Background We previously described that sensitivity to TRAIL-induced apoptosis varied in RA FLS from one patient to another, and was correlated with disease severity (1). The sensitivity of RA FLS was defined on the percentage of apoptotic cells following TRAIL stimulation: 0-10% corresponded to resistant cells and above 25% to sensitive RAFLS.
Objectives We performed transcriptomic comparison between sensitive and resistant RAFLS and examined the implication of identified candidates in the regulation of cell apoptosis and proliferation using silencing RNA.
Methods We screened for genes that were differentially expressed in RA FLS sensitive and resistant to TRAIL induced apoptosis transcriptome analysis with the DNA CHIPS “Human V4 OpArray” containing 35 035 probes representing approximately 25 100 genes and 39 600 transcripts. A gene was considered as “differentially expressed” between two subsets of cells when the probability of having a differential expression between RAFLS-S and RAFLS-R was greater than 60%. Cells were transfected with the siRNA and cultivated for 54 hours before carrying out the functional apoptosis tests. RA FLS were stimulated with TRAIL 1nM for 24 hours and apoptosis was measured by annexin-V/TOPRO-3.
Results Microarrays analysis revealed 14 genes that were differentially expressed between cells resistant and those sensitive cells to TRAIL-induced apoptosis. These factors are implicated in different functions, such as the respiratory chain (ATPase 6, NADH 3), the transport or metabolism of lipids (ORP-4, Phosopholipid transfer protein II) and the regulation of signalling pathways linked to extracellular factors (Sulfatase 2, GalNac-T1, Sialate OAE, Liprin β1). For other genes such as LARP6, the function is still not known. We confirmed differential expression for GalNAc-T1 (GALNT-1) and LARP6 by quantitative PCR. Using siRNA extinction, we demonstrated the implication of GalNac-T1, sulfatase 2 (SULF2) and LARP6 in the control of TRAIL induced responses. Indeed, the reduction in the expression of the targeted genes increased significantly TRAIL-induced apoptosis since the siRNA which target GALNT-1, SULF-2 and LARP6 significantly influence TRAIL-induced apoptosis, with a cell death of 67%, 75%, and 175%, respectively, compared to the TRAIL-induced apoptosis of non transfected cells
Conclusions We described several new potential targets controlling TRAIL induced cell death in RAFLS. These results are of particular interest since GalNac-T1 and LARP6 have been implicated in regulation of cell death and may represent interesting targets to induce apoptosis of proliferating cells.
Acknowledgements We thank Professor Chammas and Canovas for providing synovial tissues and fluids and Montpellier RIO Imaging (MRI) facility in Montpellier. This Supported by the Association pour la Recherche sur le Cancer, the Société Française de Rhumatologie, the Association Nationale de Défense contre l'Arthrite Rhumatoïde, and the Arthritis Foundation Courtin. Dr. Hahne's work was supported by INSERM (contract 05.524-DRV/MC/SS).
Disclosure of Interest None declared