Our studies demonstrated that four weeks of alcohol feeding of WT mice resulted in increased ALT, steatosis, mtDNA oxidation, caspase-3 activation, and cell cycle protein and mtDNA repair enzyme expression. In these animals, low levels of mtDNA deletions were detected while the mitochondrial membrane potential and ATP levels remained unchanged compared to that present in pair-fed mice. By contrast, IL-6 KO mice fed alcohol for 4 weeks had more severe liver injury manifested by higher levels of ALT, greater steatosis, and more TUNEL positive cells. Ethanol feeding in IL-6 KO mice induced significant mtDNA deletions that were associated with the loss of the mitochondrial membrane potential, greatly diminished levels of COI synthesized in the mitochondria, along with diminished ATP and mtDNA repair enzymes levels. Thus oxidative injury that was fairly well tolerated in WT mice fed ethanol had highly deleterious effects in IL-6 KO mice.
Elevation of serum IL-6 is generally present in patients with alcoholic liver disease (ALD) and correlates with the severity of ALD (31
). However, the findings from animal models suggest that IL-6 plays an important role in protecting against early alcoholic liver injury via multiple mechanisms. IL-6 exerts anti-apoptotic function through the gp130 protein that associates with and activates Janus kinase-signal transducer and transcription factor-3 (JAK-STAT3) (32
). Lack of IL-6/gp130/STAT3 in hepatocytes predisposes the liver to steatosis in mice (33
). Emerging evidence suggests that the hepatoprotective effect of IL-6 on ALD is mediated via induction of anti-apoptotic factors including Bcl-2 and Bcl-x(L), and anti-oxidative genes including metallothionein (23
). Here we have shown in these experiments that IL-6 is necessary to provide cell cycle check-points via induction of p21 and p53, and stimulate the transcription of DNA repair enzymes.
We observed that p21 and p53 proteins are up-regulated in WT but not IL-6 KO mice fed ethanol (). The mechanism resulting in the failure of IL-6 KO mice to increase transcription of p21 may be found in our previous studies (23
). IL-6 stimulation significantly increases phosphorylated-STAT3, which was then stimulates p21 transcription (34
). Over-expression of p21 resulted in G1 arrest. PCNA expression (G1 phase) was increased in both WT and IL-6 KO mice fed ethanol, but failed to increase Ki67 (S phase) in WT mice fed ethanol suggesting cell cycle arrest occurred. Thus, we propose that normal mice have sufficiently high levels of IL-6 during continuous moderate alcohol exposure to increase p21 production via activated STAT3 thereby leading to cell cycle (G1/S) arrest. This hypothesis is supported by the findings that pretreatment of obese animals with rIL-6 normalized proliferating cell nuclear antigen (PCNA) expression in steatotic hepatocytes but failed to increase DNA synthesis (BrdU, S phase) (36
While the activation of p53 leads to hepatocyte apoptosis its deficiency results in the abrogation of hepatocyte apoptosis and the early appearance of liver dysplasia after ethanol feeding (37
). However, the increased TUNEL reactivity in ethanol-fed IL-6 KO mice occurred in the absence of p53 and caspase-3 activation. This suggests that ethanol-induced hepatocyte apoptosis in IL-6 KO mice is independent of p53 and caspase-3. At present, the underlying mechanisms for this remain largely unknown. The facts that significant translocation of the mitochondrial intermembrane protein AIF into the nucleus was observed in the ethanol-fed IL-6 KO mice suggest that AIF translocation may contribute to ethanol-induced hepatocyte apoptosis in IL-6 KO mice as AIF has been shown to contribute to caspase-independent apoptosis (29
). Moreover, significant ATP depletion observed in ethanol-fed IL-6 KO mice appears to be one mechanisms leading to AIF translocation to the nucleus in these mice (38
The OGG1 enzyme is the primary one for the repair of 8-oxoguanine (8-oxoG), a common product of DNA oxidation, in both the nuclear and mtDNA (39
). It has been reported that the OGG1 glycosylase plays a more important role in mitochondria than in nuclear DNA repair. Under conditions of oxidative stress the accumulation of 8-oxoG in the mitochondrial DNA from OGG1−/−
(knockout) mice was much greater than in nuclear DNA when both were compared with wild type (41
). Nei-like homologs (NEILs) are a recently identified group of bi-functional DNA glycosylases, which are mammalian orthologs of the Escherichia coli MutM/Nei
. It was initially suggested that NEIL1 (42
) and NEIL2 (43
) localize exclusively to the nucleus. However, it has been recently reported that NEIL1 is also found in mouse liver mitochondria (44
). NEIL1 may initiate base excision repair of ring-fragmented purines and some saturated pyrimidines. Interestingly, NEIL1 knockout (neil1−/−) and heterozygotic (neil1+/−) mice develop severe obesity, dyslipidemia, and fatty liver disease and also have a tendency to develop hyperinsulinemia at age of 4 to 6 months (45
). Additionally, mtDNA from neil1−/− mice show increased levels of steady-state DNA damage and deletions relative to wild-type controls. Our results show that NEIL1 and OGG1 were increased in response to ethanol stimulation in wild-type mice and this was associated with much less mtDNA deletion, fatty changes and hepatocyte injury compared to IL-6 knock-out mice. IL-6 may play an essential role in the activation of DNA repair enzymes in response to ethanol stimulation.
We studied the components of cytochrome C oxidase (COI and COIV) for two reasons: first to determine if the deletions in mtDNA had functional significance resulting in a decreased level of COI and second, to compare the transcriptional capability of nuclear DNA (COIV) vs mtDNA (COI). We found that the level of mtDNA encoded COI was significantly increased in wild-type mice fed ethanol (). The increased oxidative stress may be responsible for the elevation of mtDNA encoded COI because oxidative stress has been shown to regulate the expression of COI and COII in hepatocytes (46
). The increased production of COI of the mitochondrial respiratory chain in response to ethanol in WT mice is likely to represent an adaptive response of compensatory effect of mitochondrial to oxidative injury and to re-establish ionic balance and to support the synthesis of cellular repair systems in ethanol injured cells. In contrast, oxidative mtDNA damage without repair which occurred in the IL-6 KO mice fed ethanol results in common deletions responsible for the transcription of COI. The finding that nuclear COIV synthesis was relatively unaffected after four weeks of alcohol feeding in both WT and IL-6 KO mice is the strongest argument supporting the concept that the principal target of chronic alcohol toxicity is mtDNA.
In summary, IL-6 is required to activate DNA repair enzymes (Neil1 and OGG1) and cell cycle proteins (p21 and p53) in response to chronic ethanol consumption. These newly assigned functions for IL-6 are likely to be important in other forms of injury and perhaps in other organ systems.