Identification of an element within the promoter of humanselenoprotein P responsive to transforming growth factor-b Volker Mostert1, Sandra Wolff1, Ingeborg Dreher2, Josef Ko¨hrle2and Jo
Trang 1Identification of an element within the promoter of human
selenoprotein P responsive to transforming growth factor-b
Volker Mostert1, Sandra Wolff1, Ingeborg Dreher2, Josef Ko¨hrle2and Josef Abel1
1 Medizinisches Institut fu¨r Umwelthygiene an der Heinrich-Heine-Universita¨t Du¨sseldorf Abteilung Experimentelle Toxikologie, Du¨sseldorf, Germany; 2 Medizinische Poliklinik, Universita¨t Wu¨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)-b1 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-b1and to locate the
promoter region critical for this effect As seen in
electro-phoretic-mobility-shift assays, TGF-b1 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-b1treatment Hence, we demonstrated an involvement
of Smad 3 and 4 in transcriptional regulation of SeP by TGF-b1and we were able to identify the TGF-b-responsive element in the SeP promoter
Keywords: selenoprotein P; transforming growth factor-b1; 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 50 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)-b1[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-b1is secreted by activated macrophages [10] and plays an important role in immunomodulation and wound healing [11 – 13], it is possible that TGF-b1-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
50-TG-30-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, 50-TG-30
-interacting factor; TbR, TGF-b receptor; PAI-1, plasminogen activator
inhibitor-1; SIRS, systemic inflammatory response syndrome.
Trang 2The present study investigates whether Smad proteins are
involved in the down-regulation of SeP by TGF-b1 and
explores where the critical TGF-b-responsive element is
located
M A T E R I A L S A N D M E T H O D S
Materials
Recombinant human TGF-b1 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:mL21L-glutamine, and 0.15%
NaHCO3 Cells were maintained under standard conditions
at 37 8C in 5% CO2 Forty-eight hours before treatment,
cells were cultured in RPMI 1640 medium containing 5%
BMS, 1% penicillin/streptomycin, 1 mg:mL21L-glutamine,
and 0.15% NaHCO3and 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/Kpn I – 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 (2 105 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 pMTGF-b1or 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
AGTTGTAGAAAGAAGG-30 (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 mm2 culture flasks were washed once with NaCl/Piand once with NaCl/Picontaining 1 mM
NaVO and 5 mMNaF 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.
Trang 31 mL of ice-cold buffer A (20 mM Hepes pH 7.9, 20 mM
NaF, 1 mM Na3VO4, 1 mM Na4P2O7, 1 mM EDTA, 1 mM
EGTA, 1 mMdithiothreitol, 0.5 mMphenylmethanesulfonyl
fluoride, and 1 mg:mL21of 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 mMNaCl 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
GAA-30; SBE in bold) annealed to its complementary strand
was labelled using T4 polynucleotide kinase (Amersham
Pharmacia, Freiburg, Germany) and [g-32P]ATP Binding
reactions contained 20 mg of nuclear extracts, 20 000 c.p.m
(< 10 fmol) of the labelled probe, 20 mM Hepes pH 7.9,
20 mM KCl, 4 mM MgCl2, 0.1 mM EDTA, 20% glycerol,
0.8 mM NaPi, 4 mM 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:mL21)
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 Tris/glycine/EDTA
(12.5 mM 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
R E S U L T S
Binding of a nuclear protein to a putative SBE after
treatment with TGF-b1
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
50 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-b1 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 onSeP 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.
Trang 4either 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-b1
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 50 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 pM TGF-b1 Basal promoter activity was not affected by deletion of the CAGA boxes (data not shown), whereas the marked TGF-b1 responsive-ness of the constructs BK4GL3 and DBK4GL3 was not observed with DCAGA (Table 1) demonstrating that the TGF-b1-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-b1 responsiveness is slightly enhanced rather than lowered (Table 1) Apparently, the first CAGA box is not required for the down-regulation by TGF-b1 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-b1 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-b1
D I S C U S S I O N The inhibition by TGF-b1of SeP expression is to date the only known regulation of this protein by a factor other than the availability of selenium TGF-b1has 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 processes as TGF-b1-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-b1 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
Trang 5has 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-b1 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-b1
-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-b1 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-b1
SeP is the most selenium-rich protein known to date The fact that SeP is negatively regulated by TGF-b1[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 As TGF-b1 plays an important role in regulation of inflammatory processes as well as in wound healing, an involvement of the signalling pathway unveiled in our study appears likely
A C K N O W L E D G E M E N T
S.W was supported by the Deutsche Forschungsgemeinschaft SFB 503-A5.
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