CP-25 inhibits the hyperactivation of rheumatic synoviocytes by suppressing the switch in G_s-G_i coupling to the ₂-adrenergic receptor

In essence, the beta(2) adrenergic receptor (beta(2)AR) plays an antiproliferative role by increasing the intracellular cyclic 3',5'-adenosine monophosphate (cAMP) concentration through G(alpha s) coupling, but interestingly, beta(2)AR antagonists are able to effectively inhibit fibroblast-like synoviocytes (FLSs) proliferation, thus ameliorating experimental RA, indicating that the beta(2)AR signalling pathway is impaired in RA FLSs via unknown mechanisms. The local epinephrine (Epi) level was found to be much higher in inflammatory joints than in normal joints, and high-level stimulation with Epi or isoproterenol (ISO) directly promoted FLSs proliferation and migration due to impaired beta(2)AR signalling and cAMP production. By applying inhibitor of receptor internalization, and small interfering RNA (siRNA) of G(alpha s) and G(alpha i), and by using fluorescence resonance energy transfer and coimmunoprecipitation assays, a switch in G(alpha s)-G(alpha i) coupling to beta(2)AR was observed in inflammatory FLSs as well as in FLSs with chronic ISO stimulation. This G(alpha i) coupling was then revealed to be initiated by G protein coupled receptor kinase 2 (GRK2) but not beta-arrestin2 or protein kinase A-mediated phosphorylation of beta(2)AR. Inhibiting the activity of GRK2 with the novel GRK2 inhibitor paeoniflorin-6 '-O-benzene sulfonate (CP-25), a derivative of paeoniflorin, or the accepted GRK2 inhibitor paroxetine effectively reversed the switch in G(alpha s)-G(alpha i) coupling to beta(2)AR during inflammation and restored the intracellular cAMP level in ISO-stimulated FLSs. As expected, CP-25 significantly inhibited the hyperplasia of FLSs in a collagen-induced arthritis (CIA) model (CIA FLSs) and normal FLSs stimulated with ISO and finally ameliorated CIA in rats. Together, our findings revealed the pathological changes in beta(2)AR signalling in CIA FLSs, determined the underlying mechanisms and identified the pharmacological target of the GRK2 inhibitor CP-25 in treating CIA.