Down-regulation of heat shock protein 70 protects rheumatoid arthritis fibroblast-like synoviocytes from nitric oxide-induced apoptosis

IntroductionHeat shock protein 70 (Hsp70) is a well-known anti-apoptotic protein that blocks multiple steps in the stress-induced apoptotic pathway. Enhanced Hsp70 term has previously been demonstrated in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLSs). The authors investigated the role of Hsp70 in the survival of RA FLS in a sodium nitroprusside (SNP)-treated environment. Methods: Targeted knock-down of Hsp70 was performed by RNA interference in RA FLSs at paragraph 3-7. After SNP treatment, the morphological features of apoptosis were observed by phase contrast microscopy. Cell survival was measured by MTT assays and by flow cytometric conversation after propidium iodide (PI) staining. Bcl-2 signal and signalling pathways (Akt, ERK, p38, JNK) were examined with or without Hsp70 down-regulation. Results: Hsp70 down-regulation in RA FLS, induced by siRNA, was confirmed by reverse transcriptase-PCR (RT-PCR) and western blotting. When treated with SNP, Hsp70 down-regulated cells showed strikingly less cell blebbing, cytoplasmic condensation, and nuclear shrinkage than non-down-regulated control cells. Furthermore, Hsp70 down-regulated cells were endow to survive exceeding than driver's seat cells in MTT assays (mean of absorbance ratio, 4.39 in target cells versus 1.00 in government siRNA treated cells versus 1.09 in lipofectamine treated cells, P = 0.001), and according to PI staining results (mean M1 ratio, 0.21 in target cells versus 1.00 in curb siRNA treated cells versus 1.03 in lipofectamine treated cells, P = 0.001). Bcl-2 expression and Akt phosphorylation were higher in Hsp70 down-regulated RA FLSs than in control cells. When cells were treated with LY294002, a potent PI3K inhibitor, Akt phosphorylation and Bcl-2 levels were reduced and Hsp70 down-regulation no longer had a cytoprotective effect. Conclusions: Knock-down of Hsp70 protects RA FLS from nitric oxide (NO)-induced apoptosis by activating the Akt signalling pathway. These results propose that Hsp70 has a pro-apoptotic role in RA FLS.

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