Abstract
Many neurodegenerative disorders are characterized by two pathological hallmarks: progressive loss of neurons and occurrence of inclusion bodies containing ubiquitinated proteins. Inflammation may be critical to neurodegeneration associated with ubiquitin-protein aggregates. We previously showed that prostaglandin J2 (PGJ2), one of the endogenous products of inflammation, induces neuronal death and the accumulation of ubiquitinated proteins into distinct aggregates. We now report that temporal microarray analysis of human neuroblastoma SK-N-SH revealed that PGJ2 triggered a "repair" response including increased expression of heat shock, protein folding, stress response, detoxification and cysteine metabolism genes. PGJ2 also decreased expression of cell growth/maintenance genes and increased expression of apoptotic genes. Over time pro-death responses prevailed over pro-survival responses, leading to cellular demise. Furthermore, PGJ2 increased the expression of proteasome and other ubiquitin-proteasome pathway genes. This increase failed to overcome PGJ2 inhibition of 26 S proteasome activity. Ubiquitinated proteins are degraded by the 26 S proteasome, shown here to be the most active proteasomal form in SK-N-SH cells. We demonstrate that PGJ2 impairs 26 S proteasome assembly, which is an ATP-dependent process. PGJ2 perturbs mitochondrial function, which could be critical to the observed 26 S proteasome disassembly, suggesting a cross-talk between mitochondrial and proteasomal impairment. In conclusion neurotoxic products of inflammation, such as PGJ2, may play a role in neurodegenerative disorders associated with the aggregation of ubiquitinated proteins by impairing 26 S proteasome activity and inducing a chain of events that culminates in neuronal cell death. Temporal characterization of these events is relevant to understanding the underlying mechanisms and to identifying potential early biomarkers.
Highlights
Cyclooxygenase-2 (COX-2) has emerged as a major player in inflammation
We investigated by temporal microarray analysis the gene expression profile of human neuroblastoma SKN-SH cells treated with Prostaglandin J2 (PGJ2)
Effect of PGJ2 on Differential Gene Expression in Human Neuroblastoma SK-N-SH Cells—A total of 18,664 genes were evaluated over four time points, 4, 8, 16, and 24 h after treatment with 20 M PGJ2 (Fig. 1)
Summary
Cells—Human neuroblastoma SK-N-SH cells were maintained at 37 °C and 5% CO2 in minimal essential media (MEM) with Eagle’s salts containing 2 mM L-glutamine, 1 mM sodium pyruvate, 0.4% MEM vitamins, 0.4% MEM nonessential amino acids, 100 units/ml penicillin, 100 g/ml streptomycin, and 5% normal fetal bovine serum. SK-N-SH cells were treated with Me2SO (0.5%) or 15 or 20 M PGJ2 for 24 h and analyzed for expression of genes of the ubiquitin-proteasome pathway by real time. Expression of the target genes in cells treated with PGJ2 was normalized to expression under control (0.5% Me2SO-treated) conditions by means of the comparative Ct (threshold cycle) method. Western Blotting—Following the indicated treatments, cell extracts were prepared as described previously [32], and 20 g of protein/lane were boiled for 5 min in Laemmli buffer and analyzed by SDS-PAGE on 10% polyacrylamide gels followed by Western blotting for detection of the proteasome subunits with the respective antibodies (1:1,000) obtained from BIOMOL (Plymouth Meeting, PA). Antigens were visualized by a chemiluminescent horseradish peroxidase method with the ECL reagent
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