T-cell leukemia translocation-associated gene (TCTA) protein is required for human osteoclastogenesis.
Synovial tissues of patients with rheumatoid arthritis (RA) include factors regulating bone resorption, such as receptor activator NF-kappaB ligand (RANKL), TNFalpha, IL-6, IL-17 and IFNgamma. However, in addition to these cytokines, other factors expressed in synovial tissues may play a role in resorbing bone. Here, our objective was to identify novel proteins expressed in synovial tissues of RA that regulate human osteoclastogenesis. Proteins were purified from synovial tissues of patients with RA, using gel filtration chromatography, ion-exchange chromatography, reverse-aspect HPLC, and mass spectrometry. We evaluated the effects of the purified fractions on human osteoclastogenesis induced by RANKL and M-CSF. We determined the amino acid sequences showing inhibitory activity on human osteoclastogenesis. In addition, we synthesized novel peptides from the molecule including the amino acid sequences. Then, we evaluated the effects of the peptides and antibodies against the molecule on human osteoclastogenesis from monocytes and mature osteoclasts, and on pit formation by mature osteoclasts using Osteologic discs. We examined the effect of the peptide on the expression of both mRNA and protein of NFATc1. We also examined the effect of RANKL on the expression of mRNA of the molecule on osteoclasts and macrophages. We identified a small peptide including Gly-Gln-Asn (GQN) with inhibitory activity on human osteoclastogenesis. We then found that GQN is included in the amino acid sequence of the extra-cellular domain of TCTA protein, which is expressed ubiquitously in normal human tissues, but whose function has not been clarified. We designed novel peptides, including GQN, from the sequence of TCTA protein. One of these peptides (29-mer), but not a scrambled peptide for the 29-mer peptide, potently inhibited RANKL-induced human osteoclastogenesis. The peptide also inhibited pit formation of mature human osteoclasts and suppressed the formation of large osteoclasts in the culture of mature osteoclasts. Furthermore, polyclonal antibodies against TCTA protein suppressed the formation of large osteoclasts in the cultures of both monocytes and mature osteoclasts, supporting our hypothesis. Peptide A did not significantly inhibit the expression of both mRNA and protein of NFATc1 in osteoclasts. Our novel peptide and polyclonal antibodies against the peptide inhibited human osteoclastogenesis and the function of mature osteoclasts, preventing cellular fusion by TCTA protein and a putative counterpart molecule.
Kotake
Shigeru
S
Institute of Rheumatology, Tokyo Women's Medical University, 10-22 Kawada-cho, Shinjuku, Tokyo 162-0054, Japan. skotake@ior.twmu.ac.jp
Nanke
Yuki
Y
Kawamoto
Manabu
M
Yago
Toru
T
Udagawa
Nobuyuki
N
Ichikawa
Naomi
N
Kobashigawa
Tsuyoshi
T
Saito
Seiji
S
Momohara
Shigeki
S
Kamatani
Naoyuki
N
Yamanaka
Hisashi
H
D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D015703:Antigens, CD / Q000378:metabolism; D000951:Antigens, Neoplasm / Q000378:metabolism; D001172:Arthritis, Rheumatoid / Q000378:metabolism / Q000473:pathology; D000074301:CD52 Antigen; D056657:Cathepsin K / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D002459:Cell Fusion; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D006023:Glycoproteins / Q000378:metabolism; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009000:Monocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D050778:NFATC Transcription Factors / Q000235:genetics / Q000378:metabolism; D010010:Osteoclasts / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D010012:Osteogenesis / Q000187:drug effects; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013583:Synovial Membrane / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D013601:T-Lymphocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism
D000199:Actins; D000906:Antibodies; D015703:Antigens, CD; D000951:Antigens, Neoplasm; D000074301:CD52 Antigen; C000613862:CD52 protein, human; D006023:Glycoproteins; D050778:NFATC Transcription Factors; D010455:Peptides; D011506:Proteins; D012333:RNA, Messenger; C545566:TCTA protein, human; D056657:Cathepsin K