Akt activity protects rheumatoid synovial fibroblasts from Fas-induced apoptosis by inhibition of Bid cleavage.
BACKGROUND
Synovial hyperplasia is a main feature of rheumatoid arthritis pathology that leads to cartilage and bone damage in the inflamed joints. Impaired apoptosis of resident synoviocytes is pivotal in this process. Apoptosis resistance seems to involve defects in the extrinsic and intrinsic apoptotic pathways. The aim of this study was to investigate the association of PI3Kinase/Akt and the mitochondrial apoptotic pathway in the resistance of rheumatoid arthritis (RA) fibroblast like synovial cells (FLS) to Fas-mediated apoptosis.


METHODS
Apoptosis was assessed by ELISA quantification of nucleosomal release, Hoechst staining and activated caspase-3/7 measure in cultured RA FLS stimulated with anti-Fas antibody. Two Phosphoinositol-3-kinase/protein Kinase B (PI3 Kinase) inhibitors, Wortmannine and LY294002, were used before anti-Fas stimulation. Proapoptotic BH3 interacting domain death agonist (Bid) was suppressed in RA FLS by small interfering RNA (siRNA) transfection. Bid was overexpressed by transfection with the pDsRed2-Bid vector. Phosphorylated Akt, caspase-9, and Bid expression were analysed by western blot.


RESULTS
PI3 kinase inhibition sensitizes RA FLS to Fas-induced apoptosis by increasing cleavage of Bid protein. Bid suppression completely abrogated Fas-induced apoptosis and Bid overexpression highly increased apoptotic rate of RA FLS in association with cleavage of caspase-9.


CONCLUSIONS
In RA FLS, phosphorylation of Akt protects against Fas-induced apoptosis through inhibition of Bid cleavage. The connection between the extrinsic and the intrinsic apoptotic pathways are critical in this Fas- mediated apoptosis and points to PI3Kinase as potential therapeutic target for RA.
García
Samuel
S
Research Laboratory and Rheumatology Unit, Hospital Clínico Universitario, Choupana s/n, Santiago de Compostela, 15706-Spain. samugp13@yahoo.es
Liz
Myriam
M
Gómez-Reino
Juan J
JJ
Conde
Carmen
C
D017209:Apoptosis / Q000502:physiology*; D001172:Arthritis, Rheumatoid / Q000378:metabolism*; D051023:BH3 Interacting Domain Death Agonist Protein / Q000378:metabolism*; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D005347:Fibroblasts / Q000378:metabolism*; D006801:Humans; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D034741:RNA, Small Interfering; D015398:Signal Transduction / Q000502:physiology; D013583:Synovial Membrane / Q000166:cytology / Q000378:metabolism; D014162:Transfection; D019014:fas Receptor / Q000378:metabolism*
D051023:BH3 Interacting Domain Death Agonist Protein; C494814:BID protein, human; D034741:RNA, Small Interfering; D019014:fas Receptor; D051057:Proto-Oncogene Proteins c-akt