The beginning of GPX2 and 30 years later.

We published the first paper to characterize GPX2 (aka GSHPx-GI) as a selenoenzyme with glutathione peroxidase activity in 1993. Among the four Se-GPX isozymes, GPX1-4, GPX1 and GPX2 are closely related in terms of structure, substrate specificities, and subcellular localization. What sets them apart are distinct patterns of gene regulation, tissue distribution and response to selenium. While we identified the digestive tract epithelium as the main site of GPX2 expression, later work has shown GPX2 is found more widely in epithelial tissues with concentration of expression in stem cell and proliferative compartments. GPX2 expression is regulated over a wide range of levels by many pathways, including NRF2, WNT, p53, RARE and this often results in attaching undue significance to GPX2 as GPX2 is only a part of a system of hydroperoxidase activities, including GPX1, peroxiredoxins and catalase. These other activities may play equal or greater roles, particularly in cell lines cultured without selenium supplementation and often with very low GPX2 levels. This could be assessed by examining levels of mRNA and protein among these various peroxidases at the outset of studies. As an example, it was found that GPX1 responds to the absence of GPX2 in mouse ileum and colon epithelium with higher expression. As such, both Gpx1 and Gpx2 had to be knocked out in mice to produce ileocolitis. However, we note that the actual role of GPX1 and GPX2 in relation to peroxiredoxin function is unclear. There may be an interdependence that requires only low amounts of GPX1 and/or GPX2 in a supporting role to maintain proper peroxiredoxin function. GPX2 levels may be prognostic for cancer progression in colon, breast, prostate and liver, however, there is no consistent trend for higher or lower levels to be favorable.

Control of doxorubicin-induced, reactive oxygen-related apoptosis by glutathione peroxidase 1 in cardiac fibroblasts.

Reactive oxygen formation plays a mechanistic role in the cardiotoxicity of doxorubicin, a chemotherapeutic agent that remains an important component of treatment programs for breast cancer and hematopoietic malignancies. To examine the role of doxorubicin-induced reactive oxygen species (ROS) in drug-related cardiac apoptosis, murine embryonic fibroblast cell lines were derived from the hearts of glutathione peroxidase 1 (Gpx-1) knockout mice. Cells from homozygous Gpx-1 knockout mice and parental animals were propagated with (Se+) and without (Se-) 100 nM sodium selenite. Activity levels of the peroxide detoxifying selenoprotein glutathione peroxidase (GSHPx) were marginally detectable (<1.6 nmol/min/mg) in fibroblasts from homozygous knockout animals whether or not cells were supplemented with selenium. GSHPx activity in Se- cells from parental murine fibroblasts was also <1.6 nmol/min/mg, whereas GSHPx levels in Se+ parental murine fibroblasts were 12.9 ± 2.7 nmol/min/mg (mean ± SE; P < 0.05). Catalase, superoxide dismutase, glutathione reductase, glutathione S-transferase, glucose 6-phosphate dehydrogenase, and reduced glutathione activities did not differ amongst the four cell lines. Reactive oxygen production increased from 908 ± 122 (arbitrary units) for untreated control cells to 1668 ± 54 following exposure to 1 µM doxorubicin for 24 h in parental fibroblasts not supplemented with selenium (P < 0.03); reactive oxygen formation in doxorubicin-treated parental fibroblasts propagated in selenium was 996 ± 69 (P = not significant compared to untreated control cells). Reactive oxygen levels in homozygous Gpx-1 knockout fibroblasts, irrespective of selenium supplementation status, were increased and equivalent to that in selenium deficient wild type fibroblasts. When cardiac fibroblasts were exposed to doxorubicin (0.05 µM) for 96 h and examined for cell cycle alterations by flow cytometry, and apoptosis by TUNEL assay, marked G2 arrest and TUNEL positivity were observed in knockout fibroblasts in the presence or absence of supplemental selenium, and in parental fibroblasts propagated without selenium. Parental fibroblasts propagated with selenium and exposed to the same concentration of doxorubicin demonstrated modest TUNEL positivity and substantially diminished amounts of low molecular weight DNA. These results were replicated in cardiac fibroblasts exposed to doxorubicin (1-2 µM) for 2 h (to mimic clinical drug dosing schedules) and examined 96 h following initiation of drug exposure. Doxorubicin uptake in cardiac fibroblasts was similar irrespective of the mRNA expression level or activity of GSHPx. These experiments suggest that the intracellular levels of doxorubicin-induced reactive oxygen species (ROS) are modulated by GSHPx and play an important role in doxorubicin-related apoptosis and altered cell cycle progression in murine cardiac fibroblasts.

Serotonin-Exacerbated DSS-Induced Colitis Is Associated with Increase in MMP-3 and MMP-9 Expression in the Mouse Colon.

Background. 5-HT enhances dextran sulfate sodium- (DSS-) induced colitis and is involved in inflammatory bowel disease (IBD). Matrix metalloproteinases (MMPs) play roles in the process of intestinal inflammation. Aims. To examine whether 5-HT induces MMPs expression in mouse colon to enhance DSS-induced colitis. Materials and Methods. C57BL/6J (B6) mice were treated with either low-dose (1.0 mg/kg) or high-dose (2.0 mg/kg) 5-HT by enema, low-dose (1.0%) or high-dose (2.5%) DSS, or combined low-dose (1.0%) DSS and (1.0 mg/kg) 5-HT. Mouse colitis was analyzed. MMPs and tissue inhibitors of MMPs (TIMPs) mRNA were measured by real-time quantitative RT-PCR in mouse colon and in human Caco-2 cells and neutrophils. MMP-3 and MMP-9 protein levels were quantified from immunohistochemistry (IHC) images of mouse colons. Results. 5-HT exacerbated DSS-induced colitis, low-dose 5-HT induces both MMP-3 and MMP-9, and high-dose 5-HT only increased MMP-3 mRNA expression in mouse colon. Mouse colon MMP-3 and MMP-9 protein levels were also elevated by 5-HT treatment. The MMP-2, TIMP-1, and TIMP-2 mRNA levels were increased in the inflamed colon. 5-HT induced MMP-3 and MMP-9 mRNA expression in Caco-2 and human neutrophils, respectively, in vitro. Conclusion. 5-HT induced MMP-3 and MMP-9 expression in mouse colon; these elevated MMPs may contribute to DSS-induced colitis.

Deletion of glutathione peroxidase-2 inhibits azoxymethane-induced colon cancer development.

The selenoprotein glutathione peroxidase-2 (GPx2) appears to have a dual role in carcinogenesis. While it protected mice from colon cancer in a model of inflammation-triggered carcinogenesis (azoxymethane and dextran sodium sulfate treatment), it promoted growth of xenografted tumor cells. Therefore, we analyzed the effect of GPx2 in a mouse model mimicking sporadic colorectal cancer (azoxymethane-treatment only). GPx2-knockout (KO) and wild-type (WT) mice were adjusted to an either marginally deficient (-Se), adequate (+Se), or supranutritional (++Se) selenium status and were treated six times with azoxymethane (AOM) to induce tumor development. In the -Se and ++Se groups, the number of tumors was significantly lower in GPx2-KO than in respective WT mice. On the +Se diet, the number of dysplastic crypts was reduced in GPx2-KO mice. This may be explained by more basal and AOM-induced apoptotic cell death in GPx2-KO mice that eliminates damaged or pre-malignant epithelial cells. In WT dysplastic crypts GPx2 was up-regulated in comparison to normal crypts which might be an attempt to suppress apoptosis. In contrast, in the +Se groups tumor numbers were similar in both genotypes but tumor size was larger in GPx2-KO mice. The latter was associated with an inflammatory and tumor-promoting environment as obvious from infiltrated inflammatory cells in the intestinal mucosa of GPx2-KO mice even without any treatment and characterized as low-grade inflammation. In WT mice the number of tumors tended to be lowest in +Se compared to -Se and ++Se feeding indicating that selenium might delay tumorigenesis only in the adequate status. In conclusion, the role of GPx2 and presumably also of selenium depends on the cancer stage and obviously on the involvement of inflammation.

Glutathione peroxidase-2 and selenium decreased inflammation and tumors in a mouse model of inflammation-associated carcinogenesis whereas sulforaphane effects differed with selenium supply.

Chronic inflammation and selenium deficiency are considered as risk factors for colon cancer. The protective effect of selenium might be mediated by specific selenoproteins, such as glutathione peroxidases (GPx). GPx-1 and -2 double knockout, but not single knockout mice, spontaneously develop ileocolitis and intestinal cancer. Since GPx2 is induced by the chemopreventive sulforaphane (SFN) via the nuclear factor E2-related factor 2 (Nrf2)/Keap1 system, the susceptibility of GPx2-KO and wild-type (WT) mice to azoxymethane and dextran sulfate sodium (AOM/DSS)-induced colon carcinogenesis was tested under different selenium states and SFN applications. WT and GPx2-KO mice were grown on a selenium-poor, -adequate or -supranutritional diet. SFN application started either 1 week before (SFN4) or along with (SFN3) a single AOM application followed by DSS treatment for 1 week. Mice were assessed 3 weeks after AOM for colitis and Nrf2 target gene expression and after 12 weeks for tumorigenesis. NAD(P)H:quinone oxidoreductases, thioredoxin reductases and glutathione-S-transferases were upregulated in the ileum and/or colon by SFN, as was GPx2 in WT mice. Inflammation scores were more severe in GPx2-KO mice and highest in selenium-poor groups. Inflammation was enhanced by SFN4 in both genotypes under selenium restriction but decreased in selenium adequacy. Total tumor numbers were higher in GPx2-KO mice but diminished by increasing selenium in both genotypes. SFN3 reduced inflammation and tumor multiplicity in both Se-adequate genotypes. Tumor size was smaller in Se-poor GPx2-KO mice. It is concluded that GPx2, although supporting tumor growth, inhibits inflammation-mediated tumorigenesis, but the protective effect of selenium does not strictly depend on GPx2 expression. Similarly, SFN requires selenium but not GPx2 for being protective.

A Strong Impact of Genetic Background on Gut Microflora in Mice.

Genetic background affects susceptibility to ileocolitis in mice deficient in two intracellular glutathione peroxidases, GPx1 and GPx2. The C57BL/6 (B6) GPx1/2 double-knockout (DKO) mice have mild ileocolitis, and 129S1/Sv (129) DKO mice have severe inflammation. We used diet to modulate ileocolitis; a casein-based defined diet with AIN76A micronutrients (AIN) attenuates inflammation compared to conventional LabDiets. Because luminal microbiota induce DKO ileocolitis, we assessed bacterial composition with automated ribosomal intergenic-spacer analysis (ARISA) on cecal DNA. We found that mouse strain had the strongest impact on the composition of microbiota than diet and GPx genotypes. In comparing AIN and LabDiet, DKO mice were more resistant to change than the non-DKO or WT mice. However, supplementing yeast and inulin to AIN diet greatly altered microflora profiles in the DKO mice. From 129 DKO strictly, we found overgrowth of Escherichia coli. We conclude that genetic background predisposes mice to colonization of potentially pathogenic E. coli.

Loss of GPx2 increases apoptosis, mitosis, and GPx1 expression in the intestine of mice.

Localization of glutathione peroxidase 2 (GPx2), the gastrointestinal form of GPx's, in the intestinal crypt epithelium points to a specific but so-far unknown function of this particular GPx. Therefore, the consequences of a GPx2 knockout were tested in mice fed a selenium-restricted, Se-adequate, or Se-supplemented diet. An unexpected increase in total GPx activity was found throughout the intestine in selenium-fed GPx2 knockout (KO) animals. Immunohistochemistry revealed a strong increase in GPx1 in the colon and ileum, especially in crypt bases where typically GPx2 is localized. GPx1 mRNA was not enhanced in GPx2 KO, indicating that up-regulation most probably occurs at the translational level. Loss of GPx2 was accompanied by an increase in apoptotic cells at colonic crypt bases, an area essential for the self-renewal of the intestinal epithelium, particularly under selenium restriction. Additionally, mitotic cells increased in the middle parts of the crypts, indicating an extension of the proliferative area. These findings corroborate a role for GPx2 in regulating mucosal homeostasis. In GPx2 KO mice, an increase in GPx1 can only partially compensate for GPx2, even under selenium supplementation, indicating that GPx2 is the major antiapoptotic GPx in the colon. These data explain why spontaneous ileocolitis becomes manifested only if both Gpx2 and Gpx1 are deleted.

Epithelium-specific glutathione peroxidase, Gpx2, is involved in the prevention of intestinal inflammation in selenium-deficient mice.

Mice deficient in 2 intracellular selenium (Se)-dependent glutathione peroxidases (Gpx1 and Gpx2), by genetically disrupting both alleles of the Gpx1 and Gpx2 genes (Gpx1(-/-)Gpx2(-/-)), develop ileocolitis around weaning. However, decreased Gpx activity in Se-depleted wild-type animals does not produce pathology in the gastrointestinal tract. Because a small percentage of Se-sufficient Gpx1(+/-)Gpx2(-/-) mice have mild ileocolitis, we hypothesized that Se-deficient Gpx1(+/-)Gpx2(-/-) mice will develop severe ileocolitis similarly to the Gpx1(-/-)Gpx2(-/-) mice, and even a trace amount of Gpx2 can protect intestinal mucosa against inflammation. To test our hypothesis, we fed mice at various stages of development with either Gpx1(+/)(-)Gpx2(-/-) or Gpx1(-/-)Gpx2(+/)(-) genotypes an Se-deficient diet for 4-5 wk and assessed the symptoms and pathology. Gpx1(+/)(-)Gpx2(-/-) mice that were deprived of Se in utero or at weaning (18-22 d of age), but not as young adults (31-51 d of age), manifested significantly worse pathology than their Se-sufficient counterparts. Both Gpx1 and Gpx2 activities and mRNA levels were significantly depressed in the ileum of Se-deprived mice. In mice deprived in utero, the pathology included acute inflammation with neutrophil and monocyte infiltration particularly in the colon and was externally manifested by perianal alopecia and ulceration. On the other hand, Gpx1(-/-)Gpx2(+/)(-) mice were unaffected by Se deprivation, regardless of the age of onset. The results show that a trace amount of Gpx2 is protective against ileocolitis, and Se-deficient young Gpx1(+/-)Gpx2(-/-) mice will develop pathology and symptoms similar to Se-adequate Gpx1(-/-)Gpx2(-/-) mice.

Role of Se-dependent glutathione peroxidases in gastrointestinal inflammation and cancer.

Increase in reactive oxygen species plays an integral part in the inflammatory response, and chronic inflammation increases cancer risk. Selenium-dependent glutathione peroxidase (GPX) is well recognized for its antioxidant, and thus anti-inflammatory, activity. However, due to the multiple antioxidant families present in the gastrointestinal tract, it has been difficult to demonstrate the importance of individual antioxidant enzymes. Using genetically altered mice deficient in individual Gpx genes has provided insight into the physiological functions of these genes. Insufficient GPX activity in the mucosal epithelium can trigger acute and chronic inflammation. The presence of certain microflora, such as Helicobacter species, may affect cancer risk significantly. However, when damaged cells have progressed into a precancerous status, increased GPX activity may become procarcinogenic, presumably due to inhibition of hydroperoxide-mediated apoptosis. This review summarizes the current view of GPX in inflammation and cancer with emphasis on the GI tract.

Microflora trigger colitis in mice deficient in selenium-dependent glutathione peroxidase and induce Gpx2 gene expression.

Selenium-dependent glutathione peroxidase isoenzymes-1 and -2 are the major glutathione-dependent H2O2-reducing activities in the epithelium of the mid- to lower gastrointestinal tract. The two isoenzymes protect mice against ileocolitis. We have found that luminal microflora are required for colitis to develop in mice deficient in GPX-1 and GPX-2 activity (GPX-DKO). Within 7 days of association with microflora, previously asymptomatic germ-free GPX-DKO mice developed severe acute colitis while their littermates with at least one wild-type Gpx1 or Gpx2 gene remained virtually symptom-free. Microflora also affected Gpx2 gene expression. Gpx2, but not Gpx1, mRNA levels were elevated 4-5 fold in the ileum and colon in conventionally reared or microflora-associated adult mice compared with germ-free mice. Since the gastrointestinal tract microflora undergo major changes 2-3 weeks after birth, from relatively benign to a potentially stressful composition, we examined postnatal Gpx2 gene expression. The jejunal and ileal GPX-2 activity levels were low in two to three week-old mice and increased 5-7 fold during the next two weeks. GPX-2 activity levels were correlated with the mRNA levels. Colon Gpx2 mRNA levels held steady at about 50% of adult levels from 12-21 days of age but were several times higher than ileal levels. Our results suggest that ileal Gpx2 mRNA and GPX-2 activity levels are induced by luminal microflora. This response is consistent with a role for GPX as an anti-inflammatory activity.