mechanisms and factors involved remain incompletely defined. The present study aimed to investigate whether IL-6 exerts its protective effects via arresting the cell cycle allowing base excision and repair of oxidized DNA after hepatectomy.
Following seventy percent partial hepatectomy (PH) in wild type (WT) mice IL-6 serum levels increased reaching peak levels at 3 hours. This was associated with markers of cell cycle arrest as p21 expression was increased and cyclin D1 and proliferating cell nuclear antigen (PCNA) expression decreased. In the absence of IL-6, markers of cell cycle arrest were absent and the number of bromodeoxyuridine (BrdU) positive cells was significantly higher at 28, 32 and 36 hours after PH. The mRNAs for DNA repair enzymes, including Neil-1, 8-oxodGTPase, OGG1, Apex1, and UDG (DNA glycosylase) were increased 2 to 4 fold in WT mice at 6 and/or 12 hours after PH compared to IL-6 knockout (KO) mice. The protein levels of Neil1 and OGG1 were also significantly increased in WT mice compared to KO mice. Pathological changes were far greater and survival was less in IL-6 KO mice than in WT mice. Administration of IL-6 in KO mice restored p21 and DNA repair enzyme expression to wild-type levels and survival was improved.
IL-6 caused cell cycle arrest and delayed proliferation during the first day after PH. This delay was associated with the activation of DNA repair enzymes resulting in accurate replication and restoration of hepatic mass.
KeywordsIL-6 Cell cycle arrest Oxidative DNA damage and repair Partial hepatectomy Liver regeneration Mice
Liver regeneration in response to partial resection is an efficient and precisely regulated process during which the surviving liver cells proliferate to reconstitute the liver. In rats, the two third partial hepatectomy (~70% PH) induces a compensatory hyperplasia of the remaining lobes that restores the original liver mass within 7 to 10 days . Resection of half of the human liver is followed within 2–4 weeks by full restoration of the liver structure, size, and function [2, 3]. However, even in patients without pre-existing liver disease, more significant liver resections, i.e. greater than 75%, result in an increasing perioperative mortality .
Interleukin (IL)-6 is a pleiotropic cytokine, which has been shown to provide liver protection in various settings [5–10]. Liver regeneration is impaired in IL-6 knockout mice , while IL-6 overexpression is involved in delayed liver regeneration . While IL-6 has been shown to be vital for liver regeneration [6, 13, 14], the specific mechanisms and factors involved remain incompletely defined. Identifying the molecular signals that promote regeneration may well lead to therapies that would aid recovery from massive liver injury or surgery.
Over stimulation of TNF-α and increased sensitivity to endotoxin (lipopolysacharides) have been proposed  as possible mechanisms for the intracellular production of ROS and lipid peroxidation known to follow 70% PH. While low amounts of ROS may play important roles in liver regeneration after PH, excessive amounts of reactive oxygen species (ROS) are likely to damage cellular DNA. The oxidative injury is more dramatic after large (87%) hepatectomy . Accordingly, we reasoned if the DNA is significantly oxidized and mutated, stringent, robust repair mechanisms would be required to enable injured hepatocytes to proliferate efficiently. We hypothesized that IL-6 exerts its protective effects via arresting the cell cycle allowing base excision and repair of oxidized DNA after hepatectomy.
IL-6 Is Important for Survival after 70% PH in Mice
Increased oxidative DNA damage in IL-6 KO Mice after PH
Using a competitive ELISA method, levels of 8-OHdG in total DNA were determined after PH and were found significantly and equally elevated in the DNA of the livers from both WT and IL-6 KO mice at 24 hours (Figure 2C). Interestingly, 8-OHdG levels in the DNA from WT mice decreased significantly at 48 hours after PH, whereas 8-OHdG levels from KO mice remained significantly elevated.
IL-6 Regulates cell cycle at early time after partial hepatectomy
Activation of DNA repair enzymes occurred in WT mice but not in IL-6 KO mice after 70% PH, and IL-6 pretreatment restored cell cycle arrest and increased DNA repair enzymes.
The expected burst of IL-6 secretion was present after 70% PH, but instead of finding robust measures of early proliferation we found depressed BrdU incorporation and evidence of cell cycle arrest as p21 expression was increased and cyclin-D1 and PCNA expression was depressed. IL-6 KO mice had the reverse pattern with greater early incorporation of BrdU, absent p21 and increased cyclin-D1 and PCNA levels. To explain this seeming paradox where normal mice had evidence of cellular slow down and the IL-6 deficient mice were geared to replicate, we proposed that cell cycle arrest was needed to create a check point to allow for activation of DNA repair enzymes and to have them excise and replace oxidized DNA thereby permitting accurate replication.
Using measurements of the stable oxidized adduct 8-OHdG as an indication of DNA oxidative injury, we confirmed that significant oxidative injury occurred equally in both IL-6 KO and WT mice at 24 hours after PH. However, by day two there was little 8-OHdG in the WT mice and much more in the IL-6 KO mice. This was associated with striking differences in histology as the WT mice displayed normal histology while the IL-6 KO mice had extensive areas of necrosis. The best explanation for this difference is that the absence of a check point in the IL-6 KO mice resulted in rapid but faulty DNA replication leading to apoptosis/necrosis.
The mechanism leading to cell cycle arrest is likely related to the increased expression of p21 found in the WT mice at 6 and 24 hours after 70% PH compared to IL-6 KO mice. We have shown in previous studies [16–19] that IL-6 significantly increased phosphorylated STAT3, which stimulated p21 transcription [20–23] resulting in G-1 arrest. We propose that normal mice had sufficiently high levels of IL-6 after 70% PH to increase p21 production via activated STAT3 thereby leading to cyclin D-1 and PCNA expression and to G-1/S arrest. This hypothesis is supported by the finding that rIL-6 stabilized steatotic hepatocytes from obese animals by normalizing PCNA expression (G1 phase) and not by increasing DNA synthesis (BrdU, S phase) .
We have found that the expression of p21 was significantly higher in WT mice at 6 and 24 hours, but not 12 hours after PH compared to IL-6 KO mice which had minimal if any p21 expression. Interestingly, the expression of p21 was increased only at 6 and 24 hours in IL-6 KO mice with SC injection of IL-6. It has been reported that two waves of STAT3 activation were observed after PH, the first in endothelia and the second in hepatocytes, and STAT3 activation was positively correlated with p21 protein expression . Two peaks of p21 expression at early time after PH might be due to IL-6 dependent activation of STAT3.
Direct evidence supporting the key role of DNA repair comes from the finding that the principle known enzymes for DNA repair, OGG-1, 8-oxo-GTP, Neil-1 were stimulated in the WT mice but absent in the IL-6 KO mice following 70% PH. PARP which repairs single strand breaks most commonly caused by per oxidation was also found increased at early time points in WT mice. The crucial role of IL-6 is supported by the restoration of early cell cycle arrest in IL-6 KO mice and subsequent benefit by a single injection 30 minutes before 70% PH.
Other studies have also linked IL-6 with DNA repair. Efferth et al.  reported that IL-6 improved DNA repair in melphalan-induced DNA damaged human multiple myeloma cells. Other reports have shown that IL-6 mRNA is stimulated after DNA strand breaks  and persistent DNA damage triggers senescence-associated inflammatory cytokine secretion including IL-6 [28, 29]. We have shown that activation of DNA repair enzymes occurred in WT mice and could be restored in IL-6 KO mice by IL-6 injection just before 70% PH.
In conclusion, we provide evidence that the essential role of IL-6 in liver regeneration after 70% PH is the induction of cell cycle checkpoints and activation of DNA repair enzymes in the remaining liver. IL-6 promoted DNA repair, but was not a requirement for liver cell proliferation. Cell cycle arrest permitted time for the transcription of DNA repair enzymes, DNA base excision repair and subsequent accurate cellular replication.
Materials and methods
Animals and partial hepatectomy
Male C57BL/6 J background IL-6 KO mice and control C57BL/6 J (WT) mice weighing 20–25 g were purchased from the Jackson Laboratory (Bar Harbor, ME). Animals were maintained in a pathogen-free facility of The Johns Hopkins Medical Institutions. Animals were cared for according to NIH guidelines and under a protocol approved by the Johns Hopkins University Animal Care Committee. For the IL-6-injected group, IL-6 KO mice were given a subcutaneous injection with recombinant IL-6 at a dose of 500 ng/g of animal weight thirty minutes before hepatectomy. Animals were anesthetized with isofluorane. After a midline laparotomy, the left and middle lobes (~70%) of the liver were ligated at the base and removed. The abdominal wall and skin were sutured separately. After surgery, animals were immediately given free access to water. Following surgery, mice were sacrificed, and livers and caval blood samples were collected at predetermined time points after operation. Harvested livers were weighted to assess regeneration, and portions of liver tissue were either fixed in 10% neutralized formalin for histological evaluation or were snap frozen in liquid nitrogen and maintained at −80°C until homogenization for various biochemical assays. For assessment of hepatic proliferation, BrdU was injected (50 mg/kg intraperitoneal (IP)) 2 hours prior to harvesting of liver.
Analysis of alanine aminotransaminase and aspartate aminotransaminase activity
Acute liver injury was quantified by measurement of serum alanine aminotransaminase (ALT) and aspartate aminotransaminase (AST) levels using an automated enzyme assay.
Cut sections of 4 μm were prepared from formalin-fixed paraffin-embedded tissues for immunohistochemistry. Each representative section was stained with hematoxylin-eosin (H&E). Immunohistochemical staining was performed with the avidin-biotin-peroxidase complex method, using Vectastain ABC kits (Vector Laboratories, Burlingame, CA) according to the methods described before [19, 28, 30]. The following antibodies were used: mouse anti-BrdU 1monoclonal antibody (Molecular Probes) (1:200); anti-PCNA antibody (Cell Signaling) (1: 5000); polyclonal anti-OGG1 antibody (1:100) (Novus Biological, Littleton, CO); monoclonal anti-p21 antibody (1:200) and polyclonal anti-Neil1 antibody (1:100) (Cell Signaling Technology, Boston, MA). The antibodies were incubated either at 4°C overnight or at room temperature for 1.5 hours. Immunohistochemistry staining of bromodeoxyuridine (BrdU) was performed by using a BrdU incorporation assay kit (BD Biosciences).
ELISA assay for measurement of 8-OHdG Levels in DNA
The DNA was extracted using the DNeasy tissue kit (Qiagen, Santa Clara, CA) according to the manufacturer’s instruction . The levels of 8-OHdG in nucleoside samples were used for the determination of 8-OHdG by a competitive ELISA kit (8-OHdG Check, Institute for the Control of Aging). The determination range was 0.125–10 ng/ml or 0.5–200 ng/ml. The levels of 8-OHdG were expressed as amounts of 8-OHdG (ng) per milligram of DNA.
Western blot analysis
Tissues were homogenized in lysis buffer (30 mmol/L Tris, pH 7.5, 150 mmol/L sodium chloride, 1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L sodium orthovanadate, 1% Nonidet P-40, 10% glycerol) at 4°C, vortexed and centrifuged at 16,000 rpm at 4°C for 10 minutes. The supernatants were mixed in SDS sample loading buffer (NuPAGE, Invitrogen), 70°C for 10 minutes, and then subjected to SDS-PAGE. After electrophoresis, proteins were transferred onto PVDF membranes and blotted against primary antibodies (anti-p21 (1:2000), anti-Neil1 (1:1000), anti-OGG1 (1:1000), anti-PARP (1:1000, #9542, Cell Signaling Technology) and anti-β-actin (1:1000)) for 1 hour at room temperature. Membranes were washed with TPBS (0.05% [vol/vol] Tween 20 in phosphate-buffered saline [pH 7.4]) and incubated with a 1:4000 dilution of horseradish peroxidase-conjugated secondary antibodies for 45 minutes. Protein bands were visualized by an enhanced chemiluminescence reaction (ECL-plus, Bio-Rad).
Real-Time PCR Analysis for DNA repair enzyme mRNA
The primer sets for amplification of mouse Neil1 were 5’-ACCCTGTGTCTTGCTGGAGT-3’ and 5’-TGCAGGGCCTCTAGAACTGT-3’. The primer sets for amplification of mouse OGG1 were 5’-GACTACGGCTGGCATCCTAAG-3’ and 5’-GCAAAAAGGGATCTAAGAGGC-3’. The primer sets for control amplification of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were 5’-ATTCAACGGCACAGTCAAGG-3’ and 5’- CACACCCATCACAAACATGG-3’. For the Real-Time PCR procedure: The cDNA was diluted 1:10 and 2 μl were used as template in a 25 μl qPCR reaction. The qPCR assays were performed as described previously, using SYBR Green (Applied Biosystems, Foster City, CA) on the IQ5(Bio-Rad). Amplification conditions were 95°C for 5 minutes and cycles of 95°C for 30 seconds, 59°C for 30 seconds, and 72°C for 30 minute, with a final extension at 72°C for 5 minutes. All PCR reactions were performed in triplicate. After amplification, we determined the threshold cycle (Ct) to obtain expression values of 2–Ct, as described previously. A ΔCT value was first calculated by subtracting the CT value for the housekeeping gene GAPDH from the CT value for each sample. A ΔΔCT value was then calculated by subtracting the ΔCT value of the control (normal control) from the ΔCT value of each treatment.
The results were expressed as mean values ± SEM of n independent experiments. Analyses were performed using the paired Student t test and the analysis of variance (ANOVA) with repeated measures. P values less than .05 was considered a significant difference between KO mice and WT mice.
DNA-(apurinic or apyrimidinic site) lyase
Nei endonuclease VIII-like 1
8-oxoguanine derivative of GTP
Poly ADP ribose polymerase
Proliferating cell nuclear antigen
CDK-interacting protein 1
Reactive oxygen species
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