Open AccessSphingosine kinase 1–mediated inhibition of Fas death signaling in rheumatoid arthritis B lymphoblastoid cells
Author(s) -
Pi Xiujun,
Tan ShiYu,
Hayes Michael,
Xiao Liqun,
Shayman James A.,
Ling Song,
Holoshitz Joseph
Publication year - 2006
Publication title -
arthritis & rheumatism
Language(s) - English
Resource type - Journals
eISSN - 1529-0131
pISSN - 0004-3591
DOI - 10.1002/art.21635
Subject(s) - sphingosine kinase , biology , microbiology and biotechnology , apoptosis , programmed cell death , sphingosine , terminal deoxynucleotidyl transferase , signal transduction , tunel assay , immunology , cancer research , receptor , sphingosine 1 phosphate , biochemistry
Objective It is becoming increasingly apparent that B cells play an important role in the pathogenesis of rheumatoid arthritis (RA). Due to the scarcity of B cells in RA, it has been technically difficult to functionally characterize B cell apoptosis in this disease. As a necessary first step to identify candidate aberrations, we investigated Fas‐mediated signaling events in immortalized peripheral blood B lymphoblastoid cell lines (LCLs) from patients with RA and controls. Methods Cell death was determined by the MTS assay, and apoptosis was detected by the TUNEL assay and DNA laddering. Proteolytic activation of caspase 3 was determined by immunoblotting, and its enzymatic activity was determined by a fluorometric technique. Messenger RNA (mRNA) expression was quantified by real‐time polymerase chain reaction (PCR) analysis. The functional role of sphingosine kinase (SPHK) was determined by measuring its enzymatic activity, by quantifying the levels of its product, sphingosine 1‐phosphate (S1P), and by investigating the ability of the SPHK inhibitor N , N ‐dimethylsphingosine and isozyme‐specific small interfering RNA (siRNA) oligonucleotides to reverse signaling aberrations. Results LCLs from patients with RA displayed disease‐specific Fas‐mediated signal transduction impairment with consequent resistance to cell death. RA LCLs displayed high constitutive SPHK activity and increased levels of S1P. Real‐time PCR analysis showed higher SPHK‐1 mRNA expression levels in RA patients compared with paired controls. Increased SPHK‐1 (but not SPHK‐2) mRNA levels were observed in synovial tissue from RA patients. Competitive inhibitors of SPHK reversed the resistance of RA LCLs to Fas‐induced apoptosis. Additionally, resistance to Fas‐mediated signaling was reversed by siRNA oligonucleotides specific for SPHK‐1 but not by oligonucleotides specific for SPHK‐2. Conclusion These findings demonstrate disease‐specific resistance to Fas‐mediated death signaling in patients with RA and implicate increased SPHK‐1 activity as the cause of this aberration.