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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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Venn diagrams of genes up-regulated (A) and down-regulated (B) during mineralization of VSa13 and VSa16 cells. A two class SAM test was performed; FDR and FC parameters were lower than 5% and higher than 1.5, respectively. Size of diagrams is proportional to the size of gene pools.
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Figure 2: Venn diagrams of genes up-regulated (A) and down-regulated (B) during mineralization of VSa13 and VSa16 cells. A two class SAM test was performed; FDR and FC parameters were lower than 5% and higher than 1.5, respectively. Size of diagrams is proportional to the size of gene pools.

Mentions: Confluent cultures of VSa13 and VSa16 cells were cultivated during 4 weeks under control (regular medium) or mineralizing (regular medium supplemented with calcium, phosphate and L-ascorbic acid) conditions. Deposition of mineral nodules within extracellular matrix was confirmed by von Kossa staining in cells exposed to mineralization cocktail (Figure 1) and total RNA was extracted from three biological replicates per condition. After proper amplification and labeling, each RNA sample was hybridized against the oligo-array and raw expression data were extracted and filtered using Agilent Feature Extraction 9.5.1 software, then normalized. Quantile normalization showed the highest agreement among replicates (i.e. lowest variation of normalized fluorescence distribution among replicates; data not shown) and was consequently used to normalize raw data sets. Normalized data sets were analyzed through significance analysis of microarray (SAM) and genes differentially expressed in control versus mineralizing conditions were identified. False discovery rate (FDR) threshold was set at 5% and only probes with fold change (FC) over 1.5 were considered. A total of 4,777 and 4,554 probes - corresponding to 3,011 and 3,049 unique genes - indicative of an up-regulated expression were identified from VSa13 and VSa16 RNAs, respectively. Among these genes, 1,489 were shown to be common to both cell lines (Figure 2). Similarly, 2,359 and 1,642 probes - corresponding to 1,212 and 1,098 unique genes - indicative of a down-regulated expression were identified from VSa13 and VSa16 RNAs, respectively. Among these genes, 469 were shown to be common to both cell lines (Figure 2). In VSa13 and VSa16 cells, 69% and 49% of differentially expressed genes were simultaneously detected by two non-overlapping probes, respectively, which could indicate the high occurrence of alternative splicing. It could also be the consequence of slight differences of hybridization between the two probes. Indeed, most genes detected by only one probe seem to follow the same pattern of expression when compared to the second non-significant probe. Raw and normalized fluorescence data have been deposited in the GEO database under accession numbers GSE18915 (VSa13) and GSE18941 (VSa16).


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)

Venn diagrams of genes up-regulated (A) and down-regulated (B) during mineralization of VSa13 and VSa16 cells. A two class SAM test was performed; FDR and FC parameters were lower than 5% and higher than 1.5, respectively. Size of diagrams is proportional to the size of gene pools.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Venn diagrams of genes up-regulated (A) and down-regulated (B) during mineralization of VSa13 and VSa16 cells. A two class SAM test was performed; FDR and FC parameters were lower than 5% and higher than 1.5, respectively. Size of diagrams is proportional to the size of gene pools.
Mentions: Confluent cultures of VSa13 and VSa16 cells were cultivated during 4 weeks under control (regular medium) or mineralizing (regular medium supplemented with calcium, phosphate and L-ascorbic acid) conditions. Deposition of mineral nodules within extracellular matrix was confirmed by von Kossa staining in cells exposed to mineralization cocktail (Figure 1) and total RNA was extracted from three biological replicates per condition. After proper amplification and labeling, each RNA sample was hybridized against the oligo-array and raw expression data were extracted and filtered using Agilent Feature Extraction 9.5.1 software, then normalized. Quantile normalization showed the highest agreement among replicates (i.e. lowest variation of normalized fluorescence distribution among replicates; data not shown) and was consequently used to normalize raw data sets. Normalized data sets were analyzed through significance analysis of microarray (SAM) and genes differentially expressed in control versus mineralizing conditions were identified. False discovery rate (FDR) threshold was set at 5% and only probes with fold change (FC) over 1.5 were considered. A total of 4,777 and 4,554 probes - corresponding to 3,011 and 3,049 unique genes - indicative of an up-regulated expression were identified from VSa13 and VSa16 RNAs, respectively. Among these genes, 1,489 were shown to be common to both cell lines (Figure 2). Similarly, 2,359 and 1,642 probes - corresponding to 1,212 and 1,098 unique genes - indicative of a down-regulated expression were identified from VSa13 and VSa16 RNAs, respectively. Among these genes, 469 were shown to be common to both cell lines (Figure 2). In VSa13 and VSa16 cells, 69% and 49% of differentially expressed genes were simultaneously detected by two non-overlapping probes, respectively, which could indicate the high occurrence of alternative splicing. It could also be the consequence of slight differences of hybridization between the two probes. Indeed, most genes detected by only one probe seem to follow the same pattern of expression when compared to the second non-significant probe. Raw and normalized fluorescence data have been deposited in the GEO database under accession numbers GSE18915 (VSa13) and GSE18941 (VSa16).

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