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The diabetes-linked transcription factor PAX4 promotes {beta}-cell proliferation and survival in rat and human islets.

Brun T, Franklin I, St-Onge L, Biason-Lauber A, Schoenle EJ, Wollheim CB, Gauthier BR - J. Cell Biol. (2004)

Bottom Line: Adenoviral overexpression of Pax4 caused a 3.5-fold increase in beta-cell proliferation with a concomitant 1.9-, 4-, and 5-fold increase in Bcl-xL (antiapoptotic), c-myc, and Id2 mRNA levels, respectively.Accordingly, Pax4 transactivated the Bcl-xL and c-myc promoters, whereas its diabetes-linked mutant was less efficient.We propose that Pax4 is implicated in beta-cell plasticity through the activation of c-myc and potentially protected from apoptosis through Bcl-xL gene expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Physiology and Metabolism, University Medical Center, Geneva, Switzerland. Thierry.brun@medecine.unige.ch

ABSTRACT
The mechanism by which the beta-cell transcription factor Pax4 influences cell function/mass was studied in rat and human islets of Langerhans. Pax4 transcripts were detected in adult rat islets, and levels were induced by the mitogens activin A and betacellulin. Wortmannin suppressed betacellulin-induced Pax4 expression, implicating the phosphatidylinositol 3-kinase signaling pathway. Adenoviral overexpression of Pax4 caused a 3.5-fold increase in beta-cell proliferation with a concomitant 1.9-, 4-, and 5-fold increase in Bcl-xL (antiapoptotic), c-myc, and Id2 mRNA levels, respectively. Accordingly, Pax4 transactivated the Bcl-xL and c-myc promoters, whereas its diabetes-linked mutant was less efficient. Bcl-xL activity resulted in altered mitochondrial calcium levels and ATP production, explaining impaired glucose-induced insulin secretion in transduced islets. Infection of human islets with an inducible adenoviral Pax4 construct caused proliferation and protection against cytokine-evoked apoptosis, whereas the mutant was less effective. We propose that Pax4 is implicated in beta-cell plasticity through the activation of c-myc and potentially protected from apoptosis through Bcl-xL gene expression.

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Analysis of the expression and function of Pax4 wt and its mutant R129W. (A) Immunofluorescent detection of the myc-tagged Pax4 or synaptotagmin VII proteins (red) and DAPI nuclei staining (blue) in INS-1E cells 48 h after transfection with the indicated constructs. Pax4 and synaptotagmin VII were detected via the myc epitope in the nuclei and cytoplasm of INS-1E cells, respectively. (B) EMSA using the G3 element and the recombinant proteins Pax4-myc wt (lanes 1 and 2) and Pax4-myc R129W (lanes 3 and 4). An equal amount of protein was applied in each lane (see Fig. 4 C). Pax4 wt bound to the G3 element (lane 1), whereas the binding of the R129W mutant was less efficient (lane 3). The asterisk delineates the formation of a supershift complex due to the addition of anti-myc epitope antibody (lanes 2 and 4). (C) Western blotting of the recombinant proteins Pax4-myc wt and R129W using an anti-myc epitope antibody. (D) Effects of Pax4-myc wt (▪) and its mutant R129W (□) on the human c-myc and murine Bcl-xL promoters. Cotransfection studies using BHK-21 cells were performed with increasing amounts of wt and R129W Pax4. The telomerase promoter construct (▴) was used as a negative control. The pSV-β-galactosidase control vector was used as internal control to normalize for transfection efficiency (∼15%). Data are presented as fold induction of basal luciferase activity and expressed as the mean ± SEM of four to five independent experiments. *, P < 0.05, for comparison between Pax4 wt and R129W for each of the promoter constructs. Bar, 50 μM.
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fig4: Analysis of the expression and function of Pax4 wt and its mutant R129W. (A) Immunofluorescent detection of the myc-tagged Pax4 or synaptotagmin VII proteins (red) and DAPI nuclei staining (blue) in INS-1E cells 48 h after transfection with the indicated constructs. Pax4 and synaptotagmin VII were detected via the myc epitope in the nuclei and cytoplasm of INS-1E cells, respectively. (B) EMSA using the G3 element and the recombinant proteins Pax4-myc wt (lanes 1 and 2) and Pax4-myc R129W (lanes 3 and 4). An equal amount of protein was applied in each lane (see Fig. 4 C). Pax4 wt bound to the G3 element (lane 1), whereas the binding of the R129W mutant was less efficient (lane 3). The asterisk delineates the formation of a supershift complex due to the addition of anti-myc epitope antibody (lanes 2 and 4). (C) Western blotting of the recombinant proteins Pax4-myc wt and R129W using an anti-myc epitope antibody. (D) Effects of Pax4-myc wt (▪) and its mutant R129W (□) on the human c-myc and murine Bcl-xL promoters. Cotransfection studies using BHK-21 cells were performed with increasing amounts of wt and R129W Pax4. The telomerase promoter construct (▴) was used as a negative control. The pSV-β-galactosidase control vector was used as internal control to normalize for transfection efficiency (∼15%). Data are presented as fold induction of basal luciferase activity and expressed as the mean ± SEM of four to five independent experiments. *, P < 0.05, for comparison between Pax4 wt and R129W for each of the promoter constructs. Bar, 50 μM.

Mentions: To examine whether or not Pax4 is involved in the regulation of c-myc and Bcl-xL transcription, transient transfection assays were performed in BHK-21 cells with luciferase reporter constructs harboring either gene promoter along with increasing amounts of Pax4. The impact of the type 2 diabetes–associated Pax4 mutation R129W, located in the paired DNA binding domain, was also evaluated (Shimajiri et al., 2001). We generated two expression vectors containing either a wild type (wt; Pax4-myc wt) or mutant Pax4 (Pax4-myc R129W) cDNA fused to the myc epitope. We first validated expression and localization of the proteins encoded by the two constructs in rat insulinoma INS-1E cells. Immunofluorescence using a myc antibody revealed nuclear localization of Pax4 (wt and mutant) in transfected cells (Fig. 4 A). Transfection with the vesicular protein synaptotagmin VII/myc tag resulted in cytoplasmic staining, indicating that the epitope did not interfere with proper compartmentalization (Fig. 4 A, bottom). EMSA using equal amounts of in vitro transcribed and translated recombinant proteins (verified by Western blotting, Fig. 4 C) and the G3 element confirmed the binding activity of the myc-fused wt and mutant Pax4 proteins (Fig. 4 B, lanes 1 and 3). The specificity of the complex was demonstrated by supershift assay using the myc antibody (Fig. 4 B, lanes 2 and 4). Interestingly, the G3 binding affinity of the Pax4-myc R129W protein was much weaker than the Pax4-myc wt. Transient transfections revealed that increasing amounts of the Pax4-myc wt expression vector dose dependently stimulated luciferase activity of the c-myc and Bcl-xL gene promoter constructs reaching up to 3.5- and 2.7-fold, respectively (Fig. 4 D). However, Pax4-myc R129W was less efficient in transactivating both constructs, reaching maximal induction levels of only 2.1- and 1.5-fold for the c-myc and Bcl-xL reporter constructs, respectively (Fig. 4 C). Transactivation was promoter specific because Pax4 was unable to induce the telomerase promoter Tert-Luc (Fig. 4 D). These results indicate that Pax4 regulates c-myc and Bcl-xL transcription, whereas the mutant form is less efficient in stimulating the expression of the two genes.


The diabetes-linked transcription factor PAX4 promotes {beta}-cell proliferation and survival in rat and human islets.

Brun T, Franklin I, St-Onge L, Biason-Lauber A, Schoenle EJ, Wollheim CB, Gauthier BR - J. Cell Biol. (2004)

Analysis of the expression and function of Pax4 wt and its mutant R129W. (A) Immunofluorescent detection of the myc-tagged Pax4 or synaptotagmin VII proteins (red) and DAPI nuclei staining (blue) in INS-1E cells 48 h after transfection with the indicated constructs. Pax4 and synaptotagmin VII were detected via the myc epitope in the nuclei and cytoplasm of INS-1E cells, respectively. (B) EMSA using the G3 element and the recombinant proteins Pax4-myc wt (lanes 1 and 2) and Pax4-myc R129W (lanes 3 and 4). An equal amount of protein was applied in each lane (see Fig. 4 C). Pax4 wt bound to the G3 element (lane 1), whereas the binding of the R129W mutant was less efficient (lane 3). The asterisk delineates the formation of a supershift complex due to the addition of anti-myc epitope antibody (lanes 2 and 4). (C) Western blotting of the recombinant proteins Pax4-myc wt and R129W using an anti-myc epitope antibody. (D) Effects of Pax4-myc wt (▪) and its mutant R129W (□) on the human c-myc and murine Bcl-xL promoters. Cotransfection studies using BHK-21 cells were performed with increasing amounts of wt and R129W Pax4. The telomerase promoter construct (▴) was used as a negative control. The pSV-β-galactosidase control vector was used as internal control to normalize for transfection efficiency (∼15%). Data are presented as fold induction of basal luciferase activity and expressed as the mean ± SEM of four to five independent experiments. *, P < 0.05, for comparison between Pax4 wt and R129W for each of the promoter constructs. Bar, 50 μM.
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Related In: Results  -  Collection

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fig4: Analysis of the expression and function of Pax4 wt and its mutant R129W. (A) Immunofluorescent detection of the myc-tagged Pax4 or synaptotagmin VII proteins (red) and DAPI nuclei staining (blue) in INS-1E cells 48 h after transfection with the indicated constructs. Pax4 and synaptotagmin VII were detected via the myc epitope in the nuclei and cytoplasm of INS-1E cells, respectively. (B) EMSA using the G3 element and the recombinant proteins Pax4-myc wt (lanes 1 and 2) and Pax4-myc R129W (lanes 3 and 4). An equal amount of protein was applied in each lane (see Fig. 4 C). Pax4 wt bound to the G3 element (lane 1), whereas the binding of the R129W mutant was less efficient (lane 3). The asterisk delineates the formation of a supershift complex due to the addition of anti-myc epitope antibody (lanes 2 and 4). (C) Western blotting of the recombinant proteins Pax4-myc wt and R129W using an anti-myc epitope antibody. (D) Effects of Pax4-myc wt (▪) and its mutant R129W (□) on the human c-myc and murine Bcl-xL promoters. Cotransfection studies using BHK-21 cells were performed with increasing amounts of wt and R129W Pax4. The telomerase promoter construct (▴) was used as a negative control. The pSV-β-galactosidase control vector was used as internal control to normalize for transfection efficiency (∼15%). Data are presented as fold induction of basal luciferase activity and expressed as the mean ± SEM of four to five independent experiments. *, P < 0.05, for comparison between Pax4 wt and R129W for each of the promoter constructs. Bar, 50 μM.
Mentions: To examine whether or not Pax4 is involved in the regulation of c-myc and Bcl-xL transcription, transient transfection assays were performed in BHK-21 cells with luciferase reporter constructs harboring either gene promoter along with increasing amounts of Pax4. The impact of the type 2 diabetes–associated Pax4 mutation R129W, located in the paired DNA binding domain, was also evaluated (Shimajiri et al., 2001). We generated two expression vectors containing either a wild type (wt; Pax4-myc wt) or mutant Pax4 (Pax4-myc R129W) cDNA fused to the myc epitope. We first validated expression and localization of the proteins encoded by the two constructs in rat insulinoma INS-1E cells. Immunofluorescence using a myc antibody revealed nuclear localization of Pax4 (wt and mutant) in transfected cells (Fig. 4 A). Transfection with the vesicular protein synaptotagmin VII/myc tag resulted in cytoplasmic staining, indicating that the epitope did not interfere with proper compartmentalization (Fig. 4 A, bottom). EMSA using equal amounts of in vitro transcribed and translated recombinant proteins (verified by Western blotting, Fig. 4 C) and the G3 element confirmed the binding activity of the myc-fused wt and mutant Pax4 proteins (Fig. 4 B, lanes 1 and 3). The specificity of the complex was demonstrated by supershift assay using the myc antibody (Fig. 4 B, lanes 2 and 4). Interestingly, the G3 binding affinity of the Pax4-myc R129W protein was much weaker than the Pax4-myc wt. Transient transfections revealed that increasing amounts of the Pax4-myc wt expression vector dose dependently stimulated luciferase activity of the c-myc and Bcl-xL gene promoter constructs reaching up to 3.5- and 2.7-fold, respectively (Fig. 4 D). However, Pax4-myc R129W was less efficient in transactivating both constructs, reaching maximal induction levels of only 2.1- and 1.5-fold for the c-myc and Bcl-xL reporter constructs, respectively (Fig. 4 C). Transactivation was promoter specific because Pax4 was unable to induce the telomerase promoter Tert-Luc (Fig. 4 D). These results indicate that Pax4 regulates c-myc and Bcl-xL transcription, whereas the mutant form is less efficient in stimulating the expression of the two genes.

Bottom Line: Adenoviral overexpression of Pax4 caused a 3.5-fold increase in beta-cell proliferation with a concomitant 1.9-, 4-, and 5-fold increase in Bcl-xL (antiapoptotic), c-myc, and Id2 mRNA levels, respectively.Accordingly, Pax4 transactivated the Bcl-xL and c-myc promoters, whereas its diabetes-linked mutant was less efficient.We propose that Pax4 is implicated in beta-cell plasticity through the activation of c-myc and potentially protected from apoptosis through Bcl-xL gene expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Physiology and Metabolism, University Medical Center, Geneva, Switzerland. Thierry.brun@medecine.unige.ch

ABSTRACT
The mechanism by which the beta-cell transcription factor Pax4 influences cell function/mass was studied in rat and human islets of Langerhans. Pax4 transcripts were detected in adult rat islets, and levels were induced by the mitogens activin A and betacellulin. Wortmannin suppressed betacellulin-induced Pax4 expression, implicating the phosphatidylinositol 3-kinase signaling pathway. Adenoviral overexpression of Pax4 caused a 3.5-fold increase in beta-cell proliferation with a concomitant 1.9-, 4-, and 5-fold increase in Bcl-xL (antiapoptotic), c-myc, and Id2 mRNA levels, respectively. Accordingly, Pax4 transactivated the Bcl-xL and c-myc promoters, whereas its diabetes-linked mutant was less efficient. Bcl-xL activity resulted in altered mitochondrial calcium levels and ATP production, explaining impaired glucose-induced insulin secretion in transduced islets. Infection of human islets with an inducible adenoviral Pax4 construct caused proliferation and protection against cytokine-evoked apoptosis, whereas the mutant was less effective. We propose that Pax4 is implicated in beta-cell plasticity through the activation of c-myc and potentially protected from apoptosis through Bcl-xL gene expression.

Show MeSH
Related in: MedlinePlus