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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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Effect of ParaHox overexpression (except PDX1) on the insulin promoter in the βTC6 cell line at low (0.5 mM) or high (25 mM) glucose concentration, 48 h post-transfection. The values on the x axis represent the increasing dose (in micrograms) of each transcription factor 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. 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 3: Effect of ParaHox overexpression (except PDX1) on the insulin promoter in the βTC6 cell line at low (0.5 mM) or high (25 mM) glucose concentration, 48 h post-transfection. The values on the x axis represent the increasing dose (in micrograms) of each transcription factor 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. 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: The results are summarised in the histogram diagrams of Fig. 3. All experiments were consistently replicated, and statistical significance of the observed differences was determined using the Mann-Whitney test (p<0.05, indicated by one asterisk). In summary, GSH1 and GSH2 over expression resulted in inhibition of the promoter, more pronounced for GSH2, at high glucose concentration (Fig. 3), whereas CDX1 and CDX2/3 showed a transactivation effect on the insulin promoter. CDX1, which acted in a glucose-independent manner, yielded around 2-fold increase in activity at the highest concentration of protein (Fig. 3). CDX2/3, instead, responded differently depending on the glucose concentration. While having no statitistically significant effect at low glucose levels, it was able to transactivate the insulin promoter at high glucose concentration, in accordance to previous reports 22, 23. This effect was particularly observed when high amounts of the transcription factor construct were transfected (4.4-fold increase). In contrast, CDX4, which was also sensitive to glucose levels, produced the reverse effect to that of CDX2/3. Whereas at a low glucose concentration CDX4 over expression did not affect the basal transcription of the insulin promoter, at high glucose concentration it readily inhibited transcription to nearly values in a transcription factor dose-dependent manner (Fig. 3).


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 ParaHox overexpression (except PDX1) on the insulin promoter in the βTC6 cell line at low (0.5 mM) or high (25 mM) glucose concentration, 48 h post-transfection. The values on the x axis represent the increasing dose (in micrograms) of each transcription factor 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. 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 3: Effect of ParaHox overexpression (except PDX1) on the insulin promoter in the βTC6 cell line at low (0.5 mM) or high (25 mM) glucose concentration, 48 h post-transfection. The values on the x axis represent the increasing dose (in micrograms) of each transcription factor 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. 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: The results are summarised in the histogram diagrams of Fig. 3. All experiments were consistently replicated, and statistical significance of the observed differences was determined using the Mann-Whitney test (p<0.05, indicated by one asterisk). In summary, GSH1 and GSH2 over expression resulted in inhibition of the promoter, more pronounced for GSH2, at high glucose concentration (Fig. 3), whereas CDX1 and CDX2/3 showed a transactivation effect on the insulin promoter. CDX1, which acted in a glucose-independent manner, yielded around 2-fold increase in activity at the highest concentration of protein (Fig. 3). CDX2/3, instead, responded differently depending on the glucose concentration. While having no statitistically significant effect at low glucose levels, it was able to transactivate the insulin promoter at high glucose concentration, in accordance to previous reports 22, 23. This effect was particularly observed when high amounts of the transcription factor construct were transfected (4.4-fold increase). In contrast, CDX4, which was also sensitive to glucose levels, produced the reverse effect to that of CDX2/3. Whereas at a low glucose concentration CDX4 over expression did not affect the basal transcription of the insulin promoter, at high glucose concentration it readily inhibited transcription to nearly values in a transcription factor dose-dependent manner (Fig. 3).

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