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The Synthetic Triterpenoid TP-222 Inhibits RANKL Stimulation of Osteoclastogenesis and Matrix Metalloproteinase-9 Expression
ROY A. FAVA, SARAH ELLIOTT, LAUREN RAYMOND, JESSICA MOLLMARK, EZRA HAYS, TADASHI HONDA, GORDON W. GRIBBLE, MICHAEL B. SPORN, and MATTHEW P. VINCENTI ABSTRACT. Objective. Receptor activator of nuclear factor-kB ligand (RANKL) promotes osteoclast differentiation from monocyte precursors by inducing a cohort of genes, including tartrate-resistant acid phosphatase (TRAP) and matrix metalloproteinase-9 (MMP-9). A family of synthetic triterpenoids with antiinflammatory and pro-apoptotic properties was described to modulate differentiation in monocytic cell lineages. We therefore investigated the ability of the potent and bioavailable synthetic triterpenoid TP-222 to inhibit RANKL-induced osteoclast formation and MMP-9 expression from monocytic precursor cells. Methods. Osteoclast formation was assayed by staining for TRAP-positive multinucleated cells. MMP-9 expression was measured by quantitative RT-PCR, Western blot, immunohistochemistry, and gel zymography. In vivo effects of TP-222 were assessed by daily intraperitoneal injection of 4-week-old mice for 7 days followed by measurement of osteoclast number and MMP-9 expression at the cartilage/bone junction of the epiphyseal growth plate. Results. RANKL promoted and TP-222 (300 nM) inhibited osteoclast formation in cultures of RAW264.7 cells or bone marrow-derived monocytes. RANKL also induced MMP-9 expression in RAW264.7 cells and this was reduced by concurrent or subsequent addition of TP-222. TP-222 treatment significantly reduced the mean number of osteoclasts present at the cartilage/bone interface compared to vehicle-injected control mice. Morphometric analyses of tissue sections showed that TP-222 treatment reduced the amount of immunoreactive MMP-9 present in both mononucleated pre-osteoclasts and osteoclasts. Conclusion. Our data demonstrate that TP-222 inhibits osteoclast formation and MMP-9 expression in vitro and in vivo, and suggest that triterpenoids may be useful compounds for modulating bone resorption diseases. (First Release Mar 15 2007; J Rheumatol 2007;34:1058–68) Key Indexing Terms:
OSTEOCLASTS From the Research Service, Veterans Affairs Medical Center, White River Junction, Vermont; Department of Medicine, Dartmouth Medical School, Lebanon, New Hampshire; Department of Chemistry, Dartmouth College, Hanover, New Hampshire; and Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, New Hampshire, USA. Dr. Vincenti is supported by a Merit Review Grant from the Department of Veterans Affairs and a grant from the National Institute of Arthritis and Musculoskeletal and Skin Diseases (R01-AR46977). Dr. Fava is supported by a Merit Review Grant from the Department of Veterans Affairs. Dr. Sporn is supported by funding from the National Cancer Institute (R01 78814) and from the National Foundation for Cancer Research. R.A. Fava, PhD, Research Associate, Professor of Medicine, Dartmouth Medical School, Research Biologist, Research Service, VA Medical Center White River Junction; S. Elliott, PhD, Research Associate; L. Raymond, BA, Research Assistant; J. Mollmark, BS, Research Assistant; E. Hays, BA, Research Technician, Dartmouth Medical School; T. Honda, PhD, Research Assistant Professor of Chemistry; G. Gribble, PhD, Professor of Chemistry, Dartmouth College; M. Sporn, MD, Professor of Pharmacology and Toxicology and Medicine, Dartmouth Medical School; M. Vincenti, PhD, Research Associate of Medicine, Dartmouth Medical School, Research Biologist, Research Service, VA Medical Center White River Junction. Address reprint requests to Dr. M. Vincenti, Research Service, VA Medical Center, 215 North Main Street, White River Junction, VT 05009. E-mail: mpv@dartmouth.edu Accepted for publication January 22, 2007.
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