| Abstract: | Objective Notch family proteins are transmembrane receptors that control cell fate and proliferation. Rheumatoid arthritis (RA) is characterized by activation and abnormal proliferation/differentiation of synoviocytes. We examined the expression of Notch‐1 and its role in the activation of RA synoviocytes. Methods The expression of Notch‐1 protein was detected by a specific antibody 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 antibody. To analyze the role of Notch‐1 in synoviocyte proliferation, we examined the effects of antisense Notch‐1 oligonucleotides (ODNs) and MW167, a γ‐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–Notch‐1 antibody, whereas there was predominantly cytoplasmic staining of normal and osteoarthritis (OA) synoviocytes. Western blotting showed a distinct ∼63‐kd protein detected by anti–Notch‐1 antibody in nuclear extracts from RA synoviocytes, indicating that nuclear staining of RA synovium and synoviocytes is likely to be the result of nuclear localization of Notch‐1 intracellular domain (NICD). Furthermore, tumor necrosis factor α (TNFα) increased NICD nuclear translocation in a dose‐dependent manner. Antisense Notch‐1 ODNs partially blocked the proliferation of RA synoviocytes and inhibited TNFα‐induced proliferation in both OA and RA synoviocytes. In addition, γ‐secretase inhibitor, which blocks the production of NICD, also inhibited TNFα‐induced proliferation of RA synoviocytes. Conclusion Our results demonstrate the expression of Notch‐1 in synoviocytes and the presence of Notch‐1 fragment in the nuclei of RA synoviocytes and suggest the involvement of Notch‐1 signaling in the TNFα‐induced proliferation of RA synoviocytes. |
