title: Estrogen receptor signaling in the ferutinin-induced osteoblastic differentiation of human amniotic fluid stem cells.
abstract: OBJECTIVE
  Ferutinin is a diaucane sesquiterpene with a high estrogenic activity. Since ferutinin is able to enhance osteoblastic differentiation of human amniotic fluid stem cells (hAFSCs), the aim of this study was to evaluate the role of the estrogen receptors α (ERα) and G-protein coupled receptor 30 (GPR30) in ferutinin-mediated osteoblastic differentiation. Moreover, it was investigated if MEK/ERK and PI3K/Akt signaling pathways are involved in ferutinin-induced effects.
  
  
  METHODS
  hAFSCs were cultured in a standard medium or in an osteoblastic medium for 14 or 21days and ferutinin was added at 10-8M. Immunofluorescence techniques and Western-blot 21analysis were used to study estrogen receptors and signaling pathways.
  
  
  RESULTS
  In both undifferentiated and differentiated hAFSCs we identified ERα and GPR30 with a nuclear or cytoplasmatic localization, respectively. The presence of ferutinin in the osteoblastic medium leads to an increase in ERα expression. To dissect the role of estrogen receptors, MPP and G15 were used to selectively block ERα and GPR30, respectively. Notably, ferutinin enhanced osteoblastic differentiation in cells challenged with G15. Ferutinin was able to increase ERK and Akt phosphorylations with a different timing activation. These phosphorylations were antagonized by PD0325901, a MEK inhibitor, and wortmannin, a PI3K inhibitor. Both MPP and G15 inhibited the ferutinin-induced MEK/ERK and PI3K/Akt pathway activations. In the osteoblastic condition, PD0325901, but not wortmannin, reduced the expression of OPN and RUNX-2, whereas ferutinin abrogated the down-modulation triggered by PD0325901.
  
  
  CONCLUSIONS
  PI3K/Akt pathways seems to mediate the enhancement of hAFSCs osteoblastic differentiation triggered by ferutinin through ERα.
authors.affiliation: Department of Surgical, Medical, Dental and Morphological Sciences with Interest in Transplants, Oncology and Regenerative Medicine, University of Modena and Reggio Emilia, Modena, Italy. Electronic address: manuela.zavatti@unimore.it.
authors.forename: M
authors.initials: M
authors.lastname: Zavatti
authors.affiliation: EURAC Research, Center for Biomedicine, Bolzano, Italy.
authors.forename: M
authors.initials: M
authors.lastname: Guida
authors.affiliation: Department of Surgical, Medical, Dental and Morphological Sciences with Interest in Transplants, Oncology and Regenerative Medicine, University of Modena and Reggio Emilia, Modena, Italy.
authors.forename: T
authors.initials: T
authors.lastname: Maraldi
authors.affiliation: Department of Surgical, Medical, Dental and Morphological Sciences with Interest in Transplants, Oncology and Regenerative Medicine, University of Modena and Reggio Emilia, Modena, Italy.
authors.forename: F
authors.initials: F
authors.lastname: Beretti
authors.affiliation: Department of Surgical, Medical, Dental and Morphological Sciences with Interest in Transplants, Oncology and Regenerative Medicine, University of Modena and Reggio Emilia, Modena, Italy.
authors.forename: L
authors.initials: L
authors.lastname: Bertoni
authors.affiliation: Unit of Obstetrics and Ginecology, IRCCS-Arcispedale Santa Maria Nuova, Reggio Emilia, Italy.
authors.forename: G B
authors.initials: GB
authors.lastname: La Sala
authors.affiliation: Department of Surgical, Medical, Dental and Morphological Sciences with Interest in Transplants, Oncology and Regenerative Medicine, University of Modena and Reggio Emilia, Modena, Italy.
authors.forename: A
authors.initials: A
authors.lastname: De Pol
mesh_terms: D000653:Amniotic Fluid / Q000166:cytology; D001549:Benzamides / Q000494:pharmacology; D001565:Benzoates / Q000494:pharmacology*; D001643:Bridged Bicyclo Compounds / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects* / Q000502:physiology*; D003508:Cycloheptanes / Q000494:pharmacology*; D004159:Diphenylamine / Q000031:analogs & derivatives / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D047628:Estrogen Receptor alpha / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D020935:MAP Kinase Signaling System / Q000502:physiology; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011960:Receptors, Estrogen / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D012717:Sesquiterpenes / Q000494:pharmacology*; D015398:Signal Transduction / Q000502:physiology*; D013234:Stem Cells / Q000166:cytology* / Q000201:enzymology
chemical_list: D001549:Benzamides; D001565:Benzoates; D001643:Bridged Bicyclo Compounds; D003508:Cycloheptanes; D004159:Diphenylamine; D004791:Enzyme Inhibitors; D047628:Estrogen Receptor alpha; D019869:Phosphatidylinositol 3-Kinases; D011960:Receptors, Estrogen; D043562:Receptors, G-Protein-Coupled; D012717:Sesquiterpenes; C109584:GPER1 protein, human; C111068:4-oxy-6-(4-oxybezoyloxy)dauc-8,9-en; C506614:mirdametinib
keywords: Estrogen receptor signaling; Ferutinin; Osteoblastic differentiation; hAFSCs