Rheumatoid arthritis (RA) is characterized by chronic joint inflammation and progressive destruction of cartilage and bone, which leads to severe joint pain and, ultimately, loss of function. RA synovial fibroblasts (RASFs) residing in the joint have emerged as key players in the pathogenesis of RA. RASFs are capable of producing a large set of inflammatory cytokines, chemokines, and matrix-degrading enzymes, thereby actively contributing to the inflammatory and joint-destructive state in RA ().
Since RA synovial cells produce large amounts of cytokines and enzymes, as much as 30% of all newly synthesized, endoplasmic reticulum (ER)–sorted proteins are unfolded (). When the level of unfolded proteins exceeds the capacity of this organelle, defective proteins are eliminated by a ubiquitin/proteasome-degrading process called the unfolded protein response (UPR) (). The UPR is the main nonlysosomal degradative pathway for ubiquitinated proteins. In contrast, autophagy is a highly regulated and evolutionary conserved process of lysosome-mediated degradation of organelles and cellular components that is activated by various cellular stress conditions, such as ER stress, hypoxia, starvation, heat shock, and microbial infection (). During cellular stress, large quantities of proteins are damaged, resulting in their unfolding/misfolding, polyubiquitination, aggregation, and possibly, the induction of cell death. The robust and efficient removal of these toxic factors by autophagy can help to relieve the cell of stress and reinstate homeostasis ().
Although autophagy constitutes a cytoprotective response activated by cells to cope with stress and is rather protective of cell death, induction of autophagy has also been reported to lead to cell death, generally called autophagic cell death or cell death associated with autophagy (). Increased induction of autophagy in RASFs as compared to osteoarthritis synovial fibroblasts (OASFs) was recently described (). However, the role of autophagy and its connection to death pathways in RASFs is incompletely understood. In addition, we asked whether autophagy and UPR could compensate for each other. In the present study, we investigated the functional role of autophagy activation by the accumulation of polyubiquitinated proteins in RASFs and the role of this pathway in the regulation of cell death pathways.
- Top of page
- PATIENTS AND METHODS
- AUTHOR CONTRIBUTIONS
Continued removal of unfolded and misfolded proteins by the proteasome pathway and by autophagy is essential for the survival of cells. Both pathways have been reported to be more active in RASFs as compared to control fibroblasts (). Recently, the RA synovium was reported to exhibit a highly increased ER stress–associated gene signature (), and TNFα was shown to further increase the expression of ER stress markers in RASFs (). Consistent with previous data (), we showed that autophagy induction by proteasome inhibition or ER stress is more pronounced in RASFs than in OASFs.
Although autophagy induction is mainly thought to play a protective role, induction of this pathway was also reported to be associated with autophagic cell death in a variety of cancer cells upon in vitro treatment with chemotherapeutic agents ([19-21]). Interestingly, we identified a dual role of autophagy in the regulation of cell death in RASFs. Whereas autophagy promotes cell death induced by ER stress, it plays a protective role in apoptosis induced by proteasome inhibition. We showed that OASFs were more susceptible to apoptosis induced by proteasome inhibition, a pathway that was dependent on caspase 3 activation. These data confirm those from previous studies showing an apoptosis-resistant phenotype in RASFs (). In contrast, we showed that RASFs were more sensitive to an autophagy-associated cell death pathway that was mostly independent of caspase 3.
Autophagic cell death was recently defined as cell death that is accompanied by a massive cytoplasmic vacuolization and LC3 conversion and can be suppressed by the inhibition of the autophagic pathway with the use of chemicals or by genetic means (). Such characteristics can be applied to the ER stress–induced cell death we observed in RASFs. A protective role of autophagy induction on TG-induced cell death in RASFs was previously described. However, the investigators evaluated the amount of dead cells by trypan blue exclusion and caspase 3 activity 60 hours after TG treatment (). In the present study, we showed that caspase 3 is only mildly activated and only at late stages (120 hours) in RASFs treated with TG. We also observed increased numbers of dead RASFs, as assessed by annexin V/PI staining, occurring earliest at 72 hours after TG stimulation, with a peak at 144 hours, time points that were not investigated in other studies.
Proteasome inhibition in animal models of experimental arthritis suggested proapoptotic and antiinflammatory effects in RA ([22-24]). We showed that inhibition of autophagy made RASFs more susceptible to apoptosis induced by proteasome inhibition, indicating that autophagy can at least partially compensate for a reduced clearance of polyubiquitinated proteins in the presence of impaired proteasome function. In addition, autophagy induction was recently shown to compensate for amino acid scarcity during proteasome inhibition and to rescue cells from cell death (). The modulation of autophagy activity combined with proteasome inhibition might provide new opportunities for less harmful therapies in RA. This combination was also shown to have synergistic effects in experimental anticancer therapies ([26, 27]).
Aggregation of polyubiquitinated proteins in RA has not previously been described. Aggregates of p62 containing polyubiquitinated proteins have been reported in neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease ([28, 29]), and in hepatic disorders, including alcoholic hepatitis and fatty liver disease (). Our data provide the first evidence that an imbalance in the expression of p62 and ALFY plays a critical role in the formation of polyubiquitinated protein aggregates under conditions of ER stress in RASFs and promotes cell death. It has been reported that p62, one of the known targets of autophagic degradation, played an important role in the formation of polyubiquitinated protein aggregates through its ability to interact with ubiquitin and to self-polymerize ([12, 13]). Furthermore, ALFY, one of the autophagy adaptors, has been shown to facilitate the degradation of p62-positive polyubiquitinated protein aggregates through its ability to interact with both p62 and the autophagic membrane ([15, 16]). We showed that low induction levels of ALFY after ER stress contributed to the aggregation of p62-positive polyubiquitinated proteins and the induction of autophagic cell death in RASFs in vitro. Furthermore, we detected a strong staining for ubiquitin in the lining layer of RA and OA tissue. The in vivo relevance of our data is supported by a recent study showing increased expression of the autophagy markers LC3-II and beclin 1 in the lining layer of RA tissue as compared to OA tissue, which negatively correlated with apoptosis induction in RA ().
In summary, our data provide the first evidence of a dual role of autophagy in the regulation of death pathways in RASFs. Autophagy activation exhibited a protective role in MG132-induced apoptosis and contributed to the apoptosis-resistant phenotype seen in RASFs. In contrast, RASFs were hypersensitive to autophagy under conditions of severe ER stress induced by TG, which was associated with an imbalance in the expression of p62 and ALFY, leading to the formation of polyubiquitinated protein aggregates and nonapoptotic cell death.
- Top of page
- PATIENTS AND METHODS
- AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Klein had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study conception and design. Kato, R. Gay, S. Gay, Klein.
Acquisition of data. Kato, S. Gay, Klein.
Analysis and interpretation of data. Kato, Ospelt, R. Gay, S. Gay, Klein.