The present study is part of an animal experiment in which mice were fed a selenium-adequate diet meeting the RDA for mice or a diet in which the selenium content was reduced to about 50% [28
]. Although the selenium intake was only moderately reduced, the RNA expression of four selenoproteins, SelW, GPx1, SelH and SelM, was distinctly down-regulated and genes of the Wnt pathway were up-regulated in the colon [28
]. A manual analysis of the microarray data revealed that also Nrf2 target genes and phase II enzymes were regulated by the selenium status; out of 60 genes expressed in the colon, 41 were up-regulated accounting for 68% (Table ). The expression of 12 selected genes for antioxidant defense and phase II enzymes was confirmed by qPCR (Figs. and ). The consistent up-regulation of Nrf2 target genes in marginal selenium-deficiency is highly relevant since a suboptimal selenium supply may also result from a low selenium diet often consumed by humans [45
]. The establishment of a marginal selenium-deficiency is in contrast to most of the other studies investigating the effects of either high selenium supplementation or severe selenium-deficiency.
Also Burk and colleagues, who provided the first direct link between dietary selenium-deficiency and Nrf2 activation, compared mice fed either a completely selenium-deficient or a -supplemented diet with 0.25 mg sodium selenite per kg [9
]. Under selenium-deficient conditions, ARE-driven reporter gene activity as well as GST and NQO1 activity were strongly increased in the liver of wild-type mice but were unchanged in Nrf2−/−
mice. The current study revealed that already a marginal selenium-deficiency can lead to an up-regulation of phase II enzymes underscoring how sensitive organisms are to changes in selenium homeostasis.
As Nrf2 target gene expression was unaffected by a marginal selenium-deficiency in the liver (Table ), but up-regulated in the intestine, it can be concluded that the intestine reacts more sensitively to a reduced selenium supply. In the liver of Nrf2−/−
mice, most of the known Nrf2 target genes were down-regulated, whereas mainly enzymes for detoxification were increased in Keap1 knockdown mice characterized by an increased Nrf2 activation [47
]. The authors conclude that the hepatic activation of Nrf2 is more important for detoxification than for antioxidant defense. In the current study, a distinct up-regulation of enzymes for the antioxidant defense was observed in the duodenum. Since a reduced redox state maintains intestinal epithelial cell proliferation and prevents pre-mature apoptosis [3
], a higher need for enzymes of the antioxidant defense can explain the present findings.
However, also contrary findings have been reported: Detoxifying genes were down-regulated by selenium-deficiency when feeding 0.01 mg selenium/kg diet or 1 mg/kg [44
]. Thus, the selenium content differed by a factor of 100, whereas in the present study the factor was only 2. The conflicting results (reviewed in [13
]) can be explained by the fact that a supranutritional diet can induce phase II enzymes much more efficiently than selenium-deficiency can do. Taking the supranutritional diet as reference for the deficient one, the net effect is a down-regulation in selenium-deficiency.
Underlying mechanisms for the effects of low and high selenium content appear to be different. High concentrations of certain selenium compounds or metabolites may directly activate the Nrf2 pathway by modifying critical thiols in Keap1. Reactive thiol groups can also be modified by oxidation [22
] a situation which prevails in selenium-deficiency [52
]. Whether a higher oxidative status in the moderately selenium-deficient mice was responsible for the induced Nrf2 target gene expression needs to be clarified.
The complete loss of all selenoproteins by the knockout of the gene for the Sec-specific tRNA (Trsp) in hepatocytes enhanced the expression of several phase II enzymes, indicating that at least one selenoprotein normally suppresses the activation of the Nrf2 pathway [50
]. To lower the number of putative candidates, Sengupta and colleagues used a second mouse strain in which Trsp was mutated in a way that only housekeeping but not stress-related selenoproteins were expressed. These mice did not respond with a compensatory up-regulation of phase II enzymes, indicating that a housekeeping selenoprotein must prevent Nrf2 activation [50
]. Since the thioredoxin reductase family belongs to the housekeeping selenoproteins [10
], a liver-specific TrxR1 knockout was analyzed for potential activation of the Nrf2 pathway. Indeed, the TrxR1 knockout resulted in both, the nuclear accumulation of Nrf2 and the induction of 21 Nrf2 target genes [51
]. mRNA levels of TrxR2, TrxR3 or thioredoxin were unaffected. Thus, TrxR1 appears to counteract Nrf2 activation and/or serves as a turn-off signal for Nrf2 activation. However, TrxR1-deficient livers did not show evidence of oxidative stress, leaving the underlying mechanism unclear [51
Selenium also influences the expression of multiple genes coding for non-selenoproteins, which might be involved in the specific activation of the Nrf2 pathway under marginal selenium-deficiency. In the previous study [28
], we found the Wnt pathway activated, indicated by the enhanced expression of β-catenin, Dvl2, Lef1 and c-Myc and the down-regulation of Gsk3β. GSK3β, a multifunctional serine/threonine kinase, is not only involved in glycogen metabolism and canonical Wnt signaling but has also been shown to inactivate the Nrf2 pathway [23
]. Fyn kinase is phosphorylated and thereby activated by GSK3β. Phospho-Fyn, in turn, phosphorylates Nrf2 resulting in the nuclear export of Nrf2 [23
]. The reduced activity of GSK3β, which may be deduced from the down-regulation of Gsk3β under selenium-limiting conditions in the previous study, might lower the amount of active Fyn resulting in nuclear accumulation of Nrf2.
The activation of the Nrf2 pathway in marginal selenium-deficiency is relevant in terms of colorectal cancer development. In a model of inflammation-triggered colorectal carcinogenesis, Nrf2−/−
mice showed an increased incidence, multiplicity and tumor size compared to wild type mice [25
]. On the one hand, phase II enzymes like the GST, SULT and UGT family are involved in the conjugation and excretion of xenobiotic substances. Together with the up-regulation of genes for antioxidant enzymes like peroxiredoxins, sulfiredoxin, Sod1, Hmox1 and metallothionein, the organism is protected from oxidative damage. On the other hand, the Nrf2 activation may also contribute to an increased drug export, to drug resistance and to tumor cell survival [16
]. Therefore, the consequence of an up-regulation of Nrf2 target genes for colorectal cancer in selenium-deficiency can not be easily defined. The activation of the Wnt pathway in selenium-deficiency points to a cancer-promoting function of a low-selenium status and is in accordance with the higher cancer incidence in selenium-deficiency (reviewed in [46
]). Whether the activation of the Nrf2 pathway is the attempt to compensate for the loss of selenoproteins with the strengthening of the endogenous defense system or whether it already contributes to survival of cancer cells is a challenge for further investigations.