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

Abstract

Objective Rheumatoid arthritis (RA) is characterized by a progressive destruction of joints by invasive synovial fibroblasts (SF). We searched for retroviral sequences in RA synovial fluid pellets, identified a sequence similar to that of open reading frame 2 (ORF2)/L1 retrotransposable elements, explored the expression of L1 in RA synovial tissues and cultured RA SF, and investigated the link to genomic DNA hypomethylation and the influence of functional L1 on gene expression. Methods RA synovial fluid pellets were screened by reverse transcriptase–polymerase chain reaction (RT‐PCR) using degenerated pol primers. The sequences 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 hybridization and immunohistochemistry of synovial tissues and cultured RA SF. Real‐time quantitative RT‐PCR was used for detecting ORF1 messenger RNA (mRNA). Since DNA hypomethylation occurs in inflammatory diseases, we incubated cells with the methylation inhibitor 5‐aza‐2′‐deoxycytidine (5‐azaC) and compared RA SF and osteoarthritis (OA) SF. L1‐negative RA SF were transfected with the functional L1.2 construct, and differential gene expression was analyzed by subtractive hybridization combined with nested PCR. Results RNA sequences similar to those of ORF2/L1 retrotransposable elements, THE1 transposon, human endogenous retrovirus (ERV)–E, human ERV‐HC2, and gibbon ape leukemia virus pol genes were isolated from different RA synovial fluid pellets. In RA synovial tissues, ORF2/L1 transcripts were detected in the sublining layer and at sites of cartilage and bone destruction. Galectin‐3 mRNA and L1‐related ORF1/p40 protein showed similar expression patterns. 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 RA SF (30–300‐fold the amount in normal SF), demonstrating the existence of a nondegenerated and functional L1 element. In vitro, the majority of RA SF expressed ORF2/L1 mRNA. After incubation of SF with 5‐azaC, L1 mRNA appeared in a time‐ and dose‐dependent manner. Compared with OA SF, RA SF were more sensitive to 5‐azaC. After transfection of RA SF with a functional L1.2 element, human stress‐activated protein kinase 2 delta (SAPK2δ [or SAPK4]), met protooncogene, and galectin‐3 binding protein genes were differentially expressed. The transcription of the SAPK2δ gene, favored also by DNA hypomethylation in vitro, was confirmed in RA synovial tissues. Conclusion Taken together, these data suggest that L1 elements and SAPK2δ pathways play a role in the activation of RA SF.

Tenosynovitis and osteoclast formation as the initial preclinical changes in a murine model of inflammatory arthritis

“…This is consistent with the well-documented activation of MAPK families in human RA (27)(28)(29)(30). Thus, even in the preclinical disease phase, the joints of the HuTNF-Tg mice showed an activation of these 2 MAPK members, suggesting that p38 MAPK and ERK mediate the cellular effects of TNF right from the start of molecular onset of arthritis.…”

Genetic or epigenetic difference causing discordance between monozygotic twins as a clue to molecular basis of mental disorders

“…80 Transposon may become active during cell culture, especially when the DNA methylation inhibitor, 5-aza-deoxycitidine (5-aza-dC), is applied. 81 In both cases, the possible effects of blood transfusion in utero might obscure the difference between twins. 38 Most of the above-noted problems can be overcome by using fibroblasts, which can also be cultured and stored.…”

Trex‐1 deficiency in rheumatoid arthritis synovial fibroblasts

“…Such mutations also appear to be involved in systemic lupus erythematosus (SLE) (2), in which the failure to process intermediates of nucleic acid metabolism can result in the activation of uncontrolled autoimmunity. Trex-1 prevents the accumulation of single-stranded DNA (ssDNA) fragments derived from endogenous long interspersed nuclear element 1 (LINE-1; L1) retrotransposons (3), the expression of which has been reported in rheumatoid arthritis synovial fibroblasts (RASFs) (4). The promoter of the L1 retroelement is hypomethylated in RASFs (5), reflecting global DNA hypomethylation.…”