RESUMO
BACKGROUND: 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.
Assuntos
Betaína-Homocisteína S-Metiltransferase/metabolismo , Cartilagem/enzimologia , Condrogênese , Proteínas de Peixes/metabolismo , Osteogênese , Dourada/metabolismo , Animais , Betaína-Homocisteína S-Metiltransferase/genética , Linhagem Celular , Clonagem Molecular , Evolução Molecular , Proteínas de Peixes/genética , Regulação Enzimológica da Expressão Gênica , Filogenia , Regiões Promotoras Genéticas , Dourada/genética , Transcrição Gênica , TransfecçãoRESUMO
MicroRNAs (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) 3'UTR 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.
Assuntos
Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 2/metabolismo , Calcificação Fisiológica/genética , MicroRNAs/genética , Dourada , Transdução de Sinais/genética , Animais , Sítios de Ligação , Linhagem Celular , Sequência Conservada , Evolução Molecular , Matriz Extracelular/metabolismo , Células HEK293 , Humanos , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Regulação para Cima , alfa-Galactosidase/metabolismoRESUMO
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.
Assuntos
Regulação da Expressão Gênica , Coluna Vertebral/metabolismo , Vanadatos/farmacologia , Animais , Ácido Ascórbico/farmacologia , Cálcio/farmacologia , Linhagem Celular , Peixes/genética , Análise de Sequência com Séries de Oligonucleotídeos , Fosfatos/farmacologia , Coluna Vertebral/citologiaRESUMO
Fish 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.
Assuntos
Calcificação Fisiológica/efeitos dos fármacos , Fator de Crescimento Insulin-Like I/farmacologia , Insulina/farmacologia , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Vanadatos/farmacologia , Fosfatase Alcalina/metabolismo , Animais , Bovinos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Modelos Biológicos , Osteoblastos/enzimologia , Osteocalcina/genética , Osteocalcina/metabolismoRESUMO
We recently reported proliferative and anti-mineralogenic effects of vanadate on fish chondrocytes and here we investigate the signalling pathways associated with these effects. Our data show that vanadate stimulates chondrocyte proliferation through the MAPK pathway, using signalling mechanisms similar to those used by IGF-1, while it inhibits chondrocyte differentiation/mineralization through a putative PI-3K/Ras/Erk signalling, a pathway shared with insulin. Our data also suggest that vanadate impairs ECM mineralization not only by interfering with regulatory pathways but also by inhibiting enzymatic activity of ALP. Finally, this work provides additional evidence for the conservation, throughout evolution, of mechanisms regulating chondrocyte proliferation and differentiation.
Assuntos
Proliferação de Células/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases , Fosfatidilinositol 3-Quinases/metabolismo , Vanadatos/farmacologia , Proteínas ras/metabolismo , Fosfatase Alcalina/antagonistas & inibidores , Animais , Linhagem Celular , Condrócitos/enzimologia , PeixesRESUMO
Spliced variants of insulin-like growth factor 1 (IGF-1), a small peptide with a critical role in metabolism and growth, have been identified in various vertebrate species. However, despite recent functional data in mammalian systems suggesting specific roles (e.g. in muscle formation) for their pro-peptides and/or E domains, their function remains unclear. In this study, three alternatively spliced variants of Sparus aurata proIGF-1 (1a, 1b, and 1c) were identified and their expression analyzed. In adult fish, IGF-1 gene expression was observed in various soft tissues (highest levels in liver) and calcified tissues, with IGF-1c being always the most expressed isoform. In developing larvae, each isoform presented a specific pattern of expression, characterized by different onset and extent and consistent with a possible role of IGF-1a and 1b during early post-hatching events (e.g. bone or muscle formation), while IGF-1c would be rather involved in early larvae formation but probably acts in concerted action with other isoforms at later stages. We also propose that, in adults, IGF-1a and 1b isoforms may have a local action, while isoform 1c would assume a systemic action, as its mammalian counterpart. This hypothesis was further supported by in silico analysis of isoform distribution, revealing that only IGF-1c/Ea isoform has been conserved throughout evolution and that other fish isoforms (i.e. 1a and 1b) may be associated with mechanisms of osmoregulation. We finally propose that IGF-1 variants may exhibit different modes of action (systemic or local) and may be involved in different developmental and adaptive mechanisms.
Assuntos
Processamento Alternativo , Proteínas de Peixes/genética , Fator de Crescimento Insulin-Like I/genética , Dourada/genética , Sequência de Aminoácidos , Animais , Southern Blotting , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Larva/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Dourada/crescimento & desenvolvimento , Alinhamento de SequênciaRESUMO
The use of in vitro cell culture systems to assess gene function largely depends on the successful transfer of DNA into target cells. Well developed in mammals, transfection methods are still to be optimized for non-mammalian cell culture systems, like fish. Here we describe a rapid, cost-efficient, and successful method to transfer DNA into a fish bone-derived cell line using polyethylenimine (PEI) as the DNA carrier. Using this method, DNA transfer was remarkably enhanced in comparison with commercially available reagents, as demonstrated by the increased activity of both luciferase and green fluorescent protein observed in the transfected cells. Its efficiency in transferring DNA into a wide range of cell types, including non-mammalian and hard-to-transfect cells, in addition to a low cost, show that PEI is a reagent of choice for nonviral vector transfection.
Assuntos
DNA/administração & dosagem , DNA/genética , Osteoblastos/metabolismo , Polietilenoimina/química , Dourada/genética , Dourada/metabolismo , Transfecção/métodos , Animais , Técnicas de Cultura de Células/métodos , Células Cultivadas , Materiais Revestidos Biocompatíveis/química , Portadores de Fármacos/química , Teste de MateriaisRESUMO
MGP 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.
Assuntos
Neoplasias da Mama/genética , Proteínas de Ligação ao Cálcio/genética , Carcinogênese/genética , Proteínas da Matriz Extracelular/genética , MicroRNAs/metabolismo , Transdução de Sinais , Sequência de Bases , Neoplasias da Mama/patologia , Proteínas de Ligação ao Cálcio/metabolismo , Proliferação de Células , Proteínas da Matriz Extracelular/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Células HEK293 , Humanos , Células MCF-7 , MicroRNAs/genética , Modelos Biológicos , Invasividade Neoplásica , RNA Interferente Pequeno/metabolismo , Proteína de Matriz GlaAssuntos
Calcificação Fisiológica/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Vanadatos/farmacologia , Animais , Proliferação de Células/efeitos dos fármacos , Condrócitos/citologia , Condrócitos/metabolismo , Humanos , Modelos Biológicos , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteogênese/genéticaRESUMO
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.
Assuntos
Osso e Ossos/metabolismo , Calcificação Fisiológica/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Matriz Extracelular/metabolismo , Tretinoína/farmacologia , Animais , Proteínas de Ligação ao Cálcio/biossíntese , Linhagem Celular , Proteínas da Matriz Extracelular/biossíntese , Expressão Gênica/efeitos dos fármacos , Osteocalcina/biossíntese , Receptores do Ácido Retinoico/biossíntese , Receptores X de Retinoides/biossíntese , Dourada , Proteína de Matriz GlaRESUMO
Vanadium, a trace metal known to accumulate in bone and to mimic insulin, has been shown to regulate mammalian bone formation using in vitro and in vivo systems. In the present work, short- and long-term effects of metavanadate (containing monomeric, dimeric, tetrameric and pentameric vanadate species) and decavanadate (containing decameric vanadate species) solutions on the mineralization of a fish bone-derived cell line (VSa13) were studied and compared to that of insulin. After 2 h of incubation with vanadate (10 microM in monomeric vanadate), metavanadate exhibited higher accumulation rates than decavanadate (6.85 +/- 0.40 versus 3.95 +/- 0.10 microg V/g of protein, respectively) in fish VSa13 cells and was also shown to be less toxic when applied for short periods. In longer treatments with both metavanadate and decavanadate solutions, similar effects were promoted: stimulation of cell proliferation and strong impairment (75%) of extracellular matrix (ECM) mineralization. The effect of both vanadate solutions (5 microM in monomeric vanadate), on ECM mineralization was increased in the presence of insulin (10 nM). It is concluded that chronic treatment with both vanadate solutions stimulated fish VSa13 cells proliferation and prevented ECM mineralization. Newly developed VSa13 fish cells appeared to be appropriate in the characterization of vanadate effects on vertebrate bone formation, representing a good alternative to mammalian systems.