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Global analysis of gene expression in mineralizing fish vertebra-derived cell lines: new insights into anti-mineralogenic effect of vanadate.

Tiago DM, Laizé V, Bargelloni L, Ferraresso S, Romualdi C, Cancela ML - BMC Genomics (2011)

Bottom Line: 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.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre of Marine Sciences, University of Algarve, Faro, Portugal.

ABSTRACT

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.

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Real-time PCR analysis of genes detected in vanadate-treated VSa13 cells during mineralization (M vs MV) and proliferation (P vs PV) selected for validation. Genes were selected according to the following criteria: 3 up-regulated genes with 1.5-8 FC and 3 down-regulated genes with 1.5-8 FC. All changes in gene expression evaluated by qPCR were significant according to Student's test (p < 0.05). Pearson correlation coefficients, calculated comparing probes 1 and 2 from oligo-array, and comparing probes 1 and 2 from oligo-array with qPCR, are indicated by r. Genes selected for analysis were: ependymin-related protein (ERP), arginine N-methyltransferase (RNM), cyclin-dependent kinase (CDK), signal peptide CUB and EGF-like protein (SCUBE-like), tenascin (TNS) and thrombospondin (TBSD).
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Figure 7: Real-time PCR analysis of genes detected in vanadate-treated VSa13 cells during mineralization (M vs MV) and proliferation (P vs PV) selected for validation. Genes were selected according to the following criteria: 3 up-regulated genes with 1.5-8 FC and 3 down-regulated genes with 1.5-8 FC. All changes in gene expression evaluated by qPCR were significant according to Student's test (p < 0.05). Pearson correlation coefficients, calculated comparing probes 1 and 2 from oligo-array, and comparing probes 1 and 2 from oligo-array with qPCR, are indicated by r. Genes selected for analysis were: ependymin-related protein (ERP), arginine N-methyltransferase (RNM), cyclin-dependent kinase (CDK), signal peptide CUB and EGF-like protein (SCUBE-like), tenascin (TNS) and thrombospondin (TBSD).

Mentions: Similarly, 6 genes differentially expressed in mineralizing cells exposed to vanadate (3 up- and 3 down-regulated genes) were selected for validation of microarray data by qPCR (Figure 7). Analysis of Pearson correlation revealed coefficients higher than 0.99 (strong correlation) when comparing data from microarray probes 1 and 2, and higher than 0.9 (strong correlation) when comparing data from microarray and qPCR. In general, correlation coefficients were shown to be higher among genes analyzed in vanadate-treated VSa13 cells than among those analyzed in mineralizing VSa13 and VSa16 cells. This difference could in part be explained by the lower FC values observed among genes identified in vanadate-treated cells and consequent lower tendency for FC compression of oligo-array data.


Global analysis of gene expression in mineralizing fish vertebra-derived cell lines: new insights into anti-mineralogenic effect of vanadate.

Tiago DM, Laizé V, Bargelloni L, Ferraresso S, Romualdi C, Cancela ML - BMC Genomics (2011)

Real-time PCR analysis of genes detected in vanadate-treated VSa13 cells during mineralization (M vs MV) and proliferation (P vs PV) selected for validation. Genes were selected according to the following criteria: 3 up-regulated genes with 1.5-8 FC and 3 down-regulated genes with 1.5-8 FC. All changes in gene expression evaluated by qPCR were significant according to Student's test (p < 0.05). Pearson correlation coefficients, calculated comparing probes 1 and 2 from oligo-array, and comparing probes 1 and 2 from oligo-array with qPCR, are indicated by r. Genes selected for analysis were: ependymin-related protein (ERP), arginine N-methyltransferase (RNM), cyclin-dependent kinase (CDK), signal peptide CUB and EGF-like protein (SCUBE-like), tenascin (TNS) and thrombospondin (TBSD).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3141667&req=5

Figure 7: Real-time PCR analysis of genes detected in vanadate-treated VSa13 cells during mineralization (M vs MV) and proliferation (P vs PV) selected for validation. Genes were selected according to the following criteria: 3 up-regulated genes with 1.5-8 FC and 3 down-regulated genes with 1.5-8 FC. All changes in gene expression evaluated by qPCR were significant according to Student's test (p < 0.05). Pearson correlation coefficients, calculated comparing probes 1 and 2 from oligo-array, and comparing probes 1 and 2 from oligo-array with qPCR, are indicated by r. Genes selected for analysis were: ependymin-related protein (ERP), arginine N-methyltransferase (RNM), cyclin-dependent kinase (CDK), signal peptide CUB and EGF-like protein (SCUBE-like), tenascin (TNS) and thrombospondin (TBSD).
Mentions: Similarly, 6 genes differentially expressed in mineralizing cells exposed to vanadate (3 up- and 3 down-regulated genes) were selected for validation of microarray data by qPCR (Figure 7). Analysis of Pearson correlation revealed coefficients higher than 0.99 (strong correlation) when comparing data from microarray probes 1 and 2, and higher than 0.9 (strong correlation) when comparing data from microarray and qPCR. In general, correlation coefficients were shown to be higher among genes analyzed in vanadate-treated VSa13 cells than among those analyzed in mineralizing VSa13 and VSa16 cells. This difference could in part be explained by the lower FC values observed among genes identified in vanadate-treated cells and consequent lower tendency for FC compression of oligo-array data.

Bottom Line: 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.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre of Marine Sciences, University of Algarve, Faro, Portugal.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus