Participation of estrogen receptors in the enhancement of osteoblast differentiation by TAK-778.
TAK-778 has been shown to stimulate osteogenesis both in vitro and in vivo. However, the mechanism by which TAK-778 exerts its effects is still unclear. There is evidence that TAK-778 acts via estrogen-receptor (ER)-mediated signaling; this study therefore aimed to investigate the roles that ERalpha, ERbeta, and membrane ER play in the osteogenic effect of TAK-778. To this end, human bone marrow mesenchymal cells were cultured with TAK-778 in the presence of either ICI182,780 (ERalpha and ERbeta antagonist) or MPP (ERalpha antagonist) or PD98059 (an extracellular-regulated kinase inhibitor that acts on the membrane ER pathway). The following parameters were evaluated: cell proliferation, collagen content, alkaline phosphatase (ALP) activity and bone-like formation. Data were compared using ANOVA. The effect of TAK-778 on expression of ERalpha and ERbeta was investigated by immunolabeling. In order to investigate whether TAK-778 binds to ER, an ER binding assay was performed. Both immunolabeling and binding assays were conducted using cells from human alveolar bone. The osteogenic effect of TAK-778 was inhibited by ICI182,780 and MPP; however, it was not affected by PD98059. The expression of both ERalpha and ERbeta was not affected by TAK-778. The competition curve obtained from the binding assay using TAK-778 showed maximal displacement when 10(-5) M TAK-778 was used. This study's results show that TAK-778 enhances osteoblast differentiation through an ERalpha-dependent pathway by binding to this receptor and not by increasing the expression of ER.
Department of Oral and Maxillofacial Surgery, School of Dentistry of Ribeirao Preto, University of Sao Paulo, Av do Cafe, s/n, 14040-904, Ribeirao Preto, SP, Brazil.
Márcio Mateus
MM
Beloti
Larissa Sverzut
LS
Bellesini
Paulo Tambasco
PT
de Oliveira
Adalberto Luiz
AL
Rosa
D000469:Alkaline Phosphatase / Q000378:metabolism; D001582:Benzothiepins / Q000494:pharmacology*; D002454:Cell Differentiation / Q000187:drug effects*; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism; D004338:Drug Combinations; D004958:Estradiol / Q000031:analogs & derivatives / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D000077267:Fulvestrant; D006801:Humans; D008856:Microscopy, Fluorescence; D010006:Osteoblasts / Q000187:drug effects*; D010012:Osteogenesis / Q000187:drug effects*; D011960:Receptors, Estrogen / Q000819:agonists / Q000037:antagonists & inhibitors / Q000502:physiology*
D001582:Benzothiepins; D004338:Drug Combinations; D005419:Flavonoids; D011960:Receptors, Estrogen; C120450:TAK 778; D000077267:Fulvestrant; D004958:Estradiol; D003094:Collagen; D000469:Alkaline Phosphatase; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one