14-3-3 Isoforms Differentially Regulate NFκB Signaling In The Brain After Ischemia-reperfusion

Objectives::Mammalian 14-3-3 isoforms exist predominantly in the brain and are heavily involved in neurological diseases.However,the isoform-specific role of 14-3-3 proteins in the brain remains largely unclear.Here,we investigated the role of 14-3-3 isoforms in rat brains after transient middle cerebral artery occlusion and reperfusion.Methods: Establish 14-3-3 isoforms in the ischemic brain at 12 and 24 hr of reperfusion after 1 hr of tMCAo in rats.14-3-3 isoforms were measured by immunohistochemistry 、PCR、Western blot、Bimolecular fluorescence complementation assay(Bi FC)and Glutathione S-transferase pull-down assay.We clarify the expression profiles of all 14-3-3 isoforms in the brains after I/R.At 24 hr of reperfusion after 1 hr of tMCAo,cell injury/death is a major event in the brain.Knockdown and overexpression approaches were used to study the effect of 14-3-3γ and the underlying mechanisms.Protein-protein interactions were examined by Glutathione S-transferase pull-down assay and Fluorescent immunostaining and immunohistochemistry.Results: 1.14-3-3 isoforms are differentially upregulated in the brain after ischemic reperfusion.14-3-3 ?,η,? and ? but not ? or ? were selectively upregulated in cerebral cortical neurons after ischemia-reperfusion(I/R).Only 14-3-3 ? was clearly increased in glial cells after I/R.2.Ischemic reperfusion induces nuclear translocation of 14-3-3 isoforms in the brain.Selectively,14-3-3 ?,? and ? were translocated from cytoplasm into the nuclei of neurons after I/R.3.14-3-3 bound to p65 and suppressed p65 expression in N2 a cells.In the brain,14-3-3 could either colocalize with p65 in the nuclei of neurons or segregate from p65 expression in cortical neurons after I/R.We found that nuclear transcriptional factors p65,p53 and hypoxia induced factor(HIF)-1? contained 14-3-3 binding motif with high(p65)or media(p53 and HIF1 ?)stringency selection.Results of GST pull-down confirmed that 14-3-3 bound to p65 in N2 a cells.Double fluorescent immuostaining verified that 14-3-3 ? and p65 were colocalized in the nuclei of neurons in the ipsilateral rat cerebral cortex after I/R.4.14-3-3 isoforms differentially downregulates p65 in neurons after ischemia-reperfusion.Results of Western blotting analysis and fluorescent immunostaining showed that the expression levels of p65 in the cerebral cortex was increased after I/R.Overexpression of 14-3-3 ? and ? significantly decreased p65 in N2 a cells,suggesting a differential regulation of p65 by 14-3-3 isoforms.On the contrary,knocking down of 14-3-3 ?(?-KD)significantly reduced p65 in N2 a cells.Conclusions: All evidences together suggests that 14-3-3 isoforms are differentially induced to enter into the nuclei of neurons after I/R,which might regulate NF-κB signaling directly or indirectly.Since 14-3-3 proteins are essential for cell survival and NF-κB is a key transcriptional factor,our data suggest that the 14-3-3/p65 signaling pathway might be a potential therapeutic target for stroke.