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ParaHox genes in pancreatic cell cultures: effects on the insulin promoter regulation.

Rosanas-Urgell A, Garcia-Fernàndez J, Marfany G - Int. J. Biol. Sci. (2008)

Bottom Line: The crucial role of PDX1 in pancreas development, beta-cell formation and insulin transcription regulation has long been established.There is some data on CDX2/3 function in alpha-cells, but remarkably, nothing is known on the role of the other ParaHox genes, which are also expressed in the endocrine pancreas.Homeobox transcription factors that belong to the same family show high conservation of the homeodomain and share similar target sites and oligomeric partners, and thus may act redundantly, synergistically or antagonistically on the same promoters.

View Article: PubMed Central - PubMed

Affiliation: Departament de Genètica, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain.

ABSTRACT
The gene encoding PDX1 (pancreatic duodenum homeobox 1), the main transcription factor regulating the glucose-dependent transactivation of the insulin promoter in pancreatic beta-cells, clusters with two closely related homeobox genes (Gsh1 and Cdx2/3), all of them belonging to the ParaHox gene family. The ParaHox gene evolutionary history in the vertebrate lineage involved duplications of the cluster and subsequent loss of some members, so that eventually, the human and murine genomes contain only 6 ParaHox genes. The crucial role of PDX1 in pancreas development, beta-cell formation and insulin transcription regulation has long been established. There is some data on CDX2/3 function in alpha-cells, but remarkably, nothing is known on the role of the other ParaHox genes, which are also expressed in the endocrine pancreas. Homeobox transcription factors that belong to the same family show high conservation of the homeodomain and share similar target sites and oligomeric partners, and thus may act redundantly, synergistically or antagonistically on the same promoters. Therefore, we explored the effects of the Parahox proteins (GSH1, GSH2, CDX1, CDX2/3 and CDX4) on the regulation of the insulin promoter in transfected alpha- and beta- cultured cell lines at different glucose concentrations and compared them to those of PDX1. Noticeably, several ParaHox transcription factors are able to transactivate or inhibit the insulin promoter, depending on the cell type and glucose concentration, thus suggesting their possible participation in the regulation of similar target genes, such as insulin, either by silencing or activating them, in the absence of PDX1.

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Related in: MedlinePlus

Effect of PDX1 overexpression on the insulin promoter in the βTC6 cell line at low (0.5 mM) or high (25 mM) glucose concentrations at either 24 h or 48 h post-transfection. The values on the x axis represent the increasing dose (in μg) of the PDX1 expression construct used in the transfections. The y axis represent the arbitrary transactivation values of the insulin promoter with respect to the positive control (c+), which is considered as 1. The standard deviation is also indicated. At least four independently transfected replicates of each sample were analysed. c+ : positive control (cells transfected with the –645INS-LUC construct but no transcription factor). c– : negative control (cells transfected with empty vectors). Values statistically different from the control by the Mann-Whitney test (p<0.05) are indicated by one asterisk. Statistically significant values that show a stronger effect of the transcription factor on the insulin promoter (at least two-fold or more, or half or less, than the positive controls) are indicated by two asterisks.
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Figure 2: Effect of PDX1 overexpression on the insulin promoter in the βTC6 cell line at low (0.5 mM) or high (25 mM) glucose concentrations at either 24 h or 48 h post-transfection. The values on the x axis represent the increasing dose (in μg) of the PDX1 expression construct used in the transfections. The y axis represent the arbitrary transactivation values of the insulin promoter with respect to the positive control (c+), which is considered as 1. The standard deviation is also indicated. At least four independently transfected replicates of each sample were analysed. c+ : positive control (cells transfected with the –645INS-LUC construct but no transcription factor). c– : negative control (cells transfected with empty vectors). Values statistically different from the control by the Mann-Whitney test (p<0.05) are indicated by one asterisk. Statistically significant values that show a stronger effect of the transcription factor on the insulin promoter (at least two-fold or more, or half or less, than the positive controls) are indicated by two asterisks.

Mentions: In order to set up the conditions of transfection and overexpression of all the ParaHox constructs, we first evaluated the effects of PDX1 on β-cells under high glucose concentrations, as it is well known that, under these conditions, PDX1 is able to transactivate the insulin promoter 30, 31. To determine the post-transfection time needed to observe the effects of protein overexpression, we compared the results of PDX1 overexpression in the βTC6 cell line at either 24 h or 48 h post-transfection. As depicted in Fig. 2, the results at 48 h agreed consistently with the reported effects of PDX1 upon the insulin promoter: at low glucose concentration, there is no activation (in fact, the excess of PDX1 inhibits very weakly the promoter), whereas at high glucose concentration, high transactivation levels were robustly detected (more than 10 fold), even at low DNA concentration of the construct expressing the transcription factor.


ParaHox genes in pancreatic cell cultures: effects on the insulin promoter regulation.

Rosanas-Urgell A, Garcia-Fernàndez J, Marfany G - Int. J. Biol. Sci. (2008)

Effect of PDX1 overexpression on the insulin promoter in the βTC6 cell line at low (0.5 mM) or high (25 mM) glucose concentrations at either 24 h or 48 h post-transfection. The values on the x axis represent the increasing dose (in μg) of the PDX1 expression construct used in the transfections. The y axis represent the arbitrary transactivation values of the insulin promoter with respect to the positive control (c+), which is considered as 1. The standard deviation is also indicated. At least four independently transfected replicates of each sample were analysed. c+ : positive control (cells transfected with the –645INS-LUC construct but no transcription factor). c– : negative control (cells transfected with empty vectors). Values statistically different from the control by the Mann-Whitney test (p<0.05) are indicated by one asterisk. Statistically significant values that show a stronger effect of the transcription factor on the insulin promoter (at least two-fold or more, or half or less, than the positive controls) are indicated by two asterisks.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2238182&req=5

Figure 2: Effect of PDX1 overexpression on the insulin promoter in the βTC6 cell line at low (0.5 mM) or high (25 mM) glucose concentrations at either 24 h or 48 h post-transfection. The values on the x axis represent the increasing dose (in μg) of the PDX1 expression construct used in the transfections. The y axis represent the arbitrary transactivation values of the insulin promoter with respect to the positive control (c+), which is considered as 1. The standard deviation is also indicated. At least four independently transfected replicates of each sample were analysed. c+ : positive control (cells transfected with the –645INS-LUC construct but no transcription factor). c– : negative control (cells transfected with empty vectors). Values statistically different from the control by the Mann-Whitney test (p<0.05) are indicated by one asterisk. Statistically significant values that show a stronger effect of the transcription factor on the insulin promoter (at least two-fold or more, or half or less, than the positive controls) are indicated by two asterisks.
Mentions: In order to set up the conditions of transfection and overexpression of all the ParaHox constructs, we first evaluated the effects of PDX1 on β-cells under high glucose concentrations, as it is well known that, under these conditions, PDX1 is able to transactivate the insulin promoter 30, 31. To determine the post-transfection time needed to observe the effects of protein overexpression, we compared the results of PDX1 overexpression in the βTC6 cell line at either 24 h or 48 h post-transfection. As depicted in Fig. 2, the results at 48 h agreed consistently with the reported effects of PDX1 upon the insulin promoter: at low glucose concentration, there is no activation (in fact, the excess of PDX1 inhibits very weakly the promoter), whereas at high glucose concentration, high transactivation levels were robustly detected (more than 10 fold), even at low DNA concentration of the construct expressing the transcription factor.

Bottom Line: The crucial role of PDX1 in pancreas development, beta-cell formation and insulin transcription regulation has long been established.There is some data on CDX2/3 function in alpha-cells, but remarkably, nothing is known on the role of the other ParaHox genes, which are also expressed in the endocrine pancreas.Homeobox transcription factors that belong to the same family show high conservation of the homeodomain and share similar target sites and oligomeric partners, and thus may act redundantly, synergistically or antagonistically on the same promoters.

View Article: PubMed Central - PubMed

Affiliation: Departament de Genètica, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain.

ABSTRACT
The gene encoding PDX1 (pancreatic duodenum homeobox 1), the main transcription factor regulating the glucose-dependent transactivation of the insulin promoter in pancreatic beta-cells, clusters with two closely related homeobox genes (Gsh1 and Cdx2/3), all of them belonging to the ParaHox gene family. The ParaHox gene evolutionary history in the vertebrate lineage involved duplications of the cluster and subsequent loss of some members, so that eventually, the human and murine genomes contain only 6 ParaHox genes. The crucial role of PDX1 in pancreas development, beta-cell formation and insulin transcription regulation has long been established. There is some data on CDX2/3 function in alpha-cells, but remarkably, nothing is known on the role of the other ParaHox genes, which are also expressed in the endocrine pancreas. Homeobox transcription factors that belong to the same family show high conservation of the homeodomain and share similar target sites and oligomeric partners, and thus may act redundantly, synergistically or antagonistically on the same promoters. Therefore, we explored the effects of the Parahox proteins (GSH1, GSH2, CDX1, CDX2/3 and CDX4) on the regulation of the insulin promoter in transfected alpha- and beta- cultured cell lines at different glucose concentrations and compared them to those of PDX1. Noticeably, several ParaHox transcription factors are able to transactivate or inhibit the insulin promoter, depending on the cell type and glucose concentration, thus suggesting their possible participation in the regulation of similar target genes, such as insulin, either by silencing or activating them, in the absence of PDX1.

Show MeSH
Related in: MedlinePlus