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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.
- 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.
- 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.
- 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.
- Matrix Gla protein expression: a complex process involving the use of alternative promoters, multiple splicing events and microRNAsPublication . Cancela, Leonor; Laizé, Vincent; Conceição, N.; Tiago, Daniel; Maia, Ana-Teresa; Bensimon-Brito, A.; Gavaia, Paulo J.Matrix Gla protein (MGP) is a secreted vitamin K-dependent protein (VKD) located in the extracellular matrix and capable of binding calcium through its -carboxyglutamate residues. Although identified in 1983, transcriptional and post-transcriptional mechanisms regulating its expression remain unclear.
- Matrix Gla protein repression by miR-155 promotes oncogenic signals in breast cancer MCF-7 cellsPublication . Tiago, Daniel; Conceição, Natércia; Caiado, Helena; Laizé, Vincent; Cancela, LeonorMGP is a protein that was initially associated with the inhibition of calcification in skeleton, soft tissues, and arteries, but more recently also implicated in cancer. In breast cancer, higher levels of MGP mRNA were associated with poor prognosis, but since this deregulation was never demonstrated at the protein level, we postulated the involvement of a post-transcriptional regulatory mechanism. In this work we show that MGP is significantly repressed by miR-155 in breast cancer MCF-7 cells, and concomitantly there is a stimulation of cell proliferation and cell invasiveness. This study brings new insights into the putative involvement of MGP and oncomiR-155 in breast cancer, and may contribute to develop new therapeutic strategies.
- 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, LeonorBackground 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.
- Mir-20a regulates in vitro mineralization and BMP signaling pathway by targeting BMP-2 transcript in fishPublication . Tiago, Daniel; Marques, C. L.; Roberto, Vania Palma; Cancela, Leonor; Laizé, VincentMicroRNAs (miRNAs) are important regulators of vertebrate development but their role during skeletogenesis remains unknown. In this regard, we investigated the mineralogenic activity of miR-20a, a miRNA associated with osteogenesis, in fish bone-derived cells. Expression of miR-20a was up-regulated during differentiation and its overexpression inhibited mineralization, suggesting a role in fish tissue calcification. In this regard, a conserved miR-20a binding site was identified in bone morphogenetic protein 2 (BMP-2) 30UTR and its functionality was evidenced through luciferase assays, and further confirmed by western-blot and qPCR. Type II BMP receptor (BMPR2) is also targeted by miR-20a in mammalian systems and evidence was collected for the presence of a binding site in fish sequences. We propose that miR-20a is a regulator of BMP pathway through specific action on BMP-2 and possibly BMPR2. Overexpression of miR-20a was also shown to up-regulate matrix Gla protein (MGP) transcript, a physiological inhibitor of calcification previously found to form a complex with BMP-2. We propose that MGP may play a role in the anti-mineralogenic effect promoted by miR-20a by decreasing availability of BMP-2. This study gives new insights into miRNA-mediated regulation of BMP-2, and sheds light into the potential role of miR-20a as a regulator of skeletogenesis.