Downregulation of heat shock protein 70 protects rheumatoid arthritis fibroblast-like synoviocytes from nitric oxide-induced apoptosis.
BACKGROUND
Heat shock protein 70 (Hsp70) is a well-known anti-apoptotic protein that blocks multiple steps in the stress-induced apoptotic pathway. Enhanced Hsp70 expression has previously been demonstrated in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLSs). The authors investigated the role of Hsp70 in the survival of RA FLSs in a sodium nitroprusside (SNP)-treated environment.


METHODS
Targeted knock-down of Hsp70 was performed by RNA interference in RA FLSs at passage 3-7. After SNP treatment, the morphological features of apoptosis were observed by phase-contrast microscopy. Cell survival was measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays and by flow cytometric analysis after propidium iodide (PI) staining. Bcl-2 expression and signaling pathways (Akt, extracellular signal-regulated kinase, p38, c-Jun N-terminal kinase) were examined with or without Hsp70 downregulation.


RESULTS
Hsp70 downregulation in RA FLSs, induced by small interfering RNA (siRNA), was confirmed by reverse transcriptase-polymerase chain reaction and Western blotting. When treated with SNP, Hsp70 downregulated cells showed markedly less cell blebbing, cytoplasmic condensation, and nuclear shrinkage than non-downregulated control cells. Furthermore, Hsp70 downregulated cells were found to survive better than control cells in MTT assays (mean of absorbance ratio, 4.39 in target cells versus 1.00 in control 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 control siRNA-treated cells versus 1.03 in lipofectamine-treated cells, P = 0.001). Bcl-2 expression and Akt phosphorylation were higher in Hsp70 downregulated RA FLSs than in control cells. When cells were treated with LY294002, a potent phosphoinositide 3-kinase inhibitor, Akt phosphorylation and Bcl-2 levels were reduced and Hsp70 downregulation no longer had a cytoprotective effect.


CONCLUSIONS
Knock-down of Hsp70 protects RA FLSs from nitric oxide-induced apoptosis by activating the Akt signaling pathway. These results suggest that Hsp70 has a pro-apoptotic role in RA FLSs.
Kang
Eun Ha
EH
Division of Rheumatology, Department of Internal Medicine, Rheumatism Research Institute, Seoul National University Hospital, 28 Yongun-dong, Chongno-gu, Seoul, Republic of Korea. kangeh@gmail.com
Kim
Dong Jo
DJ
Lee
Eun Young
EY
Lee
Yun Jong
YJ
Lee
Eun Bong
EB
Song
Yeong Wook
YW
D000328:Adult; D000368:Aged; D017209:Apoptosis / Q000502:physiology*; D001172:Arthritis, Rheumatoid / Q000378:metabolism*; D015153:Blotting, Western; D002478:Cells, Cultured; D015536:Down-Regulation; D005260:Female; D005347:Fibroblasts; D005434:Flow Cytometry; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008875:Middle Aged; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D009599:Nitroprusside / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology*; D013583:Synovial Membrane / Q000166:cytology* / Q000378:metabolism; D014162:Transfection
D018840:HSP70 Heat-Shock Proteins; D020030:Nitric Oxide Donors; D019253:Proto-Oncogene Proteins c-bcl-2; D034741:RNA, Small Interfering; D009599:Nitroprusside; D009569:Nitric Oxide; D051057:Proto-Oncogene Proteins c-akt