TY - JOUR
T1 - The small molecule harmine regulates NFATc1 and Id2 expression in osteoclast progenitor cells
AU - Egusa, Hiroshi
AU - Doi, Masanori
AU - Saeki, Makio
AU - Fukuyasu, Sho
AU - Akashi, Yoshihiro
AU - Yokota, Yoshifumi
AU - Yatani, Hirofumi
AU - Kamisaki, Yoshinori
N1 - Funding Information:
We are grateful to Dr. Devang Thakor (Harvard Medical School) for critical reading of the manuscript and valuable suggestions. This investigation was supported by a Grant-in-Aid for Young Scientists ( A18689045 : H.E.) from the Ministry of Education, Culture, Sports, Science and Technology, Japan . Support was also received from Grants-in-Aid for Exploratory Research ( 22659356 : H.E.) and for Scientific Research ( B19390494 : H.Y. and H.E., B22390364 : H.Y. and H.E., B20390471 : Y.K. and C20592172 : M.S.) from the Japan Society for the Promotion of Science .
PY - 2011/8
Y1 - 2011/8
N2 - Small molecule compounds that potently affect osteoclastogenesis could be useful as chemical probes for elucidating the mechanisms of various biological phenomena and as effective therapeutic strategies against bone resorption. An osteoclast progenitor cell-based high-throughput screening system was designed to target activation of NFAT, which is a key event for osteoclastogenesis. Orphan ligand library screening using this system identified the β-carboline derivative harmine, which is a highly potent inhibitor of dual-specificity tyrosine-phosphorylation regulated kinase 1A (DYRK1A), to be an NFAT regulator in osteoclasts. RAW264.7 cells highly expressed DYRK1A protein, and in vitro phosphorylation assay demonstrated that harmine directly inhibited the DYRK1A-mediated phosphorylation (in-activation) of NFATc1. Harmine promoted the dephosphorylation (activation) of NFATc1 in RAW264.7 cells within 24. h, and it significantly increased the expression of NFATc1 in RAW264.7 cells and mouse primary bone marrow macrophages (BMMs) both in the presence and absence of RANKL stimulation. Although harmine promoted NFATc1 expression and stimulated target genes for osteoclastogenesis, cell-cell fusion and the formation of TRAP-positive multinucleated osteoclasts from RAW264.7 cells and BMMs was significantly inhibited by harmine treatment. Meanwhile, harmine remarkably promoted the expression of inhibitor of DNA binding/differentiation-2 (Id2), which is a negative regulator for osteoclastogenesis, in RAW264.7 cells and BMMs. An Id2-null-mutant showed slightly increased osteoclast formation from BMMs, and the harmine-mediated inhibition of osteoclast formation was abolished in the BMMs of Id2-null-mutant mice. These results suggest that harmine is a potent activator of NFATc1 that interferes with the function of DYRK1A in osteoclast precursors and also up-regulates Id2 protein, which may dominantly inhibit expression pathways associated with cell-cell fusion, thereby leading to the disruption of the fusion events mediating osteoclastogenesis. The small molecule harmine is therefore expected to provide an experimental tool for investigating signaling cascades in osteoclastogenesis, especially those centered on DYRK1A-mediated NFATc1 and Id2 regulation.
AB - Small molecule compounds that potently affect osteoclastogenesis could be useful as chemical probes for elucidating the mechanisms of various biological phenomena and as effective therapeutic strategies against bone resorption. An osteoclast progenitor cell-based high-throughput screening system was designed to target activation of NFAT, which is a key event for osteoclastogenesis. Orphan ligand library screening using this system identified the β-carboline derivative harmine, which is a highly potent inhibitor of dual-specificity tyrosine-phosphorylation regulated kinase 1A (DYRK1A), to be an NFAT regulator in osteoclasts. RAW264.7 cells highly expressed DYRK1A protein, and in vitro phosphorylation assay demonstrated that harmine directly inhibited the DYRK1A-mediated phosphorylation (in-activation) of NFATc1. Harmine promoted the dephosphorylation (activation) of NFATc1 in RAW264.7 cells within 24. h, and it significantly increased the expression of NFATc1 in RAW264.7 cells and mouse primary bone marrow macrophages (BMMs) both in the presence and absence of RANKL stimulation. Although harmine promoted NFATc1 expression and stimulated target genes for osteoclastogenesis, cell-cell fusion and the formation of TRAP-positive multinucleated osteoclasts from RAW264.7 cells and BMMs was significantly inhibited by harmine treatment. Meanwhile, harmine remarkably promoted the expression of inhibitor of DNA binding/differentiation-2 (Id2), which is a negative regulator for osteoclastogenesis, in RAW264.7 cells and BMMs. An Id2-null-mutant showed slightly increased osteoclast formation from BMMs, and the harmine-mediated inhibition of osteoclast formation was abolished in the BMMs of Id2-null-mutant mice. These results suggest that harmine is a potent activator of NFATc1 that interferes with the function of DYRK1A in osteoclast precursors and also up-regulates Id2 protein, which may dominantly inhibit expression pathways associated with cell-cell fusion, thereby leading to the disruption of the fusion events mediating osteoclastogenesis. The small molecule harmine is therefore expected to provide an experimental tool for investigating signaling cascades in osteoclastogenesis, especially those centered on DYRK1A-mediated NFATc1 and Id2 regulation.
KW - DYRK1A
KW - Harmine
KW - Id2
KW - NFATc1
KW - Osteoclast
KW - Small molecule
UR - http://www.scopus.com/inward/record.url?scp=79958766625&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=79958766625&partnerID=8YFLogxK
U2 - 10.1016/j.bone.2011.04.003
DO - 10.1016/j.bone.2011.04.003
M3 - Article
C2 - 21504804
AN - SCOPUS:79958766625
SN - 8756-3282
VL - 49
SP - 264
EP - 274
JO - Bone
JF - Bone
IS - 2
ER -
