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The transcription factor C/EBP delta has anti-apoptotic and anti-inflammatory roles in pancreatic beta cells.

Moore F, Santin I, Nogueira TC, Gurzov EN, Marselli L, Marchetti P, Eizirik DL - PLoS ONE (2012)

Bottom Line: Most known cytokine-induced transcription factors have pro-apoptotic effects, and little is known regarding "protective" transcription factors.Interfering with C/EBPδ and CHOP or C/EBPδ and BIM in double knockdown approaches abrogated the exacerbating effects of C/EBPδ deficiency on cytokine-induced β-cell apoptosis, while C/EBPδ overexpression inhibited BIM expression and partially protected β-cells against IL-1β+IFN-γ-induced apoptosis.These observations identify a novel function of C/EBPδ as a modulatory transcription factor that inhibits the pro-apoptotic and pro-inflammatory gene networks activated by cytokines in pancreatic β-cells.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Experimental Medicine, Université Libre de Bruxelles, Brussels, Belgium. fmoore@ulb.ac.be

ABSTRACT
In the course of Type 1 diabetes pro-inflammatory cytokines (e.g., IL-1β, IFN-γ and TNF-α) produced by islet-infiltrating immune cells modify expression of key gene networks in β-cells, leading to local inflammation and β-cell apoptosis. Most known cytokine-induced transcription factors have pro-apoptotic effects, and little is known regarding "protective" transcription factors. To this end, we presently evaluated the role of the transcription factor CCAAT/enhancer binding protein delta (C/EBPδ) on β-cell apoptosis and production of inflammatory mediators in the rat insulinoma INS-1E cells, in purified primary rat β-cells and in human islets. C/EBPδ is expressed and up-regulated in response to the cytokines IL-1β and IFN-γ in rat β-cells and human islets. Small interfering RNA-mediated C/EBPδ silencing exacerbated IL-1β+IFN-γ-induced caspase 9 and 3 cleavage and apoptosis in these cells. C/EBPδ deficiency increased the up-regulation of the transcription factor CHOP in response to cytokines, enhancing expression of the pro-apoptotic Bcl-2 family member BIM. Interfering with C/EBPδ and CHOP or C/EBPδ and BIM in double knockdown approaches abrogated the exacerbating effects of C/EBPδ deficiency on cytokine-induced β-cell apoptosis, while C/EBPδ overexpression inhibited BIM expression and partially protected β-cells against IL-1β+IFN-γ-induced apoptosis. Furthermore, C/EBPδ silencing boosted cytokine-induced production of the chemokines CXCL1, 9, 10 and CCL20 in β-cells by hampering IRF-1 up-regulation and increasing STAT1 activation in response to cytokines. These observations identify a novel function of C/EBPδ as a modulatory transcription factor that inhibits the pro-apoptotic and pro-inflammatory gene networks activated by cytokines in pancreatic β-cells.

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C/EBPδ overexpression decreases BIM expression and partially protect INS-1E cells against cytokine-induced apoptosis.INS-1E cells were left untransfected (grey bars) or transfected with pCMV-Ctrl (white bars) or pCMV-C/EBPδ (black bars) and subsequently left untreated or treated with IL-1β+IFN-γ for 16 or 24 h as indicated. (A) BIM, C/EBPδ and α-tubulin expressions were evaluated by Western blot. (B) Mean optical density measurements of BIM Western blots corrected for α-tubulin (representative figure in A). (C–D) BIM and CHOP mRNA expressions were assayed by RT-PCR and normalized for the housekeeping gene GAPDH. (E) Cells were co-tranfected with the vectors as decribed above and with a BIM promoter reporter+pRL-CMV and subsequently left untreated or exposed to cytokines as indicated. Results are mean Relative Luciferase Unit (R.L.U.) ± SEM; (F) Apoptosis was assessed by HO/PI staining; (G) Cell viability was evaluated using the neutral red-based toxicology kit. Results are mean ± SEM of 4–6 experiments; **: p<0.01 and ***: p<0.001 vs untreated untransfected or transfected with the same plasmid vector; §: p<0.05, §§: p<0.01 and §§§: p<0.001 vs pCMV-Ctrl treated with cytokines at the same time point; ANOVA followed by Student's t test with Bonferroni correction.
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pone-0031062-g007: C/EBPδ overexpression decreases BIM expression and partially protect INS-1E cells against cytokine-induced apoptosis.INS-1E cells were left untransfected (grey bars) or transfected with pCMV-Ctrl (white bars) or pCMV-C/EBPδ (black bars) and subsequently left untreated or treated with IL-1β+IFN-γ for 16 or 24 h as indicated. (A) BIM, C/EBPδ and α-tubulin expressions were evaluated by Western blot. (B) Mean optical density measurements of BIM Western blots corrected for α-tubulin (representative figure in A). (C–D) BIM and CHOP mRNA expressions were assayed by RT-PCR and normalized for the housekeeping gene GAPDH. (E) Cells were co-tranfected with the vectors as decribed above and with a BIM promoter reporter+pRL-CMV and subsequently left untreated or exposed to cytokines as indicated. Results are mean Relative Luciferase Unit (R.L.U.) ± SEM; (F) Apoptosis was assessed by HO/PI staining; (G) Cell viability was evaluated using the neutral red-based toxicology kit. Results are mean ± SEM of 4–6 experiments; **: p<0.01 and ***: p<0.001 vs untreated untransfected or transfected with the same plasmid vector; §: p<0.05, §§: p<0.01 and §§§: p<0.001 vs pCMV-Ctrl treated with cytokines at the same time point; ANOVA followed by Student's t test with Bonferroni correction.

Mentions: We next tested whether C/EBPδ overexpression affects BIM expression in INS-1E cells. Interestingly, a strong overexpression of C/EBPδ (Figure 7A) reduced both basal and cytokine-induced BIM expression (Figure 7A & 7B) and also decreased IL-1β+IFN-γ-induced up-regulation of BIM and CHOP mRNA expressions (Figure 7C & 7D). The co-tranfection of the C/EBPδ overexpression vector with the rat BIM promoter also reduced cytokine-induced up-regulation of this promoter as compared to untranfected- and control-transfected counterparts (Figure 7E). Finally, viability experiments demonstrated that C/EBPδ overexpression induced a moderate protection against IL-1β+IFN-γ-induced apoptosis (Figure 7F), and increased the survival of INS-1E cells as compared to control-transfected cells (Figure 7G). Taken together, these experiments support the hypothesis that C/EBPδ inhibits BIM expression and thus partially counteracts cytokine-induced apoptosis.


The transcription factor C/EBP delta has anti-apoptotic and anti-inflammatory roles in pancreatic beta cells.

Moore F, Santin I, Nogueira TC, Gurzov EN, Marselli L, Marchetti P, Eizirik DL - PLoS ONE (2012)

C/EBPδ overexpression decreases BIM expression and partially protect INS-1E cells against cytokine-induced apoptosis.INS-1E cells were left untransfected (grey bars) or transfected with pCMV-Ctrl (white bars) or pCMV-C/EBPδ (black bars) and subsequently left untreated or treated with IL-1β+IFN-γ for 16 or 24 h as indicated. (A) BIM, C/EBPδ and α-tubulin expressions were evaluated by Western blot. (B) Mean optical density measurements of BIM Western blots corrected for α-tubulin (representative figure in A). (C–D) BIM and CHOP mRNA expressions were assayed by RT-PCR and normalized for the housekeeping gene GAPDH. (E) Cells were co-tranfected with the vectors as decribed above and with a BIM promoter reporter+pRL-CMV and subsequently left untreated or exposed to cytokines as indicated. Results are mean Relative Luciferase Unit (R.L.U.) ± SEM; (F) Apoptosis was assessed by HO/PI staining; (G) Cell viability was evaluated using the neutral red-based toxicology kit. Results are mean ± SEM of 4–6 experiments; **: p<0.01 and ***: p<0.001 vs untreated untransfected or transfected with the same plasmid vector; §: p<0.05, §§: p<0.01 and §§§: p<0.001 vs pCMV-Ctrl treated with cytokines at the same time point; ANOVA followed by Student's t test with Bonferroni correction.
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pone-0031062-g007: C/EBPδ overexpression decreases BIM expression and partially protect INS-1E cells against cytokine-induced apoptosis.INS-1E cells were left untransfected (grey bars) or transfected with pCMV-Ctrl (white bars) or pCMV-C/EBPδ (black bars) and subsequently left untreated or treated with IL-1β+IFN-γ for 16 or 24 h as indicated. (A) BIM, C/EBPδ and α-tubulin expressions were evaluated by Western blot. (B) Mean optical density measurements of BIM Western blots corrected for α-tubulin (representative figure in A). (C–D) BIM and CHOP mRNA expressions were assayed by RT-PCR and normalized for the housekeeping gene GAPDH. (E) Cells were co-tranfected with the vectors as decribed above and with a BIM promoter reporter+pRL-CMV and subsequently left untreated or exposed to cytokines as indicated. Results are mean Relative Luciferase Unit (R.L.U.) ± SEM; (F) Apoptosis was assessed by HO/PI staining; (G) Cell viability was evaluated using the neutral red-based toxicology kit. Results are mean ± SEM of 4–6 experiments; **: p<0.01 and ***: p<0.001 vs untreated untransfected or transfected with the same plasmid vector; §: p<0.05, §§: p<0.01 and §§§: p<0.001 vs pCMV-Ctrl treated with cytokines at the same time point; ANOVA followed by Student's t test with Bonferroni correction.
Mentions: We next tested whether C/EBPδ overexpression affects BIM expression in INS-1E cells. Interestingly, a strong overexpression of C/EBPδ (Figure 7A) reduced both basal and cytokine-induced BIM expression (Figure 7A & 7B) and also decreased IL-1β+IFN-γ-induced up-regulation of BIM and CHOP mRNA expressions (Figure 7C & 7D). The co-tranfection of the C/EBPδ overexpression vector with the rat BIM promoter also reduced cytokine-induced up-regulation of this promoter as compared to untranfected- and control-transfected counterparts (Figure 7E). Finally, viability experiments demonstrated that C/EBPδ overexpression induced a moderate protection against IL-1β+IFN-γ-induced apoptosis (Figure 7F), and increased the survival of INS-1E cells as compared to control-transfected cells (Figure 7G). Taken together, these experiments support the hypothesis that C/EBPδ inhibits BIM expression and thus partially counteracts cytokine-induced apoptosis.

Bottom Line: Most known cytokine-induced transcription factors have pro-apoptotic effects, and little is known regarding "protective" transcription factors.Interfering with C/EBPδ and CHOP or C/EBPδ and BIM in double knockdown approaches abrogated the exacerbating effects of C/EBPδ deficiency on cytokine-induced β-cell apoptosis, while C/EBPδ overexpression inhibited BIM expression and partially protected β-cells against IL-1β+IFN-γ-induced apoptosis.These observations identify a novel function of C/EBPδ as a modulatory transcription factor that inhibits the pro-apoptotic and pro-inflammatory gene networks activated by cytokines in pancreatic β-cells.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Experimental Medicine, Université Libre de Bruxelles, Brussels, Belgium. fmoore@ulb.ac.be

ABSTRACT
In the course of Type 1 diabetes pro-inflammatory cytokines (e.g., IL-1β, IFN-γ and TNF-α) produced by islet-infiltrating immune cells modify expression of key gene networks in β-cells, leading to local inflammation and β-cell apoptosis. Most known cytokine-induced transcription factors have pro-apoptotic effects, and little is known regarding "protective" transcription factors. To this end, we presently evaluated the role of the transcription factor CCAAT/enhancer binding protein delta (C/EBPδ) on β-cell apoptosis and production of inflammatory mediators in the rat insulinoma INS-1E cells, in purified primary rat β-cells and in human islets. C/EBPδ is expressed and up-regulated in response to the cytokines IL-1β and IFN-γ in rat β-cells and human islets. Small interfering RNA-mediated C/EBPδ silencing exacerbated IL-1β+IFN-γ-induced caspase 9 and 3 cleavage and apoptosis in these cells. C/EBPδ deficiency increased the up-regulation of the transcription factor CHOP in response to cytokines, enhancing expression of the pro-apoptotic Bcl-2 family member BIM. Interfering with C/EBPδ and CHOP or C/EBPδ and BIM in double knockdown approaches abrogated the exacerbating effects of C/EBPδ deficiency on cytokine-induced β-cell apoptosis, while C/EBPδ overexpression inhibited BIM expression and partially protected β-cells against IL-1β+IFN-γ-induced apoptosis. Furthermore, C/EBPδ silencing boosted cytokine-induced production of the chemokines CXCL1, 9, 10 and CCL20 in β-cells by hampering IRF-1 up-regulation and increasing STAT1 activation in response to cytokines. These observations identify a novel function of C/EBPδ as a modulatory transcription factor that inhibits the pro-apoptotic and pro-inflammatory gene networks activated by cytokines in pancreatic β-cells.

Show MeSH
Related in: MedlinePlus