NF‐κB is involved in the survival of cerebellar granule neurons: association of Iκβ phosphorylation with cell survival

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Abstract
The NF-κB transcription factor consists of dimeric complexes belonging to the Rel family, which include p50, p52, p65 (RelA), RelB and c-Rel. NF-κB activity is tightly controlled by IκB proteins which bind to NF-κB preventing its translocation to the nucleus. Activation of NF-κB is most often mediated by IκB degradation, which permits NF-κB to enter the nucleus. We investigated the role of NF-κB in the survival of cerebellar granule neurons. We found that survival of these neurons in high potassium medium is blocked by three separate inhibitors of NF-κB activity: SN-50, N-tosyl-l-phenylalanine chloromethyl ketone and pyrrolidinedithiocarbamate, indicating that NF-κB is required for neuronal survival. Gel-shift assays reveal three complexes that bind to the NF-κB binding site in high potassium medium. Switching these cultures to low potassium medium, a stimulus that leads to apoptotic death, causes a reduction in the level of the largest complex, which contains p65. Overexpression of p65 by transfection inhibits low potassium-induced apoptosis, whereas overexpression of IκBα promotes apoptosis even in high potassium medium. Surprisingly, however, neither the level of endogenous p65 nor that of IκBα and IκBβ is altered by low potassium treatment. Similarly, no changes are seen in the nuclear or cytoplasmic levels of p50, p52, RelB and c-Rel. Phosphorylation of p65, which can lead to its activation, is unchanged. Phosphorylation of IκBβ is, however, reduced by low potassium treatment. Besides being necessary for high potassium-mediated neuronal survival, NF-κB is also involved in the survival-promoting effects of IGF-1 and cAMP as judged by the ability of SN-50 to inhibit the actions of these survival factors and the ability of these factors to inhibit the low potassium-induced alterations in the DNA-binding activity of NF-κB. Taken together, our results show that NF-κB may represent a point of convergence in the signaling pathways activated by different survival factors and that uncommon mechanisms might be involved in NF-κB-mediated survival of cerebellar granule neurons.