Serum starvation raises turnover of phosphorylated p62/SQSTM1 (Serine 349), reveals expression of proteasome and N-glycanase1 interactive protein RAD23B and sensitizes human synovial fibroblasts to BAY 11-7085-induced cell death.
[en] Phosphorylation of p62/SQSTM1 (p62) on Serine 349 (P-Ser349 p62) as well as proteasome dysfunction have been shown to activate the cell protective Keap1/Nrf2 pathway. We showed previously that BAY 11-7085-induced human synovial fibroblast cell death includes autophagy and p62 downregulation. In this work, we have studied expression of P-Ser349 p62 in human synovial fibroblasts. Results showed that P-Ser349 p62 was not detected in synovial cell extracts unless cells were cultured in the presence of proteasome inhibitor (MG132). MG132 revealed P-Ser349 p62 turnover, that was further increased by concomitant autophagy inhibition and markedly enhanced in serum starved cells. Starvation sensitized synovial fibroblasts to BAY 11-7085 while MG132 protected both non-starved and starved cells from BAY 11-7085-induced cell death. Lentivirus mediated overexpression of phosphorylation-mimetic p62 mutant S349E markedly protected synovial fibroblasts from BAY 11-7085. Inhibitor of Keap1-P-S349 p62 interaction, K67, had synergistic effect with MG132. Starvation increased p62 molecular weight, that was reversed by serum and bovine serum albumin re-feeding. Furthermore, starvation markedly induced RAD23B. Increased endo-beta-N-acetylglucosaminidase (ENGase) turnover was detected in starved synovial fibroblasts. PNGase F treatment produced faster migration p62 form in human synovial tissue extracts but starvation-like p62 form of higher molecular weight in synovial cell extracts. Co-transfection of NGLY1, with p62 or p62 mutants S349A and S349E markedly stabilized p62 expressions in HEK293 cells. Tunicamycin upregulated p62 and protected synovial fibroblasts from BAY 11-7085-induced cell death. These results showed that P-Ser349 p62 has pro-survival role in human synovial fibroblasts and that de-glycosylation events are involved in p62 turnover.
Disciplines :
Rheumatology
Author, co-author :
RELIC, Biserka ; Centre Hospitalier Universitaire de Liège - CHU > Département de médecine interne > Service de rhumatologie
CHARLIER, Anne ; Centre Hospitalier Universitaire de Liège - CHU > Département des Services Logistiques > Secteur gardiennage
DEROYER, Céline ; Centre Hospitalier Universitaire de Liège - CHU > Département de médecine interne > Service de rhumatologie
MALAISE, Olivier ; Centre Hospitalier Universitaire de Liège - CHU > Département de médecine interne > Service de rhumatologie
Crine, Yannick
NEUVILLE, Sophie ; Centre Hospitalier Universitaire de Liège - CHU > Département de médecine interne > Service de rhumatologie
GILLET, Philippe ; Centre Hospitalier Universitaire de Liège - CHU > Département de chirurgie > Service de chirurgie de l'appareil locomoteur
DE SENY, Dominique ; Centre Hospitalier Universitaire de Liège - CHU > Département de médecine interne > Service de rhumatologie
Malaise, Michel ; Université de Liège - ULiège > Département des sciences cliniques > Rhumatologie
Language :
English
Title :
Serum starvation raises turnover of phosphorylated p62/SQSTM1 (Serine 349), reveals expression of proteasome and N-glycanase1 interactive protein RAD23B and sensitizes human synovial fibroblasts to BAY 11-7085-induced cell death.
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