Arthritis & Rheumatism, Volume 62,
November 2010 Abstract Supplement
Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Atlanta, Georgia November 6-11, 2010.
Down-Regulation of Pyrin Promotes Up-Regulation of the Anti-Apoptotic Gene, BCL2, Via the Signal Transducer, gp130, and Leads to the Activation of the PI3K/Akt Pathway.
Wood4, Geryl M., Balow Jr3, James, Sun2, Hong-Wei, Aksentijevich3, Ivona, Jin-Chae3, Jae, Shoham1, Nitza, Kastner3, Daniel L.
Allergy Immunology Laboratory, Meir Hospital, Kfar-Saba, Israel
Biodata Mining and Discovery, Office of Science and Technology, National Institutes of Health, NIAMS
Laboratory of Clinical Investigation, National Institutes of Health, NIAMS
Laboratory of Clinical Investigation, National Institutes of Health, NIAMS, Bethesda, MD
Familial Mediterranean fever (FMF) is a periodic fever syndrome characterized by episodes of self-limited fever and localized inflammation. The gene causing FMF, MEFV, encodes a protein, pyrin, which is expressed in myeloid cells and in the myeloid leukemia cell line, THP.1. Identification of several pyrin-interacting proteins has led to the conclusion that pyrin participates in at least three cellular pathways that are connected to inflammation: cell death, cytokine secretion and cytoskeletal signaling.
RNA interference (RNAi) technique was employed to compare gene expression profiles between the human myeloid leukemia cell line, THP.1, expressing endogenous pyrin (scrambled control, SC) and cells in which the gene had been knocked down (siMEFV). Affymetrix cDNA microarray analysis was used to identify potential novel pyrin-dependent pathways. Western blot and qRT-PCR analysis was used for validation. Flow cytometry and kinase inhibition assays were used to study functionality and mechanism.
Among our differentially expressed genes, we identified two up regulated genes involved in cell survival, BCL2 and gp130. Consistent with these findings, pyrin truncated mice show impaired macrophage apoptosis after IL4/LPS stimulation, independent of IL1b. Western blot and qRT-PCR analyses using independent samples, confirmed the THP.1 microarray data. Western blots comparing BCL2 expression in PBMCs from healthy controls and FMF patients revealed a significantly higher level of BCL2 in FMF patients than controls. These data were in agreement with the Bcl2 expression found in CD11b+ cells from knock-in mice expressing human mutations. The influence of pyrin knockdown on apoptosis was explored. Inducing apoptosis after staurosporine stimulation showed 1.8-fold less apoptosis in siMEFV cells compared to SC. To examine the involvement of gp130 in siMEFV up-regulation of BCL2, we co-transfected siRNA for gp130 and MEFV. We demonstrate reduction in BCL2 after the addition of siRNA for gp130 compared to siMEFV alone. Since the PI3K/Akt survival pathway is activated thru gp130 signaling, we investigated this pathway as a possible mechanism of action. Western blot detection showed no difference in expression of PI3K phosphorylation between SC and siMEFV, in contrast to Akt, which revealed increased levels of phosphorylation in siMEFV treated cells compared to SC. Also, the PI3k/Akt inhibitor LY294002 was able to reduce BCL2 up-regulation by siMEFV with further reduction evident when Akt IV, a specific Akt inhibitor was employed.
These data support the hypothesis that knocking down pyrin in myeloid cells inhibits apoptosis in part via the gp130 receptor, which activates the PI3k/Akt pathway and leads to an increase in BCL2 expression. It suggests that in some cases, the human mutations may function like the knockdown system.
To cite this abstract, please use the following information:
Wood, Geryl M., Balow Jr, James, Sun, Hong-Wei, Aksentijevich, Ivona, Jin-Chae, Jae, Shoham, Nitza, et al; Down-Regulation of Pyrin Promotes Up-Regulation of the Anti-Apoptotic Gene, BCL2, Via the Signal Transducer, gp130, and Leads to the Activation of the PI3K/Akt Pathway. [abstract]. Arthritis Rheum 2010;62 Suppl 10 :1516