CP-25 inhibits the hyperactivation of rheumatic synoviocytes by suppressing the switch in G αs -G αi coupling to the β 2 -adrenergic receptor

In essence, the β 2 adrenergic receptor (β 2 AR) plays an antiproliferative role by increasing the intracellular cyclic 3’,5’-adenosine monophosphate (cAMP) concentration through G αs coupling, but interestingly, β 2 AR antagonists are able to effectively inhibit fibroblast-like synoviocytes (FLSs) proliferation, thus ameliorating experimental RA, indicating that the β 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 β 2 AR signalling and cAMP production. By applying inhibitor of receptor internalization, and small interfering RNA (siRNA) of G αs and G αi , and by using fluorescence resonance energy transfer and coimmunoprecipitation assays, a switch in G αs -G αi coupling to β 2 AR was observed in inflammatory FLSs as well as in FLSs with chronic ISO stimulation. This G αi coupling was then revealed to be initiated by G protein coupled receptor kinase 2 (GRK2) but not β-arrestin2 or protein kinase A-mediated phosphorylation of β 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 αs -G αi coupling to β 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 β 2 AR signalling in CIA FLSs, determined the underlying mechanisms and identified the pharmacological target of the GRK2 inhibitor CP-25 in treating CIA. 7HWufKwpH5ZfY_hx8fyY6a Video Abstract