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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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Activin A and betacellulin increase Pax4 mRNA levels as well as β-cell proliferation in rat islets. (A) Pax4 is expressed in adult rat islets but not in the liver. Quantitative RT-PCR using RNA purified from freshly isolated islets and hepatocytes were performed on Pax4 and TFAM. Data are presented as relative mRNA abundance levels normalized to the transcript cyclophilin and represent the mean ± SEM of six independent experiments performed in triplicates. A representative agarose gel depicting the amplified Pax4 transcript is shown on the right. The fragment was subcloned and confirmed to be Pax4 by sequencing. White line indicates that intervening lanes have been spliced out. (B) PAX4 mRNA levels in islets treated with increasing doses of activin A, betacellulin, or TGF-β1 as indicated. (C) Islets were incubated with 0.5 nM of betacellulin in the absence or presence of 50 and 100 nM of the PI3-kinase inhibitor wortmannin. Pax4 transcript abundance levels were estimated by quantitative RT-PCR. (D) β-Cell proliferation was measured by BrdU incorporation in islets treated with the indicated growth factors at 0.5 nM. Data represent the mean ± SEM of four independent experiments, comprising more than 900 cells per condition. Statistical significance was tested by t test. *, P < 0.05; **, P < 0.01.
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fig1: Activin A and betacellulin increase Pax4 mRNA levels as well as β-cell proliferation in rat islets. (A) Pax4 is expressed in adult rat islets but not in the liver. Quantitative RT-PCR using RNA purified from freshly isolated islets and hepatocytes were performed on Pax4 and TFAM. Data are presented as relative mRNA abundance levels normalized to the transcript cyclophilin and represent the mean ± SEM of six independent experiments performed in triplicates. A representative agarose gel depicting the amplified Pax4 transcript is shown on the right. The fragment was subcloned and confirmed to be Pax4 by sequencing. White line indicates that intervening lanes have been spliced out. (B) PAX4 mRNA levels in islets treated with increasing doses of activin A, betacellulin, or TGF-β1 as indicated. (C) Islets were incubated with 0.5 nM of betacellulin in the absence or presence of 50 and 100 nM of the PI3-kinase inhibitor wortmannin. Pax4 transcript abundance levels were estimated by quantitative RT-PCR. (D) β-Cell proliferation was measured by BrdU incorporation in islets treated with the indicated growth factors at 0.5 nM. Data represent the mean ± SEM of four independent experiments, comprising more than 900 cells per condition. Statistical significance was tested by t test. *, P < 0.05; **, P < 0.01.

Mentions: Basal mRNA expression levels for Pax4 were established in islets and found to give a relative abundance value of 4.7 when normalized to the housekeeping transcript cyclophilin. In contrast, Pax4 mRNA was barely detectable in rat liver cells. The ubiquitously expressed mitochondrial transcription factor TFAM was found with similar relative abundance of 5 and 6.5 in liver and islets, confirming tissue-specific expression of Pax4 in mature islets (Fig. 1 A). Of note, Pax4 mRNA was 25-fold higher in the insulin-producing INS-1E cell line (unpublished data), which is consistent with elevated expression levels detected in human insulinomas (Miyamoto et al., 2001). The responses of the pax4 gene to activin A (a member of the TGF-β family) and betacellulin (a member of the EGF family) were investigated in rat islets (Demeterco et al., 2000). Treatment of islets for 24 h with a range of concentrations resulted in a dose-dependent increase of Pax4 mRNA levels. Maximal induction was observed with 0.5 nM of activin A or betacellulin that elicited a 4.3- and 4.2-fold increase in Pax4 mRNA, respectively (Fig. 1 B). As in insulinoma cells (Ueda, 2000), the related factor TGF-β1 had no significant effect on Pax4 expression in islets. Of note, insulin mRNA levels were unaffected by both treatments (unpublished data). The main intracellular signaling step of betacellulin via interaction with the EGF receptor is the activation of PI3-kinase. To elucidate whether or not this pathway, which has been shown to promote β-cell replication (Buteau et al., 2003), was also involved in Pax4 activation, islets were incubated with the PI3-kinase inhibitor wortmannin. The inhibitor (100 nM) almost completely abolished betacellulin-induced pax4 gene expression, suggesting that the transcription factor is a downstream target of the PI3-kinase (Fig. 1 C). In parallel, we confirmed the mitogenic effect of activin A and betacellulin by measuring β-cell replication using BrdU incorporation. Both growth factors (at 0.5 nM) increased β-cell proliferation by approximately threefold, whereas TGF-β1–treated islets remained quiescent (Fig. 1 D). Together, these results suggest that stimulation of Pax4 gene expression by activin A and betacellulin coincides with islet proliferation induced by the two mitogens.


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)

Activin A and betacellulin increase Pax4 mRNA levels as well as β-cell proliferation in rat islets. (A) Pax4 is expressed in adult rat islets but not in the liver. Quantitative RT-PCR using RNA purified from freshly isolated islets and hepatocytes were performed on Pax4 and TFAM. Data are presented as relative mRNA abundance levels normalized to the transcript cyclophilin and represent the mean ± SEM of six independent experiments performed in triplicates. A representative agarose gel depicting the amplified Pax4 transcript is shown on the right. The fragment was subcloned and confirmed to be Pax4 by sequencing. White line indicates that intervening lanes have been spliced out. (B) PAX4 mRNA levels in islets treated with increasing doses of activin A, betacellulin, or TGF-β1 as indicated. (C) Islets were incubated with 0.5 nM of betacellulin in the absence or presence of 50 and 100 nM of the PI3-kinase inhibitor wortmannin. Pax4 transcript abundance levels were estimated by quantitative RT-PCR. (D) β-Cell proliferation was measured by BrdU incorporation in islets treated with the indicated growth factors at 0.5 nM. Data represent the mean ± SEM of four independent experiments, comprising more than 900 cells per condition. Statistical significance was tested by t test. *, P < 0.05; **, P < 0.01.
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fig1: Activin A and betacellulin increase Pax4 mRNA levels as well as β-cell proliferation in rat islets. (A) Pax4 is expressed in adult rat islets but not in the liver. Quantitative RT-PCR using RNA purified from freshly isolated islets and hepatocytes were performed on Pax4 and TFAM. Data are presented as relative mRNA abundance levels normalized to the transcript cyclophilin and represent the mean ± SEM of six independent experiments performed in triplicates. A representative agarose gel depicting the amplified Pax4 transcript is shown on the right. The fragment was subcloned and confirmed to be Pax4 by sequencing. White line indicates that intervening lanes have been spliced out. (B) PAX4 mRNA levels in islets treated with increasing doses of activin A, betacellulin, or TGF-β1 as indicated. (C) Islets were incubated with 0.5 nM of betacellulin in the absence or presence of 50 and 100 nM of the PI3-kinase inhibitor wortmannin. Pax4 transcript abundance levels were estimated by quantitative RT-PCR. (D) β-Cell proliferation was measured by BrdU incorporation in islets treated with the indicated growth factors at 0.5 nM. Data represent the mean ± SEM of four independent experiments, comprising more than 900 cells per condition. Statistical significance was tested by t test. *, P < 0.05; **, P < 0.01.
Mentions: Basal mRNA expression levels for Pax4 were established in islets and found to give a relative abundance value of 4.7 when normalized to the housekeeping transcript cyclophilin. In contrast, Pax4 mRNA was barely detectable in rat liver cells. The ubiquitously expressed mitochondrial transcription factor TFAM was found with similar relative abundance of 5 and 6.5 in liver and islets, confirming tissue-specific expression of Pax4 in mature islets (Fig. 1 A). Of note, Pax4 mRNA was 25-fold higher in the insulin-producing INS-1E cell line (unpublished data), which is consistent with elevated expression levels detected in human insulinomas (Miyamoto et al., 2001). The responses of the pax4 gene to activin A (a member of the TGF-β family) and betacellulin (a member of the EGF family) were investigated in rat islets (Demeterco et al., 2000). Treatment of islets for 24 h with a range of concentrations resulted in a dose-dependent increase of Pax4 mRNA levels. Maximal induction was observed with 0.5 nM of activin A or betacellulin that elicited a 4.3- and 4.2-fold increase in Pax4 mRNA, respectively (Fig. 1 B). As in insulinoma cells (Ueda, 2000), the related factor TGF-β1 had no significant effect on Pax4 expression in islets. Of note, insulin mRNA levels were unaffected by both treatments (unpublished data). The main intracellular signaling step of betacellulin via interaction with the EGF receptor is the activation of PI3-kinase. To elucidate whether or not this pathway, which has been shown to promote β-cell replication (Buteau et al., 2003), was also involved in Pax4 activation, islets were incubated with the PI3-kinase inhibitor wortmannin. The inhibitor (100 nM) almost completely abolished betacellulin-induced pax4 gene expression, suggesting that the transcription factor is a downstream target of the PI3-kinase (Fig. 1 C). In parallel, we confirmed the mitogenic effect of activin A and betacellulin by measuring β-cell replication using BrdU incorporation. Both growth factors (at 0.5 nM) increased β-cell proliferation by approximately threefold, whereas TGF-β1–treated islets remained quiescent (Fig. 1 D). Together, these results suggest that stimulation of Pax4 gene expression by activin A and betacellulin coincides with islet proliferation induced by the two mitogens.

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