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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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Pax4 and its diabetes-linked mutant are induced by doxycycline in a dose-dependent manner in human islets. (A) Islets were coinfected with either Ad-mPax4-myc wt or R129W as described in Materials and methods. Doxycycline-dependent activation of PAX4 wt and mutant was assessed 48 h later by immunohistochemistry; myc epitope (red), insulin (green), and DAPI (blue). Arrows depict Pax4-expressing β-cells. Pax4 was detected in the nuclei of ∼70% of human islet cells cultured in the presence of doxycycline, whereas no basal induction of Pax4 was observed in the absence of doxycycline. Bar, 50 μM. (B) Western blotting of nuclear extracts derived from infected islet cells cultured in the presence of 0 (lanes 1 and 4), 0.5 (lanes 3 and 6), and 1 μg/ml (lanes 2 and 5) of doxycycline. The same myc anti-serum was used for Western blotting and immunofluorescence.
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fig7: Pax4 and its diabetes-linked mutant are induced by doxycycline in a dose-dependent manner in human islets. (A) Islets were coinfected with either Ad-mPax4-myc wt or R129W as described in Materials and methods. Doxycycline-dependent activation of PAX4 wt and mutant was assessed 48 h later by immunohistochemistry; myc epitope (red), insulin (green), and DAPI (blue). Arrows depict Pax4-expressing β-cells. Pax4 was detected in the nuclei of ∼70% of human islet cells cultured in the presence of doxycycline, whereas no basal induction of Pax4 was observed in the absence of doxycycline. Bar, 50 μM. (B) Western blotting of nuclear extracts derived from infected islet cells cultured in the presence of 0 (lanes 1 and 4), 0.5 (lanes 3 and 6), and 1 μg/ml (lanes 2 and 5) of doxycycline. The same myc anti-serum was used for Western blotting and immunofluorescence.

Mentions: Next, we assessed the impact of Pax4 and its mutant variant R129W on human islet proliferation using novel doxycycline inducible recombinant adenoviruses engineered to express these proteins tagged to the myc epitope (Ad-mPax4-myc wt or Ad-mPax4-myc R129W). In the absence of doxycycline, the immunoreactive myc epitope was not detected in transduced islet cells (Fig. 7 A). Addition of 0.5 μg/ml doxycycline resulted in the induction of mPax4-myc wt and R129W in the nuclei of ∼70% of islet cells (Fig. 7 A). Quantitative RT-PCR revealed a 10- and 20-fold increase in Pax4 transcript in islets treated with 0.5 and 1 μg/ml doxycycline, respectively (unpublished data). Similarly, a dose-dependent increase in Pax4 protein levels was detected by Western blotting using the myc epitope antibody (Fig. 7 B). To achieve physiological levels of Pax4, similar to those induced by mitogens, proliferation experiments were performed using the lower concentration of doxycycline (0.5 μg/ml). No visible proliferation was detected in the absence of doxycycline (<1%). Concomitant with induction of mPax4 wt expression, ∼10% of cells incorporated BrdU, whereas only 2% of BrdU-positive cells were detected in islets expressing Pax4 R129W (Fig. 8 A). Interestingly, 7% of cells infected with Ad-mPax4-myc wt were BrdU and insulin positive (as shown in a representative experiment in Fig. 8 B, bottom right arrow), consistent with values obtained in rat islets (Fig. 2 D). However, an additional 3% were only BrdU positive, indicating that other cell types may also be prone to proliferation in the presence of Pax4 (Fig. 8 B, top left arrows). To appraise the potential protective role conferred by Pax4-induced Bcl-xL expression observed in rat islets, transduced human islets were exposed to a mixture of cytokines (2 ng/ml IFN-γ, IL-1β, and TNF-α) to induce apoptosis. Adenoviral-infected islets in the absence of both doxycycline and cytokines exhibited a 1.3-fold increase in apoptosis as compared with control noninfected islets (Fig. 9, A and B). Treatment with cytokines provoked a further 4.4-fold increase in cell death. In contrast, Pax4 wt expression (induced by the two concentrations of doxycycline) conferred complete protection against apoptosis (Fig. 9 A). At the low dose of doxycycline, the R129W mutation showed an attenuate protection against cytokine-induced cell death (8.8% TUNEL-positive cells compared with 4% in Pax4 wt; ANOVA: P < 0.00245; Fig. 9 B). Together, our results suggest that Pax4 promotes human islet cell replication as well as conferring survival potentially through the activation of Bcl-xL. The diabetes-linked mutant form of Pax4 is incapable of reproducing these effects.


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)

Pax4 and its diabetes-linked mutant are induced by doxycycline in a dose-dependent manner in human islets. (A) Islets were coinfected with either Ad-mPax4-myc wt or R129W as described in Materials and methods. Doxycycline-dependent activation of PAX4 wt and mutant was assessed 48 h later by immunohistochemistry; myc epitope (red), insulin (green), and DAPI (blue). Arrows depict Pax4-expressing β-cells. Pax4 was detected in the nuclei of ∼70% of human islet cells cultured in the presence of doxycycline, whereas no basal induction of Pax4 was observed in the absence of doxycycline. Bar, 50 μM. (B) Western blotting of nuclear extracts derived from infected islet cells cultured in the presence of 0 (lanes 1 and 4), 0.5 (lanes 3 and 6), and 1 μg/ml (lanes 2 and 5) of doxycycline. The same myc anti-serum was used for Western blotting and immunofluorescence.
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fig7: Pax4 and its diabetes-linked mutant are induced by doxycycline in a dose-dependent manner in human islets. (A) Islets were coinfected with either Ad-mPax4-myc wt or R129W as described in Materials and methods. Doxycycline-dependent activation of PAX4 wt and mutant was assessed 48 h later by immunohistochemistry; myc epitope (red), insulin (green), and DAPI (blue). Arrows depict Pax4-expressing β-cells. Pax4 was detected in the nuclei of ∼70% of human islet cells cultured in the presence of doxycycline, whereas no basal induction of Pax4 was observed in the absence of doxycycline. Bar, 50 μM. (B) Western blotting of nuclear extracts derived from infected islet cells cultured in the presence of 0 (lanes 1 and 4), 0.5 (lanes 3 and 6), and 1 μg/ml (lanes 2 and 5) of doxycycline. The same myc anti-serum was used for Western blotting and immunofluorescence.
Mentions: Next, we assessed the impact of Pax4 and its mutant variant R129W on human islet proliferation using novel doxycycline inducible recombinant adenoviruses engineered to express these proteins tagged to the myc epitope (Ad-mPax4-myc wt or Ad-mPax4-myc R129W). In the absence of doxycycline, the immunoreactive myc epitope was not detected in transduced islet cells (Fig. 7 A). Addition of 0.5 μg/ml doxycycline resulted in the induction of mPax4-myc wt and R129W in the nuclei of ∼70% of islet cells (Fig. 7 A). Quantitative RT-PCR revealed a 10- and 20-fold increase in Pax4 transcript in islets treated with 0.5 and 1 μg/ml doxycycline, respectively (unpublished data). Similarly, a dose-dependent increase in Pax4 protein levels was detected by Western blotting using the myc epitope antibody (Fig. 7 B). To achieve physiological levels of Pax4, similar to those induced by mitogens, proliferation experiments were performed using the lower concentration of doxycycline (0.5 μg/ml). No visible proliferation was detected in the absence of doxycycline (<1%). Concomitant with induction of mPax4 wt expression, ∼10% of cells incorporated BrdU, whereas only 2% of BrdU-positive cells were detected in islets expressing Pax4 R129W (Fig. 8 A). Interestingly, 7% of cells infected with Ad-mPax4-myc wt were BrdU and insulin positive (as shown in a representative experiment in Fig. 8 B, bottom right arrow), consistent with values obtained in rat islets (Fig. 2 D). However, an additional 3% were only BrdU positive, indicating that other cell types may also be prone to proliferation in the presence of Pax4 (Fig. 8 B, top left arrows). To appraise the potential protective role conferred by Pax4-induced Bcl-xL expression observed in rat islets, transduced human islets were exposed to a mixture of cytokines (2 ng/ml IFN-γ, IL-1β, and TNF-α) to induce apoptosis. Adenoviral-infected islets in the absence of both doxycycline and cytokines exhibited a 1.3-fold increase in apoptosis as compared with control noninfected islets (Fig. 9, A and B). Treatment with cytokines provoked a further 4.4-fold increase in cell death. In contrast, Pax4 wt expression (induced by the two concentrations of doxycycline) conferred complete protection against apoptosis (Fig. 9 A). At the low dose of doxycycline, the R129W mutation showed an attenuate protection against cytokine-induced cell death (8.8% TUNEL-positive cells compared with 4% in Pax4 wt; ANOVA: P < 0.00245; Fig. 9 B). Together, our results suggest that Pax4 promotes human islet cell replication as well as conferring survival potentially through the activation of Bcl-xL. The diabetes-linked mutant form of Pax4 is incapable of reproducing these effects.

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