Zinc finger protein A20 regulates the development and progression of osteoarthritis by affecting the activity of NF-κB p65.

OBJECTIVE: To investigate the role of Zinc finger protein A20 in osteoarthritis (OA) by regulating NF-κB p65. METHODS: A20, MMP1, MMP13 and IL-1ß expressions in human OA cartilage samples were detected by qRT-PCR. IL-1ß-induced chondrocyte was treated with A20 lentivirus activation particle, pyrrolidine dithiocarbamate (PDTC, a NF-κB inhibitor) with/without A20 siRNA. IL-6, TNF-α, and PGE2 levels were measured by ELISA, and NO production by Greiss reaction. Destabilization of the medial meniscus (DMM) surgery was used to construct the OA models, followed by injection of A20 adenovirus. MMP1 and MMP13 expression was measured by immunohistochemistry. The mRNA and protein expression were performed by qRT-PCR and western blotting, respectively. RESULTS: A20 was down-regulated in human OA cartilage samples, and negatively correlated with the expressions of MMP1, MMP13 and IL-1ß. The IL-1ß-induced chondrocyte manifested decreased A20 with increased NF-κB p65 activity. A20 overexpression suppressed the NF-κB p65 activity in IL-1ß-induced chondrocyte. Furthermore, PDTC decreased IL-1ß-induced chondrocyte apoptosis with the upregulated COL1A1, COL2A1, COL10A1 and ACAN, as well as the down-regulated MMP1, MMP13, COX2, iNOS, IL-6, TNF-α, NO and PGE2, which was reversed by A20 siRNA. In vivo, OA mice gained higher OARSI score and Mankin's score, exhibited up-regulations of MMP1 and MMP13, and decreased NF-κB p65 activity, which was improved after injection of A20 adenovirus. CONCLUSION: A20 was reduced in OA cartilage samples, and its overexpression, by suppressing the activity of NF-κB p65, could improve IL-1ß-induced chondrocyte degradation and apoptosis in vitro, as well as mitigate the inflammation in OA mice.

PKCα regulates vasopressin-induced aquaporin-2 trafficking in mouse kidney collecting duct cells in vitro via altering microtubule assembly.

AIM: Aquaporin-2 (AQP2) is a vasopressin-regulated water channel located in the collecting tubule and collecting duct cells of mammalian kidney. The aim of this study is to investigate whether PKCα plays a role in vasopressin-induced AQP2 trafficking in mouse inner medullary collecting duct 3 (mIMCD3) cells. METHODS: AQP2-mIMCD3 stable cell line was constructed by transfection of mouse inner medullary collecting duct 3 (mIMCD3) cells with AQP2-GFP construct. Then the cells were transfected with PKCα shRNA, PKCα A/25E, or PKCα scrambled shRNA. The expression levels of PKCα, AQP2, and phospho-S256-AQP2 were analyzed using Western blot. The interaction between AQP2 and PKCα was examined using immunoprecipitation. The distribution of AQP2 and microtubules was studied using immunocytochemistry. The AQP2 trafficking was examined using the biotinylation of surface membranes. RESULTS: Treatment of AQP2-mIMCD3 cells with 100 µmol/L of 1-desamino-8-D-arginine vasopressin (DdAVP) for 30 min stimulated the translocation of AQP2 from the cytoplasm to plasma membrane through influencing the microtubule assembly. Upregulation of active PKCα by transfection with PKCα A/25E plasmids resulted in de-polymerization of α-tubulin and redistributed AQP2 in the cytoplasm. Down-regulation of PKCα by PKCα shRNA partially inhibited DdAVP-stimulated AQP2 trafficking without altering α-tubulin distribution. Although 100 µmol/L of DdAVP increased AQP2 phosphorylation at serine 256, down-regulation of PKCα by PKCα shRNA did not influence DdAVP-induced AQP2 phosphorylation, suggesting that AQP2 phosphorylation at serine 256 was independent of PKCα. Moreover, PKCα did not physically interact with AQP2 in the presence or absence of DdAVP. CONCLUSION: Our results suggested that PKCα regulates AQP2 trafficking induced by DdAVP via microtubule assembly.