title: Ugonin K-stimulated osteogenesis involves estrogen receptor-dependent activation of non-classical Src signaling pathway and classical pathway.
abstract: We have reported previously that ugonin K, a flavonoid isolated from Helminthostachys zeylanica (L.) Hook, potently induces cell differentiation and mineralization of MC3T3-E1 mouse osteoblast-like cells. Here we aimed to elucidate whether ugonin K evoked osteogenesis required interaction with estrogen receptor. Results showed that ugonin K induced increases in alkaline phosphatase (ALP) activity, expressions of bone sialoprotein (BSP) and osteocalcin (OCN), and subsequent bone nodule formation were concentration-dependently inhibited by estrogen receptor antagonist ICI 182,780, suggesting that an estrogen receptor-dependent pathway was involved. In the presence of ICI 182,780, ugonin K induced up-regulation of the expressions of runt-related transcription factor 2 (Runx2) and osterix was also significantly repressed. Numerous studies have demonstrated that estrogens induced rapid and transient activation of the c-Src phosphorylation cascade. We found that ugonin K indeed raised the phosphorylated level of c-Src and such phosphorylation was significantly attenuated by ICI 182,780 treatment. Application of c-Src specific inhibitor PP2 concentration-dependently repressed ugonin K-induced osteogenesis. In the nuclear translocation assay, results showed that ugonin K increased the nuclear level of estrogen receptor-α protein, suggesting that an enhanced transcriptional activity might be observed. Excepting MC3T3-E1 cells, results obtained from ALP activity assay revealed that ugonin K also stimulated osteoblastic differentiation of human MG-63 osteosarcoma cells and rat primary osteoblasts isolated from femora. Our results demonstrate that ugonin K stimulated osteogenesis might act through an estrogen receptor-dependent activation of a non-classical signaling pathway mediated by phosphorylation of c-Src. Moreover, a transactivation potential toward estrogen receptor-α through a classical pathway might not be precluded.
authors.affiliation: Department of Pharmacology, National Yang-Ming University, Taipei, Taiwan.
authors.forename: Chia-Hsin
authors.initials: CH
authors.lastname: Lee
authors.forename: Yu-Ling
authors.initials: YL
authors.lastname: Huang
authors.forename: Jyh-Fei
authors.initials: JF
authors.lastname: Liao
authors.forename: Wen-Fei
authors.initials: WF
authors.lastname: Chiou
mesh_terms: D041721:3T3-L1 Cells; D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D000081247:CSK Tyrosine-Protein Kinase; D002113:Calcification, Physiologic / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D050659:Core Binding Factor Alpha 1 Subunit / Q000235:genetics; D004958:Estradiol / Q000031:analogs & derivatives / Q000494:pharmacology; D047628:Estrogen Receptor alpha / Q000378:metabolism; D005419:Flavonoids / Q000494:pharmacology*; D000077267:Fulvestrant; D006801:Humans; D051379:Mice; D009928:Organ Specificity; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects; D010012:Osteogenesis / Q000187:drug effects*; D010766:Phosphorylation / Q000187:drug effects; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011960:Receptors, Estrogen / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects*; D000076182:Sp7 Transcription Factor; D014157:Transcription Factors / Q000235:genetics; D015854:Up-Regulation / Q000187:drug effects; D019061:src-Family Kinases
chemical_list: D050659:Core Binding Factor Alpha 1 Subunit; D047628:Estrogen Receptor alpha; D005419:Flavonoids; D011960:Receptors, Estrogen; D000076182:Sp7 Transcription Factor; C478163:SP7 protein, human; D014157:Transcription Factors; C540912:ugonin K; D000077267:Fulvestrant; D004958:Estradiol; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; C000631346:CSK protein, human; D000469:Alkaline Phosphatase