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Effects of Se-depletion on glutathione peroxidase and selenoprotein W gene expression in the colon
FEBS Letters 579 (2005) 792–796
FEBS 29196
Effects of Se-depletion on glutathione peroxidase and selenoprotein W gene expression in the colon Vasileios Pagmantidisa, Giovanna Bermanob, Stephane Villettea, Iain Broomb, John Arthurc, John Hesketha,* a
School of Cell and Molecular Biosciences, University of Newcastle upon Tyne, NE1 7RU, UK b School of Life Sciences, The Robert Gordon University, Aberdeen AB25 1HG, UK c Rowett Research Institute, Bucksburn, Aberdeen AB21 9SB, UK Received 3 September 2004; revised 22 November 2004; accepted 9 December 2004 Available online 27 December 2004 Edited by Lukas Huber
Abstract Selenium (Se)-containing proteins have important roles in protecting cells from oxidative damage. This work investigated the effects of Se-depletion on the expression of the genes encoding selenoproteins in colonic mucosa from rats fed diets of different Se content and in human intestinal Caco-2 cells grown in Se-adequate or Se-depleted culture medium. Se-depletion produced statistically significant (P < 0.05) falls in glutathione peroxidase (GPX) 1 mRNA (60–83%) and selenoprotein W mRNA (73%) levels, a small but significant fall in GPX4 mRNA (17– 25%) but no significant change in GPX2. The data show that SelW expression in the colon is highly sensitive to Se-depletion. 2004 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Colon; Glutathione peroxidase; Selenium; Selenoprotein; Caco-2; Gene array
1. Introduction The micronutrient selenium (Se) is essential for health [1]. Low intake of Se has been implicated with the increased incidence of disease and Se supplementation lowered both cancer incidence and mortality from cancer [2,3]. Se is present in several proteins, called selenoproteins, as the amino-acid selenocysteine (Se-cys) [4]. Approximately 30 selenoproteins and up to 25 selenoprotein genes have been identified in mammals and they have a variety of biological roles [4]. They include the glutathione peroxidase family of enzymes, all of which are involved in the cellÕs antioxidant systems [5], the thioredoxin reductases, which are involved in the redox regulation of gene expression [6] and a number of recently identified selenoproteins (e.g., SelX, SelN, SelW, and 15 kDa selenoprotein) that are less well characterised [4]. The glutathione peroxidases include cytosolic glutathione peroxidase (GPX1), gastrointestinal glutathione peroxidase (GPX2), plasma glutathione peroxidase (GPX3) and phospholipid hydroperoxide glutathione peroxidase (GPX4). The phenotype of knock-out mice lacking GPX1 and GPX2 includes colon pathology and increased susceptibility to colitis[7], leading to the suggestion that these enzymes are impor-
* Corresponding author. Fax: +44 0 191 222 8684. E-mail address: [email protected] (J. Hesketh).
tant in determining the level of anti-oxidant protection and inflammatory responses in the colon [8]. In addition, supplementation with Se above normal intake reduces mortality from cancer of the colon [2,3], as well as that of lung and prostate. These observations indicate that selenoproteins such as the GPXs are critical for colon function. However, little is known of how selenoprotein gene expression in colonic cells, particularly in vivo, is regulated by Se availability. The aim of the present work was to investigate how Sedeficiency modulates expression of a range of selenoprotein genes in the rat colon and a human gastrointestinal cell line. Since patterns of selenoprotein activity are accompanied by altered patterns of mRNA abundances and both are regulated by Se availability [9,10], our approach was to use a novel ÔselenoproteinÕ macroarray to screen for genes affected by Se depletion and then to extend the studies using Northern hybridisation.
2. Materials and methods 2.1. DNA probes The GPX1, GPX4 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probes were 880 bp EcoRI, 814 bp EcoRI, and 780 bp PstI/XbaI fragments, respectively, as used previously [9]. The human GPX2 probe (from Prof. R. Brigelius-Flohe) was a 663 bp EcoRI fragment [11]. The human GPX1 probe was a 300 bp BamHI/XbaI fragment [12] and the human GPX4 probe (from Prof. K. Yagi, Gifu) was an 874 bp HindIII/XbaI fragment [13]. The SelW probe (from Dr. I. Kim) was a 420 bp BamHI/XhoI fragment corresponding to the mouse brain cDNA [14]. The 18S rRNA probe was a 328 bp fragment corresponding to nucleotides 4852–5179 of the human ribosomal RNA sequence [15] cloned into the SrfI site of PCR-Script AMP vector (Stratagene) and excised using KpnI and SacI. 2.2. RNA extraction and Northern hybridisation Total RNA was extracted by the guanidinium thiocyanate/phenol/ chloroform procedure [16]. Northern blotting was carried out using standard procedures [17] and membranes pre-hybridised for 20 min at 68 C in 10 ml of QuickHyb solution (Stratagene). 25 ng of DNA probes was labelled with [32P]dCTP by random priming, as described previously [9], and hybridisation carried out for 1 h at 68 C. Non-specifically bound probe was removed from membranes by two washes in 2· SSC (1· SSC = 0.15 M NaCl/0.015 M sodium citrate)/0.1% SDS at room temperature for 15 min, followed by one wash in 0.1· SSC/0.1% SDS at 60 C for 30 min. Specific hybridisation was detected using a Canberra Packard Instantimager for quantification (results for each probe were expressed per unit of hybridisation achieved with the 18S rRNA probe to allow correction for any variation between loading of RNA on the gel or transfer to
0014-5793/$30.00 2004 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2004.12.042
V. Pagmantidis et al. / FEBS Letters 579 (2005) 792–796
793
the nylon membrane) and by autoradiography using Fujifilm Super FX at 80 C. After analysis, membranes were stripped by washing in 0.1% SDS for 10 min at 95 C before rehybridisation to other probes.
Table 1 Effects of Se-deficiency on expression of a range of genes detected by macroarray Gene name
Ratio Se/Se+
2.3. cDNA expression arrays 32 P-labelled cDNA probe synthesis from 3.5 lg total RNA and hybridisation to the Atlas Array membranes were performed according to the Atlas cDNA expression Arrays User Manual (BD Biosciences Clontech, Palo Alto, USA). Detection was performed with a phosphorimager (Molecular Dynamics, Phosphorimager Storm 860) using 24 h and 3 days exposure. Analysis of the results was performed using the AtlasImage v2.7 software (BD Biosciences Clontech) with a specific grid corresponding to our custom made ‘‘selenoprotein’’ array: the array was spotted with all available human selenoprotein cDNAs, cDNAs corresponding to genes whose products are associated with Se metabolism or arachidonate metabolism, and a series of standard housekeeping genes (Table 1 and associated legend). To analyse differential gene expression using the Atlas Array hybridisation, intensity differences were normalised by taking into account changes in the housekeeping genes.
GPX1 GPX2 GPX3 GPX4 SelP SBP1 DbpB SPS (SelD) Phospholipase A2 Cu/Zn SOD Mn SOD SOD3 (extracellular) Glutathione-S-transferase A2 Glutathione-S-transferase A4 Interleukin 1; b SelN SelW SelB (elongation factor) tRNA selenocysteine associated protein SelZ f1 TR2 TR3 MHC; class1; C b-Actin GAPDH a-Tubulin Ubiquitin Ribosomal protein L13a Ribosomal protein S9 HPRT Genomic DNA
0.46 1.63 0.67 0.76 0.90 Up 0.94 1.03 0.95 1.29 1.04 1.58 1.50 2.76 3.89 0.76 0.27 Down 1.20 Down 0.73 1.33 Up 1.08 1.11 1.20 1.09 0.90 0.96 1.67 1.19
2.4. Cell culture Stocks of human colon adenocarcinoma cells (Caco-2) were grown at 37 C in DulbeccoÕs modified EagleÕs medium (with 4.5 g/l glucose and Glutamax) supplemented with 10% (v/v) foetal calf serum (Sigma, Poole, UK), 60 lg/ml gentamycin, 1% (v/v) non-essential amino acids and penicillin/streptomycin (100 units/ml and 100 lg/ml, respectively). Cells were transferred to serum-free medium to which was added either insulin (5 lg/ml) and transferrin (5 lg/ml) (selenium-deficient cells), or insulin, transferrin and selenium as sodium selenite (7 ng/ml) (selenium-replete cells) [18]. Medium was changed every 2 days. 2.5. Animals and diets Weanling Male Hooded Lister Rats of the Rowett strain were randomly allocated to groups of six and fed ad libitum on a semi-synthetic diet containing different amounts of Se for 6 weeks. The basal diet [19] contained 8 ng of Se/g (severely Se-deficient) and the other diets contained 33, 63 (Se-deficient) or 111 (Se-adequate) ng of Se/g as sodium selenite. At the end of the 6 weeks, the animals were anaesthetised with isofluorane and blood samples were taken by cardiac puncture. The animals were killed by cervical dislocation and the entire colon dissected out and washed with cold sterile PBS; the colon was then cut open, the mucosa scraped off and then rapidly frozen in liquid nitrogen and stored at 80 C.
3. Results and discussion Caco-2 cells were grown for 2–6 days in either a Se-supplemented or Se-depleted medium and RNA extracted and analysed by Northern hybridisation. Cells grown in the Sedepleted medium showed a 60% decrease in the level of GPX1 mRNA by 2 days (P < 0.05) compared with cells grown in Se-supplemented medium (Fig. 1). This low expression remained after 4 or 6 days in the Se-depleted medium. Addition of selenite to the medium at day 6 led to an increase in GPX1 mRNA abundance such that it returned to control levels by day 8 (Fig. 1). The level of GPX1 mRNA is a consistent marker of Se status in cell and animal experiments [9,10,18,20] and these changes indicate that after 2 days in Se-depleted medium the Caco-2 cells become severely Se deficient, that the effect is not increased significantly over a further 4 days and that the effect can be reversed by readdition of selenite; this reflects closely the changes in GPX1 mRNA in H4 hepatoma cell and FRTL-5 thyroid cells grown in similar media [18,21]. Using a custom-made macroarray and RNA extracted from Caco-2 cells grown for 2 days in Se-deficient or Se-supple-
mRNA abundances were measured in Caco-2 cells after 2 days in Sedeficient or Se-replete conditions. Results are the mean values obtained from two experiments each analysed using duplicate arrays. Results are expressed as a ratio of the Se-depleted cells to Se-adequate cells. GPX1 and SelW mRNA abundances; showed greater than twofold change; which is considered to be significant. When expression levels were close to the limit of detection the ratio of expression is given as up/down and not as a precise value. The following genes were also present on the array but expression was not detected: thioredoxin reductase 1, deiodinase type I, II and III, 15 kDa selenoprotein, selenoprotein M, T, and X, mitochondrial capsule selenoprotein, SECISbinding protein 2, 5-lipoxygenase, arachidonate 15-lipoxygenase types 1 and 2, arachidonate 12-lipoxygenase 5-lipoxygenase activating protein, cyclooxygenases 1 and 2, tumor necrosis factor a and b, LTB4 receptor, thromboxane A2 receptor, prostaglandin F and D2 receptors, glutathione-S-transferase theta 1 and 2, thyroid hormone receptor a and b2, interferon gamma receptor and precursor, metallothionein isoform 1rR, interleukin 1 a and b, interleukin 2, tyrosine 3-monooxygenase. Abbreviations: Sel, selenoprotein; SOD, superoxide dismutase; TR, thioredoxin reductase; MHC, major histocompatibility complex.
mented medium, it was found that Caco-2 cells expressed ten selenoprotein mRNAs (GPX1, GPX2, GPX3, GPX4, TR2, TR3, selenoproteins N, P, W and Z) at a level that was detectable using this technology (Table 1). Expression of other selenoproteins genes on the array (see legend to Table 1) was below the level of detection. After 2 days in Se-depleted medium, only two selenoproteins showed a major change in mRNA abundance compared with Se-supplemented cells (Table 1): GPX1 and Selenoprotein W (SelW), for which the ratio of relative expression was 0.46 and 0.27, respectively. In addition, Se depletion led to increased expression of the non-selenoprotein mRNAs interleukin 1b and glutathione-Stransferase A4, for which the ratio of relative expression was
794
Fig. 1. Effect of Se-depletion on GPX1 mRNA level in Caco-2 cells. Cells were grown for 2–6 days in Se-supplemented or Se-deficient serum-free medium. After 6 days, selenite was given back to the Sedepleted cells. GPX1mRNA and 18S rRNA levels were determined by Northern hybridisation using a Canberra Packard Instantimager for quantification. Data were expressed as percentages of the mean ratio of GPX1 mRNA abundance/18S rRNA abundance obtained for Sereplete cells (day 0). Results are shown as means ± S.E.M. from two independent experiments. Statistical significance was assessed by a Mann–Whitney U test, \P < 0.05.
3.89 and 2.76, respectively. The relative expression of GPX3, GPX4 and SelN in Se-depleted cells (0.67, 0.76, and 0.76, respectively) was less than the twofold change considered to be significant but suggested that there may be a small fall in expression. There was no evidence of changes in relative expression of GPX2, SelP, TR2 and TR3 or of non-selenoprotein antioxidant enzymes such as Mn and Cu/Zn superoxide dismutases. These array data suggest that in Caco-2 cells expression of GPX1, 2 and 4 are differentially affected by Sedepletion and that the major effect of Se-depletion on selenoprotein expression in colonic epithelial cells, as assessed by mRNA abundance, is a dramatic fall in the expression of SelW and GPX1. To confirm these findings, two experiments were carried out. First, mRNA abundances were measured in Se-supplemented and deficient Caco-2 cells by Northern hybridisation. Second, and more importantly, since little was known about the effects of Se-depletion on the colon in vivo, selenoprotein gene expression was studied in rats that were fed diets that provided different levels of dietary Se. Quantification of Northern hybridisation data from cell culture experiments showed that there was no statistical significant difference in GPX2 mRNA levels compared to cells grown in medium supplemented with Se (Fig. 2). However, there was a small (17%) but statistically significant decrease (P < 0.05) in the GPX4 mRNA level in the Se-depleted cells (Fig. 2). This was in contrast with the marked decline in the level of GPX1 mRNA. These data are consistent with earlier observations that Se-supplementation of cells grown in standard medium increases GPX1 mRNA abundance but not that of GPX2 [10]. Rats were fed diets containing either an adequate supply of Se (111 ng Se/g; group D) or three levels of low Se supply ranging from a severely deficient Se supply (8 ng Se/g; group A), to deficient (33 ng Se/g; group B) or marginal supply (63 ng Se/g; group C). Previously, such diets have been found to consistently produce Se-depletion in rats [9,20]. In the present experiments measurements of liver GPX1 mRNA abundance, an
V. Pagmantidis et al. / FEBS Letters 579 (2005) 792–796
Fig. 2. Effect of Se-deficiency on the abundance of glutathione peroxidase 1, 2 and 4 mRNAs in Caco-2 cells. All lanes were loaded with 10 lg of total RNA and filters were successively hybridised with GPX1, GPX2 and GPX4 probes. The ratio of hybridisation with each probe to that with 18S rRNA probe was calculated using Canberra Packard Instantimager. Results are shown as means ± S.E.M. from two independent experiments. Statistical significance was assessed by a Mann–Whitney U test, \P < 0.05.
accepted sensitive marker of Se-depletion, were consistent with data from earlier studies (results not shown). RNA from mucosal scrapings was subjected to Northern hybridisation with probes to GPX1, 2 and 4. Quantification of the hybridisation showed that mean GPX1 mRNA abundance was decreased by 83% (P < 0.05) in the colon of severely Se-deficient rats compared with tissue from the Se-adequate rats and decreased by 60% in the colon of the deficient rats (Fig. 3). In contrast, mean GPX4 mRNA abundance showed
Fig. 3. Change of glutathione peroxidase mRNA abundances in the rat colon in response to Se supply. Samples of total RNA were analysed by Northern hybridisation with probes for GPX1, GPX2, GPX4 and GAPDH mRNAs, and for 18S rRNA. The ratio of hybridisation with each probe to that with the 18S probe was calculated and data expressed as percentages of the mean mRNA abundances obtained for animals on the Se-adequate diet (group D). Results are shown as means ± S.E.M. Results were compared by a Mann–Whitney U test, \P < 0.05.
V. Pagmantidis et al. / FEBS Letters 579 (2005) 792–796
only a small decrease (25%; P < 0.05) in the colon of the severely deficient rats and GPX2 mRNA levels showed no statistically significant change in rats fed any of the Se-deficient diets. These results confirm and extend the array data by indicating that the effect of Se in cell culture is mirrored by that in the colon in vivo and by showing that there is a differential effect of Se-depletion on GPX1, 2 and 4 in the colon. In the liver, Se-depletion has no effect on GPX4 mRNA expression although GPX1 decreases dramatically [9]. The present data show that, in the colon, GPX4 mRNA abundance decreases as well as that of GPX1, indicating that the response of glutathione peroxidase expression to Se-depletion is different in the colon from that observed previously in liver or heart [9]. This supports the contention that regulation of selenoprotein expression by Se supply is affected differentially in different tissues [9,10]. Using a mouse SelW cDNA as probe, Northern hybridisation showed a band corresponding to SelW in RNA samples from rats fed the Se-adequate diet (Fig. 4). However, the expression of SelW mRNA was decreased in the Se-depleted colon to such an extent that SelW mRNA was barely detectable in samples from animals fed the severely Se-deficient diet (Fig. 4). Density analysis of the autoradiographs showed that the ratio of SelW/GAPDH expression ranged from 1.0 to 1.9 in the rats fed the Se-adequate diet but was zero in the rats fed the severely Se-deficient diet, since the intensity of the SelW band was below the level of detection. These data indicate that SelW expression is highly sensitive to Se-depletion. SelW is expressed in muscle [22] but has not previously been shown to be expressed in either the colon or a gastrointestinal cell line. In summary, the present data obtained from both Caco-2 cells in culture and from rat colon tissue show that Se-depletion has differential effects on selenoprotein expression, in particular a much greater effect on the abundance of GPX1 and SelW mRNAs than on that of GPX4 or GPX2 mRNA. SelW is an anti-oxidant protein [14,22,23] and therefore our hypothesis is that the large effect of Se-depletion on the expression of both SelW and GPX1, together with the smaller effect on GPX4, and despite the maintenance of GPX2 expression, lowers antioxidant protection in the colon; and in turn this would increase susceptibility to development of inflammatory responses in the gastrointestinal system [8].
Fig. 4. Response of SelW mRNA levels in rat colon to Se-depletion. Total RNA isolated from rat mucosal scrapings was subjected to Northern hybridisation (10 lg in each lane). Specific hybridisation to a SelW probe was detected by autoradiography. Lanes 1–3 correspond to RNA from the severely Se-deficient group (group A) and lanes 4–8 to RNA from the Se-adequate group (group D). GAPDH mRNA was used as internal control and did not show any change due to different dietary Se.
795 Acknowledgements: This research was funded by a grant from World Cancer Research Fund to J.E.H., I.B. and G.B. J.R.A. is supported by Scottish Executive Environment and Rural Affairs Department. S.V. was supported by Food Standards Agency, which also provided funding for the macroarrays. We thank Dr. S. Dillon (Liverpool) for help with the macroarray experiments.
References [1] Rayman, M.P. (2000) The importance of selenium to human health. Lancet 356, 233–241. [2] Clark, L.C., Combs Jr., G.F., Turnbull, B.W., Slate, E., Alberts, D., Abele, D., Allison, R., Bradshaw, J., Chalker, D., Chow, J., Curtis, D., Dalen, J., Davis, L., Deal, R. and Dellasega, M. (1997) The nutritional prevention of cancer with selenium 1983–1993; a randomised controlled trial. JAMA 276, 1957–1963. [3] Whanger, P.D. (2004) Selenium and its relationship to cancer: an update. Brit. J. Nutr. 91, 11–28. [4] Kryukov, G.V., Castellano, S., Novoselov, S.V., Lobanov, A.V., Zehtab, O., Guigo, R. and Gladyshev, V.N. (2003) Characterization of mammalian selenoproteomes. Science 300, 1439–1443. [5] Brigelius-Flohe, R. (1999) Tissue-specific functions of individual glutathione peroxidases. Free Radic. Biol. Med. 27, 951–965. [6] Gasdaska, J.R., Harney, J.W., Gasdaska, P.Y., Powis, G. and Berry, M.J. (1999) Regulation of human thioredoxin reductase expression and activity by 3 0 -untranslated region selenocysteine insertion sequence and mRNA instability elements. J. Biol. Chem. 274, 25379–25385. [7] Esworthy, R.S., Aranda, R., Martin, M.G., Doroshaw, J.H., Binder, S.W. and Chu, F.-F. (2001) Mice with combined disruption of Gpx1 and Gpx2 genes have colitis. Am. J. Physiol. 281, G848–G855. [8] Chu, F.-F., Esworthy, R.S. and Doroshaw, J.H. (2004) Role of Se-dependent glutathione peroxidases in gastrointestinal inflammation and cancer. Free Radic. Biol.Med. 36, 1481–1495. [9] Bermano, G., Nicol, F., Dyer, J.A., Sunde, R.A., Beckett, G.J., Arthur, J.R. and Hesketh, J.E. (1995) Tissue-specific regulation of selenoenzyme gene expression during selenium deficiency in rats. Biochem. J. 311, 425–430. [10] Wingler, K., Bo¨cher, M., Flohe, L., Kollmus, H. and BrigeliusFlohe, R. (1999) mRNA stability and selenocysteine insertion sequence efficiency rank gastrointestinal glutathione peroxidase high in the hierarchy of selenoproteins. Eur. J. Biochem. 259, 149– 157. [11] Brigelius-Flohe, R., Muller, C., Menard, J., Florian, S., Schmehl, K. and Wingler, K. (2001) Functions of GI-GPx: Lessons from selenium-dependent expression and intracellular localization. Biofactors 14, 101–106. [12] Sukenaga, Y., Ishida, K., Takeda, T. and Takagi, K. (1987) cDNA sequence coding for human glutathione peroxidase. Nucleic Acid Res. 15, 7178–7178. [13] Yagi, K., Komura, S., Kojima, S., Sun, Q., Nagata, N., Ohishi, N.and and Nishikimi, M. (1996) Expression of human phospholipid hydroperoxide glutathione peroxidase gene for protection of host cells from lipid hydroperoxide-mediated injury. Biochem. Biophys. Res. Commun. 219, 486–491. [14] Jeong, D., Kim, T.S., Chung, Y.W., Lee, B.J. and Kim, I.Y. (2002) Selenoprotein W is a glutathione-dependent antioxidant in vivo. FEBS Lett. 517, 225–228. [15] Erickson, J.M., Rushford, C.L., Dorney, D.J., Wilson, G.N. and Schmickel, R.D. (1981) Structure and variation of human ribosomal DNA. Gene 16, 1–9. [16] Chomczynski, P. and Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidium thiocyanate–phenol–chloroform extraction. Anal. Biochem. 162, 156–159. [17] Maniatis, T., Fritsch, E.F. and Sambrook, J., Eds., (1982). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. [18] Bermano, G., Arthur, J.R. and Hesketh, J.E. (1996) Selective control of cytosolic glutathione peroxidase and phospholipid hydroperoxide peroxidase mRNA stability by selenium supply. FEBS Lett. 387, 157–160. [19] Arthur, J.R., Nicol, F., Hutchinson, A.R. and Beckett, G.J. (1990) The effects of selenium depletion and repletion on the
796 metabolism of thyroid hormones in the rat. J. Inorg. Biochem. 39, 101–108. [20] Mitchell, J.H., Nicol, F., Beckett, G.J. and Arthur, J.R. (1997) Selenium and iodine deficiencies: effects on brain and brown adipose tissue selenoenzyme activity and expression. J. Endocrinol. 155, 255–263. [21] Villettte, S., Bermano, G., Arthur, J.R. and Hesketh, J.E. (1998) Thyroid stimulating hormone and selenium supply interact to
V. Pagmantidis et al. / FEBS Letters 579 (2005) 792–796 regulate selenoenzyme gene expression in thyroid cells (FRTL-5) in culture. FEBS Lett. 438, 81–84. [22] Gu, Q.P., Ream, W. and Whanger, P.D. (2002) Selenoprotein W gene regulation by selenium in L8 cells. Biometals 15, 411– 420. [23] Ream, L.W., Vorachek, W.R. and Whanger, P.D. (2001) in: Selenium: Its Molecular Biology and Role in Human Health (Hatfield, D.L., Ed.), Kluwer Press, Boston.
FEBS 29196
Effects of Se-depletion on glutathione peroxidase and selenoprotein W gene expression in the colon Vasileios Pagmantidisa, Giovanna Bermanob, Stephane Villettea, Iain Broomb, John Arthurc, John Hesketha,* a
School of Cell and Molecular Biosciences, University of Newcastle upon Tyne, NE1 7RU, UK b School of Life Sciences, The Robert Gordon University, Aberdeen AB25 1HG, UK c Rowett Research Institute, Bucksburn, Aberdeen AB21 9SB, UK Received 3 September 2004; revised 22 November 2004; accepted 9 December 2004 Available online 27 December 2004 Edited by Lukas Huber
Abstract Selenium (Se)-containing proteins have important roles in protecting cells from oxidative damage. This work investigated the effects of Se-depletion on the expression of the genes encoding selenoproteins in colonic mucosa from rats fed diets of different Se content and in human intestinal Caco-2 cells grown in Se-adequate or Se-depleted culture medium. Se-depletion produced statistically significant (P < 0.05) falls in glutathione peroxidase (GPX) 1 mRNA (60–83%) and selenoprotein W mRNA (73%) levels, a small but significant fall in GPX4 mRNA (17– 25%) but no significant change in GPX2. The data show that SelW expression in the colon is highly sensitive to Se-depletion. 2004 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Colon; Glutathione peroxidase; Selenium; Selenoprotein; Caco-2; Gene array
1. Introduction The micronutrient selenium (Se) is essential for health [1]. Low intake of Se has been implicated with the increased incidence of disease and Se supplementation lowered both cancer incidence and mortality from cancer [2,3]. Se is present in several proteins, called selenoproteins, as the amino-acid selenocysteine (Se-cys) [4]. Approximately 30 selenoproteins and up to 25 selenoprotein genes have been identified in mammals and they have a variety of biological roles [4]. They include the glutathione peroxidase family of enzymes, all of which are involved in the cellÕs antioxidant systems [5], the thioredoxin reductases, which are involved in the redox regulation of gene expression [6] and a number of recently identified selenoproteins (e.g., SelX, SelN, SelW, and 15 kDa selenoprotein) that are less well characterised [4]. The glutathione peroxidases include cytosolic glutathione peroxidase (GPX1), gastrointestinal glutathione peroxidase (GPX2), plasma glutathione peroxidase (GPX3) and phospholipid hydroperoxide glutathione peroxidase (GPX4). The phenotype of knock-out mice lacking GPX1 and GPX2 includes colon pathology and increased susceptibility to colitis[7], leading to the suggestion that these enzymes are impor-
* Corresponding author. Fax: +44 0 191 222 8684. E-mail address: [email protected] (J. Hesketh).
tant in determining the level of anti-oxidant protection and inflammatory responses in the colon [8]. In addition, supplementation with Se above normal intake reduces mortality from cancer of the colon [2,3], as well as that of lung and prostate. These observations indicate that selenoproteins such as the GPXs are critical for colon function. However, little is known of how selenoprotein gene expression in colonic cells, particularly in vivo, is regulated by Se availability. The aim of the present work was to investigate how Sedeficiency modulates expression of a range of selenoprotein genes in the rat colon and a human gastrointestinal cell line. Since patterns of selenoprotein activity are accompanied by altered patterns of mRNA abundances and both are regulated by Se availability [9,10], our approach was to use a novel ÔselenoproteinÕ macroarray to screen for genes affected by Se depletion and then to extend the studies using Northern hybridisation.
2. Materials and methods 2.1. DNA probes The GPX1, GPX4 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probes were 880 bp EcoRI, 814 bp EcoRI, and 780 bp PstI/XbaI fragments, respectively, as used previously [9]. The human GPX2 probe (from Prof. R. Brigelius-Flohe) was a 663 bp EcoRI fragment [11]. The human GPX1 probe was a 300 bp BamHI/XbaI fragment [12] and the human GPX4 probe (from Prof. K. Yagi, Gifu) was an 874 bp HindIII/XbaI fragment [13]. The SelW probe (from Dr. I. Kim) was a 420 bp BamHI/XhoI fragment corresponding to the mouse brain cDNA [14]. The 18S rRNA probe was a 328 bp fragment corresponding to nucleotides 4852–5179 of the human ribosomal RNA sequence [15] cloned into the SrfI site of PCR-Script AMP vector (Stratagene) and excised using KpnI and SacI. 2.2. RNA extraction and Northern hybridisation Total RNA was extracted by the guanidinium thiocyanate/phenol/ chloroform procedure [16]. Northern blotting was carried out using standard procedures [17] and membranes pre-hybridised for 20 min at 68 C in 10 ml of QuickHyb solution (Stratagene). 25 ng of DNA probes was labelled with [32P]dCTP by random priming, as described previously [9], and hybridisation carried out for 1 h at 68 C. Non-specifically bound probe was removed from membranes by two washes in 2· SSC (1· SSC = 0.15 M NaCl/0.015 M sodium citrate)/0.1% SDS at room temperature for 15 min, followed by one wash in 0.1· SSC/0.1% SDS at 60 C for 30 min. Specific hybridisation was detected using a Canberra Packard Instantimager for quantification (results for each probe were expressed per unit of hybridisation achieved with the 18S rRNA probe to allow correction for any variation between loading of RNA on the gel or transfer to
0014-5793/$30.00 2004 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2004.12.042
V. Pagmantidis et al. / FEBS Letters 579 (2005) 792–796
793
the nylon membrane) and by autoradiography using Fujifilm Super FX at 80 C. After analysis, membranes were stripped by washing in 0.1% SDS for 10 min at 95 C before rehybridisation to other probes.
Table 1 Effects of Se-deficiency on expression of a range of genes detected by macroarray Gene name
Ratio Se/Se+
2.3. cDNA expression arrays 32 P-labelled cDNA probe synthesis from 3.5 lg total RNA and hybridisation to the Atlas Array membranes were performed according to the Atlas cDNA expression Arrays User Manual (BD Biosciences Clontech, Palo Alto, USA). Detection was performed with a phosphorimager (Molecular Dynamics, Phosphorimager Storm 860) using 24 h and 3 days exposure. Analysis of the results was performed using the AtlasImage v2.7 software (BD Biosciences Clontech) with a specific grid corresponding to our custom made ‘‘selenoprotein’’ array: the array was spotted with all available human selenoprotein cDNAs, cDNAs corresponding to genes whose products are associated with Se metabolism or arachidonate metabolism, and a series of standard housekeeping genes (Table 1 and associated legend). To analyse differential gene expression using the Atlas Array hybridisation, intensity differences were normalised by taking into account changes in the housekeeping genes.
GPX1 GPX2 GPX3 GPX4 SelP SBP1 DbpB SPS (SelD) Phospholipase A2 Cu/Zn SOD Mn SOD SOD3 (extracellular) Glutathione-S-transferase A2 Glutathione-S-transferase A4 Interleukin 1; b SelN SelW SelB (elongation factor) tRNA selenocysteine associated protein SelZ f1 TR2 TR3 MHC; class1; C b-Actin GAPDH a-Tubulin Ubiquitin Ribosomal protein L13a Ribosomal protein S9 HPRT Genomic DNA
0.46 1.63 0.67 0.76 0.90 Up 0.94 1.03 0.95 1.29 1.04 1.58 1.50 2.76 3.89 0.76 0.27 Down 1.20 Down 0.73 1.33 Up 1.08 1.11 1.20 1.09 0.90 0.96 1.67 1.19
2.4. Cell culture Stocks of human colon adenocarcinoma cells (Caco-2) were grown at 37 C in DulbeccoÕs modified EagleÕs medium (with 4.5 g/l glucose and Glutamax) supplemented with 10% (v/v) foetal calf serum (Sigma, Poole, UK), 60 lg/ml gentamycin, 1% (v/v) non-essential amino acids and penicillin/streptomycin (100 units/ml and 100 lg/ml, respectively). Cells were transferred to serum-free medium to which was added either insulin (5 lg/ml) and transferrin (5 lg/ml) (selenium-deficient cells), or insulin, transferrin and selenium as sodium selenite (7 ng/ml) (selenium-replete cells) [18]. Medium was changed every 2 days. 2.5. Animals and diets Weanling Male Hooded Lister Rats of the Rowett strain were randomly allocated to groups of six and fed ad libitum on a semi-synthetic diet containing different amounts of Se for 6 weeks. The basal diet [19] contained 8 ng of Se/g (severely Se-deficient) and the other diets contained 33, 63 (Se-deficient) or 111 (Se-adequate) ng of Se/g as sodium selenite. At the end of the 6 weeks, the animals were anaesthetised with isofluorane and blood samples were taken by cardiac puncture. The animals were killed by cervical dislocation and the entire colon dissected out and washed with cold sterile PBS; the colon was then cut open, the mucosa scraped off and then rapidly frozen in liquid nitrogen and stored at 80 C.
3. Results and discussion Caco-2 cells were grown for 2–6 days in either a Se-supplemented or Se-depleted medium and RNA extracted and analysed by Northern hybridisation. Cells grown in the Sedepleted medium showed a 60% decrease in the level of GPX1 mRNA by 2 days (P < 0.05) compared with cells grown in Se-supplemented medium (Fig. 1). This low expression remained after 4 or 6 days in the Se-depleted medium. Addition of selenite to the medium at day 6 led to an increase in GPX1 mRNA abundance such that it returned to control levels by day 8 (Fig. 1). The level of GPX1 mRNA is a consistent marker of Se status in cell and animal experiments [9,10,18,20] and these changes indicate that after 2 days in Se-depleted medium the Caco-2 cells become severely Se deficient, that the effect is not increased significantly over a further 4 days and that the effect can be reversed by readdition of selenite; this reflects closely the changes in GPX1 mRNA in H4 hepatoma cell and FRTL-5 thyroid cells grown in similar media [18,21]. Using a custom-made macroarray and RNA extracted from Caco-2 cells grown for 2 days in Se-deficient or Se-supple-
mRNA abundances were measured in Caco-2 cells after 2 days in Sedeficient or Se-replete conditions. Results are the mean values obtained from two experiments each analysed using duplicate arrays. Results are expressed as a ratio of the Se-depleted cells to Se-adequate cells. GPX1 and SelW mRNA abundances; showed greater than twofold change; which is considered to be significant. When expression levels were close to the limit of detection the ratio of expression is given as up/down and not as a precise value. The following genes were also present on the array but expression was not detected: thioredoxin reductase 1, deiodinase type I, II and III, 15 kDa selenoprotein, selenoprotein M, T, and X, mitochondrial capsule selenoprotein, SECISbinding protein 2, 5-lipoxygenase, arachidonate 15-lipoxygenase types 1 and 2, arachidonate 12-lipoxygenase 5-lipoxygenase activating protein, cyclooxygenases 1 and 2, tumor necrosis factor a and b, LTB4 receptor, thromboxane A2 receptor, prostaglandin F and D2 receptors, glutathione-S-transferase theta 1 and 2, thyroid hormone receptor a and b2, interferon gamma receptor and precursor, metallothionein isoform 1rR, interleukin 1 a and b, interleukin 2, tyrosine 3-monooxygenase. Abbreviations: Sel, selenoprotein; SOD, superoxide dismutase; TR, thioredoxin reductase; MHC, major histocompatibility complex.
mented medium, it was found that Caco-2 cells expressed ten selenoprotein mRNAs (GPX1, GPX2, GPX3, GPX4, TR2, TR3, selenoproteins N, P, W and Z) at a level that was detectable using this technology (Table 1). Expression of other selenoproteins genes on the array (see legend to Table 1) was below the level of detection. After 2 days in Se-depleted medium, only two selenoproteins showed a major change in mRNA abundance compared with Se-supplemented cells (Table 1): GPX1 and Selenoprotein W (SelW), for which the ratio of relative expression was 0.46 and 0.27, respectively. In addition, Se depletion led to increased expression of the non-selenoprotein mRNAs interleukin 1b and glutathione-Stransferase A4, for which the ratio of relative expression was
794
Fig. 1. Effect of Se-depletion on GPX1 mRNA level in Caco-2 cells. Cells were grown for 2–6 days in Se-supplemented or Se-deficient serum-free medium. After 6 days, selenite was given back to the Sedepleted cells. GPX1mRNA and 18S rRNA levels were determined by Northern hybridisation using a Canberra Packard Instantimager for quantification. Data were expressed as percentages of the mean ratio of GPX1 mRNA abundance/18S rRNA abundance obtained for Sereplete cells (day 0). Results are shown as means ± S.E.M. from two independent experiments. Statistical significance was assessed by a Mann–Whitney U test, \P < 0.05.
3.89 and 2.76, respectively. The relative expression of GPX3, GPX4 and SelN in Se-depleted cells (0.67, 0.76, and 0.76, respectively) was less than the twofold change considered to be significant but suggested that there may be a small fall in expression. There was no evidence of changes in relative expression of GPX2, SelP, TR2 and TR3 or of non-selenoprotein antioxidant enzymes such as Mn and Cu/Zn superoxide dismutases. These array data suggest that in Caco-2 cells expression of GPX1, 2 and 4 are differentially affected by Sedepletion and that the major effect of Se-depletion on selenoprotein expression in colonic epithelial cells, as assessed by mRNA abundance, is a dramatic fall in the expression of SelW and GPX1. To confirm these findings, two experiments were carried out. First, mRNA abundances were measured in Se-supplemented and deficient Caco-2 cells by Northern hybridisation. Second, and more importantly, since little was known about the effects of Se-depletion on the colon in vivo, selenoprotein gene expression was studied in rats that were fed diets that provided different levels of dietary Se. Quantification of Northern hybridisation data from cell culture experiments showed that there was no statistical significant difference in GPX2 mRNA levels compared to cells grown in medium supplemented with Se (Fig. 2). However, there was a small (17%) but statistically significant decrease (P < 0.05) in the GPX4 mRNA level in the Se-depleted cells (Fig. 2). This was in contrast with the marked decline in the level of GPX1 mRNA. These data are consistent with earlier observations that Se-supplementation of cells grown in standard medium increases GPX1 mRNA abundance but not that of GPX2 [10]. Rats were fed diets containing either an adequate supply of Se (111 ng Se/g; group D) or three levels of low Se supply ranging from a severely deficient Se supply (8 ng Se/g; group A), to deficient (33 ng Se/g; group B) or marginal supply (63 ng Se/g; group C). Previously, such diets have been found to consistently produce Se-depletion in rats [9,20]. In the present experiments measurements of liver GPX1 mRNA abundance, an
V. Pagmantidis et al. / FEBS Letters 579 (2005) 792–796
Fig. 2. Effect of Se-deficiency on the abundance of glutathione peroxidase 1, 2 and 4 mRNAs in Caco-2 cells. All lanes were loaded with 10 lg of total RNA and filters were successively hybridised with GPX1, GPX2 and GPX4 probes. The ratio of hybridisation with each probe to that with 18S rRNA probe was calculated using Canberra Packard Instantimager. Results are shown as means ± S.E.M. from two independent experiments. Statistical significance was assessed by a Mann–Whitney U test, \P < 0.05.
accepted sensitive marker of Se-depletion, were consistent with data from earlier studies (results not shown). RNA from mucosal scrapings was subjected to Northern hybridisation with probes to GPX1, 2 and 4. Quantification of the hybridisation showed that mean GPX1 mRNA abundance was decreased by 83% (P < 0.05) in the colon of severely Se-deficient rats compared with tissue from the Se-adequate rats and decreased by 60% in the colon of the deficient rats (Fig. 3). In contrast, mean GPX4 mRNA abundance showed
Fig. 3. Change of glutathione peroxidase mRNA abundances in the rat colon in response to Se supply. Samples of total RNA were analysed by Northern hybridisation with probes for GPX1, GPX2, GPX4 and GAPDH mRNAs, and for 18S rRNA. The ratio of hybridisation with each probe to that with the 18S probe was calculated and data expressed as percentages of the mean mRNA abundances obtained for animals on the Se-adequate diet (group D). Results are shown as means ± S.E.M. Results were compared by a Mann–Whitney U test, \P < 0.05.
V. Pagmantidis et al. / FEBS Letters 579 (2005) 792–796
only a small decrease (25%; P < 0.05) in the colon of the severely deficient rats and GPX2 mRNA levels showed no statistically significant change in rats fed any of the Se-deficient diets. These results confirm and extend the array data by indicating that the effect of Se in cell culture is mirrored by that in the colon in vivo and by showing that there is a differential effect of Se-depletion on GPX1, 2 and 4 in the colon. In the liver, Se-depletion has no effect on GPX4 mRNA expression although GPX1 decreases dramatically [9]. The present data show that, in the colon, GPX4 mRNA abundance decreases as well as that of GPX1, indicating that the response of glutathione peroxidase expression to Se-depletion is different in the colon from that observed previously in liver or heart [9]. This supports the contention that regulation of selenoprotein expression by Se supply is affected differentially in different tissues [9,10]. Using a mouse SelW cDNA as probe, Northern hybridisation showed a band corresponding to SelW in RNA samples from rats fed the Se-adequate diet (Fig. 4). However, the expression of SelW mRNA was decreased in the Se-depleted colon to such an extent that SelW mRNA was barely detectable in samples from animals fed the severely Se-deficient diet (Fig. 4). Density analysis of the autoradiographs showed that the ratio of SelW/GAPDH expression ranged from 1.0 to 1.9 in the rats fed the Se-adequate diet but was zero in the rats fed the severely Se-deficient diet, since the intensity of the SelW band was below the level of detection. These data indicate that SelW expression is highly sensitive to Se-depletion. SelW is expressed in muscle [22] but has not previously been shown to be expressed in either the colon or a gastrointestinal cell line. In summary, the present data obtained from both Caco-2 cells in culture and from rat colon tissue show that Se-depletion has differential effects on selenoprotein expression, in particular a much greater effect on the abundance of GPX1 and SelW mRNAs than on that of GPX4 or GPX2 mRNA. SelW is an anti-oxidant protein [14,22,23] and therefore our hypothesis is that the large effect of Se-depletion on the expression of both SelW and GPX1, together with the smaller effect on GPX4, and despite the maintenance of GPX2 expression, lowers antioxidant protection in the colon; and in turn this would increase susceptibility to development of inflammatory responses in the gastrointestinal system [8].
Fig. 4. Response of SelW mRNA levels in rat colon to Se-depletion. Total RNA isolated from rat mucosal scrapings was subjected to Northern hybridisation (10 lg in each lane). Specific hybridisation to a SelW probe was detected by autoradiography. Lanes 1–3 correspond to RNA from the severely Se-deficient group (group A) and lanes 4–8 to RNA from the Se-adequate group (group D). GAPDH mRNA was used as internal control and did not show any change due to different dietary Se.
795 Acknowledgements: This research was funded by a grant from World Cancer Research Fund to J.E.H., I.B. and G.B. J.R.A. is supported by Scottish Executive Environment and Rural Affairs Department. S.V. was supported by Food Standards Agency, which also provided funding for the macroarrays. We thank Dr. S. Dillon (Liverpool) for help with the macroarray experiments.
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