title: Retinoic acid differentially affects in vitro proliferation, differentiation and mineralization of two fish bone-derived cell lines: different gene expression of nuclear receptors and ECM proteins.
abstract: Retinoic acid (RA), the main active metabolite of vitamin A, regulates vertebrate morphogenesis through signaling pathways not yet fully understood. Such process involves the specific activation of retinoic acid and retinoid X receptors (RARs and RXRs), which are nuclear receptors of the steroid/thyroid hormone receptor superfamily. Teleost fish are suitable models to study vertebrate development, such as skeletogenesis. Cell systems capable of in vitro mineralization have been developed for several fish species and may provide new insights into the specific cellular and molecular events related to vitamin A activity in bone, complementary to in vivo studies. This work aims at investigating the in vitro effects of RA (0.5 and 12.5 μM) on proliferation, differentiation and extracellular matrix (ECM) mineralization of two gilthead seabream bone-derived cell lines (VSa13 and VSa16), and at identifying molecular targets of its action through gene expression analysis. RA induced phenotypic changes and cellular proliferation was inhibited in both cell lines in a cell type-dependent manner (36-59% in VSa13 and 17-46% in VSa16 cells). While RA stimulated mineral deposition in VSa13 cell cultures (50-62% stimulation), it inhibited the mineralization of extracellular matrix in VSa16 cells (11-57% inhibition). Expression of hormone receptor genes (rars and rxrs), and extracellular matrix-related genes such as matrix and bone Gla proteins (mgp and bglap), osteopontin (spp1) and type I collagen (col1a1) were differentially regulated upon exposure to RA in proliferating, differentiating and mineralizing cultures of VSa13 and VSa16 cells. Altogether, our results show: (i) RA affects proliferative and mineralogenic activities in two fish skeletal cell types and (ii) that during phenotype transitions, specific RA nuclear receptors and bone-related genes are differentially expressed in a cell type-dependent manner.
authors.affiliation: Centro de Ciências do Mar (CCMAR/CIMAR-LA), Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, Portugal; IRTA, Centre de Sant Carles de la Ràpita (IRTA-SCR), Unitat de Cultius Experimentals, Crta. del Poble Nou s/n, 43540 Sant Carles de la Ràpita, Spain. Electronic address: ivmonzon@ualg.pt.
authors.forename: Ignacio
authors.initials: I
authors.lastname: Fernández
authors.affiliation: Centro de Ciências do Mar (CCMAR/CIMAR-LA), Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, Portugal.
authors.forename: Daniel M
authors.initials: DM
authors.lastname: Tiago
authors.affiliation: Centro de Ciências do Mar (CCMAR/CIMAR-LA), Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, Portugal.
authors.forename: Vincent
authors.initials: V
authors.lastname: Laizé
authors.affiliation: Centro de Ciências do Mar (CCMAR/CIMAR-LA), Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, Portugal; Department of Biomedical Sciences and Medicine (DCBM), Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, Portugal.
authors.forename: M
authors.initials: M
authors.lastname: Leonor Cancela
authors.affiliation: IRTA, Centre de Sant Carles de la Ràpita (IRTA-SCR), Unitat de Cultius Experimentals, Crta. del Poble Nou s/n, 43540 Sant Carles de la Ràpita, Spain.
authors.forename: Enric
authors.initials: E
authors.lastname: Gisbert
mesh_terms: D000818:Animals; D001842:Bone and Bones / Q000378:metabolism*; D002113:Calcification, Physiologic / Q000187:drug effects*; D002135:Calcium-Binding Proteins / Q000096:biosynthesis; D002454:Cell Differentiation / Q000187:drug effects*; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects*; D005109:Extracellular Matrix / Q000378:metabolism*; D016326:Extracellular Matrix Proteins / Q000096:biosynthesis; D015870:Gene Expression / Q000187:drug effects; D015675:Osteocalcin / Q000096:biosynthesis; D018168:Receptors, Retinoic Acid / Q000096:biosynthesis; D047488:Retinoid X Receptors / Q000096:biosynthesis; D021541:Sea Bream; D014212:Tretinoin / Q000494:pharmacology*; D000097568:Matrix Gla Protein
chemical_list: D002135:Calcium-Binding Proteins; D016326:Extracellular Matrix Proteins; D015675:Osteocalcin; D018168:Receptors, Retinoic Acid; D047488:Retinoid X Receptors; D014212:Tretinoin; D000097568:Matrix Gla Protein
keywords: Bone-derived cell lines; Chondrocyte; Gene expression; Gilthead seabream Sparus aurata; Osteoblast; Retinoic acid; Vitamin A