2.1. The Effect of Alcohol on H2O2 Production and Hepcidin Expression in Catalase−/− and Gpx-1−/− Mice
In order to study the synergistic action of alcohol and hydrogen peroxide (H
2O
2), transgenic mice, lacking the expression of antioxidant enzymes, glutathione peroxidase-1 (gpx-1
−/−) or catalase (catalase
−/−) on both alleles, were fed with plain water (control) or ethanol for 1 week. This feeding protocol did not alter liver histology or serum ALT levels, and the body weights were similar between experimental groups at the beginning and end of the 7 day period, as reported previously [
7,
8]. Both wild-type and transgenic mice consumed around 4 mL of 10% ethanol or 5 mL of plain water per day. The blood alcohol levels in wild-type, catalase
−/− and gpx-1
−/− mice were similar and matched the values we have published previously with 129/Sv strain mice [
8] (
Table 1). 7 day-long alcohol feeding induced a weak but significant increase in CYP2E1 activity, which was similar in wild-type and both knockout mice (
Table 1). These findings suggest that alcohol exposure and/or the deficiency of catalase or gpx-1 does not induce liver injury in our experimental model.
Table 1.
The levels of blood alcohol and liver CYP2E1 enzyme activity in wild-type and knockout mice were measured, as described in the experimental section. (N.D. = not detectable).
Table 1.
The levels of blood alcohol and liver CYP2E1 enzyme activity in wild-type and knockout mice were measured, as described in the experimental section. (N.D. = not detectable).
Mouse identity | Blood alcohol (mg/dL) | CYP2E1 activity (nmole 4-nc/h/mg protein) |
---|
Wild-type water-fed | N.D. | 57 ± 3 |
Wild-type alcohol-fed | 123 ± 16 | 92 ± 3.9 |
Catalase−/− water-fed | N.D. | 61 ± 6 |
Catalase−/− alcohol-fed | 125 ± 13 | 100 ± 2 |
Gpx-1−/− water-fed | N.D. | 62 ± 4 |
Gpx-1−/− alcohol-fed | 127 ± 14 | 100 ± 3 |
The effect of alcohol exposure on H
2O
2 production was determined by 2'-7'-dichlorodihydrofluorescein diacetate (DCFH-DA) assays. For these experiments, hepatocytes, freshly isolated by perfusion from the livers of control and ethanol-fed mice, were incubated with DCFH-DA, as described in Experimental Section. After DCFH-DA enters the cell, it is deacetylated to DCFH by intracellular esterases, and then oxidized by peroxides to highly fluorescent 2'-7'-dichlorodihydrofluorescein (DCF) [
28]. Significantly higher levels of DCF fluorescence were observed in the hepatocytes of untreated catalase
−/− and gpx-1
−/− mice than in untreated wild-type mice (
Figure 1A). Gpx-1
−/− mice exhibited the most prominent hepatic H
2O
2 accumulation. DFC fluorescence in gpx-1
−/− mice was 2-fold higher than wild-type mice (
Figure 1A). Similarly, the level of fluorescence in gpx-1
−/− was also significantly greater than that in catalase
−/− mice (
Figure 1A). In contrast, alcohol exposure elevated hepatic H
2O
2 levels in wild-type and catalase
−/−, but not in gpx-1
−/−, mice compared to their water-fed counterparts (
Figure 1B). Alcohol-induced increase in hepatic H
2O
2 content was observed most significantly in catalase
−/− mice. Untreated or alcohol-fed wild-type and gpx-1
−/− mice exhibited significantly less fluorescence intensity than alcohol-fed catalase
−/− mice (
Figure 1B). Furthermore, the amount of hepatic H
2O
2 in alcohol-fed wild-type mice was similar to that in gpx-1
−/− mice (
Figure 1B).
The combined effect of alcohol and H
2O
2 in the regulation of hepcidin gene expression was determined by real-time PCR, as described in Experimental Section. Alcohol inhibited hepcidin mRNA expression in the livers of wild-type mice (
Figure 2A). The deletion of catalase gene in catalase
−/− transgenic mice did not significantly alter the basal level of hepcidin expression in the liver (
Figure 2A). Similar to wild-type mice, hepcidin expression in the livers of catalase
−/− mice was also significantly inhibited by alcohol (
Figure 2A). Contrary to catalase
−/−, the basal level of hepcidin mRNA expression was decreased by two-fold in gpx-1
−/− mice compared to wild-type mice (
Figure 2A). Alcohol however up-regulated hepcidin gene expression in gpx-1
−/− mice over two-fold compared to that in water-fed gpx-1
−/− mice (
Figure 2B).
Figure 1.
Intracellular H2O2 levels in hepatocytes freshly isolated by perfusion from the livers of untreated wild-type and catalase−/− or gpx-1−/− transgenic mice (A), and mice fed with 10% ethanol (alc.) or plain water (H2O) for 1 week (B) were measured by 2'-7'-dichlorodihydrofluorescein (DCF) fluorescence. DCF fluorescence detected by spectrophotometer in cells incubated with 2'-7'-dichlorodihydrofluorescein diacetate (DCFH-DA) was normalized to that in cells incubated with 0.1% DMSO (control), and expressed as arbitrary fluorescence per 0.5 × 106 hepatocytes. Asterisks indicate statistical significance (p < 0.05).
Figure 1.
Intracellular H2O2 levels in hepatocytes freshly isolated by perfusion from the livers of untreated wild-type and catalase−/− or gpx-1−/− transgenic mice (A), and mice fed with 10% ethanol (alc.) or plain water (H2O) for 1 week (B) were measured by 2'-7'-dichlorodihydrofluorescein (DCF) fluorescence. DCF fluorescence detected by spectrophotometer in cells incubated with 2'-7'-dichlorodihydrofluorescein diacetate (DCFH-DA) was normalized to that in cells incubated with 0.1% DMSO (control), and expressed as arbitrary fluorescence per 0.5 × 106 hepatocytes. Asterisks indicate statistical significance (p < 0.05).
Our knockout mice studies indicated a correlation between H
2O
2 and the inhibition of hepcidin expression in the liver. Namely, H
2O
2 accumulation in the livers of untreated gpx-1
−/− and alcohol-fed wild-type or catalase
−/− mice was accompanied by a significant decrease in hepcidin expression. This inhibition might be concentration-dependent because the level of hepatic H
2O
2 in untreated catalase
−/− mice, which was significantly lower than that in untreated gpx-1
−/−, was not sufficient to inhibit hepcidin. Miura
et al. [
26] and Millonig
et al. [
27] have reported the regulation of hepcidin expression by H
2O
2 in tissue culture cells. Millonig
et al. have shown an induction and inhibition of hepcidin expression by low and high concentrations of H
2O
2, respectively. They suggested the involvement of Stat3 in the stimulation and cytotoxicity in the inhibition of hepcidin expression by H
2O
2 in vitro [
27]. Miura
et al. however have reported a role for elevated histone deacetylase activity in H
2O
2-mediated inhibition of hepatic hepcidin in Huh7 cells [
26]. Although antioxidant defense mechanisms in the liver
in vivo are more elaborate than in cultured hepatoma cells, these studies show a concentration-dependent effect of H
2O
2 on liver hepcidin expression. Given the fact that the intracellular localization of catalase and gpx-1 are not similar, our study also pointed out the spatial dynamics of H
2O
2 in hepcidin regulation. Namely, the regulation of hepcidin expression was different in catalase
−/− and gpx-1
−/− mice. Unlike catalase, which is primarily expressed in peroxisomes, gpx-1 is expressed both in cytosol and mitochondria [
13]. We have previously reported that mitochondrial superoxide is not involved in inhibition of hepcidin expression by alcohol [
29]. Attenuation of hepcidin expression in untreated gpx-1
−/− mice, which displayed a higher H
2O
2 content than untreated catalase
−/− mice, suggested that cytosolic and mitochondrial H
2O
2 might be involved in inhibition of hepcidin expression. However, upon alcohol exposure, gpx-1
−/− mice displayed induction of hepcidin expression without changes in H
2O
2 levels. Nevertheless, our knockout mice studies suggested that both subcellular location and concentration of H
2O
2 in hepatocytes are equally important in differential regulation of hepcidin expression.
Figure 2.
Hepcidin mRNA expression in the liver. cDNA synthesized from liver RNA of transgenic mice, lacking the expression of either glutathione peroxidase-1 (gpx-1−/−) or catalase (catalase−/−), and wild-type mice, fed with plain water (H2O) or ethanol (alc.), was employed to determine hepcidin mRNA expression by Taqman real-time PCR. (A) Hepcidin gene expression in water or alcohol-fed transgenic mice and alcohol-fed wild-type mice was expressed as-fold hepcidin expression of that in wild-type mice fed with water. (B) Hepcidin gene expression in alcohol-fed gpx-1−/− mice was expressed as-fold hepcidin expression of that in gpx-1−/− mice fed with water. Asterisks indicate statistical significance (p < 0.05).
Figure 2.
Hepcidin mRNA expression in the liver. cDNA synthesized from liver RNA of transgenic mice, lacking the expression of either glutathione peroxidase-1 (gpx-1−/−) or catalase (catalase−/−), and wild-type mice, fed with plain water (H2O) or ethanol (alc.), was employed to determine hepcidin mRNA expression by Taqman real-time PCR. (A) Hepcidin gene expression in water or alcohol-fed transgenic mice and alcohol-fed wild-type mice was expressed as-fold hepcidin expression of that in wild-type mice fed with water. (B) Hepcidin gene expression in alcohol-fed gpx-1−/− mice was expressed as-fold hepcidin expression of that in gpx-1−/− mice fed with water. Asterisks indicate statistical significance (p < 0.05).
2.2. The Effect of Alcohol and H2O2 on ER Stress in the Liver
Elevated hepcidin expression in alcohol-fed gpx-1
−/− mice may involve various mechanisms such as inflammation or ER stress, which are known to induce hepcidin expression [
24,
25]. Our previous studies have however shown that alcohol can inhibit liver hepcidin expression in the presence of inflammation [
30,
31]. We have also shown the involvement of alcohol-mediated TLR4 and NF-κB activation in this process [
31]. It is therefore not feasible that the induction of hepcidin in alcohol-treated gpx-1
−/− mice is mediated by inflammation. We then studied the role of ER stress in this process.
Iron-regulatory genes have been reported to be the downstream targets of the transcription factor, CHOP (GADD153), which is involved in ER stress [
24,
32]. In untreated cells, CHOP is expressed at very low levels and its expression is induced by stress [
21]. We therefore determined the expression level of CHOP in the livers of untreated and alcohol-treated wild-type, gpx-1
−/− and catalase
−/− mice by western blotting (
Figure 3A). CHOP protein expression was significantly elevated in the livers of untreated gpx-1
−/−, but not catalase
−/− mice, compared to untreated wild-type mice. Alcohol induced liver CHOP expression strongly in gpx-1
−/−, and only marginally and not significantly in catalase
−/− mice, compared to wild-type mice (
Figure 3A,C). To further examine ER stress, the expression of chaperone protein, GRP78 (BiP) in the livers of untreated and alcohol-treated wild-type, catalase
−/− and gpx-1
−/− mice was detected by western blotting (
Figure 3B). GRP78 protein expression was significantly elevated in the livers of untreated gpx-1, but not catalase, knockout mice, compared to untreated wild-type mice. Alcohol treatment further induced liver GRP78 expression significantly in gpx-1
−/−, but not catalase
−/− or wild-type, mice (
Figure 3B,D).
Figure 3.
Liver CHOP (GADD153) and GRP78 (BiP) protein expression. Total cell lysates isolated from the livers of H2O or ethanol (alc.)-fed wild-type and gpx-1−/− or catalase−/− transgenic mice were employed to detect CHOP (A) or GRP78/BiP (B) protein expression by western blotting. An anti-gapdh antibody was used to demonstrate equal protein loading. CHOP (C) and GRP78 (D) expression was quantified by densitometric analysis and normalized to gapdh expression, respectively. Normalized expression in H2O or alcohol-fed knockout and alcohol-fed wild-type mice was expressed as fold expression of that in H2O-fed wild-type mice. Asterisks indicate statistical significance (p < 0.05).
Figure 3.
Liver CHOP (GADD153) and GRP78 (BiP) protein expression. Total cell lysates isolated from the livers of H2O or ethanol (alc.)-fed wild-type and gpx-1−/− or catalase−/− transgenic mice were employed to detect CHOP (A) or GRP78/BiP (B) protein expression by western blotting. An anti-gapdh antibody was used to demonstrate equal protein loading. CHOP (C) and GRP78 (D) expression was quantified by densitometric analysis and normalized to gapdh expression, respectively. Normalized expression in H2O or alcohol-fed knockout and alcohol-fed wild-type mice was expressed as fold expression of that in H2O-fed wild-type mice. Asterisks indicate statistical significance (p < 0.05).
CHOP acts as a negative regulator of the C/EBP family of transcription factors. We have previously shown that alcohol inhibits C/EBPα in wild-type mice livers [
8]. Since CHOP was induced by alcohol in gpx-1 mice, we determined nuclear C/EBPα expression in gpx-1
−/− mice livers by western blotting. The absence of gpx-1 did not significantly alter the basal expression level of liver C/EBPα protein in gpx-1
−/− mice compared to wild-type mice (
Figure 4A,B). Alcohol inhibited C/EBPα expression to the same extent in gpx-1
−/− and wild-type mice (
Figure 4A,B).
Figure 4.
Liver C/EBPα protein expression. (A) Nuclear cell lysates isolated from the livers of H2O or ethanol (alc.)-fed wild-type and gpx-1−/− transgenic mice were employed to detect C/EBPα protein expression by western blotting. An anti-TATA-binding protein (TBP) antibody was used to demonstrate equal nuclear protein loading. (B) C/EBPα protein (30 kDa and 42 kDa) expression was quantified by densitometric analysis and normalized to TBP expression. Normalized expression in H2O or alcohol-fed gpx-1−/− and alcohol-fed wild-type mice was expressed as fold expression of that in H2O-fed wild-type mice.
Figure 4.
Liver C/EBPα protein expression. (A) Nuclear cell lysates isolated from the livers of H2O or ethanol (alc.)-fed wild-type and gpx-1−/− transgenic mice were employed to detect C/EBPα protein expression by western blotting. An anti-TATA-binding protein (TBP) antibody was used to demonstrate equal nuclear protein loading. (B) C/EBPα protein (30 kDa and 42 kDa) expression was quantified by densitometric analysis and normalized to TBP expression. Normalized expression in H2O or alcohol-fed gpx-1−/− and alcohol-fed wild-type mice was expressed as fold expression of that in H2O-fed wild-type mice.
The up-regulation of CHOP and Grp78 protein expression in the livers of gpx-1, but not catalase, knockout mice, suggest that accumulation of H
2O
2 in cytosol and mitochondria of hepatocytes together with alcohol metabolism can cause ER stress, thereby leading to the induction of hepcidin gene expression. C/EBPα, which is known to be inhibited by CHOP, is involved in the regulation of hepcidin gene expression [
22,
33]. We have previously shown that alcohol suppresses liver hepcidin expression via the inhibition of C/EBPα [
7,
8]. Oliveira
et al. have also suggested that ER-stress-mediated biphasic regulation of hepcidin
in vitro involves CHOP and C/EBPα [
24]. However, our western blot analysis did not establish a correlation between CHOP and C/EBPα protein expression patterns in the livers of alcohol-fed mice. Namely, independent of differences in CHOP expression levels, both wild-type and gpx-1
−/− mice displayed inhibition of nuclear C/EBPα expression upon alcohol exposure. Based on these data and previous studies by us and others, we believe that CHOP does not associate with C/EBPα in alcohol-mediated induction of hepcidin expression in gpx-1 knockout mice (see
Scheme 1 below). Similarly, Miura
et al. have also shown that inhibition of C/EBPα by hepatitis C viral proteins does not involve CHOP [
26]. Our findings nonetheless suggest that ER stress signaling may be involved in induction of liver hepcidin expression by alcohol in gpx-1
−/− mice.
The effect of H
2O
2 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 H
2O
2 did not activate the unfolded protein response transducer, IRE1 in the liver.
Figure 5.
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.
Figure 5.
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.
The transcription factors, ATF4 and ATF6, which are activated by ER stress, have been shown to regulate CHOP expression [
21]. The level of ATF4 and ATF6 mRNA expression in the livers of untreated and alcohol-treated transgenic and wild-type mice was determined by real-time PCR (
Figure 6). The expression of ATF4 in the liver was significantly increased in gpx-1
−/− and was unchanged in catalase
−/− mice, compared to wild-type mice (
Figure 6A). Alcohol however significantly inhibited ATF4 mRNA expression in gpx-1
−/−, but not catalase
−/− or wild-type, mice (
Figure 6A). No significant changes in ATF6 mRNA expression were observed in transgenic mice compared to wild-type mice (
Figure 6B). Similarly, ATF6 mRNA expression was not altered by alcohol treatment in wild-type or transgenic mice (
Figure 6B).
The transcription factor, cyclic AMP-responsive element binding protein H (CREBH) has been shown to be involved in ER stress-mediated regulation of hepcidin transcription. To study the effect of alcohol and/or H
2O
2 on hepcidin gene promoter, chromatin immunoprecipitation (CHIP) assays were performed, as described in Experimental Section. The occupancy of mouse hepcidin gene promoter by CREBH in the livers of untreated and alcohol-fed gpx-1
−/−, catalase
−/− and wild-type mice was determined (
Figure 7). Alcohol and/or H
2O
2 did not stimulate the binding of CREBH to hepcidin gene promoter in the livers of transgenic or wild-type mice (
Figure 7). The amplification of total input DNA was similar in all the samples confirming that different liver chromatins contained equal amounts of DNA (
Figure 7).
Figure 6.
Liver ATF4 and ATF6 mRNA expression. cDNA synthesized from liver RNA of wild-type and catalase−/− or gpx-1−/− transgenic mice, fed with plain water (H2O) or ethanol (alc.), was employed to determine ATF4 (A) and ATF6 (B) mRNA expression by real-time PCR. Gene expression levels in alcohol fed wild-type and untreated or alcohol-fed transgenic mice were expressed as-fold ATF expression of that in wild-type mice fed with water. Asterisks indicate statistical significance (p < 0.05).
Figure 6.
Liver ATF4 and ATF6 mRNA expression. cDNA synthesized from liver RNA of wild-type and catalase−/− or gpx-1−/− transgenic mice, fed with plain water (H2O) or ethanol (alc.), was employed to determine ATF4 (A) and ATF6 (B) mRNA expression by real-time PCR. Gene expression levels in alcohol fed wild-type and untreated or alcohol-fed transgenic mice were expressed as-fold ATF expression of that in wild-type mice fed with water. Asterisks indicate statistical significance (p < 0.05).
Figure 7.
CREBH binding to hepcidin gene promoter. Chromatin isolated from the livers of water (H2O) or ethanol (alc.)-fed wild-type mice and gpx-1−/− or catalase−/− transgenic mice were immunoprecipitated by an anti-CREBH antibody or normal rabbit IgG, as control. The co-immunoprecipitated and total input (control) DNA were used as templates in PCR to amplify a 321 bp mouse hepcidin gene promoter region, which harbors CREBH DNA-binding site, as described in Experimental Section.
Figure 7.
CREBH binding to hepcidin gene promoter. Chromatin isolated from the livers of water (H2O) or ethanol (alc.)-fed wild-type mice and gpx-1−/− or catalase−/− transgenic mice were immunoprecipitated by an anti-CREBH antibody or normal rabbit IgG, as control. The co-immunoprecipitated and total input (control) DNA were used as templates in PCR to amplify a 321 bp mouse hepcidin gene promoter region, which harbors CREBH DNA-binding site, as described in Experimental Section.
The elevation of ER stress is characterized by the activation of three distinct signaling pathways mediated by IRE1, ATF6 and PERK/eIF2α [
34]. However, the lack of XBP1 splicing in the livers of knockout and wild-type mice suggests that the IRE1 arm of ER stress signaling is not activated by alcohol in these mice. Transcription factors, ATF4 and ATF6 are involved in the regulation of CHOP expression [
21]. The expression of ATF4, but not ATF6, was elevated in gpx-1
−/− mice livers. It is therefore feasible that CHOP induction in untreated gpx-1
−/− mice is regulated by ATF4. However, alcohol inhibited ATF4 expression but induced CHOP expression in gpx-1
−/− mice suggesting the presence of other mechanisms. Different stress conditions have been shown to induce variable ATF4 expression [
35]. Furthermore, ATF4 and CHOP are regulated at both the transcriptional and posttranscriptional levels [
35,
36]. The transcription factor, CREBH has also been reported to play a role in hepcidin up-regulation by tunicamycin-mediated ER stress [
25]. The binding of CREBH to hepcidin gene promoter in gpx-1
−/−, catalase
−/− or wild-type mice was however not stimulated by alcohol exposure, as shown by our CHIP assays. Compared to experimental ER stress models, other transcription factors besides CREBH may play a role in the regulation of hepcidin gene expression by ER stress in liver diseases. Future studies will investigate the mechanisms involved in synergistic regulation of CHOP by alcohol and H
2O
2.