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

Abstract

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. 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 gamma-secretase inhibitor. 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 approximately 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 alpha (TNFalpha) increased NICD nuclear translocation in a dose-dependent manner. Antisense Notch-1 ODNs partially blocked the proliferation of RA synoviocytes and inhibited TNFalpha-induced proliferation in both OA and RA synoviocytes. In addition, gamma-secretase inhibitor, which blocks the production of NICD, also inhibited TNFalpha-induced proliferation of RA synoviocytes. 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 TNFalpha-induced proliferation of RA synoviocytes.