Trex‐1 deficiency in rheumatoid arthritis synovial fibroblasts

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 p38␦ 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.Trex-1 is the major 3Ј35Ј DNA exonuclease in mammalian cells, and mutations in the human trex-1 gene can cause Aicardi-Goutières syndrome (1), a disease that is characterized by perturbed immunity. 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. The L1-related p40 protein (L1-ORF1p) has nucleic acid binding properties and favors the transcription of specific genes (6). The L1-encoded p150 protein (L1-ORF2p) has a reverse transcription acti...

Modes of Retrotransposition of Long Interspersed Element-1 by Environmental Factors

“…Moreover, single-stranded DNA derived from endogenous retroelements accumulated in Trex1 -deficient cells, and Trex1 overexpression blocked RTP of L1 (Stetson et al, 2008). Consistent with these experimental data, Trex1 activity was reduced in the synovial fibroblasts of rheumatoid arthritis patients (Neidhart et al, 2010). These observations suggest that RTP-induced production of DNA derived from endogenous retroelements is a potential molecular mechanism for the development of autoimmune disorders.…”

Expression and Regulation of PIWIL-Proteins and PIWI-Interacting RNAs in Rheumatoid Arthritis

“…This is interesting in light of the fact that LINE-1 is known to be deregulated in RASF [3] and the PIWIL/piRNA system is believed to have a main function in silencing of transponsable elements. [6] However there is another system to control LINE-1 expression, namely TREX-1 which has been found to be deficient in RASF,[27] suggesting a redundancy in LINE-1 silencing mechanisms with apparently less importance of the PIWIL/piRNA system in RASF.…”

The Exonuclease Trex1 Restrains Macrophage Proinflammatory Activation

“…Second, alternative evidence shows that ssDNA originates from the replication of endogenous retroelements being accumulated in Trex1-deficient mice (13). In fact, it has been found that Trex1 also neutralizes part of the function of endogenous retroelements in synovial fibroblasts, which in consequence leads to rheumatoid arthritis when Trex1 is not present (14). The IFN-ab production, which contributes to the above-mentioned, has been shown to be dependent on stimulator of IFN genes (STING) pathway (15).…”