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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.

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PI3 kinase inhibition increases Bid cleavage. Rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) were left untreated (Basal) or treated for 12 hours with 1 μg/ml anti-Fas antibody or pretreated for one hour with phosphoinositol-3 (PI3) kinase inhibitor Wortmannine (Wort) before Fas stimulation. Expression of (a) phosphoAkt and (b) Bid was analysed by western blot. Data are expressed as mean (standard error of the mean) of FLS from six different RA patients. * indicates P < 0.05, ** indicates P < 0.01 and *** indicates P < 0.001. (c) A representative blot is shown.
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Figure 3: PI3 kinase inhibition increases Bid cleavage. Rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) were left untreated (Basal) or treated for 12 hours with 1 μg/ml anti-Fas antibody or pretreated for one hour with phosphoinositol-3 (PI3) kinase inhibitor Wortmannine (Wort) before Fas stimulation. Expression of (a) phosphoAkt and (b) Bid was analysed by western blot. Data are expressed as mean (standard error of the mean) of FLS from six different RA patients. * indicates P < 0.05, ** indicates P < 0.01 and *** indicates P < 0.001. (c) A representative blot is shown.

Mentions: Given the above results, it seemed possible that RA FLS could resemble human prostate cancer lines, in which the PI3K/Akt pathway interferes with TRAIL-mediated apoptosis by inhibiting the cleavage of Bid [29,30]. To test whether a similar mechanism was at play in RA FLS, we analysed the effect of Akt inhibition on Bid expression. For this, RA FLS from six different patients were treated with the PI3 kinase inhibitor Wort for one hour before the addition of anti-Fas antibody. As shown in Figure 3, this treatment significantly reduced the level of Akt phosphorylation and markedly increased the cleavage of Bid in comparison to that observed after anti-Fas alone. This later effect was demonstrated by a marked reduction of cellular Bid protein expression.


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)

PI3 kinase inhibition increases Bid cleavage. Rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) were left untreated (Basal) or treated for 12 hours with 1 μg/ml anti-Fas antibody or pretreated for one hour with phosphoinositol-3 (PI3) kinase inhibitor Wortmannine (Wort) before Fas stimulation. Expression of (a) phosphoAkt and (b) Bid was analysed by western blot. Data are expressed as mean (standard error of the mean) of FLS from six different RA patients. * indicates P < 0.05, ** indicates P < 0.01 and *** indicates P < 0.001. (c) A representative blot is shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: PI3 kinase inhibition increases Bid cleavage. Rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) were left untreated (Basal) or treated for 12 hours with 1 μg/ml anti-Fas antibody or pretreated for one hour with phosphoinositol-3 (PI3) kinase inhibitor Wortmannine (Wort) before Fas stimulation. Expression of (a) phosphoAkt and (b) Bid was analysed by western blot. Data are expressed as mean (standard error of the mean) of FLS from six different RA patients. * indicates P < 0.05, ** indicates P < 0.01 and *** indicates P < 0.001. (c) A representative blot is shown.
Mentions: Given the above results, it seemed possible that RA FLS could resemble human prostate cancer lines, in which the PI3K/Akt pathway interferes with TRAIL-mediated apoptosis by inhibiting the cleavage of Bid [29,30]. To test whether a similar mechanism was at play in RA FLS, we analysed the effect of Akt inhibition on Bid expression. For this, RA FLS from six different patients were treated with the PI3 kinase inhibitor Wort for one hour before the addition of anti-Fas antibody. As shown in Figure 3, this treatment significantly reduced the level of Akt phosphorylation and markedly increased the cleavage of Bid in comparison to that observed after anti-Fas alone. This later effect was demonstrated by a marked reduction of cellular Bid protein expression.

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