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Cyclo(His-Pro) promotes cytoprotection by activating Nrf2-mediated up-regulation of antioxidant defence.

Minelli A, Conte C, Grottelli S, Bellezza I, Cacciatore I, Bolaños JP - J. Cell. Mol. Med. (2008)

Bottom Line: Here, we addressed this issue and found that cyclo(His-Pro) triggered nuclear accumulation of NF-E2-related factor-2 (Nrf2), a transcription factor that up-regulates antioxidant-/electrophile-responsive element (ARE-EpRE)-related genes, in PC12 cells.Furthermore, these effects were abolished by RNA interference-mediated Nrf2 knockdown.These results suggest that the signalling mechanism responsible for the cytoprotective actions of cyclo(His-Pro) would involve p-38 MAPK activation leading to Nrf2-mediated up-regulation of antioxidant cellular defence.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento Medicina Sperimentale Scienze Biochimiche, Università di Perugia, Italy. aminelli@unipg.it <aminelli@unipg.it>

ABSTRACT
Hystidyl-proline [cyclo(His-Pro)] is an endogenous cyclic dipeptide produced by the cleavage of thyrotropin releasing hormone. Previous studies have shown that cyclo(His-Pro) protects against oxidative stress, although the underlying mechanism has remained elusive. Here, we addressed this issue and found that cyclo(His-Pro) triggered nuclear accumulation of NF-E2-related factor-2 (Nrf2), a transcription factor that up-regulates antioxidant-/electrophile-responsive element (ARE-EpRE)-related genes, in PC12 cells. Cyclo(His-Pro) attenuated reactive oxygen species production, and prevented glutathione depletion caused by glutamate, rotenone, paraquat and beta-amyloid treatment. Moreover, real-time PCR analyses revealed that cyclo(His-Pro) induced the expression of a number of ARE-related genes and protected cells against hydrogen peroxide-mediated apoptotic death. Furthermore, these effects were abolished by RNA interference-mediated Nrf2 knockdown. Finally, pharmacological inhibition of p-38 MAPK partially prevented both cyclo(His-Pro)-mediated Nrf2 activation and cellular protection. These results suggest that the signalling mechanism responsible for the cytoprotective actions of cyclo(His-Pro) would involve p-38 MAPK activation leading to Nrf2-mediated up-regulation of antioxidant cellular defence.

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Cyclo(His-Pro) triggers nuclear translocation of Nrf2 leading to changes in the antioxidant gene expression pattern in PC12 cells. Cells were incubated with 50 μM cyclo(His-Pro) for 24 hrs prior to treatment with 100 μM H2O2. (A) Nrf2 protein in cytosol (white bars) and nuclear (grey bars) fractions. The nuclear and cytosolic extracts containing 40 /ag of proteins were subjected to Western blotting analysis with the indicated antibodies. Anti-α-tubulin and anti-lamin B antibodies were used as markers for the cytosolic and nuclear extracts, respectively. Quantification of the scanned band intensities revealed a ∼2-fold increase in Nrf2 intensity in the nuclear fraction (lower panel). (B) Real-time PCR analyses of the samples revealing changes in the expression of several genes, whose values were normalized to β-actin expression, and presented as 2−ΔΔ. Relative mRNA abundance of each gene in untreated cells was assumed to be 1.0 (control). Results are given as mean ± S.D. values for n= 3 independent experiments. *P< 0.05 versus control; #P< 0.05 versus H2O2-treated cells.
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fig03: Cyclo(His-Pro) triggers nuclear translocation of Nrf2 leading to changes in the antioxidant gene expression pattern in PC12 cells. Cells were incubated with 50 μM cyclo(His-Pro) for 24 hrs prior to treatment with 100 μM H2O2. (A) Nrf2 protein in cytosol (white bars) and nuclear (grey bars) fractions. The nuclear and cytosolic extracts containing 40 /ag of proteins were subjected to Western blotting analysis with the indicated antibodies. Anti-α-tubulin and anti-lamin B antibodies were used as markers for the cytosolic and nuclear extracts, respectively. Quantification of the scanned band intensities revealed a ∼2-fold increase in Nrf2 intensity in the nuclear fraction (lower panel). (B) Real-time PCR analyses of the samples revealing changes in the expression of several genes, whose values were normalized to β-actin expression, and presented as 2−ΔΔ. Relative mRNA abundance of each gene in untreated cells was assumed to be 1.0 (control). Results are given as mean ± S.D. values for n= 3 independent experiments. *P< 0.05 versus control; #P< 0.05 versus H2O2-treated cells.

Mentions: To investigate the molecular mechanism of the protection exerted by cyclo(His-Pro), we focused on Nrf2, a transcription factor responsible for ARE-mediated gene expression. As shown in Figure 3A, H2O2 triggered a slight, not significant increase in nuclear levels of Nrf2 protein, whereas cyclo(His-Pro) promoted a significant increase in Nrf2. Cyclo(His-Pro) alone had no effect when compared with PC12 cells maintained in standard conditions (data not shown). Real-time PCR analyses showed that the treatment of the cells with cyclo(His-Pro) enhanced mRNA expression of Nrf2-regulated genes (Fig. 3B).


Cyclo(His-Pro) promotes cytoprotection by activating Nrf2-mediated up-regulation of antioxidant defence.

Minelli A, Conte C, Grottelli S, Bellezza I, Cacciatore I, Bolaños JP - J. Cell. Mol. Med. (2008)

Cyclo(His-Pro) triggers nuclear translocation of Nrf2 leading to changes in the antioxidant gene expression pattern in PC12 cells. Cells were incubated with 50 μM cyclo(His-Pro) for 24 hrs prior to treatment with 100 μM H2O2. (A) Nrf2 protein in cytosol (white bars) and nuclear (grey bars) fractions. The nuclear and cytosolic extracts containing 40 /ag of proteins were subjected to Western blotting analysis with the indicated antibodies. Anti-α-tubulin and anti-lamin B antibodies were used as markers for the cytosolic and nuclear extracts, respectively. Quantification of the scanned band intensities revealed a ∼2-fold increase in Nrf2 intensity in the nuclear fraction (lower panel). (B) Real-time PCR analyses of the samples revealing changes in the expression of several genes, whose values were normalized to β-actin expression, and presented as 2−ΔΔ. Relative mRNA abundance of each gene in untreated cells was assumed to be 1.0 (control). Results are given as mean ± S.D. values for n= 3 independent experiments. *P< 0.05 versus control; #P< 0.05 versus H2O2-treated cells.
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Related In: Results  -  Collection

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fig03: Cyclo(His-Pro) triggers nuclear translocation of Nrf2 leading to changes in the antioxidant gene expression pattern in PC12 cells. Cells were incubated with 50 μM cyclo(His-Pro) for 24 hrs prior to treatment with 100 μM H2O2. (A) Nrf2 protein in cytosol (white bars) and nuclear (grey bars) fractions. The nuclear and cytosolic extracts containing 40 /ag of proteins were subjected to Western blotting analysis with the indicated antibodies. Anti-α-tubulin and anti-lamin B antibodies were used as markers for the cytosolic and nuclear extracts, respectively. Quantification of the scanned band intensities revealed a ∼2-fold increase in Nrf2 intensity in the nuclear fraction (lower panel). (B) Real-time PCR analyses of the samples revealing changes in the expression of several genes, whose values were normalized to β-actin expression, and presented as 2−ΔΔ. Relative mRNA abundance of each gene in untreated cells was assumed to be 1.0 (control). Results are given as mean ± S.D. values for n= 3 independent experiments. *P< 0.05 versus control; #P< 0.05 versus H2O2-treated cells.
Mentions: To investigate the molecular mechanism of the protection exerted by cyclo(His-Pro), we focused on Nrf2, a transcription factor responsible for ARE-mediated gene expression. As shown in Figure 3A, H2O2 triggered a slight, not significant increase in nuclear levels of Nrf2 protein, whereas cyclo(His-Pro) promoted a significant increase in Nrf2. Cyclo(His-Pro) alone had no effect when compared with PC12 cells maintained in standard conditions (data not shown). Real-time PCR analyses showed that the treatment of the cells with cyclo(His-Pro) enhanced mRNA expression of Nrf2-regulated genes (Fig. 3B).

Bottom Line: Here, we addressed this issue and found that cyclo(His-Pro) triggered nuclear accumulation of NF-E2-related factor-2 (Nrf2), a transcription factor that up-regulates antioxidant-/electrophile-responsive element (ARE-EpRE)-related genes, in PC12 cells.Furthermore, these effects were abolished by RNA interference-mediated Nrf2 knockdown.These results suggest that the signalling mechanism responsible for the cytoprotective actions of cyclo(His-Pro) would involve p-38 MAPK activation leading to Nrf2-mediated up-regulation of antioxidant cellular defence.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento Medicina Sperimentale Scienze Biochimiche, Università di Perugia, Italy. aminelli@unipg.it <aminelli@unipg.it>

ABSTRACT
Hystidyl-proline [cyclo(His-Pro)] is an endogenous cyclic dipeptide produced by the cleavage of thyrotropin releasing hormone. Previous studies have shown that cyclo(His-Pro) protects against oxidative stress, although the underlying mechanism has remained elusive. Here, we addressed this issue and found that cyclo(His-Pro) triggered nuclear accumulation of NF-E2-related factor-2 (Nrf2), a transcription factor that up-regulates antioxidant-/electrophile-responsive element (ARE-EpRE)-related genes, in PC12 cells. Cyclo(His-Pro) attenuated reactive oxygen species production, and prevented glutathione depletion caused by glutamate, rotenone, paraquat and beta-amyloid treatment. Moreover, real-time PCR analyses revealed that cyclo(His-Pro) induced the expression of a number of ARE-related genes and protected cells against hydrogen peroxide-mediated apoptotic death. Furthermore, these effects were abolished by RNA interference-mediated Nrf2 knockdown. Finally, pharmacological inhibition of p-38 MAPK partially prevented both cyclo(His-Pro)-mediated Nrf2 activation and cellular protection. These results suggest that the signalling mechanism responsible for the cytoprotective actions of cyclo(His-Pro) would involve p-38 MAPK activation leading to Nrf2-mediated up-regulation of antioxidant cellular defence.

Show MeSH
Related in: MedlinePlus