Rheumatoid arthritis as a hyper-endoplasmic reticulum-associated degradation disease
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We introduce Synoviolin as a novel pathogenic factor in rheumatoid arthritis (RA). Experimental studies indicate that this endoplasmic reticulum (ER)-resident E3 ubiquitin ligase has important functions in the ER-associated degradation (ERAD) system, an essential system for ER homeostasis. Overexpression of Synoviolin in mice causes arthropathy with synovial hyperplasia, whereas heterozygous knockdown results in increased apoptosis of synovial cells and resistance to collagen-induced arthritis in mice. On the basis of these experimental data, we propose that excess elimination of unfolded proteins (that is, 'hyper-ERAD') by overexpression of Synoviolin triggers synovial cell overgrowth and hence a worsening of RA. Further analysis of the hyper-ERAD system may permit the complex pathomechanisms of RA to be uncovered.
KeywordsRheumatoid Arthritis Endoplasmic Reticulum Endoplasmic Reticulum Stress Unfold Protein Response Synovial Cell
endoplasmic reticulum-associated degradation
rheumatoid synovial cells
small interfering RNA
tumor necrosis factor
TdT-mediated dUTP nick end labelling
unfolded protein response.
There is a general agreement that synovial cells have a crucial function in rheumatoid arthritis (RA) by forming a mass of synovial tissue, which promotes the production of matrix-degrading proteases and osteoclast activation that lead to joint destruction [1, 2, 3, 4, 5, 6]. In a series of experiments that focused on synovial cells, we determined that human T cell leukemia virus type I (HTLV-I) causes arthropathy , and that tax, the viral transforming gene of HTLV-1, and its product, pp40Tax, could transform synovial cells of patients as well as those of tax-overexpressing mice [8, 9, 10]. These results suggest that synoviocytes can acquire the ability to overgrow autonomously in RA.
Here we discuss the role of a novel pathogenic factor for RA named 'Synoviolin' (GenBank accession no. AB024690) . This novel molecule is an endoplasmic reticulum (ER)-resident ubiquitin ligase and is involved in the ER-associated degradation (ERAD) system [12, 13, 14, 15, 16, 17]. ERAD is an important processing system for ER homeostasis, and its disruption is known to result in cellular apoptosis . Surprisingly, both the amount and enzymatic activity of Synoviolin regulate synovial cell proliferation and apoptosis, at least in mice .
Cloning of Synoviolin
Synoviolin transgenic mice and arthropathy
To study the role of Synoviolin in RA, we reported previously the establishment of Synoviolin-overexpressing and Synoviolin knockout mice . Analyses of these mice demonstrated both the induction of arthritis by overexpression and the inhibition of arthritis by knockout of a single gene, that encoding Synoviolin.
First, we established human Synoviolin-overexpressing mice by using a β-actin promoter to drive systemic expression of the gene, because a northern blot analysis demonstrated that the tissue distribution of Synoviolin in the mouse is ubiquitous. Surprisingly, 10 of 33 Synoviolin-overexpressing mice developed spontaneous arthropathy after 20 weeks of age , and a histological analysis of joints of these mice demonstrated synovial cell hyperplasia and bone destruction, which resembled typical pathological features of RA joints. It should be noted that no other abnormalities are apparent in these mice throughout their life.
Next, we attempted to verify the hypothesis that Synoviolin is important for the development of arthritis using Synoviolin-knockout mice; that is, a 'loss-of-function' study. Collagen injection can induce arthritis in experimental mice, a model known as collagen-induced arthritis (CIA). Because Synoviolin homozygous knockout (syno-/-) mice die in utero , the 'loss-of-function' experiments were conducted in Synoviolin heterozygous knockout (syno+/-) mice. The incidence of arthritis in syno+/- mice (7%) was significantly lower than that in wild-type counterparts (syno+/+) (65%). Examination of the joints by soft X-ray revealed that bone destruction in syno+/- mice was much milder than that in syno+/+ mice. Immunological responses, including the production of type II collagen antibody, inflammatory cell infiltration, and elevation of inflammatory cytokine levels, were not impaired in syno+/- mice. Histological analysis of synovial tissues showed marked differences between syno+/+ and syno+/- mice. No advanced synovial cell hyperplasia was detected in CIA-syno+/- mice, even though inflammatory cell infiltration was clearly observed in them. Detailed analysis of synovial tissues showed that the number of proliferating-cell nuclear antigen (PCNA)-positive cells in CIA-syno+/- mice was not different from that in syno+/+ mice, but TdT-mediated dUTP nick end labelling (TUNEL) analysis demonstrated a significant increment of apoptotic cells in CIA-syno+/- mice. Consistent with these results, synoviolin homozygous knockout was associated with aberrantly increased apoptosis of liver and severe impairment of erythrogenesis, and embryonic death . These data suggested the importance of Synoviolin in inhibiting apoptosis.
Synoviolin in human synovial cells
Because Synoviolin-overexpressing mice show synovial cell hyperplasia, and syno+/- mice are resistant to CIA because of increased apoptosis of synovial cells, we expected that Synoviolin has both cell-proliferating and anti-apoptotic effects. In a small-scale study we showed that suppression of Synoviolin by small interfering RNA (siRNA) inhibited the growth of RSC, even under mitogenic stimulation by tumor necrosis factor (TNF)-α and interleukin-1β . These results suggested the possible role of Synoviolin in cell proliferation. We also examined the effect of tunicamycin (a glycosylation inhibitor that inhibits proper protein folding in ER) on RSC treated with siRNA to test whether the downregulation of Synoviolin increases their susceptibility to apoptosis caused by disruption of ER function. TUNEL staining of RSC revealed enhanced susceptibility to tunicamycin-induced apoptosis, similar to Synoviolin knock-down , implicating the anti-apoptotic effect of Synoviolin in ER stress. Further, larger, studies are needed to confirm the relevance of Synoviolin to human RA. It is also important to explain the molecular basis of these Synoviolin-induced cellular regulatory processes to determine the underlying pathomechanism of synovial cell overgrowth in RA.
Results of a preliminary study from our laboratories suggest that RSC are basically refractory to ER stress-induced apoptosis: the concentration of tunicamycin necessary to induce apoptosis of RSC was about tenfold that required by other human cell lines such as HEK-293 or HeLa cells. In addition, among synovial cells, RA synovial cells (n = 5) were more refractory to ER stress-induced apoptosis than OA synovial cells (n = 5) (Yamasaki S, Yagishita N, Tsuchimochi K, Kato Y, Sasaki T, Amano T, Beppu M, Nakamura H, Nishioka K, Nakajima T, unpublished data). These results suggest that RA synovial cells are refractory to ER stress-induced apoptosis. Accordingly, our working hypothesis in human RA is that Synoviolin promotes synovial cell proliferation and inhibits ER stress-induced apoptosis, leading to RA progression.
Hyper-ERAD in RA
It is generally accepted that ER-resident E3 ubiquitin ligases including Synoviolin are inherently crucial in the ERAD system, a process indispensable for elimination of unfolded proteins in the ER [11, 12, 13, 14, 15, 16, 17]. Furthermore, other studies showed that disruption of the ERAD system (a hypo-ERAD system) causes cell apoptosis and can induce various human diseases such as neurodegenerative diseases [25, 26, 27]. What are the consequences of an aberrantly upregulated ERAD (that is, a hyper-ERAD system), induced by Synoviolin overexpression in synovial cells, on the pathological process of RA?
Second, a hyper-ERAD status could keep synovial cell functioning even in the hostile milieu of inflamed RA synovia . Elevated temperature, starvation, and hypoxia increase the amount of unfolded proteins in organelles [32, 33, 34, 35], which has occasionally been observed in the RA joint. In fact, the existence of ER stress in arthritic joints has been demonstrated by the activation of activating transcription factor 6 (ATF6), an ER-resident transcriptional factor, in the nuclei of synoviocytes, because ATF6 is cleaved from ER membrane after the induction of ER stress and is translocated into the nucleus [11, 36]. It is therefore possible that hyper-ERAD could keep the ER of synovial cells functioning in inflamed joints by overcoming the environmental challenges that cause ER stress.
Third, a hyper-ERAD system could work as an anti-apoptosis system in RA synovial cells. Our previous experimental studies conducted in mice with CIA demonstrated that the downregulation of the synoviolin gene promoted the apoptosis of synovial cells in the arthritic joints . Studies by other researchers also confirmed that several E3 ubiquitin ligases (such as Parkin) exhibit a protective function against ER stress-induced apoptosis in neuronal cells . It is possible that Synoviolin also acts as an anti-apoptotic factor, and thus hyper-ERAD could prevent ER stress-induced apoptosis. Support for this conclusion is also provided by Synoviolin knockout; mouse embryonic fibroblasts lacking Synoviolin showed increased susceptibility to ER stress-induced apoptosis as observed in Synoviolin-ablated synovial cells [11, 24].
In all, there seems to be sufficient experimental evidence for the following consequences of a hyper-ERAD status: first, enhanced protein production and cell overgrowth; second, maintenance of ER function of synovial cells despite ER stress in the milieu of inflamed joints; and third, prevention of apoptosis induced by ER stress. Consequently, these processes could worsen the pathological process of RA.
The immunological aspects of RA have been studied extensively over the past several years. However, understanding these processes and their implementation in the design of new therapies for RA have not been completely successful [37, 38, 39, 40]. Here we propose a novel hypothesis for RA pathogenesis: 'hyper-ERAD', which may alter the characteristics of synovial cells in RA. Because Synoviolin knock-down does not affect the immunological pathway , this novel concept might explain the underlying pathogenic processes in RA, especially in patients with RA refractory to anti-TNF-α therapies. It is therefore important to investigate the expression of Synoviolin or the status of the ERAD system especially in these patients.
In this review we have presented a new concept of the hyper-ERAD system in the pathogenic process of RA. Although this concept was formulated through several years of research involving laboratory animals and a limited number of patients with RA, the relevance to human disease remains somewhat speculative at present. More time and efforts are needed to understand the role of the ERAD system in human RA and to define other as yet unknown aspects of RA before the design of any ERAD-based therapy for the disease.
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