The L1 retroelement-related p40 protein induces p38delta MAP kinase.

We characterized a full length L1 mRNA in a rheumatoid arthritis (RA) synovial tissue and determined the degree of methylation of its 5'-UTR. We asked whether not only intact but also altered L1s can exert biological activities by transfecting RA synovial fibroblasts (SF) with either retrotransposition-competent or incompetent L1s and examined their capacity to induce p38delta. Total RNA was isolated from the synovial tissue of a 35-year-old woman with highly destructive RA. A complete L1 sequence was obtained by 3'/5'-RACE. Methylation of the genomic 5'-UTR was determined by the sodium-disulfide/PCR method. RA-SF were transfected by lipofection with either a functional L1 or an ORF2-mutated L1 element. The expression of p38delta was measured by RT-PCR and Western blot. The full length L1 mRNA included a 5'-UTR, an ORF1 and an ORF2. Three of five CpG islands (60%) of the genomic L1 5'-UTR were hypomethylated and the ORF2 was deactivated by the insertion of stop codons. Both, intact and ORF2-mutated L1 vectors, induced the expression of p38delta. Thus, even an ORF2-mutated L1 element, as expressed in RA, is biologically active and both L1 ORF1 and p38delta transcripts may appear as a consequence of genomic hypomethylation. The induction of p38delta appears to be mediated by an ORF1/p40-dependent process. This is the first indication of a p40 mediated transactivation.

Cartilage destruction mediated by synovial fibroblasts does not depend on proliferation in rheumatoid arthritis.

The aim of the study was to investigate the relationship between invasion and proliferation in rheumatoid arthritis synovial fibroblasts (RASFs). In vitro, RASFs, normal synovial fibroblasts (NSFs), and RASFs transformed with SV40 T-antigen (RASF(SV40)) were analyzed for the expression of cell surface markers (Thy1, VCAM-1, ICAM-1, CD40, CD44) and their proliferation by flow cytometry. Furthermore, colony-forming unit assays were performed and the expression of matrix metalloproteinases (MMP)-14 and cathepsin K mRNA were determined by real-time polymerase chain reaction. In vivo, in the severe combined immunodeficiency (SCID) mouse co-implantation model, RASFs, NSFs, and RASF(SV40) were tested for cartilage invasion, cellular density, and for their expression of the cell cycle-associated protein Ki67. In the SCID mouse co-implantation model, RASFs invaded significantly stronger into the cartilage than NSFs and RASF(SV40). Of note, RASF(SV40) cells formed tumor-like tissues, and the cellular density adjacent to the cartilage was significantly higher than in RASFs or NSFs. In turn, the proliferation marker Ki67 was strongly expressed in the SV40-transformed synoviocytes in SCID mice, but not in RASFs, and specifically not at sites of cartilage invasion. Using the colony-forming unit assay, RASFs and NSFs did not form colonies, whereas RASF(SV40) lost contact inhibition. In vitro, the proliferative rate of RASFs was low (4.3% S phase) in contrast to RASF(SV40) (24.4%). Expression of VCAM-1 was significantly higher, whereas of ICAM-1 was significantly lower, in RASFs than in RASF(SV40). CD40 was significantly stronger expressed in RASF(SV40), whereas CD44 and AS02 were present at the same degree in almost all synoviocytes. Expression of cathepsin K and matrix metalloproteinase-14 mRNA was significantly higher in RASFs than in the RASF(SV40). Our data demonstrate clearly that invasion of cartilage is mediated by activated RASFs characterized by increased expression of adhesion molecules, matrix-degrading enzymes, but does not depend on cellular proliferation, suggesting the dissociation of invasion and proliferation in RASFs.

FLICE-inhibitory protein expression in synovial fibroblasts and at sites of cartilage and bone erosion in rheumatoid arthritis.

OBJECTIVE: Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by a hyperplastic synovial tissue, inflammatory infiltrates, and a progressive destruction of cartilage and bone. FLICE-inhibitory protein (FLIP) prevents the association of caspase 8 with FADD and thus exerts an antiapoptotic effect through inhibition of Fas-mediated apoptosis. We undertook this study to examine the expression of FLIP in RA, osteoarthritic (OA), and normal synovial tissues. METHODS: We investigated the expression of FLIP (long form) in 5 RA, 2 OA, and 2 normal synovial tissue samples. A 393-bp fragment was amplified from complementary DNA obtained from cultured RA synovial fibroblasts (RASF) by reverse transcription-polymerase chain reaction (RT-PCR). Using in situ hybridization, the expression of FLIP messenger RNA (mRNA) in paraffin-embedded synovial tissue sections was investigated semiquantitatively by analyzing the lining layer, the sublining, and sites of invasion. Immunohistochemistry with anti-CD68 antibodies was performed on serial tissue sections to further characterize the cell types expressing FLIP. In addition, quantitative expression of FLIP was measured by real-time PCR. RESULTS: RT-PCR revealed the expression of FLIP mRNA in all RA and OA samples tested. Using in situ hybridization in synovial tissue, FLIP was detected in all 5 RA samples and in 1 of 2 OA samples, but in neither of the 2 normal control samples. In RA, FLIP expression could be found in both the lining and sublining layers; most importantly, it could also be identified at sites of cartilage invasion and bone destruction. Moreover, quantitative PCR analysis showed 50% higher FLIP expression in RASF than in OASF. CONCLUSION: The expression of antiapoptotic FLIP in RA synovial tissue and in synovial fibroblasts suggests the idea of a novel pathway in RA that potentially extends the lifespan of cartilage- and bone-degrading synovial cells, thus contributing to the progression of joint destruction.