Session Type: Abstract Submissions (ACR)
Chondrocyte stress responses to biomechanical injury and joint inflammation, and associated changes in differentiation and function, provide a foundation upon which cartilage injury and OA can be triggered and accelerated. Fundamental proteostasis responses by which cells normally resolve stress include the unfolded protein response (UPR), which restores equilibrium to the stressed ER via a reprogrammed proteome, rich in chaperones and protein folding catalysts. The UPR also regulates oxidative stress responses, inflammation, and cell fate including autophagy. Chondrocyte autophagy is impaired in biomechanical cartilage explant injury, and in aging and OA cartilage. Autophagy repairs damaged cell organelles and is anti-inflammatory, in part by degrading pro-IL-1beta. The UPR can promote autophagy and autophagy promotes normal UPR and mitochondrial functional. UPR-specific XBP1 alternative mRNA splicing generates the potent transcriptional activator XBP1s, which can exert either promote or inhibit autophagy or inflammation depending on cell type and context. In this study, we tested the hypothesis that XBP1 activation is noxious in chondrocytes by impairing autophagy.
We knocked down XBP1 expression in cultured normal human chondrocytes (passage 1) via siRNA transfection. After 48 hours, the XBP1 knockdown chondrocytes were subjected to SDS-PAGE/Western blot analysis for phosphorylation of the autophagy promoter AMPKα, expression of FOXO1, transglutaminase 2, and the autophagy proteins LC3, and p62. XBP1 knockdown chondrocytes were also treated with IL-1β for 18 hours, and nitric oxide (NO) production and MMP-3 release were measured from the conditioned media by Griess reaction method and Western blot, respectively.
We observed markedly increased activation of XBP1 in human knee OA chondrocytes. In XBP1 knockdown human knee chondrocytes, we observed increased basal levels of expression of the autophagy promoter FOXO1, and increasing LC3 conversion from I to II, and decreased basal levels of expression of p62, indicating induction of autophagy. This was linked with increased basal level of phosphorylation of the autophagy promoter, anti-inflammatory mediator and metabolic super-regulator AMPK in the XBP1 knockdown chondrocytes. Conversely, IL-1β-induced release of NO and MMP-3 was significantly inhibited in the XBP1 knockdown chondrocytes. Last, basal expression of transglutaminase 2, which transduced inflammatory responses of IL-1β and chemokines in chondrocytes, was blunted in the XBP1 knockdown chondrocytes.
This study demonstrated increased UPR-specific XBP1 activation in human knee OA cartilage, and that XBP1 critically transduces noxious functions in chondrocytes, including impairment of autophagy, and pro-catabolic activities, mediated by regulation of AMPK and transglutaminase activity. These findings identify XBP1 as a novel target to limit OA progression.
R. L. Bryan,
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/inhibition-of-x-box-binding-protein-1-xbp1-is-chondroprotective-by-promoting-autophagy-and-inhibiting-catabolic-responses/