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Ectopic expression of the beta-cell specific transcription factor Pdx1 inhibits glucagon gene transcription
B Ritz-Laser1, B R Gauthier, A Estreicher
1Diabetes Unit, University Hospital Geneva, 24, rue Micheli-du-Crest, 1211 Geneva 14, Switzerland. Beate.Laser@medecine.unige.ch
Aims/Hypothesis:
The transcription factor Pdx1 is required for the development and differentiation of all pancreatic cells. Beta-cell specific inactivation of Pdx1 in developing or adult mice leads to an increase in glucagon-expressing cells, suggesting that absence of Pdx1could favour glucagon gene expression by a default mechanism.
Method:
We investigated the inhibitory role of Pdx1 on glucagon gene expression in vitro. The glucagonoma cell line InR1G9 was transduced with a Pdx1-encoding lentiviral vector and insulin and glucagon mRNA levels were analysed by northern blot and real-time PCR. To understand the mechanism by which Pdx1 inhibits glucagon gene expression, we studied its effect on glucagon promoter activity in non-islet cells using transient transfections and gel-shift analysis.
Results:
In glucagonoma cells transduced with a Pdx1-encoding lentiviral vector, insulin gene expression was induced while glucagon mRNA levels were reduced by 50 to 60%. In the heterologous cell line BHK-21, Pdx1 inhibited by 60 to 80% the activation of the alpha-cell specific element G1 conferred by Pax-6 and/or Cdx-2/3. Although Pdx1 could bind three AT-rich motifs within G1, two of which are binding sites for Pax-6 and Cdx-2/3, the affinity of Pdx1 for G1 was much lower as compared to Pax-6. In addition, Pdx1 inhibited Pax-6 mediated activation through G3, to which Pdx1 was unable to bind. Moreover, a mutation impairing DNA binding of Pdx1 had no effect on its inhibition on Cdx-2/3. Since Pdx1 interacts directly with Pax-6 and Cdx-2/3 forming heterodimers, we suggest that Pdx1 inhibits glucagon gene transcription through protein to protein interactions with Pax-6 and Cdx-2/3.
Conclusion/Interpretation:
Cell-specific expression of the glucagon gene can only occur when Pdx1 expression extinguishes from the early alpha cell precursor.
Insights
The transcription factor Pdx1 inhibits glucagon gene expression. Pdx1 achieves this by interacting with Pax-6 and Cdx-2/3, preventing glucagon production in pancreatic alpha cells.
Area of Science:
- Endocrinology
- Molecular Biology
- Developmental Biology
Background:
- The transcription factor Pdx1 is crucial for pancreatic cell development and differentiation.
- Loss of Pdx1 in beta cells increases glucagon-expressing cells, suggesting Pdx1 normally suppresses glucagon.
- Understanding Pdx1's role in regulating glucagon is key to controlling pancreatic cell function.
Purpose of the Study:
- To investigate the inhibitory effect of Pdx1 on glucagon gene expression.
- To elucidate the molecular mechanism by which Pdx1 regulates glucagon transcription.
- To determine if Pdx1 directly binds the glucagon promoter or interacts with other transcription factors.
Main Methods:
- Utilized a glucagonoma cell line (InR1G9) transduced with a Pdx1-expressing lentiviral vector.
- Analyzed insulin and glucagon mRNA levels using northern blot and real-time PCR.
- Investigated Pdx1's effect on glucagon promoter activity in non-islet cells via transient transfections and gel-shift analysis.
Main Results:
- Pdx1 expression in glucagonoma cells induced insulin and reduced glucagon mRNA by 50-60%.
- Pdx1 inhibited the activation of the alpha-cell specific element G1 by Pax-6 and/or Cdx-2/3 in BHK-21 cells.
- Pdx1 inhibited Pax-6 mediated activation through G3, suggesting a mechanism involving protein-protein interactions with Pax-6 and Cdx-2/3.
Conclusions:
- Pdx1 actively suppresses glucagon gene expression.
- Pdx1 inhibits glucagon transcription primarily through protein-protein interactions with transcription factors Pax-6 and Cdx-2/3.
- Extinguishment of Pdx1 expression is necessary for cell-specific glucagon gene expression in early alpha cell precursors.