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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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Expression of the ParaHox constructs in αTC1 and βTC6 cell lines. Cells were transfected with 0.8 μg of the construct for each transcription factor, allowed to grow in high glucose media (25mM glucose) and harvested 48 hours post-transfections. Cells were harvested from at least three replicates and two different experiments for each construct, and pooled to obtain the first strand cDNA. Specific primers and PCR conditions allowed the detection of each ParaHox gene expression. GAPDH was used as an internal control. –RT: transfected cells with the Pdx1 transcription factor, but in which no reverse transcription was performed (negative control of the RT-PCR, as there is no amplification if no cDNA is previously obtained). C: empty vector transfected cells, which also serve as positive control of the RT-PCR, as Pdx1 is endogenously expressed (at low levels in αTC1 cells and at much higher levels in βTC6 cells, as expected).
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Figure 1: Expression of the ParaHox constructs in αTC1 and βTC6 cell lines. Cells were transfected with 0.8 μg of the construct for each transcription factor, allowed to grow in high glucose media (25mM glucose) and harvested 48 hours post-transfections. Cells were harvested from at least three replicates and two different experiments for each construct, and pooled to obtain the first strand cDNA. Specific primers and PCR conditions allowed the detection of each ParaHox gene expression. GAPDH was used as an internal control. –RT: transfected cells with the Pdx1 transcription factor, but in which no reverse transcription was performed (negative control of the RT-PCR, as there is no amplification if no cDNA is previously obtained). C: empty vector transfected cells, which also serve as positive control of the RT-PCR, as Pdx1 is endogenously expressed (at low levels in αTC1 cells and at much higher levels in βTC6 cells, as expected).

Mentions: After transfection, the expression levels of each Parahox construct were verified by RT-PCR. Cells from at least three replicates for each construct and cell line were pooled and, total RNA was obtained to synthesise the first strand cDNA with a mixture of poly-d(T) and random hexamers. Subsequently, the cDNA from each Parahox was amplified with specific primers from sequences of different exons (Fig. 1). GAPDH was used as an internal control. As expected, no expression was detected in the reverse transcription negative control (–RT lane, Fig.1), whereas we amplified the endogenously expressed Pdx1 in β-cells transfected with empty-vector (positive control, C lane in Fig.1). Much lower, although detectable, levels of Pdx1 were also observed in αTC1 cells, in agreement with microarray expression reports comparing β- and α-derived cells 28.


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)

Expression of the ParaHox constructs in αTC1 and βTC6 cell lines. Cells were transfected with 0.8 μg of the construct for each transcription factor, allowed to grow in high glucose media (25mM glucose) and harvested 48 hours post-transfections. Cells were harvested from at least three replicates and two different experiments for each construct, and pooled to obtain the first strand cDNA. Specific primers and PCR conditions allowed the detection of each ParaHox gene expression. GAPDH was used as an internal control. –RT: transfected cells with the Pdx1 transcription factor, but in which no reverse transcription was performed (negative control of the RT-PCR, as there is no amplification if no cDNA is previously obtained). C: empty vector transfected cells, which also serve as positive control of the RT-PCR, as Pdx1 is endogenously expressed (at low levels in αTC1 cells and at much higher levels in βTC6 cells, as expected).
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Related In: Results  -  Collection

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

Figure 1: Expression of the ParaHox constructs in αTC1 and βTC6 cell lines. Cells were transfected with 0.8 μg of the construct for each transcription factor, allowed to grow in high glucose media (25mM glucose) and harvested 48 hours post-transfections. Cells were harvested from at least three replicates and two different experiments for each construct, and pooled to obtain the first strand cDNA. Specific primers and PCR conditions allowed the detection of each ParaHox gene expression. GAPDH was used as an internal control. –RT: transfected cells with the Pdx1 transcription factor, but in which no reverse transcription was performed (negative control of the RT-PCR, as there is no amplification if no cDNA is previously obtained). C: empty vector transfected cells, which also serve as positive control of the RT-PCR, as Pdx1 is endogenously expressed (at low levels in αTC1 cells and at much higher levels in βTC6 cells, as expected).
Mentions: After transfection, the expression levels of each Parahox construct were verified by RT-PCR. Cells from at least three replicates for each construct and cell line were pooled and, total RNA was obtained to synthesise the first strand cDNA with a mixture of poly-d(T) and random hexamers. Subsequently, the cDNA from each Parahox was amplified with specific primers from sequences of different exons (Fig. 1). GAPDH was used as an internal control. As expected, no expression was detected in the reverse transcription negative control (–RT lane, Fig.1), whereas we amplified the endogenously expressed Pdx1 in β-cells transfected with empty-vector (positive control, C lane in Fig.1). Much lower, although detectable, levels of Pdx1 were also observed in αTC1 cells, in agreement with microarray expression reports comparing β- and α-derived cells 28.

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