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Proteostasis is critical for cells to maintain the balance between protein synthesis, quality control, and degradation. This is particularly important for myeloid cells of the central nervous system as their immunological function relies on proper intracellular protein turnover by the ubiquitin-proteasome system. Accordingly, disruption of proteasome activity due to, e.g., loss-of-function mutations within genes encoding proteasome subunits, results in systemic autoinflammation. On the molecular level, pharmacological inhibition of proteasome results in endoplasmic reticulum (ER) stress-activated unfolded protein response (UPR) as well as an induction of type I interferons (IFN). Nevertheless, our understanding as to whether and to which extent UPR signaling regulates type I IFN response is limited. To address this issue, we have tested the effects of proteasome dysfunction upon treatment with proteasome inhibitors in primary murine microglia and microglia-like cell line BV-2. Our data show that proteasome impairment by bortezomib is a stimulus that activates all three intracellular ER-stress transducers activation transcription factor 6, protein kinase R-like endoplasmic reticulum kinase and inositol-requiring protein 1 alpha (IRE1α), causing a full activation of the UPR. We further demonstrate that impaired proteasome activity in microglia cells triggers an induction of IFNβ1 in an IRE1-dependent manner. An inhibition of the IRE1 endoribonuclease activity significantly attenuates TANK-binding kinase 1-mediated activation of type I IFN. Moreover, interfering with TANK-binding kinase 1 activity also compromised the expression of C/EBP homologous protein 10, thereby emphasizing a multilayered interplay between UPR and type IFN response pathway. Interestingly, the induced protein kinase R-like endoplasmic reticulum kinase-activation transcription factor 4-C/EBP homologous protein 10 and IRE1-X-box-binding protein 1 axes caused a significant upregulation of proinflammatory cytokine interleukin 6 expression that exacerbates STAT1/STAT3 signaling in cells with dysfunctional proteasomes. Altogether, these findings indicate that proteasome impairment disrupts ER homeostasis and triggers a complex interchange between ER-stress sensors and type I IFN signaling, thus inducing in myeloid cells a state of chronic inflammation.
Molecular Mechanisms of Bortezomib Action: Novel Evidence for the miRNA−mRNA Interaction Involvement
(2020)
Bortezomib is an anti-tumor agent, which inhibits 26S proteasome degrading ubiquitinated
proteins. While apoptotic transcription-associated activation in response to bortezomib has been
suggested, mechanisms related to its influence on post-transcriptional gene silencing mediated
regulation by non-coding RNAs remain not fully elucidated. In the present study, we examined
changes in global gene and miRNA expression and analyzed the identified miRNA–mRNA interactions
after bortezomib exposure in human neuroblastoma cells to define pathways affected by this agent in
this type of cells. Cell viability assays were performed to assess cytotoxicity of bortezomib. Global gene
and miRNA expression profiles of neuroblastoma cells after 24-h incubation with bortezomib were
determined using genome-wide RNA and miRNA microarray technology. Obtained results were
then confirmed by qRT-PCR and Western blot. Further bioinformatical analysis was performed
to identify affected biological processes and pathways. In total, 719 genes and 28 miRNAs were
downregulated, and 319 genes and 61 miRNAs were upregulated in neuroblastoma cells treated with
bortezomib. Possible interactions between dysregulated miRNA/mRNA, which could be linked to
bortezomib-induced neurotoxicity, affect neurogenesis, cellular calcium transport, and neuron death.
Bortezomib might exert toxic effects on neuroblastoma cells and regulate miRNA–mRNA interactions
influencing vital cellular functions. Further studies on the role of specific miRNA–mRNA interactions
are needed to elucidate mechanisms of bortezomib action.