Date: Sunday, November 8, 2015
Session Type: ACR Poster Session A
Session Time: 9:00AM-11:00AM
Background/Purpose: BAY 11-7085 is an inhibitor of IκBα phosphorylation that leads to NF-κB inactivation and downregulation of inflammation in mouse model of asthma, in articular chondrocytes as well as in synovial fibroblasts. Proliferation of synovial fibroblasts is one of the major causes of cartilage destruction in rheumatic diseases that needs to be counteracted. In our previous work, we observed that BAY 11-7085 induces apoptosis by inactivating PPAR-γ in human synovial fibroblasts. In this work, we investigated if autophagy was also involved in BAY 11-7085-induced synovial fibroblast cell death.
Methods: Primary human synovial fibroblasts were isolated from synovial membrane of osteoarthritis patients after joint surgery. Cells were seeded within 0.5 ml of DMEM medium with 10% FCS or without FCS in starvation condition. Synovial fibroblasts were stimulated with 15d-PGJ2, prednisolone, mifepristone, pepstatin A, rapamycin or NH4Cl for 24 h, then BAY 11-7085 was added for two additional hours. MTS was performed for survival assay. Proteins such as LC3B, atg3, p62, glucocorticoid receptor (GR), phospho-glucocorticoid receptor pSer211, PPAR-γ, phospho-PPAR-γ pSer112, phospho-ERK1/2 on Tyr-204, caspase 8, α-tubulin and GAPDH were analyzed by western blotting. pPPAR-γ1, pCMV-MEK1 and pEGFP were overexpressed in HEK293 cells. P values were obtained using the Mann-Whitney test. A value of P< 0.05 was considered as statistically significant.
Results: Results have shown that BAY 11-7085 could induce autophagy within 2 hours of cell treatment and that it preceded BAY 11-7085-induced apoptosis. Of interest, BAY 11-7085 induced Serine 211 phosphorylation and degradation of glucocorticoid receptor (GR), suggesting that GR activation is involved in BAY 11-7085-induced autophagy. Glucocorticoid prednisolone induced both activation and degradation of GR, as well as autophagy in synovial fibroblasts. Of interest, BAY 11-7085-induced cell death was significantly decreased with glucocorticoid inhibitor mifepristone and with inhibitors of autophagy. BAY 11-7085-induced autophagy and GR activation were both downregulated by PPAR-γ agonists such as 15d-PGJ2 and MEK/ERK inhibitor UO126. Inhibition of autophagy markedly decreased endogenous and BAY 11-7085-induced ERK phosphorylation, suggesting a positive feed back loop between ERK activation and autophagy in synovial fibroblasts. Co-transfection of MEK1 with PPAR-γ1 in HEK293 cells caused known inhibitory phosphorylation of PPAR-γ1 (Serine 112) and enhanced GR degradation, in the absence or presence of prednisolone. Furthermore, GR was phosphorylated on Serine 211 and downregulated in synovial fibroblasts during serum starvation-induced autophagy.
Conclusion: BAY 11-7085 induced human synovial fibroblast autophagy that acts as an agonist to promote BAY 11-7085-induced apoptosis. Furthermore, our results suggest that BAY 11-7085-induced autophagy in synovial fibroblasts is mediated through GR activation and PPAR-γ inactivation. Thus, modulation of autophagy and inflammation through GR and PPAR-γ regulation is a challenging approach to be further studied in rheumatic diseases.
To cite this abstract in AMA style:Relic B, Charlier E, Deroyer C, Malaise O, Neuville S, Desoroux A, Gillet P, Malaise MG, de Seny D. Bay 11-7085 Induces Glucocorticoid Receptor Activation and Autophagy to Initiate Human Synovial Fibroblast Cell Death [abstract]. Arthritis Rheumatol. 2015; 67 (suppl 10). https://acrabstracts.org/abstract/bay-11-7085-induces-glucocorticoid-receptor-activation-and-autophagy-to-initiate-human-synovial-fibroblast-cell-death/. Accessed September 22, 2019.
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/bay-11-7085-induces-glucocorticoid-receptor-activation-and-autophagy-to-initiate-human-synovial-fibroblast-cell-death/