IL-1 activation of NF-kB contributes to perinatal hypoxia/ischemia induced cell death



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IL-1 activity has been implicated in perinatal hypoxia/ischemia (HI) brain damage without the underlying mechanisms being characterized. We used a 7-day-old rat model to elucidate the role of NF-kB in HI stimulation of IL-1 signaling. HI was induced by permanent ligation of the left carotid artery followed by 90 minutes of hypoxia (7.8% O2). We observed increased cell death and caspase 3 activity in the hippocampus and the cortex 3 to 48h post-HI. IL-1beta protein expression increased began at 3h after HI and lasted until 24h post-HI in the hippocampus and 12h post-HI in the cortex. Intracerebroventricular injection of 2mg IL-1 receptor antagonist (IL-1Ra) 2h post-HI significantly reduced cell death and caspase 3 activity. EMSA analyses for NF-kB activity showed increased p65/p50 DNA-binding activity at 24h post-HI. Western blot analyses and immunofluorescent staining showed significant nuclear translocation of p65. Protein expression levels of iNOS and COX2, known to be transcriptionally regulated by NF-kB, also increased at 24h post-HI. All these changes were reversed by IL-1Ra blockade of IL-1, consistent with IL-1 triggering of inflammatory apoptotic outcomes via NF-kB transcriptional activation. The observed increase in cytoplasmic phosphorylated IkBa and nuclear translocation of Bcl-3, was also attenuated by IL-1Ra blockade, suggesting that HI-induced IL-1 activation of NF-kB is via both degradation of IkBalpha and nuclear translocation of Bcl-3. \r\nInhibition of NF-kB via decoys containing NF-kB binding consensus sequences present in IgG-kB promoter showed specific inhibition of p65/p50 binding activity, while Bcl-x decoys specifically inhibited c-Rel/p50 (p52) binding activity. RPAs showed that IgG-kB decoys significantly decreased IL-1alpha, TNF-alpha and TNF-beta mRNA levels compared to minimal changes after Bcl-x or scrambled decoy treatment, indicating a specific IgG-kB decoy effect on inflammation post-HI. Microarray data indicated that 1) Genes that were significantly downregulated by IgG-kB decoys were not affected by Bcl-x decoys and vice versa, another piece of evidence for selective effects of different decoys. 2) A large number of cell death/survival related genes were affected by IgG-kB decoys, confounding the final outcomes. Our results suggest that IgG-kB decoys selectively inhibit inflammatory responses to HI. However, careful design of decoy sequences is essential to acquire selective effects on cell death.



IL-1, decoys, hypoxia/ischemia