Objective
Osteoarthritis (OA) chondrocytes have impaired autophagy, one arm of the proteostasis network that coordinates proteome and organelle quality control and degradation. Deficient proteostasis impacts differentiation and viability, and inflammatory processes in aging and disease. Studying OA chondrocytes, we assessed ubiquitin proteasome system proteasomal function.
Methods
We evaluated human knee cartilages by immunohistochemistry, and assessed proteasomal function, levels of proteasomal core subunits and chaperones, and autophagy in cultured chondrocytes. Assays included Western blotting, quantitative RT-PCR, proteasomal protease activity and cell immunofluorescence.
Results
Human knee OA cartilages demonstrated polyubiquitin accumulation, with increased ubiquitin K48-linked polyubiquitinated proteins in situ, suggesting proteasomal impairment. Cultured OA chondrocytes demonstrated accumulation of K48 polyubiquitinated proteins, significantly decreased 20S proteasome core protease activity, and decreased levels of phosphorylated FOXO4 and PSMD11, a FOXO4-inducible promoter of proteasomal activation. Levels of proteasomal core subunits PSMB3, 5, 6, and assembly chaperone PSMG1 were not decreased in OA chondrocytes. In normal chondrocytes, PSMD11 siRNA knockdown stimulated certain autophagy machinery elements, elevated nitric oxide (NO) release, and decreased levels of chondrocytic master transcription factor SOX9 protein (and mRNA) and aggrecan (AGC1) mRNA. PSMD11 gain of function by transfection increased proteasomal function, raised levels of SOX9-induced AGC1 mRNA, stimulated elements of the autophagic machinery, and inhibited IL-1-induced NO and MMP13 release in OA chondrocytes.
Conclusion
Deficient PSMD11, associated with less phosphorylated FOXO4, promotes impaired proteasomal function in OA chondrocytes, dysregulated chondrocytic homeostasis, and decreased levels of SOX9 mRNA, SOX9 protein, and AGC1 mRNA. Chondrocyte proteasomal impairment may be a therapy target for OA.