ABSTRACT
Podocyte injury or dysfunction can lead to proteinuria and glomerulosclerosis. Zonula occludens 1 (ZO-1) is a tight junction protein which connects slit diaphragm (SD) proteins to the actin cytoskeleton. Previous studies have shown that the expression of ZO-1 is decreased in chronic kidney disease (CKD). Thus, elucidation of the regulation mechanism of ZO-1 has considerable clinical importance. Triptolide (TP) has been reported to exert a strong antiproteinuric effect by inhibiting podocyte epithelial mesenchymal transition (EMT) and inflammatory response. However, the underlying mechanisms are still unclear. We found that TP upregulates ZO-1 expression and increases the fluorescence intensity of ZO-1 in a puromycin aminonucleoside (PAN)-induced podocyte injury model. Permeablity assay showed TP decreases podocyte permeability in PAN-treated podocyte. TP also upregulates the DNA demethylase TET2. Our results showed that treatment with the DNA methyltransferase inhibitors 5-azacytidine (5-AzaC) and RG108 significantly increased ZO-1 expression in PAN-treated podocytes. Methylated DNA immunoprecipitation (MeDIP) and hydroxymethylated DNA immunoprecipitation (hMeDIP) results showed that TP regulates the methylation status of the ZO-1 promoter. Knockdown of TET2 decreased ZO-1 expression and increased methylation of its promoter, resulting in the increase of podocyte permeability. Altogether, these results indicate that TP upregulates the expression of ZO-1 and decreases podocyte permeability through TET2-mediated 5 mC demethylation. These findings suggest that TP may alleviate podocyte permeability through TET2-mediated hydroxymethylation of ZO-1.
Subject(s)
Dioxygenases , Diterpenes , Epoxy Compounds , Phenanthrenes , Podocytes , Zonula Occludens-1 Protein , Podocytes/metabolism , Podocytes/drug effects , Podocytes/pathology , Zonula Occludens-1 Protein/metabolism , Phenanthrenes/pharmacology , Diterpenes/pharmacology , Epoxy Compounds/pharmacology , Dioxygenases/metabolism , Animals , DNA-Binding Proteins/metabolism , Mice , Proto-Oncogene Proteins/metabolism , Permeability/drug effects , Humans , DNA Methylation/drug effectsABSTRACT
Deficiency of G protein-coupled receptor kinase 2 (GRK2) was found to protect mice from dextran sulfate sodium (DSS)-induced colitis. Paeoniflorin-6'-O-benzene sulfonate (CP-25) has been shown to exert anti-inflammatory immune regulatory effects in animal models of inflammatory autoimmune disease. This study aimed to investigate the of GRK2 in the pathogenesis of ulcerative colitis (UC) and its effects on macrophage polarization, macrophage subtype regulation of intestinal barrier function, and therapeutic effects of CP-25 in mice with DSS-induced colitis. We found imbalanced macrophage polarization, intestinal barrier dysfunction, and abnormal activation of GRK2 and TLR4-NF-κB-NLRP3 inflammasome signaling pathway in the colonic mucosa of patients with UC. CP-25, restored the damaged intestinal barrier function by inhibiting the transmembrane region of GRK2 in macrophages stimulated by lipopolysaccharides. CP-25 exerted therapeutic effects by ameliorating clinical manifestation, regulating macrophage polarization, and restoring abnormally activated TLR4-NF-κB-NLRP3 inflammasome signaling pathway by inhibiting GRK2. These data suggest the pathogenesis of UC may be related to the imbalance of macrophage polarization, which leads to abnormal activation of TLR4-NF-κB-NLRP3 inflammasome signaling pathway mediated by GRK2 and destruction of the intestinal mucosal barrier. CP-25 confers therapeutic effects on colitis by inhibiting GRK2 translocation to induce the downregulation of TLR4-NF-κB-NLRP3 inflammasome signaling in macrophages.
Subject(s)
Colitis , Inflammasomes , Animals , Colitis/chemically induced , Colitis/drug therapy , Colitis/genetics , Dextran Sulfate/toxicity , Disease Models, Animal , Humans , Macrophages/metabolism , Mice , Mice, Inbred C57BL , NF-kappa B/genetics , NF-kappa B/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Signal Transduction , Sulfates , Toll-Like Receptor 4/genetics , Toll-Like Receptor 4/metabolismABSTRACT
OBJECTIVE: This study aims to explore the effect of paeoniflorin-6'-O-benzene sulfonate (CP-25) on the migration of fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA) and the mechanism focused on CXCR4-Gßγ-PI3K/AKT signaling. METHODS: Human synovial tissues were collected from RA and osteoarthritis (OA) patients. Immunohistochemistry (IHC) and Western blot were used to detect the protein expression of CXCR4, GRK2, Gßγ, PI3K, p-PI3K, AKT and p-AKT. Transwell was adopted to analyse the migration of fibroblast-like synoviocytes (FLS). Co-immunoprecipitation (Co-IP) and laser scanning confocal microscopy (LSCM) were used to detect the combination of GRK2 and Gßγ, the combination of PI3K and Gßγ. RESULTS: The expression level of CXCR4, GRK2, Gßγ, p-p85 and p-AKT were increased in RA synovial tissue according to the results of IHC and Western blot. In vitro, the migration of FLS was increased after stimulation of CXCL12, inhibition of Gßγ suppressed the migration and phosphorylation of p85 and AKT induced by CXCL12 in FLS, and CP-25 had the same effect as inhibition of Gßγ. The membrane expression of GRK2, Gßγ, PI3K and the combination of GRK2 and Gßγ, the combination of PI3K and Gßγ in FLS were increased after the stimulation of CXCL12, and CP-25 had an ability in reducing the membrane expression and the combination of these proteins. CONCLUSION: Excessive migration of FLS in RA was associated with over-activation of PI3K/AKT signaling, and the activity of Gßγ was involved in the over-activation of PI3K/AKT. CP-25 down-regulated CXCR4-Gßγ-PI3K/AKT signals by inhibiting GRK2-Gßγ complex membrane translocation.
