NOD Mice Are Defective in Proteasome Production and Activation of NF-κB

Type I diabetes is an autoimmune disease with genetic risk factors that map to the major histocompatibility complex (MHC) region of the genome. In both human type I diabetes and animal models of the disease (the nonobese diabetic [NOD] mouse and the BB rat), the insulin-producing β cells in the pancreatic islets of Langerhans are selectively destroyed by the abnormal immune responses (15, 63). The MHC region of the genome contains immune response genes that are important in T-cell education and in antigen presentation through either MHC class I or class II molecules. Two such genes, encoding the proteasome components LMP2 and LMP7, are located in this genomic region. The proteasome is a multiprotein complex that catalyzes the ATP-dependent processing or degradation of intracellular proteins in eukaryotic cells (14, 31). Proteasome-mediated protein cleavage plays important roles in the regulation of cell growth, metabolism, and function. Cleavage of endogenous proteins by the proteasome generates small self-peptide fragments that contribute to T-cell education after presentation by MHC class I molecules. Although, in general, the proteasome exhibits minimal variability in substrate selectivity and subunit composition, gamma interferon (IFN-γ) induces the expression of the LMP2 and LMP7 subunits. Incorporation of these subunits into the proteasome alters its specificity for self-proteins in such a manner in that suitability of the generated peptides for presentation in the peptide-binding groove of molecules is increased (7, 29). The proteasome also plays an important role in the processing and activation of the transcription factor nuclear factor κB (NF-κB). NF-κB is activated in response to various extracellular stimuli, including interleukin (IL-1), lipopolysaccharide (LPS), and tumor necrosis factor alpha (TNF-α) (3, 4, 78, 82). Activated NF-κB, in turn, has been implicated in the regulation of genes that contribute to cytokine generation, expression of cell surface adhesion epitopes, lymphocyte maturation, protection from apoptosis, and MHC class I antigen processing and presentation (5, 9, 16, 76, 80). Active NF-κB exists predominantly as a heterodimer composed of either a p50 or p52 subunit and p65 (RelA). The p50 and p52 subunits of NF-κB are the products of processing of p105 and p100 precursors, respectively, by the proteasome (13, 19, 61, 67, 68). Additionally, cotranslational processing by the ubiquitin-proteasome pathway is an alternative mechanism for p50 generation (50). In the cytoplasm of resting cells, NF-κB associates with the inhibitory protein IκBα. Cell stimulation results in the degradation of phosphorylated IκBα by the proteasome, thereby allowing the p50-p65 or p52-p65 dimers to translocate to the nucleus and initiate the transcription of target genes. The p105, p100, and IκBα proteins are thought to typically undergo phosphorylation and ubiquitination prior to proteasome degradation (8, 51, 61). Studies of knockout mice lacking IκB kinase revealed that phosphorylation of IκBα requires the IκB kinase IKK-2 (18, 54, 64, 83). Recent reports demonstrate that Rel complexes sequestered by p105 or p100 are not rapidly mobilized to the nucleus in response to these same signals (50, 74). Insights into the various biological functions of NF-κB have been obtained by the generation of knockout mice lacking NF-κB subunits or linked regulatory proteins in all tissues (6, 10–12, 24, 41, 44, 79, 88). For example, mice lacking the p50 subunit exhibit multifocal defects in immune responses, including defects specific to B lymphocytes. B cells derived from p50−/− mice do not proliferate in response to either CD40L (L indicates ligand) or bacterial LPS, and they exhibit differentiation defects, an altered pattern of cytokine secretion, and abnormal germ line immunoglobulin class switching (70, 72). The phenotype of the IKK-1 knockout mouse is principally a defect in epidermal skin development; the limb defect in IKK-1−/− mice is believed to be a product of this epidermal abnormality (38, 48, 75). In contrast, ablation of IKK-2 causes severe TNF-α sensitivity , as well as liver abnormalities due to apoptosis, and mimics the phenotype of p65-deficient mice (6, 37, 47, 59, 77). Humans and animals with autoimmune diabetes exhibit symptomatology indicative of possible NF-κB and proteasome dysfunction, although there is no evidence supporting a generalized proteasome defect or implicating NF-κB dysregulation. In both affected humans and NOD mice, impaired antigen presentation by MHC class I molecules is due to a defect in the generation of self-peptides, suggestive of altered immune peptide generation or transport (21, 27, 45). Errors in lymphocyte development, often characterized by an overabundance of naive-lymphocyte subsets, are also characteristic of humans with diabetes and of rodent (BB rat and NOD mouse) models of autoimmune diabetes (15, 20, 22, 40, 66, 69, 71). Moreover, humans with type I diabetes and NOD mice appear to be defective in the production of LMP2 mRNA, possibly implying deranged immune peptide generation by the proteasome (28, 91). Humans and rodents with autoimmune diabetes also exhibit defects in cytokine gene regulation and secretion (63). We have now investigated the activity and subunit composition of NF-κB as an indicator of general proteasome function and diverse protein processing errors in NOD mice. The activity of NF-κB in NOD mouse lymphocytes was shown to be markedly impaired because of a pronounced defect in the proteolytic processing of p50 and p52 and degradation of phosphorylated IκBα by the ubiquitin-proteasome pathway. The virtual inability of TNF-α to activate NF-κB in NOD mouse spleen cells was associated with an increased susceptibility to TNF-α-induced apoptosis. The defect in proteasome function in NOD mouse splenocytes was cellular and developmentally specific and resulted from the lack of the LMP2 subunit protein. Studies of mutant cell lines confirmed that LMP proteins are obligatory for diverse protein processing events beyond peptide presentation for class I antigen processing. We propose that proteasome dysfunction caused by a defect in the expression of the MHC-linked Lmp2 gene is an important factor predisposing NOD mice to autoimmune diabetes. Deranged immune and nonimmune protein processing by the NOD proteasome is tissue and developmental stage specific for cells and likely contributes to the diverse symptomatology that relates to disease expression. The NOD mouse represents a novel spontaneous-disease model of disrupted intracellular protein production and processing. These observations provide an explanation for the sex and age differences observed at the onset of insulin-dependent diabetes mellitus (type I diabetes).