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


CONCLUSIONS
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.
Nakazawa
M
M
St Marianna University School of Medicine, Kanagawa, Japan.
Ishii
H
H
Aono
H
H
Takai
M
M
Honda
T
T
Aratani
S
S
Fukamizu
A
A
Nakamura
H
H
Yoshino
S
S
Kobata
T
T
Nishioka
K
K
Nakajima
T
T
D053829:Amyloid Precursor Protein Secretases; D016375:Antisense Elements (Genetics); D001172:Arthritis, Rheumatoid / Q000473:pathology* / Q000503:physiopathology; D016282:Aspartic Acid Endopeptidases; D015152:Blotting, Northern; D015153:Blotting, Western; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002467:Cell Nucleus / Q000737:chemistry; D002478:Cells, Cultured; D010450:Endopeptidases / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D008565:Membrane Proteins / Q000032:analysis / Q000737:chemistry* / Q000235:genetics*; D020370:Osteoarthritis, Knee / Q000473:pathology* / Q000503:physiopathology; D010455:Peptides; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000032:analysis; D051881:Receptor, Notch1; D011956:Receptors, Cell Surface; D015398:Signal Transduction / Q000502:physiology; D013583:Synovial Membrane / Q000737:chemistry / Q000473:pathology*; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D016375:Antisense Elements (Genetics); D004791:Enzyme Inhibitors; C432344:MW167; D008565:Membrane Proteins; C497761:NOTCH1 protein, human; D010455:Peptides; D012333:RNA, Messenger; D051881:Receptor, Notch1; D011956:Receptors, Cell Surface; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human