| Objective The study was designed to investigate the role of the endoplasmic reticulum stress (ERS) in senescence and apoptosis of bone marrow-mesenchymal stem cells (BM-MSCs) from systemic lupus erythematosus patients (SLE), and we also explored the possible mechanisms.Methods BM-MSCs were isolated from bone marrow of SLE patients and healthy subjects, then cultured in vitro. The surface markers were detected by flow cytometry. Electron microscopy was used to evaluate the ultrastructural changes of ER in the BM-MSCs. Western blotting (WB) was used to assay the expressions of Glucose Regulated Protein 78 (GRP 78), inositol-requiring protein-1 (IRE-1), phosphorylated protein kinase RNA-like ER kinase (PERK) and activation of downstream target eukaryotic translation initiator factor 2a (eIF 2a) and CCAAT/enhancer-binding protein (C/EBP)-homologous protein (CHOP) in BM-MSCs. Cell cycle was tested by flow cytometry. SA-?-gal assays were used to detect senescent BM-MSCs. FCM analysis was used to assess apoptotic cells. The localization of CHOP and cleaved caspase-3 were observed by Immunofluorescence staning. WB was used to assay the expressions of p27 and Skp2 in BM-MSCs. The localization of GRP 78, p27, Skp2 and F-actin were observed by Immunofluorescence staning. Reverse transcription polymerase chain reaction (RT-PCR) were used to detect the expression of p27 and Skp2 in BM-MSCs. BM-MSCs from SLE patients were treated with inhibition of ERS 4-phenylbutyric acid (4-PBA). WB and Immunofluorescence staning were used to detect the expression of GRP 78, p27 and Skp2 in BM-MSCs from SLE patients. SA-0-gal activity, Cell cycle and F-actin were observed again. Small interfering RNA (siRNA) was used to disturb the expression of p27. Expression of p27 and Cell cycle were observed again after silencing p27 with siRNA. Western blot analysis with anti-ubiquitin antibody was detected polyubiquitinated forms of p27. WB and Immunofluorescence staning were also used to detect the expression of p-IRE-1, p-PERK, cleaved caspase-3, Bcl-2 and BAX. We further used a siRNA for CHOP and p-PERK, respectively. WB was used to assay the expression of p-PERK, CHOP and Bcl-2. Then we detected the expression of p-JNK1/2, p-ERK1/2 and p-p38 in treated with or without 4-PBA.We also used a siRNA for p-IRE-1 and p-JNK1/2, respectively. WB was used to assay the expression of p-IRE-1, p-JNK1/2 and BAX.Results Dilated, distorted and swollen ER and up-regulated expression of GRP78 phosphorylated PERK and IRE-1 were observed in BM-MSCs from SLE patients. We then found that, in BM-MSCs from SLE patients,4-PBA could partly reduce the senescent and situation, such as increased SA-?-gal-positive cells, cell cycle arrest and disordered F-actin distribution. Further, we found that ERS led to the accumulation of p27 through inhibiting the ubiquitin-dependent proteasomal degradation, which contributed to ERS-induced G1 cell cycle arrest in BM-MSCs from SLE patients. Interestingly, we discovered that 4-phenylbutyric acid (4-PBA), a selective inhibitor of ERS, blocked the apoptosis of BM-MSCs from SLE patients and alleviated the level of Jun N-terminal kinase1/2 (JNK1/2) and CHOP. Furthermore, blockage of PERK signaling expression by siRNA significantly reduced the expression of CHOP, on the contrary, activated the anti-apoptotic regulator B-cell lymphoma-2 (BCL-2). Blockage of IRE-1 or JNK1/2 by siRNA resulted in the decreased expression of JNK1/2 and pro-apoptosis protein Bcl-2 associated protein X (BAX). These results implicated that ERS-mediated apoptosis was a critical determinant of BM-MSCs from SLE patients.Conclusions We provide evidence that ERS participate in the process of the senescence and apoptosis of BM-MSCs from SLE patients, which may implicate the possible involvement of BM-MSCs in the pathogenesis of SLE. |
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