title: Calcium sensing receptor activation by calcimimetic R-568 in human amniotic fluid mesenchymal stem cells: correlation with osteogenic differentiation.
abstract: Human amniotic fluid mesenchymal stem cells (hAFMSCs) are promising for therapeutic applications in bone damage. Calcium sensing receptor (CaSR), a G protein-coupled receptor, plays a physiological role in the regulation of bone metabolism. Thus, the bone CaSR could be targeted by calcimimetic agonists, which may be potentially helpful in treating bone diseases. The aim of our study was to characterize CaSR expression in hAFMSCs and to assess the activity of calcimimetic R-568 during in vitro osteogenesis. Using western blotting, immunofluorescence, and flow cytometry, we consistently observed constitutive CaSR in osteo-differentiating hAFMSCs. Notably, both R-568 and calcium significantly enhanced hAFMSC osteogenic differentiation after exposure to osteogenic medium. To provide further evidence of the involvement of CaSR in osteogenesis, we correlated its expression with that of established osteogenic markers, that is, alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), and osteopontin (OPN), and novel, not yet completely defined regulators of osteogenesis. Among these are β-catenin and Slug, which are mediators of Wnt signaling, and nuclear factor of activated T cells c1 (NFATc1), which plays a critical role in calcium/calcineurin signaling. Taken together, our results demonstrate that CaSR is expressed in hAFMSCs, positively correlates with osteogenic markers, and is activated by R-568. Notably, downregulation of CaSR by RNA interference supports the conclusion that CaSR activation plays a central role in hAFMSC osteogenesis. Thus, this study provides significant information on the mechanisms of hAFMSC osteogenesis, which could provide additional molecular basis for the use of calcimimetics in bone regenerative medicine.
authors.affiliation: 1 Department of Experimental and Clinical Sciences, School of Medicine and Health Sciences, "G. d'Annunzio" University Chieti-Pescara , Chieti, Italy .
authors.forename: Caterina
authors.initials: C
authors.lastname: Pipino
authors.forename: Pamela
authors.initials: P
authors.lastname: Di Tomo
authors.forename: Domitilla
authors.initials: D
authors.lastname: Mandatori
authors.forename: Eleonora
authors.initials: E
authors.lastname: Cianci
authors.forename: Paola
authors.initials: P
authors.lastname: Lanuti
authors.forename: Meritxell B
authors.initials: MB
authors.lastname: Cutrona
authors.forename: Letizia
authors.initials: L
authors.lastname: Penolazzi
authors.forename: Laura
authors.initials: L
authors.lastname: Pierdomenico
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Lambertini
authors.forename: Ivana
authors.initials: I
authors.lastname: Antonucci
authors.forename: Vittorio
authors.initials: V
authors.lastname: Sirolli
authors.forename: Mario
authors.initials: M
authors.lastname: Bonomini
authors.forename: Mario
authors.initials: M
authors.lastname: Romano
authors.forename: Roberta
authors.initials: R
authors.lastname: Piva
authors.forename: Marco
authors.initials: M
authors.lastname: Marchisio
authors.forename: Assunta
authors.initials: A
authors.lastname: Pandolfi
mesh_terms: D000653:Amniotic Fluid / Q000166:cytology*; D000814:Aniline Compounds / Q000494:pharmacology*; D002478:Cells, Cultured; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D010012:Osteogenesis; D010627:Phenethylamines; D011437:Propylamines; D044169:Receptors, Calcium-Sensing / Q000819:agonists / Q000378:metabolism*
chemical_list: D000814:Aniline Compounds; C107873:N-(2-chlorophenylpropyl)-1-(3-methoxyphenyl)ethylamine; D010627:Phenethylamines; D011437:Propylamines; D044169:Receptors, Calcium-Sensing