Artikel
In-vitro Inhibition of mdm2 with anti-inflammatory effects in RA fibroblasts like synoviocytes
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Autoren
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Gunter Assmann
- José Carreras Center for Immuno- and Gene Therapy, University Medical School of Saarland, Department of Internal Medicine I, Homburg/Saar
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Martin Eggimann
- University Medical School of Saarland, Internal Medicine I, Homburg/Saar
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Christoph Rittich
- University Medical School of Saarland, Internal Medicine I, Homburg/Saar
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Michael Pfreundschuh
- José Carreras Center for Immuno- and Gene Therapy, University Medical School of Saarland, Department of Internal Medicine I, Homburg/Saar
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Dieter Kohn
- University Medical School of Saarland, Orthopedics, Homburg/Saar
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Kristina Heyne
- José Carreras Center for Immuno- and Gene Therapy, University Medical School of Saarland, Homburg/Saar
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Klaus Roemer
- José Carreras Center for Immuno- and Gene Therapy, University Medical School of Saarland, Homburg/Saar
| Veröffentlicht: | 1. September 2015 |
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Gliederung
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Introduction: Fibroblasts like synoviocytes (FLS) have been proved to be involved in chronic inflammation and growth of synovial tissue of rheumatoid arthritis (RA). Reduced apoptotic activity due to alterations in the p53-pathway together with higher expression of proinflammatory NFKb signaling has been demonstrated in in-vitro experiments of RA FLS of synovial tissue. Recent studies investigating tumor cell lines have shown that the oncoprotein mdm2 (murine-double-minute2) regulates negatively p53-expression and interacts with NFKb-activation.
Methods: From joint surgery in 7 RA patients and 12 osteoarthritis (OA) patients FLS were isolated; the FLS from 4 joint/gender/age matched RA and 4 OA patients were cultured for 4 passages with stimulation by 5 ng/ml of TNF alpha and 2.5 ng/ml of Il-1beta. Expression levels of p53, p65 (NFKb), interleukin 6 (IL6), mdm2, IKB alpha, PUMA (p53 upregulating modulator of apoptosis) and p21 were evaluated in western blot technique* as well as mRNA expression# profiling with and without mdm2 knock-down through transfection with si-RNA. Differences in the expression patterns were determinated by densiometry techniques.
Results: RA FLS showed significantly lower expression of p53 (0.47 vs. 0.66*) and PUMA compared to OA FLS and significantly lower levels of IKB alpha (0.72 vs. 0.91#); no significant differences were detected in expression of mdm2 and p65 (NFKb) between RA and OA (beta actin or Gapdh, respectively, as control=1). However, after mdm2 knock-down a significant decrease of p65 (NFKb) (0.99 vs. 0.73*) and IL6 expression were detected in RA but not in OA, a remarkable reconstitution of p53 expression were achieved in RA (0.09 to 0.45) and OA FLS (0.39 to 0.75*), and the diminished IKB alpha expression in RA reached again comparable levels like in OA FLS [IMG1].
Conclusion: Compared to OA mdm2 has to be considered to develop higher activity in RA with subsequent escalation of NFKb activity through reduced expression of the NFKb Inhibitor IKB alpha. Furthermore, relevant anti-apoptotic effects of mdm2 could be demonstrated in RA FLS in the contrast to OA. In what way Mdm2 inhibition could be a new target of therapeutic intervention has to be further evaluated.
