Identification of an element within the promoter of human selenoprotein P responsive to transforming growth factor-b Volker Mostert 1 , Sandra Wolff 1 , Ingeborg Dreher 2 , Josef Ko¨ hrle 2 and Josef Abel 1 1 Medizinisches Institut fu ¨ r Umwelthygiene an der Heinrich-Heine-Universita ¨ tDu ¨ sseldorf Abteilung Experimentelle Toxikologie, Du ¨ sseldorf, Germany; 2 Medizinische Poliklinik, Universita ¨ tWu ¨ rzburg, Wu ¨ rzburg, Germany Selenoprotein P (SeP) is a plasma protein that contains up to 10 selenocysteine residues and accounts for about 50% of total selenium in human plasma. We have previously shown that SeP expression in the human liver cell line HepG2 is inhibited by transforming growth factor (TGF)-b 1 on a transcriptional level. Smad proteins are the transcriptional mediators of TGF-b signalling and putative Smad-binding elements (SBE) comprising the core sequence CAGACA are present at two positions in the SeP promoter. The aim of our study was to investigate whether Smad molecules are involved in inhibition of SeP expression by TGF-b 1 and to locate the promoter region critical for this effect. As seen in electro- phoretic-mobility-shift assays, TGF-b 1 treatment led to enhanced binding of nuclear proteins to a putative SBE from the SeP promoter. Overexpression of Smad 3 and 4, but not of Smad 2, resulted in a marked down-regulation of SeP mRNA expression. Similar effects were observed for luciferase expression under control of a human SeP- promoter construct. Deletion as well as point-mutation of putative SBEs led to a loss of promoter sensitivity towards TGF-b 1 treatment. Hence, we demonstrated an involvement of Smad 3 and 4 in transcriptional regulation of SeP by TGF-b 1 and we were able to identify the TGF-b-responsive element in the SeP promoter. Keywords: selenoprotein P; transforming growth factor-b 1 ; Smad; promoter; CAGA box. Selenoprotein P (SeP) is a selenocysteine-containing glyco- protein that accounts for about 50% of total plasma selenium in humans (reviewed in [1]). It differs from all other mam- malian selenoproteins identified so far by its high selenium content. From the mRNA of human SeP, it is predicted selenocysteine will occur at 10 positions [2] whereas most other selenoproteins contain only one selenocysteine residue per subunit. The physiological function of SeP is not com- pletely understood, but a role as a part of the body’s anti- oxidant defence system seems most likely. SeP is secreted into plasma mainly by the liver, but it is also highly expressed in a number of different tissues indicating that they also secrete it [3]. In vitro, SeP in human plasma can act as a protective agent against the oxidation and nitration reactions mediated by peroxynitrite, a potent oxidant generated in vivo [4]. Another in vitro study revealed a reducing activity of SeP against phospholipid hydroperoxides, albeit with low efficiency compared to the selenoenzyme phospholipid hydroperoxide glutathione peroxidase [5]. The 5 0 flanking region of the human SeP gene was shown to contain a functional interferon (IFN)-g responsive element (GAS) and negative regulation of the SeP promoter by pro-inflamma- tory cytokines such as interleukin (IL)-1b and TNF-a was found [6]. Expression of SeP mRNA and protein in human hepatoma cells HepG2 is efficiently inhibited on a tran- scriptional level by the anti-inflammatory cytokine trans- forming growth factor (TGF)-b 1 [7]. Thus, SeP might be considered as a negative acute-phase protein. Consistent with this assumption, selenium levels in human plasma are drastically diminished in conditions of systemic inflammatory response, sepsis, and burn injuries [8,9]. As TGF-b 1 is secreted by activated macrophages [10] and plays an important role in immunomodulation and wound healing [11–13], it is possible that TGF-b 1 -mediated down-regulation of SeP contributes largely to the decline in plasma selenium observed in pathological conditions. The Smad family of proteins has been found to transduce TGF-b signals into the nucleus after being activated by the TGF-b-receptor (TbR) complex [14]. Subsequently, they bind directly to critical Smad-binding elements (SBEs) in the promoter of TGF-b-inducible genes [15–18]. The human SeP promoter (GenBank accession no. Y12262) also contains two putative SBEs, therefore we sought to elucidate whether these elements are functional at mediating the inhibitory effect of TGF-b. SBEs have mostly been described for TGF-b-inducible promoters, so far. However, Smad proteins are also able to recruit transcriptional corepressors such as 5 0 -TG-3 0 -interacting factor (TGIF) [19] or c-Ski [20], which could repress the transcriptional activity of promoters bearing SBEs. In addition, we showed very recently the involvement of TGIF, Smads and an SBE in the down- regulation by TGF-b of the human aryl hydrocarbon receptor [21]. Correspondence to J. Abel, Abteilung Experimentelle Toxikologie, Medizinisches Institut fu ¨ r Umwelthygiene, Postfach 103751, D-40028 Du ¨ sseldorf, Germany. Fax: 1 49 211 3190 910, Tel.: 1 49 211 3389 204, E-mail: josef.abel@uni-duesseldorf.de (Received 6 June 2001, revised 28 August 2001, accepted 01 October 2001) Abbreviations: BMS, basal medium supplement; EMSA, electrophoretic-mobility-shift assay; GAS, IFN-g-activation site; IFN, interferon; IL, interleukin; SBE, Smad-binding element; SeP, selenoprotein P; TGF, transforming growth factor; TGIF, 5 0 -TG-3 0 - interacting factor; TbR, TGF-b receptor; PAI-1, plasminogen activator inhibitor-1; SIRS, systemic inflammatory response syndrome. Eur. J. Biochem. 268, 6176–6181 (2001) q FEBS 2001 The present study investigates whether Smad proteins are involved in the down-regulation of SeP by TGF-b 1 and explores where the critical TGF-b-responsive element is located. MATERIALS AND METHODS Materials Recombinant human TGF-b 1 and sodium selenite were supplied by Sigma (Taufkirchen, Germany). RPMI 1640 medium was from PAA (Linz, Austria), penicillin/strepto- mycin, basal medium supplement (BMS), fetal bovine serum, sodium hydrogen carbonate, and L-glutamine pur- chased from Biochrom (Berlin, Germany). Other chemicals were from Sigma unless otherwise stated. Cell culture and treatment The human hepatocarcinoma line HepG2 was cultured in RPMI 1640 medium containing 10% fetal bovine serum, 1% penicillin/streptomycin, 1 mg : mL 21 L-glutamine, and 0.15% NaHCO 3 . Cells were maintained under standard conditions at 37 8Cin5%CO 2 . Forty-eight hours before treatment, cells were cultured in RPMI 1640 medium containing 5% BMS, 1% penicillin/streptomycin, 1 mg : mL 21 L-glutamine, and 0.15% NaHCO 3 and 250 nM sodium selenite to allow selenoprotein expression. Cells were then treated as indicated. Plasmids and cell culture for transfection The 1800-bp Bgl II/KpnI–SeP-promoter fragment was isolated from the plasmid pBK15 [6] and subcloned into the luciferase reporter gene plasmid pGL3basic revealing plasmid BK4GL3. Restriction digestion with Kpn I/Pst I, blunt end generation and religation removes the first CAGA-box and 59 bp of a sequence belonging to the pBluescriptSKII 1 plasmid used as a cloning vector during generation of pBK15 [6]. The resulting plasmid is termed DBK4GL3 and possesses only one complete CAGA box (Fig. 1) Digestion of BK4GL3 with Kpn I/Sac I, blunt end gener- ation with the Klenow fragment of DNA polymerase and religation leads to removal of nucleotides 21868 to 21725, yielding the construct termed DCAGA (Fig. 1). Expression vectors for Smad 2, 3, and 4 in pcDNA3.1 (Invitrogen, Karlsruhe, Germany) were a generous gift from G. Gross (Gesellschaft fu ¨ r Biotechnologische Forschung, Braun- schweig, Germany). Transfections for reporter gene assays were performed using the Dual Luciferase system (Promega, Mannheim, Germany) using a reporter construct expressing Photinus pyralis luciferase and the pRL-TK plasmid, which bears an expression vector for Renilla reniformis luciferase as inter- nal control for transfection efficiency [22]. The reporter construct was present at 1 mg per well together with 0.15 mg pRL-TK and 5 mg Transfectam reagent per well. In co- transfection experiments, each Smad expression plasmid was present at 0.5 mg per well, empty pGL3basic plasmid was added to give a final plasmid concentration of 2 mg where appropriate. HepG2 cells were seeded into six-well plates (2 Â 10 5 cells per well). After 24 h, cells were transfected with the indicated plasmids in 1 mL RPMI 1640 medium containing 5% BMS and allowed to rest for 4 h. Medium was then replaced by 2 mL RPMI 1640 and after an additional 24 h cells were either washed and lysed for luciferase assay or treated with 100 p M TGF-b 1 or vehicle (0.1% BSA in 4 mM HCl) for 16 h prior to lysis. For RT-PCR analysis, cells were allowed to rest for 48 h before RNA preparation. Site-directed mutagenesis The sequence CAGACA at position 21797 comprising the putative SBE in the plasmid DBK4GL3 was mutated into the sequence TACATA with a PCR-based strategy using the QuikChange Site-Directed-Mutagenesis Kit (Stratagene, La Jolla, CA, USA) according to the manufacturer’s instruc- tion. Mutagenesis was achieved using the 45-nucleotide primer 5 0 -GATAGGTACACAAAACCTTTTACATACTG AGTTGTAGAAAGAAGG-3 0 (exchanged nucleotides are in bold) and its complementary sequence. Luciferase assays Cell lysis was performed in 200 mL of passive lysis buffer (Promega). Luciferase activities in cell lysates were deter- mined using a LB 9505C luminometer (Berthold, Bad Wildbad, Germany). Finally, P. pyralis luciferase activities were normalized to R. reniformis luciferase activity and protein content. RT-PCR For isolation of RNA from transfected cells, cells from three wells treated equally were pooled prior to lysis. Semi- quantitative RT-PCR was performed as described previously [7]. SeP and GAPDH transcripts were coamplified using gene-specific primers. Quantitative analyses were performed using a phosphorimaging system and QUANTITY ONE software (Bio-Rad, Hercules, CA, USA). Nuclear extracts Nuclear extracts were prepared from HepG2 cells using a modified protocol according to Dennler et al. [15]. Briefly, confluent cells from 100 mm 2 culture flasks were washed once with NaCl/P i and once with NaCl/P i containing 1 mM Na 3 VO 4 and 5 mM NaF. After that, cells were scraped into Fig. 1. Schematic representation of the SeP promoter constructs used in this study. The numbering given refers to the major transcription start site of the human SeP gene [6]. For simplicity, the numbering is continued beyond the first nucleotide of the known SeP promoter sequence at 21811. q FEBS 2001 A TGF-b-responsive element in the SeP promoter (Eur. J. Biochem. 268) 6177 1 mL of ice-cold buffer A (20 mM Hepes pH 7.9, 20 mM NaF, 1 mM Na 3 VO 4 ,1mM Na 4 P 2 O 7 ,1mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.5 mM phenylmethanesulfonyl fluoride, and 1 mg : mL 21 of each leupeptin and aprotinin). The cells were allowed to swell on ice for 15 min and then lysed by 30 strokes with a Teflon pestle. Nuclei were pelleted by centrifugation (16 000 g, 30 s) and resuspended in 150 mL buffer B (buffer A containing 420 m M NaCl and 20% glycerol). The nuclear membrane was disrupted by 15 strokes with a Teflon pestle. The resulting suspension was shaken for 30 min at 4 8C. After centrifugation (16 000 g, 20 min), the supernatants were aliquoted and stored at 280 8C until use. Electrophoretic mobility shift assays A putative SBE from the SeP promoter (nucleotides 21808 to 21778, 5 0 -ACAAAACCTTTCAGACACTGAGTTGTA GAA-3 0 ; SBE in bold) annealed to its complementary strand was labelled using T4 polynucleotide kinase (Amersham Pharmacia, Freiburg, Germany) and [g- 32 P]ATP. Binding reactions contained 20 mg of nuclear extracts, 20 000 c.p.m. (< 10 fmol) of the labelled probe, 20 m M Hepes pH 7.9, 20 m M KCl, 4 mM MgCl 2 , 0.1 mM EDTA, 20% glycerol, 0.8 m M NaP i ,4mM spermidine, and 3 mg poly(dI : dC). Where indicated, 5 pmol unlabelled probe was included in the binding reaction. Binding was performed for 15 min, at this point 1 mL anti-(Smad-2-, 3-, or 4) IgG (500 mg : mL 21 ) were added when appropriate and incubated for another 15 min. Anti-Smad sera were a kind gift from P. ten Dijke, Ludwig Institute for Cancer Research, Uppsala, Sweden [23]. After binding, samples were separated on a 5% poly- acrylamide gel containing 0.5 Â Tris/glycine/EDTA (12.5 m M Tris/HCl, pH 8.3, 95 mM glycine, 0.5 nM EDTA). Radioactivity in the dried gel was visualized by exposure to X-ray film. RESULTS Binding of a nuclear protein to a putative SBE after treatment with TGF-b 1 Besides binding sites for well-established basal and regu- latory transcription factors, which have been described previously by Dreher et al. [6], the Smad-binding consensus sequence CAGACA is present twice in the human SeP promoter at position 21811 and 21797, respectively (Fig. 1). As the CAGA box at position 21811 is located at the very margin of the known SeP promoter sequence, the 5 0 flanking bases do not represent the context of the genomic promoter but the sequence of the pBluescriptSKII 1 plasmid. Hence, we deemed the second CAGA-box at 21797 and its flanking sequences more suitable to design a representative probe. Treatment with TGF-b 1 leads to formation of a complex between a nuclear protein and the labelled 30-bp DNA probe enclosing the putative SBE (Fig. 2). The complex formation is apparent after 30 min and further intensifies during 1 h of treatment. The specific complex could be identified by displacement of the labelled probe with a 500-fold molar excess of unlabelled probe (lane 4). As the so-called CAGA box has been described as a Smad-binding element by several authors [15,16,24], we sought to identify the protein component of the complex by inducing a supershifted complex with Smad-specific antibodies (Fig. 2, lanes 5–7). We were, however, not successful using various commercially available antibodies against human Smad isoforms or preparations provided from published sources [23]. As it has been shown by other groups that Smad oligomers can associate with different partners [25,26], we hypothesized that Smad molecules in the nuclear extract might be inaccessible to the antibody. Effect of Smad overexpression on SeP mRNA levels To examine whether Smad proteins as mediators of TGF-b are capable of suppressing SeP mRNA expression, we transfected HepG2 cells with Smad expression plasmids and analysed SeP mRNA expression by RT-PCR. As shown in Fig. 3, Smad 2 overexpression has no effect on SeP mRNA expression. In contrast, Smad 3 and 4 alone and in combination suppress the expression of SeP with the most substantial effect exerted by Smad 3. Densitometric evaluation reveals a suppression to < 26% of control values after transfection with Smad 3. In this assay, no synergistic effect of Smad 3 and 4 was observed but a weaker inhibition of around 50%, similar to the effect of Smad 4 alone. Transfection of Smad 2 along with Smad 4 abolished to some extent the inhibition observed after transfection with Smad 4 alone. RT-PCR analyses of Smad mRNA expression, however, revealed that the expression levels of the individual Smads did not vary substantially when Fig. 2. Electrophoretic-mobility-shift assay with a probe encom- passing the putative SBE from the SeP promoter. Lane 1 contains nuclear extracts from untreated HepG2 cells, lane 2 and 3 include extracts from cells treated with 100 p M TGF-b 1 for 30 and 60 min, respectively. Lane 4 depicts the protein preparation from lane 3 containing 500-fold molar excess of unlabelled probe. Lane 5, 6 and 7 each contain 500 ng of anti-Smad 2, 3 and 4-specific antibodies, respectively, together with extracts from 60 min TGF-b 1 treatment. 6178 V. Mostert et al. (Eur. J. Biochem. 268) q FEBS 2001 either of the other isoforms were cotransfected (data not shown). Modulation of SeP promoter activity by overexpression of Smad 2, 3, and 4 Overexpression of Smad proteins has been shown to activate promoters of TGF-b-responsive genes [15,27]. As shown above, overexpression of certain Smad proteins results in inhibition of SeP mRNA expression. We investigated whether Smad overexpression results in similar effects on the SeP promoter construct BK4GL3 as does TGF-b 1 treatment [7]. Smad 2 did not significantly diminish luciferase activity of the BK4GL3 construct whereas Smad 3 and 4 alone as well as in combination caused a marked inhibitory effect on luciferase expression (Fig. 4). Mutagenesis of the putative SBEs Restriction digestion of BK4GL3 with Sac I/Kpn I removes 91 bp from the 5 0 end of the SeP promoter sequence along with a 53-bp stretch belonging to the pBluescriptSKII 1 plasmid. Religation yields a truncated promoter construct without any CAGA boxes termed DCAGA. Cells transfected with either BK4GL3 or DCAGA for 24 h were treated for additional 16 h with 100 p M TGF-b 1 . Basal promoter activity was not affected by deletion of the CAGA boxes (data not shown), whereas the marked TGF-b 1 responsive- ness of the constructs BK4GL3 and DBK4GL3 was not observed with DCAGA (Table 1) demonstrating that the TGF-b 1 -responsive region of the SeP promoter resides in this sequence stretch. Furthermore, the DCAGA construct is insensitive to Smad overexpression (Fig. 4). It is also noteworthy that when the first CAGA box in BK4GL3 is destroyed by Kpn I/Pst I digestion yielding DBK4GL3, TGF-b 1 responsiveness is slightly enhanced rather than lowered (Table 1). Apparently, the first CAGA box is not required for the down-regulation by TGF-b 1 of the SeP promoter. To define the actual TGF-b-responsive element more accurately, we mutated three nucleotides of the remaining functional CAGA box in the DBK4GL3 construct yielding a construct we termed mDBK4GL3 (Fig. 1). The response of mDBK4GL3 towards TGF-b 1 was only 19% of that observed for DBK4GL3 (Table 1), indicating that the presence of an intact consensus sequence CAGACA in the SeP promoter is required for its down-regulation by TGF-b 1 . DISCUSSION The inhibition by TGF-b 1 of SeP expression is to date the only known regulation of this protein by a factor other than the availability of selenium. TGF-b 1 has a variety of target genes resulting in a manifold of effects on gene expression and cell cycle (reviewed in [28]). The most significant function of TGF-b appears to be the control of inflammatory processesasTGF-b 1 -deficient mice show multifocal inflammatory lesions as the main phenotype [29,30]. The Smad family of proteins has been characterized as mediators of TGF-b signalling in recent years [14,23]. After binding of TGF-b 1 to its cognate receptor, TbR, the so- called activating Smads, which are represented by Smad 2 and 3 in mammals, are phosphorylated by the intrinsic Ser/ Thr-kinase activity of the activated receptor. Once phosphorylated, Smads 2/3 bind to the ‘common pathway’ Smad 4 followed by translocation of this heterodimer into the nucleus where target genes are modulated in their transcriptional activity. Overexpression of Smad proteins Fig. 4. Effect of transient Smad overexpression on the luciferase expression of BK4GL3 (hatched bars) and DCAGA (open bars). Cells were transfected as described in the legend to Fig. 3 except that transfection time was only 24 h. Corrected luciferase activities are given as percent of control transfection (means ^ SE, n ¼ 9). Asterisks indicate statistical significance according to Student’s t-test (P , 0.001). Fig. 3. Effect of transient Smad overexpression on the expression of SeP mRNA. SeP and GAPDH mRNA expression was analysed by semiquantitative RT-PCR. Where indicated, cells were transfected for 48 h with 0.5 mg of the indicated plasmids. When appropriate, empty pGL3basic plasmid was added to give a total plasmid concentration of 1 mg per well. The figures in the bottom row are means of two independent replicates and represent densitometric values normalized to the intensity of the coamplified GAPDH fragment. Table 1. Relative TGF-b 1 responsiveness of the different reporter constructs used in this study. HepG2 cells were transfected with 1 mg of the indicated construct for 24 h and subsequently treated with 100 p M TGF-b 1 for 16 h. The response of the individual reporter constructs were normalized to the response of the construct DBK4GL3. Values represent the means of two independent experiments. Construct Insert length (bp) Relative TGF-b 1 response BK4GL3 1855 0.79 DBK4GL3 1799 1.00 mDBK4GL3 1799 0.19 DCAGA 1711 0.00 q FEBS 2001 A TGF-b-responsive element in the SeP promoter (Eur. J. Biochem. 268) 6179 has been shown to mimic the activation of the TbR by its agonists regarding the effects on target gene expression [31]. The sequence CAGACA has recently been described as the consensus motif forming the Smad-binding element (SBE), also known as the CAGA box. Among the genes bearing functional SBEs in their promoter regions are plasminogen activator inhibitor-1 (PAI-1) [15,32], JunB [16], collagen type VII [17], and Smad 7 [18] all of them being strongly induced by TGF-b 1 . The reports about functional SBEs in these genes included electrophoretic-mobility-shift assays (EMSAs) with successful supershift studies using anti-Smad Ig. The fact that we were not able to unambiguously prove a participation of Smad proteins in our EMSA by addition of Smad-specific antibodies points towards the involvement of additional nuclear factors which ultimately mediate the transcriptional repression and conceal the antigenic deter- minants of the Smad molecules. Numerous interactions between Smads and other signalling molecules have been reported (reviewed in [33]), therefore a variety of pathways are worth considering for the interaction between Smad proteins and the SeP promoter. For example, in TGF-b 1 - treated human lung cancer cells A549, a recruitment of histone deacetylase by Smad 2 was observed [26]. Histone deacetylation leads to a tighter nucleosomal packing with a general attenuation of the transcription of downstream sequences. It is unclear whether Smad 3 or 4 can act in a comparable fashion in HepG2 cells. The oncoprotein c-Ski also binds to Smad 2/3 and recruits histone deacetylase resulting in suppressed transcription [20,34]. However, antibodies to these potential corepressors are not commer- cially available so that we could not probe their involvement directly using an EMSA approach. Here, we describe the presence of a TGF-b-responsive element in the promoter of the SeP gene. In addition, we found the expression of SeP mRNA being attenuated by overexpression of Smad 3 and 4 which mimics activation of the TbR by TGF-b. We found that Smad 3 and 4, but not Smad 2, confer inhibition of both SeP transcription and promoter activity. This is in agreement with previous studies showing that Smad 3 and 4, unlike Smad 2, bind to the CAGA box [15,24]. In contrast, cotransfection with Smad 2 seems to interfere with the repression exerted by Smad 3 or 4 alone (Figs 3,4). As previous reports have demonstrated the inability of Smad 2 to bind to the CAGA box [15,16], one could speculate that Smad 2 binds transcriptional corepressors without directing them to the CAGA box, thereby reducing the amount of repressor molecules avail- able for Smad 3 and 4 to recruit. Also, overexpression of Smad 4 along with Smad 3 seems to attenuate the inhibitory effect of the latter on SeP mRNA expression (Fig. 3). On the other hand, in the luciferase reporter assays we conducted, Smad 4 did not interfere with the inhibition of luciferase activity by Smad 3 (Fig. 4). However, major differences between the assessment of the SeP mRNA expression and the measurement of luciferase activity exist. For example, whereas in the first case the species assessed is the mRNA (i.e. the respective cDNA) of SeP, the latter assay gauges the activity of an enzyme derived from an unrelated mRNA, namely that of luciferase. If the SeP mRNA would be subject to any post-transcriptional regulation, it would not be reflected by a change in luciferase activity and vice versa. Thus, there is the potential for a different outcome of mRNA expression studies and reporter gene assays. In addition to the identification of Smad 3 and 4 as regulators of SeP expression, we were able to demonstrate that the presence of an intact CAGACA motif is required for the inhibition of SeP promoter activity by TGF-b 1 . It is also noteworthy that disruption of the first CAGA box (transition from BK4GL3 to DBK4GL3) did not lead to an impaired response to TGF-b. Furthermore, the total absence of CAGA boxes in the construct DCAGA renders it insensitive towards ectopical expression of Smad 3 and 4. This suggests a key role of the CAGA box for the inhibition of SeP by Smads as mediators of TGF-b 1 . SeP is the most selenium-rich protein known to date. The fact that SeP is negatively regulated by TGF-b 1 [7] and that SeP levels in intensive care patients suffering from sepsis or systemic inflammatory response syndrome (SIRS) show drastically decreased plasma SeP levels [35] suggests that SeP is regulated in a manner similar to a negative acute- phase protein. In SIRS patients, low plasma selenium levels were scarcely elevated after selenium supplementation [9] indicating that a prime target protein of supplemental selenium could be absent, which would be SeP in plasma because of its large proportion among selenium-containing plasma proteins. Furthermore, patients with major burns exhibit an early and drastic decrease in plasma selenium levels to around 35% of control values [8]. This decrease by about 65% fairly corresponds to the proportion of plasma selenium represented by SeP [36], thus, this effect could principally be attributed to a decrease in SeP. 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Identification of an element within the promoter of human selenoprotein P responsive to transforming growth factor-b Volker Mostert 1 , Sandra Wolff 1 , Ingeborg Dreher 2 , Josef Ko¨ hrle 2 and. SeP against phospholipid hydroperoxides, albeit with low efficiency compared to the selenoenzyme phospholipid hydroperoxide glutathione peroxidase [5]. The 5 0 flanking region of the human SeP gene