Differential Regulation Ofβ-arrestins On Inflammation In Rheumatoid Atrhritis

Rheumatoid arthritis (RA) is a chronic autoimmune disease of unknownetiology that predominantly presents with chronic synovial inflammation. With theability to ultimately destroy synovium, cartilage and bone tissue, RA affectsapproximately 1% of the global population and induces significant morbidity withassociated economic cost. Pro-inflammatory cytokines and chemokines play criticalroles in RA. Tumor necrosis factor (TNF)α, interleukin (IL)-1βand IL-6 contribute tothe development of RA. A number of agents that block the production of TNFα, IL-1βor IL-6 have been introduced into clinical practice or are currently in clinical trials.Interpretation of the signaling pathways regulating pro-inflammatory cytokine andchemokine production in RA may provide us with novel therapeutic approaches.The Toll-like receptors (TLRs) signaling pathway is a major pathway for theregulation of pro-inflammatory gene expression. Recent studies have addressed thepotential role of TLRs in RA. Involvement of various TLRs in the onset andperpetuation of experimental arthritis has been convincingly demonstrated. TLRs andseveral of their ligands, such as low molecular weight hyaluronan (LMW-HA) andhigh-mobility group box 1 (HMGB1), are highly expressed in the synovial tissue fromRA patients compared with that from healthy donors. TLR4-mediated activation ofdendritic cells (DCs) from RA patients induced significantly increased levels of keypro-inflammatory cytokines compared with DCs from healthy volunteers. Injection ofthe TLR4 ligand bacterial endotoxin lipopolysaccharide (LPS) into murine synovialjoints induced experimental arthritis that shares similar immunological andpathological features with human RA. These evidences therefore suggest that theTLRs signaling pathway play critical roles in RA development.β-arrestin 1 and 2 are ubiquitously expressed adaptor proteins that regulate Gprotein function by forming complexes with G protein-coupled receptor (GPCR)sfollowing agonist binding that lead to termination of G protein activation anddesensitization of GPCRs.β-arrestins also mediate GPCR endocytosis by interacting with clathrin in clathrin-coated pits (CCP). However, in addition to GPCRdesensitization, it has been also shown thatβ-arrestins function as multifunctionalscaffold/adaptor proteins for GPCR activation of MAP kinases, including ERK1/2,JNK, p38, and Src family kinases.β-arrestins also regulate cellular survival/apoptosispathways such as ERK, JNK, p38 and PI3K-mediated signaling. Recently,β-arrestin1 and 2 were reported to be involved in regulation of inflammation. Substantialevidence confirmed thatβ-arrestins are essential regulators of chemotaxis. In addition,recent studies have implicatedβ-arrestins participate in TLRs signaling. It has beenshown thatβ-arrestin 1 and 2 differentially mediate TLR4-induced activation ofERK1/2 but suppress NF-κB (nuclear factor kappa B) activation. NF-κB is inhibitedas a consequence ofβ-arrestins interacting with TNF receptor-associated factor(TRAF) 6 and inhibiting TRAF6 ubiquitination.β-arrestins also negatively regulateNF-κB activation by binding and preventing the degradation of IκBα(inhibitor kappaBα). It was concluded thatβ-arrestins are essential regulators of TLRs signaling.In this project, we hypothesized thatβ-arrestin 1 and 2 play critical roles inRA. We first determined the expression ofβ-arrestin 1 and 2 in spleen tissue andspecific splenocytes in collagen-induced arthritis (CIA) mice. Compared with thecontrol mice,β-arrestin 1 expression showed no change in spleen tissue from CIAmice, whileβ-arrestin 2 expression was increased. Augmentedβ-arrestin 1 and 2expressions were found in CD4+T cells and dendritic cells. However,β-arrestin 1expression was significantly decreased whileβ-arrestin 2 expression was unaffectedin CD8+T cells and macrophages. Since fibroblast-like synoviocytes (FLS) insynovium are the primary effectors of cartilage destruction and inflammatorypathogenesis, the hind knee joint tissue and ex vivo FLS were isolated from the CIAmice and human TNFαtransgenic (TNFtg) mice. Then theβ-arrestin 1 and 2expression were examined in the hind knee joint tissue and FLS. Our resultsdemonstrated for the first time thatβ-arrestin 1 and 2 expression were significantlyincreased in the joint tissue and ex vivo FLS from CIA and TNFtg mice. In subsequentstudies, TNFα, LMW-HA or HMGB1 was used to stimulate FLS in vitro. Our datashowed that TNFαor LMW-HA significantly increasedβ-arrestin 1 and 2 expressionin FLS. Interestingly, HMGB1 only increasedβ-arrestin 1 but notβ-arrestin 2expression in FLS. By employing MAP kinase inhibitors, we found that TNFα- or LMW-HA- inducedβ-arrestin 2 expression was blocked by a p38 inhibitor SB203580,which suggested thatβ-arrestin 2 expression in FLS may be mediated by p38 pathway.To examine the role ofβ-arrestins in the regulation of inflammation in RA, the effectsofβ-arrestins overexpression/deficience on LMW-HA induction of inflammatorymediators were determined. We observed that LMW-HA-induced TNFαand IL-6production were increased byβ-arrestin 1 overexpression, but decreased byβ-arrestin1 deficiency. However,β-arrestin 2 overexpression significantly decreased LMW-HA-induced TNFαand IL-6 production. To further examine the role ofβ-arrestin 2 inthe pathogenesis of RA, WT andβ-arrestin 2 KO mice were subjected to the collagenantibody-induced arthritis (CAIA). Our data demonstrated thatβ-arrestin 2 KO miceexhibit more severe arthritis in CAIA. These collectively studies demonstrated for thefirst time thatβ-arrestin 1 and 2 differentially regulate FLS inflammatory mediatorproduction, andβ-arrestin 2 may negatively regulate the inflammation ofexperimental arthritis.In conclusion, increasedβ-arrestin 1 and 2 expressions were found in specificsplenocytes, the joint tissue and FLS in arthritic mice. Moreover, theβ-arrestin 2expression in FLS seems to be regulated by p38 pathway. However, increasedβ-arrestin 1 and 2 expression appear to regulate arthritis development differentially.β-arrestin 2 may negatively regulate experimental arthritis, whereasβ-arrestin 1 maycontribute to the development of arthritis. The newly discovered isoform specific roleofβ-arrestins provides novel insights into molecular mechanisms of regulatinginflammatory respose in RA pathogenesis.β-arrestins may become potentialtherapeutic targets for RA and other autoimmune diseases.