Role of Notch-1 intracellular domain in activation of rheumatoid synoviocytes

Objective. Notch family proteins are transmembrane receptors that control c ell fate and proliferation. Rheumatoid arthritis (RA) is characterized by a ctivation and abnormal proliferation/differentiation of synoviocytes. We ex amined the expression of Notch-1 and its role in the activation of RA synov iocytes. Methods. The expression of Notch-1 protein was detected by a specific antib ody raised against the Notch-1 intracellular domain. Notch-1 messenger RNA (mRNA) expression in synoviocytes was analyzed by Northern blotting. Notch- 1 protein expression was confirmed by Western blotting with anti-Notch-1 an tibody. To analyze the role of Notch-1 in synoviocyte proliferation, we exa mined the effects of antisense Notch-1 oligonucleotides (ODNs) and MW167, a gamma -secretase inhibitor. Results. Notch-1 protein and mRNA were detected in synovium from all study subjects. The nucleus of RA synoviocytes showed strong staining with anti-N otch-1 antibody, whereas there was predominantly cytoplasmic staining of no rmal and osteoarthritis (OA) synoviocytes. Western blotting showed a distin ct similar to 63-kd protein detected by anti-Notch-1 antibody in nuclear ex tracts from RA synoviocytes, indicating that nuclear staining of RA synoviu m and synoviocytes is likely to be the result of nuclear localization of No tch-1 intracellular domain (NICD). Furthermore, tumor necrosis factor alpha (TNF alpha) increased NICD nuclear translocation in a dose-dependent manne r. Antisense Notch-1 ODNs partially blocked the proliferation of RA synovio cytes and inhibited TNF alpha -induced proliferation in both OA and RA syno viocytes. In addition, gamma -secretase inhibitor, which blocks the product ion of NICD, also inhibited TNF alpha -induced proliferation of RA synovioc ytes. Conclusion. Our results demonstrate the expression of Notch-1 in synoviocyt es and the presence of Notch-1 fragment in the nuclei of RA synoviocytes an d suggest the involvement of Notch-1 signaling in the TNF alpha -induced pr oliferation of RA synoviocytes.