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Expression of leukemia inhibitory factor in Müller glia cells is regulated by a redox-dependent mRNA stability mechanism.

Agca C, Boldt K, Gubler A, Meneau I, Corpet A, Samardzija M, Stucki M, Ueffing M, Grimm C - BMC Biol. (2015)

Bottom Line: Affinity purification combined with quantitative mass spectrometry identified several proteins that bound to these AREs.Additionally we show that KH-type splicing regulatory protein (KHSRP) was crucial for maintaining basal Lif expression levels in non-stressed Müller cells.Generation of H2O2 by injured photoreceptors may thus enhance stability of Lif mRNA and therefore augment neuroprotective LIF signaling during degenerative conditions in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Lab for Retinal Cell Biology, University of Zurich, Wagistrasse 14, Zurich, 8091, Switzerland. cavitagca@gmail.com.

ABSTRACT

Background: Photoreceptor degeneration is a main hallmark of many blinding diseases making protection of photoreceptors crucial to prevent vision loss. Thus, regulation of endogenous neuroprotective factors may be key for cell survival and attenuation of disease progression. Important neuroprotective factors in the retina include H2O2 generated by injured photoreceptors, and leukemia inhibitory factor (LIF) expressed in Müller glia cells in response to photoreceptor damage.

Results: We present evidence that H2O2 connects to the LIF response by inducing stabilization of Lif transcripts in Müller cells. This process was independent of active gene transcription and p38 MAPK, but relied on AU-rich elements (AREs), which we identified within the highly conserved Lif 3'UTR. Affinity purification combined with quantitative mass spectrometry identified several proteins that bound to these AREs. Among those, interleukin enhancer binding factor 3 (ILF3) was confirmed to participate in the redox-dependent Lif mRNA stabilization. Additionally we show that KH-type splicing regulatory protein (KHSRP) was crucial for maintaining basal Lif expression levels in non-stressed Müller cells.

Conclusions: Our results suggest that H2O2-induced redox signaling increases Lif transcript levels through ILF3 mediated mRNA stabilization. Generation of H2O2 by injured photoreceptors may thus enhance stability of Lif mRNA and therefore augment neuroprotective LIF signaling during degenerative conditions in vivo.

No MeSH data available.


Related in: MedlinePlus

ILF3 regulates redox dependent mRNA stabilization in rMC-1 Müller cells. A) rMC-1 cells were transfected with siRNAs directed against Ilf3, Elavl1 or Hnrnpd, as indicated. Scrambled siRNA was used as control (Ctrl). Medium was changed 48 hours after transfection and then cells were (SD) or were not (C) serum deprived. H2O2 (50 μM) was added to the indicated samples. RNA levels were determined by real-time PCR two hours after treatment and expressed relative to untreated cells (C), which were set to 1 for each knockdown series. N = 4 to 8. B) Top panel: rMC-1 cells were transfected with siRNAs directed against Ilf3, Elavl1 or Hnrnpd, as indicated. Scrambled siRNA was used as control (Ctrl). Medium was changed 48 hours after transfection and respective target RNA levels were determined after two hours by real-time PCR. Bottom panels show protein levels at 48 hours after siRNA transfection. N = 4. C) rMC-1 cells were transfected with siRNAs directed against Ilf3, Elavl1 or Hnrnpd, as indicated. Scrambled siRNA was used as control (Ctrl). Medium was changed 48 hours after transfection and Lif RNA levels were determined after two hours by real-time PCR. To knockdown Ilf3 expression, two different siRNAs (Ilf3 (si1), Ilf3 (si2)) were used to exclude off-target effects. Shown are means ± SEM of N = 4. One-way ANOVA with Dunnett’s posttests was used to compare Lif levels after SD + H2O2 treatments (A) and in untreated knockdowns against control (C). Student’s t-test was used to compare downregulation of target genes (B). (**) P <0.01, (***) P <0.001. ANOVA, analysis of variance; ILF3, interleukin enhancer binding factor 3; SEM, standard error of the mean.
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Fig6: ILF3 regulates redox dependent mRNA stabilization in rMC-1 Müller cells. A) rMC-1 cells were transfected with siRNAs directed against Ilf3, Elavl1 or Hnrnpd, as indicated. Scrambled siRNA was used as control (Ctrl). Medium was changed 48 hours after transfection and then cells were (SD) or were not (C) serum deprived. H2O2 (50 μM) was added to the indicated samples. RNA levels were determined by real-time PCR two hours after treatment and expressed relative to untreated cells (C), which were set to 1 for each knockdown series. N = 4 to 8. B) Top panel: rMC-1 cells were transfected with siRNAs directed against Ilf3, Elavl1 or Hnrnpd, as indicated. Scrambled siRNA was used as control (Ctrl). Medium was changed 48 hours after transfection and respective target RNA levels were determined after two hours by real-time PCR. Bottom panels show protein levels at 48 hours after siRNA transfection. N = 4. C) rMC-1 cells were transfected with siRNAs directed against Ilf3, Elavl1 or Hnrnpd, as indicated. Scrambled siRNA was used as control (Ctrl). Medium was changed 48 hours after transfection and Lif RNA levels were determined after two hours by real-time PCR. To knockdown Ilf3 expression, two different siRNAs (Ilf3 (si1), Ilf3 (si2)) were used to exclude off-target effects. Shown are means ± SEM of N = 4. One-way ANOVA with Dunnett’s posttests was used to compare Lif levels after SD + H2O2 treatments (A) and in untreated knockdowns against control (C). Student’s t-test was used to compare downregulation of target genes (B). (**) P <0.01, (***) P <0.001. ANOVA, analysis of variance; ILF3, interleukin enhancer binding factor 3; SEM, standard error of the mean.

Mentions: To test whether the identified proteins are indeed involved in redox regulation of Lif mRNA stability we silenced their expression in rMC-1 cells and analyzed the effect of H2O2 on Lif mRNA levels. Ilf3 silencing by two different siRNAs significantly impaired Lif mRNA stabilization in H2O2-treated serum-deprived cells by about 50%. Knockdown of Elavl1 or Hnrnpd, however, had no significant effect (Figure 6A). Controls showed that transfection of siRNA decreased expression levels of target mRNAs by 86% (Elavl1), 79% (Ilf3; si1), 52% (Ilf3; si2) and 91% (Hnrnpd), as compared to cells transfected with scrambled siRNA (Ctrl, Figure 6B). Similarly, protein levels of ELAVL1 and ILF3 (even in the case of Ilf3 siRNA2) were severely reduced in cells treated with the respective siRNAs (Figure 6B, lower panels). Silencing of either gene did not significantly alter Lif expression in untreated Müller cells (Figure 6C).Figure 6


Expression of leukemia inhibitory factor in Müller glia cells is regulated by a redox-dependent mRNA stability mechanism.

Agca C, Boldt K, Gubler A, Meneau I, Corpet A, Samardzija M, Stucki M, Ueffing M, Grimm C - BMC Biol. (2015)

ILF3 regulates redox dependent mRNA stabilization in rMC-1 Müller cells. A) rMC-1 cells were transfected with siRNAs directed against Ilf3, Elavl1 or Hnrnpd, as indicated. Scrambled siRNA was used as control (Ctrl). Medium was changed 48 hours after transfection and then cells were (SD) or were not (C) serum deprived. H2O2 (50 μM) was added to the indicated samples. RNA levels were determined by real-time PCR two hours after treatment and expressed relative to untreated cells (C), which were set to 1 for each knockdown series. N = 4 to 8. B) Top panel: rMC-1 cells were transfected with siRNAs directed against Ilf3, Elavl1 or Hnrnpd, as indicated. Scrambled siRNA was used as control (Ctrl). Medium was changed 48 hours after transfection and respective target RNA levels were determined after two hours by real-time PCR. Bottom panels show protein levels at 48 hours after siRNA transfection. N = 4. C) rMC-1 cells were transfected with siRNAs directed against Ilf3, Elavl1 or Hnrnpd, as indicated. Scrambled siRNA was used as control (Ctrl). Medium was changed 48 hours after transfection and Lif RNA levels were determined after two hours by real-time PCR. To knockdown Ilf3 expression, two different siRNAs (Ilf3 (si1), Ilf3 (si2)) were used to exclude off-target effects. Shown are means ± SEM of N = 4. One-way ANOVA with Dunnett’s posttests was used to compare Lif levels after SD + H2O2 treatments (A) and in untreated knockdowns against control (C). Student’s t-test was used to compare downregulation of target genes (B). (**) P <0.01, (***) P <0.001. ANOVA, analysis of variance; ILF3, interleukin enhancer binding factor 3; SEM, standard error of the mean.
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Related In: Results  -  Collection

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Fig6: ILF3 regulates redox dependent mRNA stabilization in rMC-1 Müller cells. A) rMC-1 cells were transfected with siRNAs directed against Ilf3, Elavl1 or Hnrnpd, as indicated. Scrambled siRNA was used as control (Ctrl). Medium was changed 48 hours after transfection and then cells were (SD) or were not (C) serum deprived. H2O2 (50 μM) was added to the indicated samples. RNA levels were determined by real-time PCR two hours after treatment and expressed relative to untreated cells (C), which were set to 1 for each knockdown series. N = 4 to 8. B) Top panel: rMC-1 cells were transfected with siRNAs directed against Ilf3, Elavl1 or Hnrnpd, as indicated. Scrambled siRNA was used as control (Ctrl). Medium was changed 48 hours after transfection and respective target RNA levels were determined after two hours by real-time PCR. Bottom panels show protein levels at 48 hours after siRNA transfection. N = 4. C) rMC-1 cells were transfected with siRNAs directed against Ilf3, Elavl1 or Hnrnpd, as indicated. Scrambled siRNA was used as control (Ctrl). Medium was changed 48 hours after transfection and Lif RNA levels were determined after two hours by real-time PCR. To knockdown Ilf3 expression, two different siRNAs (Ilf3 (si1), Ilf3 (si2)) were used to exclude off-target effects. Shown are means ± SEM of N = 4. One-way ANOVA with Dunnett’s posttests was used to compare Lif levels after SD + H2O2 treatments (A) and in untreated knockdowns against control (C). Student’s t-test was used to compare downregulation of target genes (B). (**) P <0.01, (***) P <0.001. ANOVA, analysis of variance; ILF3, interleukin enhancer binding factor 3; SEM, standard error of the mean.
Mentions: To test whether the identified proteins are indeed involved in redox regulation of Lif mRNA stability we silenced their expression in rMC-1 cells and analyzed the effect of H2O2 on Lif mRNA levels. Ilf3 silencing by two different siRNAs significantly impaired Lif mRNA stabilization in H2O2-treated serum-deprived cells by about 50%. Knockdown of Elavl1 or Hnrnpd, however, had no significant effect (Figure 6A). Controls showed that transfection of siRNA decreased expression levels of target mRNAs by 86% (Elavl1), 79% (Ilf3; si1), 52% (Ilf3; si2) and 91% (Hnrnpd), as compared to cells transfected with scrambled siRNA (Ctrl, Figure 6B). Similarly, protein levels of ELAVL1 and ILF3 (even in the case of Ilf3 siRNA2) were severely reduced in cells treated with the respective siRNAs (Figure 6B, lower panels). Silencing of either gene did not significantly alter Lif expression in untreated Müller cells (Figure 6C).Figure 6

Bottom Line: Affinity purification combined with quantitative mass spectrometry identified several proteins that bound to these AREs.Additionally we show that KH-type splicing regulatory protein (KHSRP) was crucial for maintaining basal Lif expression levels in non-stressed Müller cells.Generation of H2O2 by injured photoreceptors may thus enhance stability of Lif mRNA and therefore augment neuroprotective LIF signaling during degenerative conditions in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Lab for Retinal Cell Biology, University of Zurich, Wagistrasse 14, Zurich, 8091, Switzerland. cavitagca@gmail.com.

ABSTRACT

Background: Photoreceptor degeneration is a main hallmark of many blinding diseases making protection of photoreceptors crucial to prevent vision loss. Thus, regulation of endogenous neuroprotective factors may be key for cell survival and attenuation of disease progression. Important neuroprotective factors in the retina include H2O2 generated by injured photoreceptors, and leukemia inhibitory factor (LIF) expressed in Müller glia cells in response to photoreceptor damage.

Results: We present evidence that H2O2 connects to the LIF response by inducing stabilization of Lif transcripts in Müller cells. This process was independent of active gene transcription and p38 MAPK, but relied on AU-rich elements (AREs), which we identified within the highly conserved Lif 3'UTR. Affinity purification combined with quantitative mass spectrometry identified several proteins that bound to these AREs. Among those, interleukin enhancer binding factor 3 (ILF3) was confirmed to participate in the redox-dependent Lif mRNA stabilization. Additionally we show that KH-type splicing regulatory protein (KHSRP) was crucial for maintaining basal Lif expression levels in non-stressed Müller cells.

Conclusions: Our results suggest that H2O2-induced redox signaling increases Lif transcript levels through ILF3 mediated mRNA stabilization. Generation of H2O2 by injured photoreceptors may thus enhance stability of Lif mRNA and therefore augment neuroprotective LIF signaling during degenerative conditions in vivo.

No MeSH data available.


Related in: MedlinePlus