Trex-1 deficiency in rheumatoid arthritis synovial fibroblasts.
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.


CONCLUSIONS
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.
Neidhart
Michel
M
Center for Experimental Rheumatology, Zurich Center for Integrative Human Physiology, Zurich, Switzerland. michel.neidhart@usz.ch
Karouzakis
Emmanuel
E
Schumann
Gerald G
GG
Gay
Renate E
RE
Gay
Steffen
S
D001172:Arthritis, Rheumatoid / Q000378:metabolism* / Q000473:pathology; D001374:Azacitidine / Q000494:pharmacology; D002478:Cells, Cultured; D015536:Down-Regulation; D005090:Exodeoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D005434:Flow Cytometry; D015870:Gene Expression; D020868:Gene Silencing; D006801:Humans; D007150:Immunohistochemistry; D020084:Long Interspersed Nucleotide Elements / Q000235:genetics*; D020411:Oligonucleotide Array Sequence Analysis; D010003:Osteoarthritis / Q000378:metabolism* / Q000473:pathology; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D013583:Synovial Membrane / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D014162:Transfection / Q000379:methods
D010750:Phosphoproteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D005090:Exodeoxyribonucleases; C429526:three prime repair exonuclease 1; D001374:Azacitidine