Ubiquitin-proteasome system (UPS) is the primary mechanism for intracellular metabolism of proteins, thus regulating numerous cellular processes. The key enzyme in UPS is constitutive proteasome, which maintains cellular homeostasis via degradation of misfolded, damaged or regulatory proteins and consequently affects signalling pathways, cellular cycle and other processes. Recent research emphasizes the role of these signaling pathways in mesenchymal stromal/stem cells (MSCs). Studies show constitutive proteasome can shift into immunoproteasome under inflammatory or hypoxic conditions, which changes MSC phenotype, increases their immunogenicity and susceptibility to rejection in allogeneic transplantation.
The goal of this master's thesis was to evaluate effect of selective inhibition of catalytic subunits β5i or β1i in MSC, acquired from either sinovial fluid or periosteum, on their viability or phenotype. We consequently exposed cells to selective inhibitors of specific subunits. We used a clinically-used pan-proteosomal inhibitor carfilzomib and two other compounds, which inhibit either β1i or β5i immunoproteasome subunit. After exposure, we evaluated cells' viability and potential shift in phenotype of clusters of CD14, CD19, CD45, CD73 and CD90. Under set experimental conditions we weren't able to detect any statistically significant changes neither on viability neither on phenotype. Furthermore, we isolated cell supernatants in order to study cytokine secretion in cells' microenviroment with emphasis on most common cytokines in MSC secretome. Flow cytometry analysis showed no statistically significant effect of proteasome inhibitors on cytokine secretion. Lastly, we used quantitative real-time polymerase chain reaction (qPCR) to assess changes in expression of genes encoding vascular endothelial growth factor (VEGFA), indolamine 2,3-dioxygenase 1 (IDO1) and transforming growth factor β1 (TGFB1). Statistical analysis showed effects of inhibition only on expression of IDO1.
The conclussions of our study are preliminary and are based on small sample size. We would continue this study with increasing sample size to reach better statistical representativeness. We would also investigate changes in cytokine and gene expression after exposing MSC to inflammatory factors and other stress conditions, which should promote shift towards immunomodulatory MSC phenotype.
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