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The effect of alcohol and hydrogen peroxide on liver hepcidin gene expression in mice lacking antioxidant enzymes, glutathione peroxidase-1 or catalase.

Harrison-Findik DD, Lu S - Biomolecules (2015)

Bottom Line: Gpx-1(-/-) displayed significantly higher hepatic H2O2 levels than catalase(-/-) compared to wild-type mice, as measured by 2'-7'-dichlorodihydrofluorescein diacetate (DCFH-DA).In conclusion, our findings strongly suggest that H2O2 inhibits hepcidin expression in vivo.Synergistic induction of CHOP by alcohol and H2O2, in the absence of gpx-1, stimulates liver hepcidin gene expression by ER stress independent of CREBH.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, Division of Gastroenterology/Hepatology, University of Nebraska Medical Center, Omaha, NE 68198, USA. dharrisonfindik@unmc.edu.

ABSTRACT
This study investigates the regulation of hepcidin, the key iron-regulatory molecule, by alcohol and hydrogen peroxide (H2O2) in glutathione peroxidase-1 (gpx-1(-/-)) and catalase (catalase(-/-)) knockout mice. For alcohol studies, 10% ethanol was administered in the drinking water for 7 days. Gpx-1(-/-) displayed significantly higher hepatic H2O2 levels than catalase(-/-) compared to wild-type mice, as measured by 2'-7'-dichlorodihydrofluorescein diacetate (DCFH-DA). The basal level of liver hepcidin expression was attenuated in gpx-1(-/-) mice. Alcohol increased H2O2 production in catalase(-/-) and wild-type, but not gpx-1(-/-), mice. Hepcidin expression was inhibited in alcohol-fed catalase(-/-) and wild-type mice. In contrast, alcohol elevated hepcidin expression in gpx-1(-/-) mice. Gpx-1(-/-) mice also displayed higher level of basal liver CHOP protein expression than catalase(-/-) mice. Alcohol induced CHOP and to a lesser extent GRP78/BiP expression, but not XBP1 splicing or binding of CREBH to hepcidin gene promoter, in gpx-1(-/-) mice. The up-regulation of hepatic ATF4 mRNA levels, which was observed in gpx-1(-/-) mice, was attenuated by alcohol. In conclusion, our findings strongly suggest that H2O2 inhibits hepcidin expression in vivo. Synergistic induction of CHOP by alcohol and H2O2, in the absence of gpx-1, stimulates liver hepcidin gene expression by ER stress independent of CREBH.

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XBP1 mRNA splicing in the livers of H2O or ethanol (alc.)-fed wild-type and catalase−/− or gpx-1−/− transgenic mice was determined by RT-PCR and Pst1 restriction enzyme digestion. Wild-type mice injected with dextrose as control (Dext.) or tunicamycin (Tunic.) were used as controls. 454 bp, and 291bp and 189 bp amplicons refer to spliced (Pst1-resistant) and unspliced XBP1, respectively.
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biomolecules-05-00793-f005: XBP1 mRNA splicing in the livers of H2O or ethanol (alc.)-fed wild-type and catalase−/− or gpx-1−/− transgenic mice was determined by RT-PCR and Pst1 restriction enzyme digestion. Wild-type mice injected with dextrose as control (Dext.) or tunicamycin (Tunic.) were used as controls. 454 bp, and 291bp and 189 bp amplicons refer to spliced (Pst1-resistant) and unspliced XBP1, respectively.

Mentions: The effect of H2O2 on ER stress was further analyzed by determining the splicing of transcription factor, X-box binding protein 1 (XBP1), as described in Experimental Section (Figure 5). XBP1 is a specific substrate of the endoribonuclease, inositol-requiring enzyme 1 (IRE1). XBP1 mRNA splicing is therefore used as a marker for IRE1 activation. Splicing of XBP1 alters Pst1 recognition sequence present within the IRE1 excision site, and spliced XBP1 becomes resistant to Pst1 digestion. XBP1 was not spliced in the livers of untreated or alcohol-fed catalase−/−, gpx-1−/− and wild-type mice, as confirmed by the presence of Pst1 digested 291 and 189 bp XBP1 DNA fragments (Figure 5A,B). In contrast, XBP1 was spliced in the livers of wild-type mice injected with an ER-inducer, tunicamycin, but not with dextrose (as control), as shown by the presence of a Pst1-resistant 454 bp XBP1 amplicon, and thereby validating our XBP1 splicing assay (Figure 5A,B). These findings show that unlike tunicamycin, alcohol and/or H2O2 did not activate the unfolded protein response transducer, IRE1 in the liver.


The effect of alcohol and hydrogen peroxide on liver hepcidin gene expression in mice lacking antioxidant enzymes, glutathione peroxidase-1 or catalase.

Harrison-Findik DD, Lu S - Biomolecules (2015)

XBP1 mRNA splicing in the livers of H2O or ethanol (alc.)-fed wild-type and catalase−/− or gpx-1−/− transgenic mice was determined by RT-PCR and Pst1 restriction enzyme digestion. Wild-type mice injected with dextrose as control (Dext.) or tunicamycin (Tunic.) were used as controls. 454 bp, and 291bp and 189 bp amplicons refer to spliced (Pst1-resistant) and unspliced XBP1, respectively.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496697&req=5

biomolecules-05-00793-f005: XBP1 mRNA splicing in the livers of H2O or ethanol (alc.)-fed wild-type and catalase−/− or gpx-1−/− transgenic mice was determined by RT-PCR and Pst1 restriction enzyme digestion. Wild-type mice injected with dextrose as control (Dext.) or tunicamycin (Tunic.) were used as controls. 454 bp, and 291bp and 189 bp amplicons refer to spliced (Pst1-resistant) and unspliced XBP1, respectively.
Mentions: The effect of H2O2 on ER stress was further analyzed by determining the splicing of transcription factor, X-box binding protein 1 (XBP1), as described in Experimental Section (Figure 5). XBP1 is a specific substrate of the endoribonuclease, inositol-requiring enzyme 1 (IRE1). XBP1 mRNA splicing is therefore used as a marker for IRE1 activation. Splicing of XBP1 alters Pst1 recognition sequence present within the IRE1 excision site, and spliced XBP1 becomes resistant to Pst1 digestion. XBP1 was not spliced in the livers of untreated or alcohol-fed catalase−/−, gpx-1−/− and wild-type mice, as confirmed by the presence of Pst1 digested 291 and 189 bp XBP1 DNA fragments (Figure 5A,B). In contrast, XBP1 was spliced in the livers of wild-type mice injected with an ER-inducer, tunicamycin, but not with dextrose (as control), as shown by the presence of a Pst1-resistant 454 bp XBP1 amplicon, and thereby validating our XBP1 splicing assay (Figure 5A,B). These findings show that unlike tunicamycin, alcohol and/or H2O2 did not activate the unfolded protein response transducer, IRE1 in the liver.

Bottom Line: Gpx-1(-/-) displayed significantly higher hepatic H2O2 levels than catalase(-/-) compared to wild-type mice, as measured by 2'-7'-dichlorodihydrofluorescein diacetate (DCFH-DA).In conclusion, our findings strongly suggest that H2O2 inhibits hepcidin expression in vivo.Synergistic induction of CHOP by alcohol and H2O2, in the absence of gpx-1, stimulates liver hepcidin gene expression by ER stress independent of CREBH.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, Division of Gastroenterology/Hepatology, University of Nebraska Medical Center, Omaha, NE 68198, USA. dharrisonfindik@unmc.edu.

ABSTRACT
This study investigates the regulation of hepcidin, the key iron-regulatory molecule, by alcohol and hydrogen peroxide (H2O2) in glutathione peroxidase-1 (gpx-1(-/-)) and catalase (catalase(-/-)) knockout mice. For alcohol studies, 10% ethanol was administered in the drinking water for 7 days. Gpx-1(-/-) displayed significantly higher hepatic H2O2 levels than catalase(-/-) compared to wild-type mice, as measured by 2'-7'-dichlorodihydrofluorescein diacetate (DCFH-DA). The basal level of liver hepcidin expression was attenuated in gpx-1(-/-) mice. Alcohol increased H2O2 production in catalase(-/-) and wild-type, but not gpx-1(-/-), mice. Hepcidin expression was inhibited in alcohol-fed catalase(-/-) and wild-type mice. In contrast, alcohol elevated hepcidin expression in gpx-1(-/-) mice. Gpx-1(-/-) mice also displayed higher level of basal liver CHOP protein expression than catalase(-/-) mice. Alcohol induced CHOP and to a lesser extent GRP78/BiP expression, but not XBP1 splicing or binding of CREBH to hepcidin gene promoter, in gpx-1(-/-) mice. The up-regulation of hepatic ATF4 mRNA levels, which was observed in gpx-1(-/-) mice, was attenuated by alcohol. In conclusion, our findings strongly suggest that H2O2 inhibits hepcidin expression in vivo. Synergistic induction of CHOP by alcohol and H2O2, in the absence of gpx-1, stimulates liver hepcidin gene expression by ER stress independent of CREBH.

Show MeSH
Related in: MedlinePlus