Modification of nuclear PML protein by SUMO-1 regulates Fas-induced apoptosis in rheumatoid arthritis synovial fibroblasts

Abstract

The small ubiquitin-like modifier (SUMO)-1 is an important posttranslational regulator of different signaling pathways and involved in the formation of promyelocytic leukemia (PML) protein nuclear bodies (NBs). Overexpression of SUMO-1 has been associated with alterations in apoptosis, but the underlying mechanisms and their relevance for human diseases are not clear. Here, we show that the increased expression of SUMO-1 in rheumatoid arthritis (RA) synovial fibroblasts (SFs) contributes to the resistance of these cells against Fas-induced apoptosis through increased SUMOylation of nuclear PML protein and increased recruitment of the transcriptional repressor DAXX to PML NBs. We also show that the nuclear SUMO-protease SENP1, which is found at lower levels in RA SFs, can revert the apoptosis-inhibiting effects of SUMO-1 by releasing DAXX from PML NBs. Our findings indicate that in RA SFs overexpression of SENP1 can alter the SUMO-1-mediated recruitment of DAXX to PML NBs, thus influencing the proapoptotic effects of DAXX. Accumulation of DAXX in PML NBs by SUMO-1 may, therefore, contribute to the pathogenesis of inflammatory disorders.inflammation ͉ autoimmunity ͉ DAXX ͉ SENP

The small ubiquitin‐like modifier mediates the resistance of prosthesis‐loosening fibroblast‐like synoviocytes against fas‐induced apoptosis

“…SUMO-1 can covalently modify a variety of proteins and modulates the downstream pathways of Fas signaling (5-7). Interestingly, it was shown recently that SUMO-1 is overexpressed in RA FLS when compared with osteoarthritic (OA) fibroblasts and contributes to the resistance of these cells to Fas-induced apoptosis (8). This study demonstrated that rather than interfering directly with the Fas molecule, sumoylation of the promyelocytic leukemia protein nuclear bodies and subsequent recruitment of the proapoptotic molecule Daxx into promyelocytic leukemia protein nuclear bodies constitute the mechanism by which SUMO-1 inhibits Fasinduced apoptosis in RA FLS.…”

“…FLS were grown in Dulbecco's modified Eagle's medium (DMEM; 10% fetal calf serum [FCS]) until passages 4-6, as previously described (8). Bone marrow-derived stromal cells were isolated by gradient centrifugation and plastic adherence.…”

“…Protein levels were monitored by Western blotting, using monoclonal anti-SUMO-1 (clone 21C7; Zymed, Burlingame, CA) or anti-␤-actin (Sigma-Aldrich, Poole, UK) antisera. Subcellular distribution was studied using mouse monoclonal anti-SUMO-1 antiserum (Alexis Biochemicals, Grünberg, Germany) and rabbit polyclonal anti-promyelocytic leukemia protein antiserum (Santa Cruz Biotechnology, Santa Cruz, CA), visualized with secondary fluorescein isothiocyanateconjugated and tetramethylrhodamine isothiocyanateconjugated antisera (Jackson ImmunoResearch, Hamburg, Germany), as previously described (8). The experiments were analyzed, and photographs were obtained using a confocal microscope (Leica DMIRE2 TCS SP2; Leica, Wetzlar, Germany).…”

“…SUMO-1 siRNA (5Ј-CUGGGAAU-GGAGGAAGAAG-3Ј) and mismatched, scrambled oligonucleotides (Eurogentec, Luik, Belgium) were used as recommended. Transfections were performed using an Amaxa electroporation kit (Amaxa, Cologne, Germany), as previously described (8).…”

“…Apoptosis was induced by incubation with 100 ng/ml recombinant human FasL (rHuFasL) for 16 hours, and cell death was determined using a histone fragmentation assay (Cell Death Detection ELISA plus ; Roche, Mannheim, Germany), as previously described (8). 2066 MEINECKE ET AL…”