Abstract
BackgroundIschemia and reperfusion (I/R) induces cytokines, and up-regulates inducible nitric oxide synthase (iNOS), interferon regulatory factor-1(IRF1) and p53 up-regulated modulator of apoptosis (PUMA), which contribute to cell death and tissue injury. However, the mechanisms that I/R induces IRF1-PUMA through iNOS/NO is still unknown.MethodsIschemia was induced by occluding structures in the portal triad (hepatic artery, portal vein, and bile duct) to the left and median liver lobes for 60 min, and reperfusion was initiated by removal of the clamp. Induction of iNOS, IRF1 and PUMA in response to I/R were analyzed. I/R induced IRF1 and PUMA expression were compared between iNOS wild-type and iNOS knockout (KO) mice. Human iNOS gene transfected-cells were used to determine iNOS/NO signals targeting IRF1. To test whether HDAC2 was involved in the mediation of iNOS/NO-induced IRF1 transcriptional activities and its target gene (PUMA and p21) expression, NO donors were used in vitro and in vivo.ResultsIRF1 nuclear translocation and PUMA transcription elevation were markedly induced following I/R in the liver of iNOS wild-type mice compared with that in knock-out mice. Furthermore, I/R induced hepatic HDAC2 expression and activation, and decreased H3AcK9 expression in iNOS wild-type mice, but not in the knock-out mice. Mechanistically, over-expression of human iNOS gene increased IRF1 transcriptional activity and PUMA expression, while iNOS inhibitor L-NIL reversed these effects. Cytokine-induced PUMA through IRF1 was p53 dependent. IRF1 and p53 synergistically up-regulated PUMA expression. iNOS/NO-induced HDAC2 mediated histone H3 deacetylation and promoted IRF1 transcriptional activity. Moreover, treating the cells with romidepsin, an HDAC1/2 inhibitor decreased NO-induced IRF1 and PUMA expression.ConclusionsThis study demonstrates a novel mechanism that iNOS/NO is required for IRF1/PUMA signaling through a positive-feedback loop between iNOS and IRF1, in which HDAC2-mediated histone modification is involved to up-regulate IRF1 in response to I/R in mice.
Highlights
Interferon regulatory factor-1 (IRF1) is a transcription factor up-regulated in response to various stimuli such as cytokines, double stranded RNA and hormones (Kroger et al 2002)
p53 up-regulated modulator of apoptosis (PUMA) induction is dependent on inducible nitric oxide synthase (iNOS) in response to ischemia-reperfusion We and others previously described that Ischemia and reperfusion (I/R) was known to induce cytokines and iNOS in liver (Ueki et al 2010; Hamada et al 2009; Tsung et al 2006; Lee et al 2001), as well as PUMA expression in intestine and heart (Wu et al 2007; Toth et al 2006)
Hepatic iNOS mRNA was induced in a time-dependent manner in iNOS+/+, but not in iNOS−/− mice, with peak mRNA seen at 12 h after partial warm I/R (Fig. 1b)
Summary
Interferon regulatory factor-1 (IRF1) is a transcription factor up-regulated in response to various stimuli such as cytokines, double stranded RNA and hormones (Kroger et al 2002). Our previous study identified a critical role for IRF1 in regulation of cell death in liver transplant ischemia and reperfusion (I/R) (Ueki et al 2010). PUMA expression, transcriptionally regulated by p53 (Nakano et al 2001; Yu and Zhang 2003), NF-κB (Wu et al 2007), forkhead box protein O1 (FOXO1) (Hughes et al 2011), FOXO3a (You et al 2006), IRF1 (Gao et al 2010) and others, is a key step in pathogenesis of IRI in intestine and heart (Wu et al 2007; Toth et al 2006). Ischemia and reperfusion (I/R) induces cytokines, and up-regulates inducible nitric oxide synthase (iNOS), interferon regulatory factor-1(IRF1) and p53 up-regulated modulator of apoptosis (PUMA), which contribute to cell death and tissue injury. The mechanisms that I/R induces IRF1-PUMA through iNOS/NO is still unknown
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