Retrotransposable L1 elements expressed in rheumatoid arthritis synovial tissue - Association with genomic DNA hypomethylation and influence on gene expression

Objective. Rheumatoid arthritis (RA) is characterized by a progressive dest ruction of joints by invasive synovial fibroblasts (SF), We searched for re troviral sequences in Ri synovial: fluid pellets, identified a sequence sim ilar to that: of open reading frame 2 (ORF2)/L1 retrotransposable elements, explored the expression of L1 in RA synovial tissues and cultured RA SF, a nd investigated the link to genomic DNA hypomethylation and the influence o f functional L1 on gene expression. Methods. RA synovial fluid pellets were screened by re verse transcriptase- polymerase chain reaction (KT-PCR) using degenerated pol primers. The seque nces were identified by GenBank search. Riboprobes to ORF2/L1 and galectin- 3 and antibodies to the ORF1/L1-related p40 protein were used for in situ h ybridization and immunohistochemistry of synovial tissues and cultured RA S F, Real-time quantitative RT-PCR was used for: detecting ORF1 messenger RNA (mRNA), Since DNA hypomethylation occurs in inflammatory diseases, we incu bated cells with the methylation inhibitor 5-aza-2'-deoxycytidine (5-azaC) and compared RA SF and osteoarthritis (OA) SF, L1-negative RA SP were trans fected with the functional L1,2 construct, and differential gene expression was analyzed by subtractive hybridization combined,vith nested PCR. Results, RNA sequences similar to those of ORF2/L1 retrotransposable elemen ts, THE1 transposon, human endogenous retrovirus (ERV)-E, human ERV-HC2, an d gibbon ape leukemia virus pol genes were isolated from different RA synov ial fluid pellets. In RA synovial tissues, ORF2/L1 transcripts were detecte d in the sublining layer and at sites of cartilage and bone destruction. Ga lectin-3 mRNA and L1-related ORF1/p40 protein showed similar expression pat terns. In contrast, OA synovial tissues in situ and cultures in vitro were negative. Real-time quantitative RT-PCR confirmed the presence of ORF1 mRNA in cultured EPA SF (30-300-fold the amount in normal SF), demonstrating th e existence of a nondegenerated and functional L1 element. In vitro, the ma jority of RA SF expressed ORF2/L1 mRNA, After incubation of SF with 5-azaC, LI mRNA appeared in a time- and dose-dependent manner. Compared with OA SP , RA SF were more sensitive to 5-azaC, After transfection of RA SF with a f unctional L1.2 element, human stress-activated protein kinase 2 delta (SAPK 2 delta [or SAPK4]), met protooncogene, and galectin-3 binding protein gene s were differentially expressed. The transcription of the SAPK2 delta gene, favored also by DNA hypomethylation in vitro, was confirmed in RA synovial tissues. Conclusion. Taken together, these data suggest that L1 elements and SAPK2 d elta pathways play a role in the activation of RA SF.