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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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AdCMVPax4IRESGFP-transduced rat islets express Pax4 and exhibit increased β-cell replication. (A) Immunofluorescent detection of EGFP (green) and insulin (red) as well as DAPI nuclei staining (blue) in dispersed islet cells 48 h after infection with the indicated doses of adenovirus. Pax4 is identified via the reporter cotranslated EGFP in insulin-positive cells (arrows). (B) EMSA using a radiolabeled G3 element of the glucagon gene promoter and full-length mouse Pax4, produced by the coupled TNT system (lanes 1–3), as well as 6 μg of nuclear protein extracts from infected rat islets (lanes 4–8). Infection for 48 h with the indicated amounts of the adenovirus increased Pax4 DNA binding activity to the G3 element in a dose-dependent manner (lanes 5–7). The asterisk delineates the formation of a supershift complex in the presence of anti-Pax4 serum (lanes 2 and 8). White line indicates that intervening lanes have been spliced out. (C) β-Cell proliferation was measured by BrdU incorporation in islets infected either with AdCaLacZ or AdCMVPax4IRESGFP (2.4 × 107 pfu/ml). A representative composite image of an islet immunostained for BrdU (green), insulin (red), and DAPI (blue) is shown. (D) Dispersed β-cells immunostained for both insulin and BrdU were counted under a fluorescent microscope and results are depicted as a percentage of BrdU/insulin-positive cells over the total amount of insulin-positive cells. Data show the mean ± SEM of five independent experiments, each representing more than 1,000 cells per condition. **, P < 0.01. Bars, 50 μM.
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fig2: AdCMVPax4IRESGFP-transduced rat islets express Pax4 and exhibit increased β-cell replication. (A) Immunofluorescent detection of EGFP (green) and insulin (red) as well as DAPI nuclei staining (blue) in dispersed islet cells 48 h after infection with the indicated doses of adenovirus. Pax4 is identified via the reporter cotranslated EGFP in insulin-positive cells (arrows). (B) EMSA using a radiolabeled G3 element of the glucagon gene promoter and full-length mouse Pax4, produced by the coupled TNT system (lanes 1–3), as well as 6 μg of nuclear protein extracts from infected rat islets (lanes 4–8). Infection for 48 h with the indicated amounts of the adenovirus increased Pax4 DNA binding activity to the G3 element in a dose-dependent manner (lanes 5–7). The asterisk delineates the formation of a supershift complex in the presence of anti-Pax4 serum (lanes 2 and 8). White line indicates that intervening lanes have been spliced out. (C) β-Cell proliferation was measured by BrdU incorporation in islets infected either with AdCaLacZ or AdCMVPax4IRESGFP (2.4 × 107 pfu/ml). A representative composite image of an islet immunostained for BrdU (green), insulin (red), and DAPI (blue) is shown. (D) Dispersed β-cells immunostained for both insulin and BrdU were counted under a fluorescent microscope and results are depicted as a percentage of BrdU/insulin-positive cells over the total amount of insulin-positive cells. Data show the mean ± SEM of five independent experiments, each representing more than 1,000 cells per condition. **, P < 0.01. Bars, 50 μM.

Mentions: To evaluate the importance of Pax4 in β-cell replication, isolated islets were infected with a CMV promoter–driven Pax4/GFP-expressing adenovirus (AdCMVPax4IRESGFP) or control adenovirus (AdCAlacZ). Because the antibody against Pax4 is unable to detect the protein by immunohistochemistry or by Western blotting (unpublished data), we monitored its overexpression via the reporter GFP cotranslated from a bi-cistronic transcript. Approximately 25% and 50% of β-cells expressed GFP 48 h after infection with 1 and 2.4 × 107 pfu/ml of AdCMVPax4IRESGFP, respectively (Fig. 2 A). Pax4 transcript was estimated to reach levels 22 ± fivefold higher (n = 3) than those found in control AdCALacZ-infected islets (unpublished data). Like mitogen-stimulated islets, insulin mRNA levels (Fig. 3 C) were unchanged, indicating that Pax4 overexpression did not alter the phenotypic profile of the β-cell. Production of a functional protein was confirmed by electrophoretic mobility shift assay (EMSA) using a cognate radiolabeled G3 element of the glucagon gene promoter (Ritz-Laser et al., 2002). A single complex previously identified as Pax6 (Ritz-Laser et al., 2002) was observed in nuclear extracts derived from AdCaLacZ-infected islets (Fig. 2 B, lane 4). An additional complex of similar migration pattern to that produced by recombinant Pax4 was generated in islets infected with increasing amounts of AdCMVPax4IRESGFP (Fig. 2 B, lanes 1 and 5–7). This complex was supershifted by the Pax4 antiserum, confirming the binding of Pax4 to this site (Fig. 2 B, lane 2 and 8). The capacity of Pax4 to promote islet proliferation was then evaluated by BrdU incorporation (Fig. 2 C). Quantification revealed a 3.5-fold increase in BrdU labeling of β-cells expressing Pax4 as compared with AdCALacZ-transduced islets. In contrast, proliferation was unaffected by overexpression of Pax6 and neurogenin3, confirming the specificity of Pax4-associated β-cell replication (Fig. 2 D). Thus, forced expression of Pax4 specifically induced DNA synthesis in β-cells, recapitulating the effect observed with both activin A and betacellulin.


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)

AdCMVPax4IRESGFP-transduced rat islets express Pax4 and exhibit increased β-cell replication. (A) Immunofluorescent detection of EGFP (green) and insulin (red) as well as DAPI nuclei staining (blue) in dispersed islet cells 48 h after infection with the indicated doses of adenovirus. Pax4 is identified via the reporter cotranslated EGFP in insulin-positive cells (arrows). (B) EMSA using a radiolabeled G3 element of the glucagon gene promoter and full-length mouse Pax4, produced by the coupled TNT system (lanes 1–3), as well as 6 μg of nuclear protein extracts from infected rat islets (lanes 4–8). Infection for 48 h with the indicated amounts of the adenovirus increased Pax4 DNA binding activity to the G3 element in a dose-dependent manner (lanes 5–7). The asterisk delineates the formation of a supershift complex in the presence of anti-Pax4 serum (lanes 2 and 8). White line indicates that intervening lanes have been spliced out. (C) β-Cell proliferation was measured by BrdU incorporation in islets infected either with AdCaLacZ or AdCMVPax4IRESGFP (2.4 × 107 pfu/ml). A representative composite image of an islet immunostained for BrdU (green), insulin (red), and DAPI (blue) is shown. (D) Dispersed β-cells immunostained for both insulin and BrdU were counted under a fluorescent microscope and results are depicted as a percentage of BrdU/insulin-positive cells over the total amount of insulin-positive cells. Data show the mean ± SEM of five independent experiments, each representing more than 1,000 cells per condition. **, P < 0.01. Bars, 50 μM.
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fig2: AdCMVPax4IRESGFP-transduced rat islets express Pax4 and exhibit increased β-cell replication. (A) Immunofluorescent detection of EGFP (green) and insulin (red) as well as DAPI nuclei staining (blue) in dispersed islet cells 48 h after infection with the indicated doses of adenovirus. Pax4 is identified via the reporter cotranslated EGFP in insulin-positive cells (arrows). (B) EMSA using a radiolabeled G3 element of the glucagon gene promoter and full-length mouse Pax4, produced by the coupled TNT system (lanes 1–3), as well as 6 μg of nuclear protein extracts from infected rat islets (lanes 4–8). Infection for 48 h with the indicated amounts of the adenovirus increased Pax4 DNA binding activity to the G3 element in a dose-dependent manner (lanes 5–7). The asterisk delineates the formation of a supershift complex in the presence of anti-Pax4 serum (lanes 2 and 8). White line indicates that intervening lanes have been spliced out. (C) β-Cell proliferation was measured by BrdU incorporation in islets infected either with AdCaLacZ or AdCMVPax4IRESGFP (2.4 × 107 pfu/ml). A representative composite image of an islet immunostained for BrdU (green), insulin (red), and DAPI (blue) is shown. (D) Dispersed β-cells immunostained for both insulin and BrdU were counted under a fluorescent microscope and results are depicted as a percentage of BrdU/insulin-positive cells over the total amount of insulin-positive cells. Data show the mean ± SEM of five independent experiments, each representing more than 1,000 cells per condition. **, P < 0.01. Bars, 50 μM.
Mentions: To evaluate the importance of Pax4 in β-cell replication, isolated islets were infected with a CMV promoter–driven Pax4/GFP-expressing adenovirus (AdCMVPax4IRESGFP) or control adenovirus (AdCAlacZ). Because the antibody against Pax4 is unable to detect the protein by immunohistochemistry or by Western blotting (unpublished data), we monitored its overexpression via the reporter GFP cotranslated from a bi-cistronic transcript. Approximately 25% and 50% of β-cells expressed GFP 48 h after infection with 1 and 2.4 × 107 pfu/ml of AdCMVPax4IRESGFP, respectively (Fig. 2 A). Pax4 transcript was estimated to reach levels 22 ± fivefold higher (n = 3) than those found in control AdCALacZ-infected islets (unpublished data). Like mitogen-stimulated islets, insulin mRNA levels (Fig. 3 C) were unchanged, indicating that Pax4 overexpression did not alter the phenotypic profile of the β-cell. Production of a functional protein was confirmed by electrophoretic mobility shift assay (EMSA) using a cognate radiolabeled G3 element of the glucagon gene promoter (Ritz-Laser et al., 2002). A single complex previously identified as Pax6 (Ritz-Laser et al., 2002) was observed in nuclear extracts derived from AdCaLacZ-infected islets (Fig. 2 B, lane 4). An additional complex of similar migration pattern to that produced by recombinant Pax4 was generated in islets infected with increasing amounts of AdCMVPax4IRESGFP (Fig. 2 B, lanes 1 and 5–7). This complex was supershifted by the Pax4 antiserum, confirming the binding of Pax4 to this site (Fig. 2 B, lane 2 and 8). The capacity of Pax4 to promote islet proliferation was then evaluated by BrdU incorporation (Fig. 2 C). Quantification revealed a 3.5-fold increase in BrdU labeling of β-cells expressing Pax4 as compared with AdCALacZ-transduced islets. In contrast, proliferation was unaffected by overexpression of Pax6 and neurogenin3, confirming the specificity of Pax4-associated β-cell replication (Fig. 2 D). Thus, forced expression of Pax4 specifically induced DNA synthesis in β-cells, recapitulating the effect observed with both activin A and betacellulin.

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