| Abstract: | Objective To explore whether the increased expression of long interspersed nuclear element 1 (LINE‐1; L1) messenger RNA (mRNA) and protein in rheumatoid arthritis synovial fibroblasts (RASFs) is associated with decreased expression of Trex‐1, an exonuclease involved in the metabolization of L1 DNA:RNA hybrids. Methods Chromatin immunoprecipitation was used to detect L1‐related p40 protein (L1‐ORF1p) binding sequences in RASFs. Luciferase activity was measured in the synovial fibroblasts following cotransfection of the episomal plasmid with pJM105 expressing L1‐ORF1p and pGL3‐TS3 carrying the target sequence for L1‐ORF1p. This luciferase reporter assay was used to compare the activity between RASFs and osteoarthritis synovial fibroblasts (OASFs) and to assess correlations of luciferase activity with the expression of Trex‐1 measured by flow cytometry. The expression of Trex‐1 mRNA and protein was also compared using real‐time polymerase chain reaction, immunohistochemistry, and Western blot analyses. The role of Trex‐1 in the L1‐ORF1p–mediated luciferase activity assay was studied using interfering RNAs (iRNA) and a Trex‐1 expression vector. Results Increased luciferase activity occurred after cotransfection of synovial fibroblasts with pJM105 and pGL3‐TS3. L1‐ORF1p activity was increased in RASFs as compared with OASFs, and this was correlated inversely with the expression of Trex‐1. Levels of Trex‐1 mRNA and protein were lower in RASFs than in OASFs. After transfection of the L1 expression plasmid, Trex‐1 mRNA levels increased in OASFs, but not in RASFs. The addition of iRNA against Trex‐1, however, resulted in an enhancement of L1‐ORF1p activity in OASFs to the levels measured in RASFs. Overexpression of Trex‐1 inhibited 5‐azacytidine–induced expression of p38δ MAPK, a gene carrying the TS3 sequence. Conclusion The deficiency of Trex‐1 in RASFs allows a longer half‐life of gene products encoded by active endogenous L1 retrotransposons. This pathway may play a role in diseases in which the cells exhibit a “spontaneous” aggressive behavior. |
