Inhibition of GRK2 ameliorates the pristane-induced mouse SLE model by suppressing plasma cells differentiation.

Systemic lupus erythematosus (SLE) is a multifaceted autoimmune disorder characterized by diverse clinical manifestations and organ damage. Despite its elusive etiology, dysregulated subsets and functions of B cells are pivotal in SLE pathogenesis. Peoniflorin-6'-O-benzene sulfonate (CP-25), an esterification modification of Paeoniflorin, exhibits potent anti-inflammatory and immunomodulatory properties in autoimmune diseases (AID). However, the involvement of CP-25 and its target, GRK2, in SLE development has not been explored. In this study, we demonstrate that both genetic deficiency and pharmacological inhibition of GRK2 attenuate autoantibodies production, reduce systemic inflammation, and mitigate histopathological alterations in the spleen and kidney in the pristane-induced mouse SLE model. Importantly, our findings highlight that both genetic deficiency and pharmacological inhibition of GRK2 suppress plasma cells generation and restore dysregulated B-cell subsets by modulating two crucial transcription factors, Blimp1 and IRF4. Collectively, targeting GRK2 with CP-25 emerges as a promising therapeutic approach for SLE.

GRK2 mediated degradation of SAV1 initiates hyperplasia of fibroblast-like synoviocytes in rheumatoid arthritis.

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 E2 (PGE2) in RA. This process further contributes to the decreased phosphorylation, nuclear translocation, and transcriptional potency of YAP, and leads to aberrant FLSs proliferation. Genetic depletion of GRK2 or inhibition of GRK2 by paroxetine rescued SAV1 expression and restored YAP phosphorylation and finally inhibited RA FLSs proliferation and migration. Similarly, paroxetine treatment effectively reduced the abnormal proliferation of FLSs in a rat model of collagen-induced arthritis which was accompanied by a significant improvement in clinical manifestations. Collectively, these results elucidate the significance of GRK2 regulation of Hippo-YAP signaling in FLSs proliferation and migration and the potential application of GRK2 inhibition in the treatment of FLSs-driven joint destruction in RA.

Chronic ß-adrenergic stress contributes to cardiomyopathy in rodents with collagen-induced arthritis.

Patients with rheumatoid arthritis (RA) have a much higher incidence of cardiac dysfunction, which contributes to the high mortality rate of RA despite anti-arthritic drug therapy. In this study, we investigated dynamic changes in cardiac function in classic animal models of RA and examined the potential effectors of RA-induced heart failure (HF). Collagen-induced arthritis (CIA) models were established in rats and mice. The cardiac function of CIA animals was dynamically monitored using echocardiography and haemodynamics. We showed that cardiac diastolic and systolic dysfunction occurred in CIA animals and persisted after joint inflammation and that serum proinflammatory cytokine (IL-1ß, TNF-α) levels were decreased. We did not find evidence of atherosclerosis (AS) in arthritic animals even though cardiomyopathy was significant. We observed that an impaired cardiac ß1AR-excitation contraction coupling signal was accompanied by sustained increases in blood epinephrine levels in CIA rats. Furthermore, serum epinephrine concentrations were positively correlated with the heart failure biomarker NT-proBNP in RA patients (r2 = +0.53, P < 0.0001). In CIA mice, treatment with the nonselective ßAR blocker carvedilol (2.5 mg·kg-1·d-1, for 4 weeks) or the specific GRK2 inhibitor paroxetine (2.5 mg·kg-1·d-1, for 4 weeks) effectively rescued heart function. We conclude that chronic and persistent ß-adrenergic stress in CIA animals is a significant contributor to cardiomyopathy, which may be a potential target for protecting RA patients against HF.

A novel GRK2 inhibitor alleviates experimental arthritis through restraining Th17 cell differentiation.

T helper type 17 (Th17) cell which is induced by interleukine-6 (IL-6)-signal transducers and activators of transcription 3 (STAT3) signaling is a central pro-inflammatory T cell subtype in rheumatoid arthritis (RA) and could be significantly reduced by paeoniflorin-6'-O-benzene sulfonate (CP-25) treatment with unclear mechanisms. This study was aimed to found out the mechanism of CP-25 in hampering Th17 cells differentiation in arthritic animals thus explore more therapeutic targets for RA. In mice with collagen-induced arthritis (CIA), both circulating and splenic Th17 subsets were expanded with increased STAT3 phosphorylation and decreased Src homology 2 domain-containing protein tyrosine phosphatase 1 (SHP1)-ß-arrestin2 (arrb2)-STAT3 interaction in CD4+ helper T (Th) cells. Either CP-25 or paroxetine (PAR), an established G protein coupled receptor kinase 2 (GRK2) inhibitor treatment effectively relieved the joints inflammation of CIA mice with substantially reduced Th17 cell population through inhibiting STAT3 and restoring the SHP1-arrb2-STAT3 complex. Knockout of arrb2 exacerbated the clinical manifestations of collagen antibody-induced arthritis with upregulated Th17 cells. In vitro studies revealed that depletion of arrb2 or inhibition of SHP1 promoted Th17 cell differentiation. Moreover, stimulation of adenosine A3 receptor (A3AR) simultaneously promoted Th17 cell differentiation via accelerating abbr2-A3AR binding, which could be prevented through inhibiting GRK2 phosphorylation by CP-25 or PAR, or genetically reducing GRK2. This work has demonstrated that CP-25 or PAR treatment recovers the SHP1-arrb2-STAT3 complex which prevents STAT3 activation in Th cells through reducing arrb2 recruitment to A3AR by inhibiting GRK2 phosphorylation, leading to the reduction in Th17 cell differentiation and arthritis attenuation.

Regulating Th17/Treg Balance Contributes to the Therapeutic Effect of Ziyuglycoside I on Collagen-Induced Arthritis.

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-ß) 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.

Functions of G protein-coupled receptor 56 in health and disease.

Human G protein-coupled receptor 56 (GPR56) is encoded by gene ADGRG1 from chromosome 16q21 and is homologously encoded in mice, at chromosome 8. Both 687 and 693 splice forms are present in humans and mice. GPR56 has a 381 amino acid-long N-terminal extracellular segment and a GPCR proteolysis site upstream from the first transmembrane domain. GPR56 is mainly expressed in the heart, brain, thyroid, platelets, and peripheral blood mononuclear cells. Accumulating evidence indicates that GPR56 promotes the formation of myelin sheaths and the development of oligodendrocytes in the cerebral cortex of the central nervous system. Moreover, GPR56 contributes to the development and differentiation of hematopoietic stem cells, induces adipogenesis, and regulates the function of immune cells. The lack of GPR56 leads to nervous system dysfunction, platelet disorders, and infertility. Abnormal expression of GPR56 is related to the malignant transformation and tumor metastasis of several cancers including melanoma, neuroglioma, and gastrointestinal cancer. Metabolic disorders and cardiovascular diseases are also associated with dysregulation of GPR56 expression, and GPR56 is involved in the pharmacological resistance to some antidepressant and cancer drug treatments. In this review, the molecular structure, expression profile, and signal transduction of GPR56 are introduced, and physiological and pathological functions of GRP56 are comprehensively summarized. Attributing to its significant biological functions and its long N-terminal extracellular region that interacts with multiple ligands, GPR56 is becoming an attractive therapeutic target in treating neurological and hematopoietic diseases.

Ziyuglycoside I attenuates collagen-induced arthritis through inhibiting plasma cell expansion.

ETHNOBOTANICAL RELEVANCE: With most of the anti-rheumatic drugs having severe adverse drug reactions and poor tolerance, the active components from natural herbs provides a repository for novel, safe, and effective drug development. Sanguisorba officinalis L. exhibits definite anti-inflammatory capacity, however, whether it has anti-rheumatic effects has not been revealed. AIM OF THE STUDY: In the present study, the effect of Ziyuglycoside I (Ziyu I), one of the most important active components in Sanguisorba officinalis L., was investigated in treating collagen-induced arthritis (CIA), illuminating its potential pharmacological mechanisms. MATERIAL AND METHODS: CIA mice were treated with 5, 10, or 20 mg/kg of Ziyu I or 2 mg/kg of MTX, and clinical manifestations as well as pathological changes were observed. T and B cell viability was determined using cell counting kit-8, plasma autoantibodies and cytokines were tested with ELISA, T and B cell subsets were identified by flow cytometry, Blimp1 expression was detected by RT-qPCR and in situ immunofluorescence. The expression of activation-induced cytidine deaminase (AID) was detected by immunohistochemistry. ERK activation in B cells was verified through western blotting and immunofluorescence. Meanwhile, bioinformatics retrieval and molecular docking/molecular dynamics were used to predict the relationship between Blimp1, ERK and Ziyu I with the pharmacokinetics and toxicity of Ziyu I being evaluated in the ADMETlab Web platform. RESULTS: Ziyu I treatment effectively alleviated the joint inflammatory manifestation including arthritis index, global scores, swollen joint count and body weight of CIA mice. It improved the pathological changes of joint and spleen of arthritic mice, especially in germinal center formation. Ziyu I displayed a moderate regulatory effect on T cell activation, the percentage of total T and helper T cells, and tumor necrosis factor-α, but transforming growth factor-ß was not restored. Increased spleen index, B cell viability and plasma auto-antibody production in CIA mice were significantly reduced by Ziyu I therapy. Of note, we found that Ziyu I administration substantially inhibited the excessive expansion of plasma cells in spleen through preventing the expression of B lymphocyte induced maturation protein 1 (Blimp1) and AID in B cells. Ziyu I was predicted in silico to directly interact with ERK2, and reduce ERK2 activation, contributing to the depressed expression of Blimp1. Moreover, Ziyu I was predicted to have a favorable pharmacokinetic profile and low toxicity. CONCLUSION: Ziyu I effectively ameliorates CIA in mice by inhibiting plasma cell generation through prevention of ERK2-mediated Blimp1 expression in B cells. Therefore, Ziyu I is a promising candidate for anti-arthritic drug development.

G protein-coupled receptor kinase type 2 and ß-arrestin2: Key players in immune cell functions and inflammation.

G protein-coupled receptor kinase type 2 (GRK2) and ß-arrestin2 are representative proteins that regulate the transduction and trafficking of G protein-coupled receptor (GPCR) signaling. The kinase GRK2 and the multifunctional scaffolding protein ß-arrestin2 are key integrated signaling nodes in various biological processes, and both of them regulate cell proliferation and promote cell invasion and migration. GRK2/ß-arrestin2 play multiple roles in the pathological mechanisms of a wide range of diseases including heart failure, cancer, and inflammatory diseases. This review summarizes the roles of GRK2/ß-arrestin2 in immune cell function and focuses on the pathological implications of GRK2/ß-arrestin2 in various inflammatory diseases.

IgD promotes pannus formation by activating Wnt5A-Fzd5-CTHRC1-NF-κB signaling pathway in FLS of CIA rats and the regulation of IgD-Fc-Ig fusion protein.

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by joint inflammation, synovial hyperplasia, cartilage degeneration, bone erosion, and pannus. Immunoglobulin D (IgD) plays an important role in autoimmune diseases although the content of it in vivo is low. Increased concentrations of anti-IgD autoantibodies have been detected in many RA patients. IgD-Fc-Ig fusion protein is constructed by connecting human IgD Fc domain and IgG1 Fc domain, which specifically block the IgD/ IgDR pathway and regulate the function of cells expressing IgDR to treat RA. The expression levels of Wnt5A and Frizzled 5 are higher in RA synovial tissue specimens. The complex of Wnt5A-Fzd5-LRP5/6-CTHRC1 promotes the expression of hypoxia inducible factor-1α by activating nuclear factor kappa-B (NF-κB), leading to high expression of VEGF and participating in angiogenesis. VEGF is the strongest angiogenic factor found so far. Here, we aimed to explore whether IgD participates in synovitis by binding to IgDR and regulating the activation of Wnt5A-Fzd5-CTHRC1-NF-κB signaling pathway in fibroblast synovial cells (FLSs), whether IgD-Fc-Ig fusion protein inhibits VEGF production in FLS of CIA and explore mechanism. We found that IgDR is expressed on MH7A and FLS. IgD promotes VEGF expression by activating Wnt5A-Fzd5-CTHRC1-NF-κB signaling pathway in MH7A and FLS. After activation of Fzd5 with Wnt5A, IgD-Fc-Ig reduced VEGF-A level in the culture supernatant of MH7A stimulation by IgD. The expressions of CTHRC1, Fzd5, p-P65 and VEGF in MH7A and FLSs were down-regulated after IgD-Fc-Ig treatment. IgD-Fc-Ig suppressed the combination of CTHRC1 and Fzd5 as well. By using the animal model, we demonstrated that IgD-Fc-Ig suppress ankle CTHRC1 and Fzd5 production resulted in inhibition of index of joint inflammation of CIA rats, which were consistent with vitro results. Conclusively, IgD-Fc-Ig inhibits IgD and Wnt5A-induced angiogenesis and joint inflammation by suppressing the combination of CTHRC1 and Fzd5. Our results show that IgD-Fc-Ig exerts its suppressive effect on IgD and Wnt5A by Wnt5A-Fzd5-CTHRC1-NF-κB signaling pathway.