TNF-α impairs EP4 signaling through the association of TRAF2-GRK2 in primary fibroblast-like synoviocytes.

Our previous study showed that chronic treatment with tumor necrosis factor-α (TNF-α) decreased cAMP concentration in fibroblast-like synoviocytes (FLSs) of collagen-induced arthritis (CIA) rats. In this study we investigated how TNF-α impairs cAMP homeostasis, particularly clarifying the potential downstream molecules of TNF-α and prostaglandin receptor 4 (EP4) signaling that would interact with each other. Using a cAMP FRET biosensor PM-ICUE3, we demonstrated that TNF-α (20 ng/mL) blocked ONO-4819-triggered EP4 signaling, but not Butaprost-triggered EP2 signaling in normal rat FLSs. We showed that TNF-α (0.02-20 ng/mL) dose-dependently reduced EP4 membrane distribution in normal rat FLS. TNF-α significantly increased TNF receptor 2 (TNFR2) expression and stimulated proliferation in human FLS (hFLS) via ecruiting TNF receptor-associated factor 2 (TRAF2) to cell membrane. More interestingly, we revealed that TRAF2 interacted with G protein-coupled receptor kinase (GRK2) in the cytoplasm of primary hFLS and helped to bring GRK2 to cell membrane in response of TNF-α stimulation, the complex of TRAF2 and GRK2 then separated on the membrane, and translocated GRK2 induced the desensitization and internalization of EP4, leading to reduced production of intracellular cAMP. Silencing of TRAF2 by siRNA substantially diminished TRAF2-GRK2 interaction, blocked the translocation of GRK2, and resulted in upregulated expression of membrane EP4 and intracellular cAMP. In CIA rats, administration of paroxetine to inhibit GRK2 effectively improved the symptoms and clinic parameters with significantly reduced joint synovium inflammation and bone destruction. These results elucidate a novel form of cross-talk between TNFR (a cytokine receptor) and EP4 (a typical G protein-coupled receptor) signaling pathways. The interaction between TRAF2 and GRK2 may become a potential new drug target for the treatment of inflammatory diseases.

Deficiency of ß-arrestin2 exacerbates inflammatory arthritis by facilitating plasma cell formation.

ß-arrestin2 (ß-arr2) is, a key protein that mediates desensitization and internalization of G protein-coupled receptors and participates in inflammatory and immune responses. Deficiency of ß-arr2 has been found to exacerbate collagen antibody-induced arthritis (CAIA) through unclear mechanisms. In this study we tried to elucidate the molecular mechanisms underlying ß-arr2 depletion-induced exacerbation of CAIA. CAIA was induced in ß-arr2-/- and wild-type (WT) mice by injection of collagen antibodies and LPS. The mice were sacrificed on d 13 after the injection, spleen, thymus and left ankle joints were collected for analysis. Arthritis index (AI) was evaluated every day or every 2 days. We showed that ß-arr2-/- mice with CAIA had a further increase in the percentage of plasma cells in spleen as compared with WT mice with CAIA, which was in accordance with elevated serum IgG1 and IgG2A expression and aggravating clinical performances, pathologic changes in joints and spleen, joint effusion, and joint blood flow. Both LPS stimulation of isolated B lymphocytes in vitro and TNP-LPS challenge in vivo led to significantly higher plasma cell formation and antibodies production in ß-arr2-/- mice as compared with WT mice. LPS treatment induced membrane distribution of toll-like receptor 4 (TLR4) on B lymphocytes, accordingly promoted the nuclear translocation of NF-κB and the transcription of Blimp1. Immunofluorescence analysis confirmed that more TLR4 colocalized with ß-arr2 in B lymphocytes in response to LPS stimulation. Depletion of ß-arr2 restrained TLR4 on B lymphocyte membrane after LPS treatment and further enhanced downstream NF-κB signaling leading to additional increment in plasma cell formation. In summary, ß-arr2 depletion exacerbates CAIA and further increases plasma cell differentiation and antibody production through inhibiting TLR4 endocytosis and aggravating NF-κB signaling.