Limits...
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.

Show MeSH

Related in: MedlinePlus

Time-dependent gene expression profiling of Pax4-overexpressing islets. (A) EMSA using 6 μg of nuclear protein extracts from AdCMVPax4IRESGFP-transduced rat islets cultured in RPMI 1640 medium over a period of 6 d. Pax4 DNA binding activity to the G3 element is maximal 1 d after infection. The asterisk represents the supershifted complex in the presence of anti-Pax4 serum. (B and C) Quantitative RT-PCR analysis performed on RNA isolated from AdCaLacZ (LacZ; •)- and AdCMVPaxIRESGFP (PAX4; ▪)-infected islets (2.4 × 107 pfu/ml, 50% infectibility). Transcript levels were grouped into four categories: proliferative genes comprising c-myc and Id2; apoptotic genes composed of Bcl-xL, Bcl-2, and caspase-3; the transcription factor Pdx-1; and endocrine hormone genes comprising insulin, glucagon, and somatostatin. Expression patterns were measured over a period of 6 d. Each value represents mean ± SEM of three independent experiments. Statistical significance was tested between LacZ- and PAX4-infected islets by unpaired t test. *, P < 0.05; **, P < 0.01.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172618&req=5

fig3: Time-dependent gene expression profiling of Pax4-overexpressing islets. (A) EMSA using 6 μg of nuclear protein extracts from AdCMVPax4IRESGFP-transduced rat islets cultured in RPMI 1640 medium over a period of 6 d. Pax4 DNA binding activity to the G3 element is maximal 1 d after infection. The asterisk represents the supershifted complex in the presence of anti-Pax4 serum. (B and C) Quantitative RT-PCR analysis performed on RNA isolated from AdCaLacZ (LacZ; •)- and AdCMVPaxIRESGFP (PAX4; ▪)-infected islets (2.4 × 107 pfu/ml, 50% infectibility). Transcript levels were grouped into four categories: proliferative genes comprising c-myc and Id2; apoptotic genes composed of Bcl-xL, Bcl-2, and caspase-3; the transcription factor Pdx-1; and endocrine hormone genes comprising insulin, glucagon, and somatostatin. Expression patterns were measured over a period of 6 d. Each value represents mean ± SEM of three independent experiments. Statistical significance was tested between LacZ- and PAX4-infected islets by unpaired t test. *, P < 0.05; **, P < 0.01.

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)

Time-dependent gene expression profiling of Pax4-overexpressing islets. (A) EMSA using 6 μg of nuclear protein extracts from AdCMVPax4IRESGFP-transduced rat islets cultured in RPMI 1640 medium over a period of 6 d. Pax4 DNA binding activity to the G3 element is maximal 1 d after infection. The asterisk represents the supershifted complex in the presence of anti-Pax4 serum. (B and C) Quantitative RT-PCR analysis performed on RNA isolated from AdCaLacZ (LacZ; •)- and AdCMVPaxIRESGFP (PAX4; ▪)-infected islets (2.4 × 107 pfu/ml, 50% infectibility). Transcript levels were grouped into four categories: proliferative genes comprising c-myc and Id2; apoptotic genes composed of Bcl-xL, Bcl-2, and caspase-3; the transcription factor Pdx-1; and endocrine hormone genes comprising insulin, glucagon, and somatostatin. Expression patterns were measured over a period of 6 d. Each value represents mean ± SEM of three independent experiments. Statistical significance was tested between LacZ- and PAX4-infected islets by unpaired t test. *, P < 0.05; **, P < 0.01.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172618&req=5

fig3: Time-dependent gene expression profiling of Pax4-overexpressing islets. (A) EMSA using 6 μg of nuclear protein extracts from AdCMVPax4IRESGFP-transduced rat islets cultured in RPMI 1640 medium over a period of 6 d. Pax4 DNA binding activity to the G3 element is maximal 1 d after infection. The asterisk represents the supershifted complex in the presence of anti-Pax4 serum. (B and C) Quantitative RT-PCR analysis performed on RNA isolated from AdCaLacZ (LacZ; •)- and AdCMVPaxIRESGFP (PAX4; ▪)-infected islets (2.4 × 107 pfu/ml, 50% infectibility). Transcript levels were grouped into four categories: proliferative genes comprising c-myc and Id2; apoptotic genes composed of Bcl-xL, Bcl-2, and caspase-3; the transcription factor Pdx-1; and endocrine hormone genes comprising insulin, glucagon, and somatostatin. Expression patterns were measured over a period of 6 d. Each value represents mean ± SEM of three independent experiments. Statistical significance was tested between LacZ- and PAX4-infected islets by unpaired t test. *, P < 0.05; **, P < 0.01.
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