Abstract 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.
PDE4D has been reported to exhibit significantly elevated levels in the synovium of RA patients compared with OA, yet its role in RA remains underexplored. This study aimed to elucidate the role of the GRK2-PDE4D axis in FLSs and explore its potential as a therapeutic target for RA. Abundant expression of both PDE4D and GRK2 was observed in synovial tissues from both experimental arthritis animals and RA patients, with synchronized expression noted in RA patients. Global deletion of Pde4d reduced disease incidence and alleviated arthritis in CIA mice. TNF-α upregulated PDE4D expression, causing abnormal FLSs activation and hyperproliferation. Inhibiting PDE4D restored cAMP levels, thereby reducing FLSs hyperproliferation, migration, and anti-apoptosis. Mechanistically, TNF-α-induced PDE4D upregulation was dependent on GRK2. Inhibition of GRK2 with CP-25, an esterification modification of paeoniflorin, reduced PDE4D expression and FLSs proliferation, while restoring cAMP levels. Both genetic deficiency and pharmacological inhibition of GRK2 decreased PDE4D expression, ameliorating arthritis severity in animal models. This is the first study to investigate the role of PDE4D in RA and to clarify that it can be regulated by GRK2. These findings suggest that targeting the GRK2-PDE4D axis represents a promising therapeutic strategy for RA.
To investigate the therapeutic effect and primary pharmacological mechanism of Ziyuglycoside I (Ziyu I) on collagen-induced arthritis (CIA) mice. CIA mice were treated with 5, 10, or 20 mg/kg of Ziyu I or 2 mg/kg of methotrexate (MTX), and clinical manifestations, as well as pathological changes, were observed. T cell viability and subset type were determined, and serum levels of transforming growth factor-beta (TGF-β) and interleukin-17 (IL-17) were detected. The mRNA expression of retinoid-related orphan receptor-γt (RORγt) and transcription factor forkhead box protein 3 (Foxp3) in mouse spleen lymphocytes was ascertained by the real-time reverse transcriptase-polymerase chain reaction (RT-qPCR). Molecular docking was used to detect whether there was a molecular interaction between Ziyu I and protein kinase B (Akt). The activation of mechanistic target of rapamycin (mTOR) in T cells was verified by Western blotting or immunofluorescence. Ziyu I treatment effectively alleviated arthritis symptoms of CIA mice, including body weight, global score, arthritis index, and a number of swollen joints. Similarly, pathological changes of joints and spleens in arthritic mice were improved. The thymic index, T cell activity, and RORγt production of Ziyu I-treated mice were significantly reduced. Notably, through molecular docking, western blotting, and immunofluorescence data analysis, it was found that Ziyu I could interact directly with Akt to reduce downstream mTOR activation and inhibit helper T cell 17 (Th17) differentiation, thereby regulating Th17/regulatory T cell (Treg) balance and improving arthritis symptoms. Ziyu I effectively improves arthritic symptoms in CIA mice by inhibiting mTOR activation, thereby affecting Th17 differentiation and regulating Th17/Treg balance.
Hyperplasia and migration of fibroblast-like synoviocytes (FLSs) are the key drivers in the pathogenesis of rheumatoid arthritis (RA) and joint destruction. Abundant Yes-associated protein (YAP), which is a powerful transcription co-activator for proliferative genes, was observed in the nucleus of inflammatory FLSs with unknown upstream mechanisms. Using Gene Expression Omnibus database analysis, it was found that Salvador homolog-1 (SAV1), the pivotal negative regulator of the Hippo-YAP pathway, was slightly downregulated in RA synovium. However, SAV1 protein expression is extremely reduced. Subsequently, it was revealed that SAV1 is phosphorylated, ubiquitinated, and degraded by interacting with an important serine-threonine kinase, G protein-coupled receptor (GPCR) kinase 2 (GRK2), which was predominately upregulated by GPCR activation induced by ligands such as prostaglandin E
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