Limits...
Akt activity protects rheumatoid synovial fibroblasts from Fas-induced apoptosis by inhibition of Bid cleavage.

García S, Liz M, Gómez-Reino JJ, Conde C - Arthritis Res. Ther. (2010)

Bottom Line: Bid suppression completely abrogated Fas-induced apoptosis and Bid overexpression highly increased apoptotic rate of RA FLS in association with cleavage of caspase-9.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Research Laboratory and Rheumatology Unit, Hospital Clínico Universitario, Choupana s/n, Santiago de Compostela, 15706-Spain. samugp13@yahoo.es

ABSTRACT

Introduction: 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.

Show MeSH

Related in: MedlinePlus

Effect of Bid overexpression on anti-Fas induced apoptosis of RA FLS. Rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) were transfected with pDsRed2-Bid or pDsRed2 control vector, 48 hours after transfection Bid expression was analysed by (a) immunofluorescence and (b) western blot. Representative experiments are shown. (c) After transfection, cells were left untreated (Basal) or treated for 12 hours with 1 μg/ml anti-Fas antibody or pretreated for one hour with Wortmannine (Wort) or with Wort and the caspase-9 inhibitor, Z-LE(OMe) HD (O Me) FMK (LEHD), before Fas stimulation. Apoptosis was analysed by ELISA. Data are the mean (standard error of the mean) of six RA FLS lines. * indicates P < 0.05 versus basal, # indicates P < 0.05 versus anti-Fas-only treatment and † indicates P < 0.05 versus anti-Fas and Wort treatment.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2875667&req=5

Figure 4: Effect of Bid overexpression on anti-Fas induced apoptosis of RA FLS. Rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) were transfected with pDsRed2-Bid or pDsRed2 control vector, 48 hours after transfection Bid expression was analysed by (a) immunofluorescence and (b) western blot. Representative experiments are shown. (c) After transfection, cells were left untreated (Basal) or treated for 12 hours with 1 μg/ml anti-Fas antibody or pretreated for one hour with Wortmannine (Wort) or with Wort and the caspase-9 inhibitor, Z-LE(OMe) HD (O Me) FMK (LEHD), before Fas stimulation. Apoptosis was analysed by ELISA. Data are the mean (standard error of the mean) of six RA FLS lines. * indicates P < 0.05 versus basal, # indicates P < 0.05 versus anti-Fas-only treatment and † indicates P < 0.05 versus anti-Fas and Wort treatment.

Mentions: The high cleavage of Bid shown after blocking Akt phosphorylation was accompanied by a modest increase in Fas-induced apoptosis. We wondered whether availability of Bid could limit the extent of apoptosis in a way reminiscent of the resistance mediated by increased expression of anti-apoptotic molecules [6-11]. To test this possibility, cells from six different patients were transiently transfected with full-length Bid vector (pDs Red2-Bid) or pDsRed2 control vector. The efficiency of transfection was analysed by immunofluorescence assays and western blot as shown in Figures 4a and 4b. As observed in Figure 4c, the treatment with Wort alone did not alter cell viability. Interestingly, Bid overexpression highly increased Fas-induced apoptosis compared with cells transfected with pDs2Red2 control vector (P = 0.01, Figure 4c), indicating that the amount of Bid contributed to resistance to apoptosis. Pre-treatment with Wort further sensitizes to apoptosis the Bid-overexpressing FLS cells, indicating that in spite of the high levels of Bid, they were still regulated by phosphorylated Akt.


Akt activity protects rheumatoid synovial fibroblasts from Fas-induced apoptosis by inhibition of Bid cleavage.

García S, Liz M, Gómez-Reino JJ, Conde C - Arthritis Res. Ther. (2010)

Effect of Bid overexpression on anti-Fas induced apoptosis of RA FLS. Rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) were transfected with pDsRed2-Bid or pDsRed2 control vector, 48 hours after transfection Bid expression was analysed by (a) immunofluorescence and (b) western blot. Representative experiments are shown. (c) After transfection, cells were left untreated (Basal) or treated for 12 hours with 1 μg/ml anti-Fas antibody or pretreated for one hour with Wortmannine (Wort) or with Wort and the caspase-9 inhibitor, Z-LE(OMe) HD (O Me) FMK (LEHD), before Fas stimulation. Apoptosis was analysed by ELISA. Data are the mean (standard error of the mean) of six RA FLS lines. * indicates P < 0.05 versus basal, # indicates P < 0.05 versus anti-Fas-only treatment and † indicates P < 0.05 versus anti-Fas and Wort treatment.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2875667&req=5

Figure 4: Effect of Bid overexpression on anti-Fas induced apoptosis of RA FLS. Rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) were transfected with pDsRed2-Bid or pDsRed2 control vector, 48 hours after transfection Bid expression was analysed by (a) immunofluorescence and (b) western blot. Representative experiments are shown. (c) After transfection, cells were left untreated (Basal) or treated for 12 hours with 1 μg/ml anti-Fas antibody or pretreated for one hour with Wortmannine (Wort) or with Wort and the caspase-9 inhibitor, Z-LE(OMe) HD (O Me) FMK (LEHD), before Fas stimulation. Apoptosis was analysed by ELISA. Data are the mean (standard error of the mean) of six RA FLS lines. * indicates P < 0.05 versus basal, # indicates P < 0.05 versus anti-Fas-only treatment and † indicates P < 0.05 versus anti-Fas and Wort treatment.
Mentions: The high cleavage of Bid shown after blocking Akt phosphorylation was accompanied by a modest increase in Fas-induced apoptosis. We wondered whether availability of Bid could limit the extent of apoptosis in a way reminiscent of the resistance mediated by increased expression of anti-apoptotic molecules [6-11]. To test this possibility, cells from six different patients were transiently transfected with full-length Bid vector (pDs Red2-Bid) or pDsRed2 control vector. The efficiency of transfection was analysed by immunofluorescence assays and western blot as shown in Figures 4a and 4b. As observed in Figure 4c, the treatment with Wort alone did not alter cell viability. Interestingly, Bid overexpression highly increased Fas-induced apoptosis compared with cells transfected with pDs2Red2 control vector (P = 0.01, Figure 4c), indicating that the amount of Bid contributed to resistance to apoptosis. Pre-treatment with Wort further sensitizes to apoptosis the Bid-overexpressing FLS cells, indicating that in spite of the high levels of Bid, they were still regulated by phosphorylated Akt.

Bottom Line: Bid suppression completely abrogated Fas-induced apoptosis and Bid overexpression highly increased apoptotic rate of RA FLS in association with cleavage of caspase-9.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Research Laboratory and Rheumatology Unit, Hospital Clínico Universitario, Choupana s/n, Santiago de Compostela, 15706-Spain. samugp13@yahoo.es

ABSTRACT

Introduction: 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.

Show MeSH
Related in: MedlinePlus