Characteristics And Implication Of ER Stress And ARMET Expression In Antigen-induced Arthritis

Rheumatoid arthritis (RA) is an inflammatory, progressive, destructive autoimmune disease with synovial hyperplasia followed by impairment of quality of life. However, its exact mechanism(s) is not fully understood. The unfolded protein response (UPR) of the endoplasmic reticulum (ER) is a fundamental stress response used by eukaryotic cells to match protein synthesis demand to its capability to fold proteins within the ER to maintain cellular homeostasis. A variety of human disorders, including neurodegenerative diseases, autoimmune diseases and diabetes mellitus, are known to involve ER stress. Recent studies have been proposed that ER stress and the related signal pathway maybe involved in the pathogenesis of RA. However, the crucial questions about the role of ER stress in inflammation response of RA have not yet been answered. Armet (Arginine-Rich, Mutated in Early stage Tumors) gene is a commonly upregulated genes under various forms of ER stress in different cell types using microarray analysis. The recent studies show ARMET to be a secreted protein, and ER stress upregulates its expression and secretion. It was also found that ARMET is widely expressed in mammalian tissues and differently regulated by epileptic and ischemic insults in rodent brain and heart. These results suggest that ARMET may have important functions both under normal and pathological conditions. Therefore, we speculate up-regulated ARMET expression may be self-protective regulation, then, ARMET may play a role in inhibiting inflammation under inflammation-induced ER stress status.Objective:The purposes of this study were to investigate the ER stress activation and ARMET expression in antigen-induced arthritis model, analysis the relationship between ARMET expression and inflammation evolution. Furthermore, the possible effects of ER stress and ARMET on inflammation in antigen-induced arthritis were also explored.Methods:Methylated bovine serum albumin (mBSA) and Freund's complete adjuvant (FCA) were employed to establish rabbit AIA model. Joint lesions were assessed by MRI and hematoxylin-erosin (HE) staining. Injection of FCA in rats was employed to establish AA model, hind paw volumes of rats were measured by volume meter, arthritis index (AI) was assayed every two days after immunization, pathological changes were observed by HE staining. Fibroblasts-like synovial cells (FLS) and peritoneal macrophage (PMΦ) were isolated and cultured at 37°C with 5% CO2. Small interference RNA for ARMET was systhesized from Genepharm. Co. LTD. Synoviocytes were then transfected with pCIneo-ARMET-FLAG or siRNA-ARMET respectively. MTT assay was used to determine cell proliferation. Cell morphologies and intracellular protein localization were examed by immunocytochemistry and immunofluorescence. Reverse transcription polymerase chain reaction (RT-PCR) and western blot (WB) were used to detect mRNA and protein level respectively. Radioimmunoassay (RIA) was used to detect the levels of pro-inflammatory cytokines IL-1βand TNF-αsecreted by FLS. ELISA assay was used to determine the levels of ARMET,IL-1β,TNF-αand CRP in peripheral blood serum of AA rats.Results:1. Establishment of antigen-induced arthritis model1.1 mBSA induced rabbit arthritis model: In the arthritis model group, MRI scanning showed marked thickening of joint synoviums. HE staining showed synovium thickening and inflammatory cells infiltration. The expressions of CD68 (activated macrophage marker) and fibroblast markerα-SMA were significantly increased in AIA rabbit synoviums.1.2 AA rat model: AA rats were established and hind volumes of rats were measured by volume meter. Inflammatory polyarthritis was induced in all immunization. Synoviocytes were proliferated three to ten layers and articular cartilages were destructed and infiltrated with inflammatory cells. The levels of TNF-αand IL-1βin peripheral blood serum of AA rats significantly increased. Above results suggest that arthritis models were established successfully.2. Diverse regulation of synovium inflammation by ER stress inducible proteins in antigen-induced arthritis2.1 ER stress-related proteins were upregulated in antigen-induced arthritis: BiP expression was remarkably up-regulated in thickened synoviums, while the total level of CHOP was slightly increased in the AA synovium. Additionally, unlike BiP expressing in bothα-SMA-positive fibroblasts and CD68-positive microphages, CHOP expression only was induced inα-SMA-positive fibroblasts, but not CD68-positive microphages. We also observed expressions of BiP and CHOP in PMΦderived from AA rats. 2.2 Effects of ER stress on FLS proliferation and cytokine production:ER stress inducer tunicamycin significantly decreased the proliferation of normal FLS, while have little effect on proliferation of inflammatory FLS. Furthermore, Synoviolin was remarkably up-regulated in thickened synovial membrane, suggested inflammatory FLS overexpressing Synoviolin can resistance to ER stress-induced cell death. Additionally, tunicamycin significantly increases the expression and secretion of inflammatory cytokines IL-1β, TNF-αin AIA rabbits-derived FLS. These results suggest that ER stress response may be induced in the inflammatory status, which may contribute to the pathogenesis of AA by increased expression of genes encoding pro-inflammatory cytokines.3. Characteristics and implication of ARMET expression in antigen-induced arthritis3.1 Inflammation up-regulated ARMET expression : ARMET expression was remarkably up-regulated in inflammatory synovial tissue compared to normal controls. More interestingly, ARMET expression only was induced inα-SMA-positive fibroblasts, but not CD68-positive microphages. We also observed ARMET expression in PMΦderived from AA rats. These results suggest that inflammation up-regulated ARMET expression in FLS and PMΦ.3.2 Induction profile of ER stress and ARMET expression in synovial tissue during different phases of inflammation:BiP expression was increased significantly early in primary inflammation response (d2 ~ d7), then remained at a high level, then decreased slightly until d28; expression of ARMET showed a significant increase at d14 and then decreased slightly. CHOP expression increased slowly during inflammation development.3.3 The level of ARMET in peripheral blood serum is correlated with the degree of arthritis inflammation. The levels of ARMET in peripheral blood serum of AA rats increased significantly at d14, while serum CRP also exhibited a similar trend of expression. Correlation analysis revealed that arthritis symptoms, serum IL-1β, TNF-αlevels were negatively correlated with the level of ARMET during second inflammation phase.4. Effects of ARMET on FLS proliferation and inflammatory cytokine production Over-expression of ARMET inhibited cell proliferation under ER stress status. Inhibition of endogenous ARMET expression increased cell proliferation, promoted IL-1β,TNF-αexpression and secretion in AA rats-derived FLS. Similarly, exogenous ARMET protein can dose-dependently inhibited cell proliferation, and reduced IL-1βand TNF-αexpression and secretion in AA rats-derived FLS.5. ER stress induces nuclear translocation of ARMET in FLS Intriguingly,ARMET was found expressed in the nucleus of FLS whileα-SMA activated greatly, and tunicamycin induced nuclear translocation of ARMET in FLS. The data ruled out the possibility that ARMET is activated under ER stress and translocated to nucleus as a transcription factor,regulates downstream gene expression through specific binding with promoter sequences.Conclusions:1. ER stress may be induced by inflammation, which may contribute to the pathogenesis of arthritis by increased expression and secretion of pro-inflammatory cytokines;2. Inflammation up-regulated ARMET expression, and fibroblasts of the myofibroblastic phenotype seem to be the major cell type expressing ARMET in synovial tissues;3. ARMET is involved in the development of AA and more sensitive to inflammatory stimulus than BiP and CHOP;4. Inflammation induced ER stress and ARMET expression in AA PMΦ, suggesting different tissues-derived macrophages exhibited different responses to ER stress;5. The level of ARMET in PB of AA rats is increased and correlated with arthritis symptoms, pro-inflammatory cytokine, and CRP, suggesting ARMET is closely related to the progression of arthritis;6. ARMET plays a protective role in controlling inflammation through inhibiting synoviocytes proliferation and inflammatory cytokines production, and these effects may related to ER stress induced nuclear translocation of ARMET.