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- Central role of betaine-homocysteine S-methyltransferase 3 in chondral ossification and evidence for sub-functionalization in neoteleost fishPublication . Rosa, Joana; Tiago, Daniel; Marques, Cátia L.; Vijayakumar, Parameswaran; Fonseca, Luís; Cancela, Leonor; Laizé, VincentBackground: To better understand the complex mechanisms of bone formation it is fundamental that genes central to signaling/regulatory pathways and matrix formation are identified. Cell systems were used to analyze genes differentially expressed during extracellular matrix mineralization and bhmt3, coding for a betaine-homocysteine S-methyltransferase, was shown to be down-regulated in mineralizing gilthead seabream cells.Methods: Levels and sites of bhmt3 expression were determined by qPCR and in situ hybridization throughout seabream development and in adult tissues. Transcriptional regulation of bhmt3 was assessed from the activity of promoter constructs controlling luciferase gene expression. Molecular phylogeny of vertebrate BHMT was determined from maximum likelihood analysis of available sequences.Results: bhmt3 transcript is abundant in calcified tissues and localized in cartilaginous structures undergoing endo/perichondral ossification. Promoter activity is regulated by transcription factors involved in bone and cartilage development, further demonstrating the central role of Bhmt3 in chondrogenesis and/or osteogenesis. Molecular phylogeny revealed the explosive diversity of bhmt genes in neoteleost fish, while tissue distribution of bhmt genes in seabream suggested that neoteleostean Bhmt may have undergone several steps of sub-functionalization.Conclusions: Data on bhmt3 gene expression and promoter activity evidences a novel function for betaine-homocysteine S-methyltransferase in bone and cartilage development, while phylogenetic analysis provides new insights into the evolution of vertebrate BHMTs and suggests that multiple gene duplication events occurred in neoteleost fish lineage.General significance: High and specific expression of Bhmt3 in gilthead seabream calcified tissues suggests that bone-specific betaine-homocysteine S-methyltransferases could represent a suitable marker of chondral ossification.
- Warfarin-exposed zebrafish embryos resembles human warfarin embryopathy in a dose and developmental-time dependent manner - From molecular mechanisms to environmental concernsPublication . Granadeiro, Luis; Dirks, Ron P.; Ortiz-Delgado, Juan B.; J. Gavaia, Paulo; Sarasquete, Carmen; Laizé, Vincent; Leonor Cancela, M.; I, FernándezWarfarin is the most worldwide used anticoagulant drug and rodenticide. Since it crosses placental barrier it can induce warfarin embryopathy (WE), a fetal mortality in neonates characterized by skeletal deformities in addition to brain hemorrhages. Although the effects of warfarin exposure in aquatic off target species were already described, the particular molecular toxicological mechanisms during early development are still unclear. Here, we used zebrafish (Danio rerio) to describe and compare the developmental effects of warfarin exposure (0, 15.13, 75.68 and 378.43 mM) on two distinct early developmental phases (embryos and eleuthero-embryos). Although exposure to both developmental phases induced fish mortality, only embryos exposed to the highest warfarin level exhibited features mimicking mammalian WE, e.g. high mortality, higher incidence of hemorrhages and altered skeletal development, among other effects. To gain insights into the toxic mechanisms underlying warfarin exposure, the transcriptome of embryos exposed to warfarin was explored through RNA-Seq and compared to that of control embryos. 766 differentially expressed (564 up- and 202 down-regulated) genes were identified. Gene Ontology analysis revealed particular cellular components (cytoplasm, extracellular matrix, lysosome and vacuole), biological processes (mainly amino acid and lipid metabolism and response to stimulus) and pathways (oxidative stress response and apoptosis signaling pathways) being significantly over-represented in zebrafish embryos upon warfarin exposure. Protein-protein interaction further evidenced an altered redox system, blood coagulation and vasculogenesis, visual phototransduction and collagen formation upon warfarin exposure. The present study not only describes for the first time the WE in zebrafish, it provides new insights for a better risk assessment, and highlights the need for programming the rat eradication actions outside the fish spawning season to avoid an impact on off target fish community. The urge for the development of more species-specific anticoagulants for rodent pest control is also highlighted.
- Overexpression of four and a half LIM domains protein 2 promotes epithelial-mesenchymal transition-like phenotype in fish pre-osteoblastsPublication . Rafael, Marta S.; Laizé, Vincent; Florindo, C.; Ferraresso, Serena; Bargelloni, Luca; Cancela, LeonorFHL2 is a multifunctional protein involved in gene transcription regulation and cytoarchitecture modulation that has been recently associated with epithelial-mesenchymal transition (EMT) in colon cancer. Overexpression of FHL2 in a fish pre-osteoblastic cell line promoted cell dedifferentiation and impaired its extracellular matrix mineralization capacity. Cell cultures also acquired a novel threedimensional structure organization, their proliferation rate was enhanced and gene expression profile was altered in agreement with an EMT-like phenotype upon overexpression of FHL2. Altogether, our results provide additional support to the relevance of FHL2 for cell differentiation and its association with hallmarks of cancer phenotype.
- Proliferative and mineralogenic effects of insulin, IGF-1, and vanadate in fish osteoblast-like cellsPublication . Tiago, Daniel; Cancela, Leonor; Laizé, VincentFish have recently been recognized as a suitable model and a promising alternative to mammalian systems to study skeletogenesis. In this regard, several fish bone-derived cell lines have been developed and are being used to investigate mechanisms associated with insulin-like action of vanadium on extracellular matrix (ECM) mineralization. Although proliferative and mineralogenic effects of vanadate, insulin-like growth factor 1 (IGF-1), and insulin have recently been evaluated in a fish prechondrocyte cell line, no data are available in fish bone-forming cells, the osteoblasts. Using fish preosteoblast cells, we showed that IGF-1, but not insulin or vanadate, stimulated cell proliferation through the mitogen-activated protein kinase (MAPK) pathway, while both IGF-1 and vanadate inhibited cell differentiation/ECM mineralization through the same mechanism. Our data also indicated that the phosphatidyl inositol-3 kinase (PI-3K) pathway stimulates differentiation/ECM mineralization in osteoblasts and could represent a way to balance MAPK pathway action. The comparison of these new data obtained in fish with those available in mammals clearly evidenced a conservation of regulatory mechanisms among vertebrate bone-derived systems, although different players are involved.
- Effect of variable levels of dietary cholesterol and plant sterols on the growth performance and bone metabolism in gilthead seabream (Sparus aurata) juvenilesPublication . Dias, J.; Colen, Rita; Rodrigues, V.; Aragão, C.; Engrola, S.; Viegas, Michael; Laizé, Vincent; Gavaia, Paulo J.; Cancela, LeonorCholesterol is found in all animal tissues and is an important component of biological cell membranes with functions such as precursor to bile acids, hormones and vitamins. Fish meal and fish oil are cholesterol-rich ingredients. Replacement of these marine-derived ingredients by plant proteins and vegetable oils tends to reduce dietary cholesterol levels.
- 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 proteinsPublication . Fernández, Ignacio; Tiago, Daniel; Laizé, Vincent; Cancela, Leonor; Gisbert, EnricRetinoic 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.
- Sturgeon osteocalcin shares structural features with matrix gla protein evolutionary relationship and functional implicationsPublication . S B Viegas, Carla; Simes, Dina; Williamson, Matthew K.; Cavaco, Sofia; Laizé, Vincent; Price, Paul A.; Leonor Cancela, M.Osteocalcin (OC) and matrix Gla protein (MGP) are considered evolutionarily related because they share key structural features, although they have been described to exert different functions. In this work, we report the identification and characterization of both OC and MGP from the Adriatic sturgeon, a ray-finned fish characterized by a slow evolution and the retention of many ancestral features. Sturgeon MGP shows a primary structure, post-translation modifications, and patterns of mRNA/protein distribution and accumulation typical of known MGPs, and it contains seven possible Gla residues that would make the sturgeon protein the most gamma-carboxylated among known MGPs. In contrast, sturgeon OC was found to present a hybrid structure. Indeed, although exhibiting protein domains typical of known OCs, it also contains structural features usually found in MGPs (e. g. a putative phosphorylated propeptide). Moreover, patterns of OC gene expression and protein accumulation overlap with those reported for MGP; OC was detected in bone cells and mineralized structures but also in soft and cartilaginous tissues. We propose that, in a context of a reduced rate of evolution, sturgeon OC has retained structural features of the ancestral protein that emerged millions of years ago from the duplication of an ancient MGP gene and may exhibit intermediate functional features.
- Global analysis of gene expression in mineralizing fish vertebra-derived cell lines: new insights into anti-mineralogenic effect of vanadatePublication . Tiago, Daniel; Laizé, Vincent; Bargelloni, Luca; Ferraresso, Serena; Romualdi, Chiara; Cancela, LeonorAbstract Background Fish has been deemed suitable to study the complex mechanisms of vertebrate skeletogenesis and gilthead seabream (Sparus aurata), a marine teleost with acellular bone, has been successfully used in recent years to study the function and regulation of bone and cartilage related genes during development and in adult animals. Tools recently developed for gilthead seabream, e.g. mineralogenic cell lines and a 4 × 44K Agilent oligo-array, were used to identify molecular determinants of in vitro mineralization and genes involved in anti-mineralogenic action of vanadate. Results Global analysis of gene expression identified 4,223 and 4,147 genes differentially expressed (fold change - FC > 1.5) during in vitro mineralization of VSa13 (pre-chondrocyte) and VSa16 (pre-osteoblast) cells, respectively. Comparative analysis indicated that nearly 45% of these genes are common to both cell lines and gene ontology (GO) classification is also similar for both cell types. Up-regulated genes (FC > 10) were mainly associated with transport, matrix/membrane, metabolism and signaling, while down-regulated genes were mainly associated with metabolism, calcium binding, transport and signaling. Analysis of gene expression in proliferative and mineralizing cells exposed to vanadate revealed 1,779 and 1,136 differentially expressed genes, respectively. Of these genes, 67 exhibited reverse patterns of expression upon vanadate treatment during proliferation or mineralization. Conclusions Comparative analysis of expression data from fish and data available in the literature for mammalian cell systems (bone-derived cells undergoing differentiation) indicate that the same type of genes, and in some cases the same orthologs, are involved in mechanisms of in vitro mineralization, suggesting their conservation throughout vertebrate evolution and across cell types. Array technology also allowed identification of genes differentially expressed upon exposure of fish cell lines to vanadate and likely involved in its anti-mineralogenic activity. Many were found to be unknown or they were never associated to bone homeostasis previously, thus providing a set of potential candidates whose study will likely bring insights into the complex mechanisms of tissue mineralization and bone formation.
- Serum-specific stimulation of proliferation and mineralization of fish bone-derived cellsPublication . Rosa, Joana; Tiago, Daniel; Dias, J.; Cancela, Leonor; Laizé, VincentTeleost fish have recently been implemented as suitable model organisms to study vertebrate development, in particular skeletogenesis. In vitro cell systems derived from fish bone have been successfully established, although their development has been hampered by the limited availability of fish serum to supplement culture medium. Commercially available sera are mostly of mammalian origin and thus not necessarily adequate to fish cell growth. The main objective of this work was to compare proliferative and mineralogenic potential of bovine and fish sera using fish bone-derived cell lines VSa13 and VSa16. Fish serum was shown to (i) strongly stimulate cell proliferation in an apparent dose-dependent and cell type-specific manner, (ii) induce morphological changes, and (iii) enhance extracellular matrix mineralization of bone cells, although cytotoxic for fish osteoblast-like cells at the concentration tested. To better understand mechanisms underlying mineralogenic effect of fish serum in fish chondrocytes, expression of several mineralization-related genes was evaluated by qPCR. Regulation of matrix Gla protein (MGP) and bone morphogenetic protein 2 (BMP2) gene expression was modified upon culture with fish serum in a way compatible with an early onset and an increase in mineralization. In conclusion, fish serum was shown to be more adequate to proliferation and differentiation/mineralization of fish bone-derived cells.
- Gla-rich protein, a new player in tissue calcification?Publication . Cancela, Leonor; Conceição, N.; Laizé, VincentA novel g-carboxyglutamate (Gla)-containing protein, named Gla-rich protein (GRP) after its high content in Gla residues or upper zone of growth plate and cartilage matrix associated protein after its preferential expression by cartilage chondrocyte, was recently identified in sturgeon, mice, and humans through independent studies. GRP is the most densely g-carboxylated protein identified to date and its structure has been remarkably conserved throughout vertebrate evolution but is apparently absent from bird genomes. Several transcript and genomic variants affecting key protein features or regulatory elements were described and 2 paralogs were identified in the teleost fish genome. In the skeleton, most relevant levels of GRP gene expression were observed in cartilaginous tissues and associated with chondrocytes, suggesting a role in chondrogenesis. But GRP expression was also detected in bone cells, indicative of a more widespread role for the protein throughout skeletal formation. Although the molecular function of GRP is yet unknown, the high content of Gla residues and its accumulation at sites of pathological calcification in different human pathologies affecting skin or the vascular system and in breast cancer tumors suggest that GRP may function as a modulator of calcium availability. Because of its association with fibrillar collagens, GRP could also be involved in the organization and/or stabilization of cartilage matrix. Although transgenic mice did not reveal obvious phenotypic alterations in skeletal development or structure, zebrafish morphants lack craniofacial cartilage and exhibit limited calcification, suggesting a role for GRP during skeletal development, but additional functional data are required to understand its function.