NF-kappaB is involved in the survival of cerebellar granule neurons: association of IkappaBbeta [correction of Ikappabeta] phosphorylation with cell survival.

The NF-kappaB transcription factor consists of dimeric complexes belonging to the Rel family, which include p50, p52, p65 (RelA), RelB and c-Rel. NF-kappaB activity is tightly controlled by IkappaB proteins which bind to NF-kappaB preventing its translocation to the nucleus. Activation of NF-kappaB is most often mediated by IkappaB degradation, which permits NF-kappaB to enter the nucleus. We investigated the role of NF-kappaB 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-kappaB activity: SN-50, N-tosyl-L-phenylalanine chloromethyl ketone and pyrrolidinedithiocarbamate, indicating that NF-kappaB is required for neuronal survival. Gel-shift assays reveal three complexes that bind to the NF-kappaB 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 IkappaBalpha promotes apoptosis even in high potassium medium. Surprisingly, however, neither the level of endogenous p65 nor that of IkappaBalpha and IkappaBbeta 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 IkappaBbeta is, however, reduced by low potassium treatment. Besides being necessary for high potassium-mediated neuronal survival, NF-kappaB 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-kappaB. Taken together, our results show that NF-kappaB may represent a point of convergence in the signaling pathways activated by different survival factors and that uncommon mechanisms might be involved in NF-kappaB-mediated survival of cerebellar granule neurons.

Ly49I NK cell receptor transgene inhibition of rejection of H2b mouse bone marrow transplants.

The Ly49 family of genes encode NK cell receptors that bind class I MHC Ags and transmit negative signals if the cytoplasmic domains have immunoregulatory tyrosine-based inhibitory motifs (ITIMs). 5E6 mAbs recognize Ly49C and Ly49I receptors and depletion of 5E6+ NK cells prevents rejection of allogeneic or parental-strain H2d bone marrow cell (BMC) grafts. To determine the function of the Ly49I gene in the rejection of BMC grafts, we transfected fertilized eggs of FVB mice with a vector containing DNA for B6 strain Ly49I (Ly49IB6). Ly49IB6 is ITIM+ and is recognized by 5E6 as well as Ly49I-specific 8H7 mAbs. Normal FVB H2q mice reject H2b but not H2d BMC allografts, and the rejection of H2b BMC was inhibited partially by anti-NK1.1 and completely by anti-asialo GM1, but not by anti-CD8, Abs. In FVB mice, NK1.1 is expressed on only 60% NK cells. FVB. Ly49IB6 hosts failed to reject H2d or H2b BMC, but did reject class I-deficient TAP-1-/- BMC, indicating that NK cells were functional. Nondepleting doses of anti-Ly49I Abs reversed the acceptance of H2b BMC by FVB.Ly49IB6 mice. FVB.Ly49IB6+/- mice were crossed and back-crossed with 129 mice-H2b, 5E6-, poor responders to H2d BMC grafts. While transgene-negative H2b/q F1 or first-generation back-crossed mice rejected H2b marrow grafts (hybrid resistance), transgene-positive mice did not. Thus B6 strain Ly49I receptors transmit inhibitory signals from H2b MHC class I molecules. Moreover, Ly49IB6 has no positive influence on the rejection of H2d allografts.