BACKGROUND
gene zfp <dig> encodes for tristetraprolin .
the gene product is the prototype of the tandem ccch zinc finger protein family, called tis <dig>  which includes four structurally and sequentially related proteins - ttp, brf- <dig>  brf- <dig> and zfp36l <dig>  <cit> .
tis <dig> family members feature a conserved non-typical tandem zinc finger domain that mediates its interaction with target rna species  <cit> .
ttp is the most thoroughly described member of the tis <dig> family, and has been identified as a nucleo-cytoplasmic protein that specifically binds mrnas containing adenine/uridine-rich elements  in their 3'-utrs  and directs them to exosome- or p-body-mediated degradation  <cit> .
genome-wide experiments have revealed many potential targets for ttp-mediated degradation.
a more direct approach confirmed that ttp interacts with transcripts encoding for a number of cytokines , pro-inflammatory factors , proteins which have important roles in breast cancer invasion and metastasis , immediate-early response proteins like c-fos and tristetraprolin itself  <cit> .
the activity of ttp is regulated predominantly via its phosphorylation by map kinases , kinases downstream of mapks  or akt in response to pro-inflammatory cytokines , lps and anti-inflammatory factors .
the ttp protein sequence contains several key serine/threonine residues, the modification of which was previously shown to determine the ability of ttp to recruit the mrna-degradation machinery, bind transcripts, be shuttled to p-bodies or stress granules and its nuclear/cytoplasmic localization and protein stability  <cit> .
less is known about the regulation of ttp expression.
both mrna and protein are induced rapidly after stimulation of the cells with pro- and anti-inflammatory factors   <cit> .
the structure of the murine zfp <dig> gene promoter has been characterized, with the identification of a conserved proximal egr- <dig> transcription factor binding site, ap <dig>  sp <dig>  ttp promoter element  <dig>  stat <dig> and smad biding sites and a functional gas element.
the first intron has also been shown to play a key role in mitogen-induced expression of zfp <dig>  <cit> .
the elk- <dig> transcription factor is a representative member of ets protein family characterized by the presence of the evolutionary conserved ets domain stabilized by three key tryptophan residues and responsible for the interaction with dna  <cit> .
the elk- <dig> domain structure includes an ets domain  at the n-terminus, box b domain in the middle part of the sequence and c-terminal transactivation domain .
box b is also found in other members of tcf  subfamily  and is relevant for the formation of ternary complex with srf  on sres   <cit> .
box a is a site of recruitment of the msin3a/hdac <dig> complex, which confers the repressor function of elk- <dig>  <cit> .
hdac- <dig> is recruited to elk- <dig> through the repressive r-motif in sumo-dependent manner.
the phosphorylation of the tad serine/threonine residues is crucial for switching from repression to activation of transcription  <cit> .
the phosphorylation is catalyzed mainly by mapks such as erk1/ <dig> and ser <dig> phosphorylation serves as a hallmark of elk- <dig> activation.
only a handful of the genes directly targeted by elk- <dig> are known.
among them egr- <dig> and fos seem to have the most important function in the regulation of the immediate-early cell response and widen the spectrum of elk-1-regulated genes  <cit> .
the role of elk- <dig> in the regulation of immunological response has also been emphasized  <cit> .
RESULTS
egf regulates tristetraprolin expression in erk1/2-dependent manner
stimulation of the human breast cancer mcf- <dig> cell line with egf resulted in a rapid induction of ttp expression, the maximal effect being observed  <dig> minutes after egf treatment.
the erk1/ <dig> pathway inhibitor, u <dig>  inhibited this process .
in order to confirm the involvement of egf in the activation of erk1/ <dig> in the mcf- <dig> cell line we performed western blot analysis using anti-phospho erk1/ <dig>  anti-phospho p <dig> and anti-phospho jnk antibodies.
we found that egf was a specific activator of erk1/ <dig> phosphorylation in this system .
we were unable to detect the phosphorylation of jnk or p <dig> after egf treatment whereas pma treatment induced phosphorylation of all tested map kinases .
we concluded that egf activates the erk1/ <dig> pathway in the mcf- <dig> cell line and that activation of this pathway resulted in increase of ttp mrna.
regulation of zfp <dig> promoter by egf
we decided to test the hypothesis that erk-mediated expression of ttp is regulated at the promoter level.
we therefore generated a reporter construct containing the zfp <dig> promoter fragment , further abbreviated as zfp <dig>  the promoter fragment included the first exon, intron and the upstream promoter sequence of zfp <dig> gene.
this promoter fragment was activated after egf treatment, and this activation was blocked by the erk1/ <dig> pathway inhibitor, u <dig> .
elk- <dig>  a well-characterized substrate of erk1/ <dig>  is phosphorylated on ser <dig> after erk1/ <dig> activation.
we therefore investigated elk- <dig> ser <dig> phosphorylation status upon egf treatment, and observed an increase after  <dig> minutes of egf stimulation .
as all canonical mapk are capable of phosphorylating elk- <dig> on ser <dig>  we found that inhibition of erk1/ <dig> using u <dig> abrogated the observed phosphorylation.
we also confirmed the expression of elk- <dig> in mcf- <dig> cells at the protein level, and found that elk- <dig> level was relatively high in comparison to other tested cell lines .
having shown that elk- <dig> is activated by erk1/ <dig> in response to egf stimulation, we checked whether the promoter of zfp <dig> is the target of elk- <dig> regulation.
for this purpose we used the zfp <dig> reporter vector and expression vectors for constitutively active and dominant-negative forms of elk- <dig>  elk-vp <dig> and elk-en, respectively.
due to the presence of strong and non-facultative activation  or repression  domains fused in frame with c-terminus of elk- <dig>  the transcription factor acts independently of mapk activation while preserving the dna binding specificity  <cit> .
zfp <dig> is activated by elk-vp <dig> and repressed by elk-en in a dose-dependent manner .
the intensity of activation of the promoter by elk-vp <dig> is much stronger than the intensity of activation observed after egf treatment.
one possible explanation of this phenomenon may be that vp <dig> domain conjugated to elk- <dig> is more powerful on zfp <dig> promoter than phosphorylated form of elk- <dig>  dose-dependent regulation of activation/repression of zfp <dig> promoter by elk-vp <dig> or elk-en suggests that observed regulation is specific.
in the mock-transfected mcf- <dig> cells, egf stimulation caused an increase in promoter activity, which was completely abolished by the inhibition of erk1/ <dig> .
the transient overexpression of native elk- <dig> did not change the fold of stimulation by egf in mcf- <dig> cell line, which already expresses high levels of endogenous elk- <dig> .
in hek <dig> cell line with very low level of elk- <dig>  the overexpression of this transcription factor restored the responsiveness of zfp <dig> promoter to egf.
in mcf- <dig> as well as elk- <dig> transfected hek <dig> cells the egf-dependent activation of the promoter was blocked by u <dig>  this made us conclude, that elk- <dig> can be engaged in the regulation of zfp <dig> promoter after egf stimulation.
these findings were further supported by the results obtained by qpcr.
in elk-1-limited hek <dig> cells the stimulation of ttp expression by egf is not observed .
sequences in zfp <dig> promoter responsible for elk- <dig> regulation
in order to find sequences involved in the observed regulation of zfp <dig> promoter by elk- <dig> we designed a series of truncation mutants.
deletion of two regions, - <dig> to - <dig> bp and + <dig> to + <dig> bp, has resulted in a substantial decrease in promoter activation by elk-vp <dig> .
the region - <dig> to - <dig> bp contains two ebs sites , namely ebs <dig> and ebs <dig> and one ap- <dig> binding site .
we introduced point-mutations into each of these sites  in the full-length zfp <dig> promoter.
results of these experiments exclude the role of ap- <dig> and ebs <dig> binding sites in the investigated regulation , despite the fact that elk- <dig> can stimulate the expression of both c-fos  and egr- <dig> in mcf- <dig> cell line after egf treatment .
only the mutation of ebs <dig> sequence resulted in 30% decrease in zfp <dig> promoter activation by elk-vp <dig> .
comparison of the sequence of ttp gene in different species revealed the presence of conservative elements in this region .
importance of murine homologue of human ebs <dig> in serum responsiveness was already shown earlier  <cit> .
we have confirmed the involvement of egr- <dig> in the regulation of zfp <dig> promoter by experiments with sirna against egr- <dig>  the knockdown of egr- <dig> in mcf- <dig> cells caused the lack of activation of zfp <dig> promoter by egf .
taken together, we conclude that egr- <dig> by possible interaction with ebs <dig> site can upregulate the activity of zfp <dig> promoter.
the region + <dig> to + <dig> bp contains three ets sequences  which potentially can bind transcription factors from the ets family and ebs <dig> sequence which can potentially interact with egr- <dig> .
we have generated point-mutations of ets <dig>  ets <dig> or ebs <dig> and deletion mutation of ets <dig> in the full-length zfp <dig> promoter .
despite high degree  of conservation of ebs <dig> sequence among analyzed species , its mutation did not influence the activation of zfp <dig> promoter by elk-vp <dig>  also mutation of ets <dig> did not result in decrease of promoter activation.
mutations of ets <dig> and ets <dig> sequences leaded to about 50% reduction of elk-vp16-induced up regulation of zfp <dig> promoter activity .
these results suggest that ets <dig> and ets <dig> can participate in the regulation of zfp <dig> promoter activity by elk- <dig>
since deletions of the regions containing ebs <dig> or ets4/ets <dig>  did not result in a loss of dose-dependent responsiveness to elk-vp <dig> we decided to check whether deletion of both regions  will abolish this regulation.
the results indicate that both investigated regions  are jointly needed for the regulation of zfp <dig>  removing of both of them resulted in a loss of dose-dependent regulation of zfp <dig> promoter by elk-vp <dig> .
to confirm the binding of egr- <dig> to the sequence located - <dig> to - <dig> bp and the binding of elk- <dig> to the sequence located + <dig> to + <dig> bp chromatin immunoprecipitation was performed.
the lysates from mcf- <dig> cells were immunoprecipitated with anti-egr- <dig>  anti-elk- <dig> or nonspecific antibody.
by pcr with primers flanking the investigated sequences, the levels of immunoprecipitated promoter sequences was analyzed.
we have observed increased level of - <dig> to - <dig> bp amplicon after immunoprecipitation with anti-egr- <dig> antibody, in comparison to the level of template immunoprecipitated with anti-elk- <dig> or nonspecific igg .
when the primers flanking the region + <dig> to + <dig> bp were used, we have observed a higher amplification in samples immunoprecipitated with anti-elk- <dig> antibody .
these results made us conclude that in vivo egr- <dig> interacts with promoter sequence at the region - <dig> to - <dig> bp and elk- <dig> interacts with the region + <dig> to + <dig> bp.
discussion
our research focused on the mechanisms of ttp transcript induction by egf in a cellular model of human breast cancer, using mcf- <dig> cell line.
our data indicate that the expression is under a stringent control of erk1/2-dependent pathway.
the activation of zfp <dig> promoter by egf is abolished when erk1/ <dig> pathway inhibitor, u <dig>  is present .
dose-dependent regulation of investigated promoter  by constitutively active  and dominant-negative  forms of elk- <dig>  suggests involvement of this transcription factor in the regulation of ttp transcription .
the dose-dependent activation of the investigated promoter by elk-vp <dig> is lost when both regions of the promoter  are removed .
the region + <dig> to + <dig> bp contains two ets sites  important for the activation of the zfp <dig> promoter by elk-vp <dig>  the sequence of ets <dig> is gcggaa, whereas the most frequent motif recognized by elk- <dig> is ccggaa.
such motif was characterized in egr- <dig>  tr <dig>  pip <dig>  mcl- <dig> and srf promoters  <cit> .
however in other known elk- <dig> target genes modifications of this canonical sequence are present.
in c-fos promoter elk- <dig> binds to caggat, in nur <dig> promoter to gaggaa, in mcpip- <dig> and pai- <dig> to caggaa  <cit> .
in the sequence of ets <dig> , the gga core is preserved but the rest of the sequence is changed in respect to the canonical one .
the ets <dig> and ets <dig> sequences are located in close proximity and the disruption of any of them results in the similar effect , which may suggest that ets <dig> and ets <dig> are in a functional relationship.
a cooperation between proteins from ets family was already described for promoters of stromelisine- <dig> and p <dig>  where two ets- <dig> proteins have to bind to the promoters to obtain their full activation.
recognition of ets sequence through first ets- <dig> molecule and its interaction with the second ets- <dig> molecule results in conformational changes and formation of complex with dna.
this type of interaction between proteins and dna enhances the affinity of the second transcription factor, even to the sequence which is not canonical  <cit> .
it could not be excluded that elk- <dig> forms a complex with other protein from ets family on the zfp <dig> promoter, especially that such complexes were described on other promoters  <cit> .
for instance, the formation of heterodimer between ets- <dig> and elk- <dig> is a key step in the regulation of dpp-iii gene expression.
the c-terminal domain of elk- <dig> and the n-terminal domain of ets- <dig> are engaged in this interaction so that ets sequences participating in this process have to be arranged in the orientation "head to tail".
such orientation of ets <dig> and ets <dig> is present in zfp <dig> promoter which rises the possibility of such heterodimer formation on the investigated promoter.
ebs <dig> located at - <dig> to - <dig> bp turned out to be another sequence important for the regulation of human zfp <dig> promoter by elk- <dig> .
murine ebs <dig> homologue was already shown to play a role in the regulation of zfp <dig> promoter after serum stimulation  <cit> .
elk- <dig> activates zfp <dig> promoter through ebs <dig> indirectly by stimulation of egr- <dig> transcription which in turn binds to ebs <dig>  knockdown of egr- <dig> in mcf- <dig> cells abrogates the activation of zfp <dig> promoter by egf .
two other investigated ebs sequences  do not take part in the regulation of zfp <dig> promoter by egf.
also ap- <dig> binding site, despite activation of c-fos by egf in mcf- <dig> cell line , is not important for the activation of ttp promoter by egf.
lai et al  <cit>  described the contribution of ebs <dig>  ap <dig> and tpe <dig>  to the serum induction of murine zfp <dig> promoter.
despite very high degree of conservation of all these elements in human and murine promoter we have detected only the importance of ebs <dig> in the regulation of human zfp <dig> promoter by egf.
we hypothesize that the regions containing ebs <dig> and ets4/ets <dig> are equally important for the stimulation of ttp expression by egf.
removing of both regions resulted in a complete loss of dose-dependent regulation of the promoter by elk-vp <dig>  and point mutations of any of these sites abrogated the egf-dependent promoter activation .
elimination of egr- <dig> from the cells causes the same effect .
neither ebs <dig> nor ets4/ <dig> site is sufficient enough to drive the activation of zfp <dig> promoter alone.
the binding of elk- <dig> and egr- <dig> to zfp <dig> promoter detected by means of chromatin immunoprecipitation confirmed involvement of these transcription factors in the regulation of ttp expression.
CONCLUSIONS
egf regulates zfp <dig> expression through activation of transcription factor elk- <dig>  elk- <dig> binds directly to the zfp <dig> promoter through the sequences localized at +  <dig> to + <dig> bp.
elk- <dig> induces also the expression of another transcription factor egr- <dig> which as well binds to the zfp <dig> promoter to the sequence at - <dig> to - <dig> bp .
ttp was shown to negatively modulate a number of factors connected with mammary gland tumor progression.
among them il- <dig>  cox- <dig>  c-fos, urokinase, urokinase receptor, metalloproteinase- <dig> can be pointed out and notably, all of them are down-modulated at their mrna level by tristetraprolin  <cit> .
our results demonstrate that the expression of zfp <dig> is stimulated by egf.
the results show complex influence of egf on the development of breast cancer.
egf is well-known as a factor which promotes tumor growth and survival.
this growth factor is able to induce heterodimerization between her-2/neu  and its exclusive receptor c-erbb <dig>  elevated levels of c-erbb <dig> and her- <dig> in breast cancer is correlated with high disease recurrence rates and decreased patient survival  <cit> .
upregulation of ttp expression by egf described in this paper reveals unexpected influence of egf on breast cancer development.
ttp expression is diminished in many cancers and overexpression of ttp in tumors delayed tumor growth and vascularization  <cit> .
thus induction of ttp expression by egf can be classified as a anti-tumor activity of this growth factor.
materials and methods
cell culture
adenocarcinoma cell line mcf- <dig> , human embryonic kidney epithelium cell line hek <dig> , human epithelium cell line hela  and human hepatoma cell line  were cultured at 37°c and 5% co <dig>  mcf- <dig> cells in minimal eagle's medium   supplemented with 10% fbs and bovine insuline , hek <dig>  hela and hepg <dig> in dulbecco's modified eagle's minimum essential medium  with  <dig> g/l d-glucose supplemented with 10% fbs.
reagents and cell stimulation
cells were stimulated with egf  or pma  .
when applied, the inhibitor of mek1/ <dig>  u <dig>   was added to the medium  <dig> min prior stimulation.
pre-designed oligonucleotide silencer sirna targeted to egr- <dig>  and control one  were supplied by ambion.
rna preparation and northern blot analysis
total rna isolation and northern blot analysis was performed as described previously  <cit> .
plasmid constructs
pef1/myc-his/lacz is a control vector containing the gene for β-galactosidase .
pelk-vp <dig>  pelk-en were described before  <cit> .
pzfp <dig> containing human zfp <dig> promoter fragment  was generated by two step pcr, using total dna isolated from mcf- <dig> cells.
the first round pcr was carried out with the primer forward: 5' gtcttccctcccttcctcac 3' and reverse 5' gtcagggctcagcgacag 3'.
then the second round was performed with nested primers introducing saci and bglii restriction sites: forward: 5' tcgagctcttcctcaccctgtctatc 3' and reverse: 5' tcagatctcaggaggctctgcggaaatg 3'.
the introduced restriction sites were used during cloning of pcr product to pgl2-basic reporter vector .
the set of deletion mutants was prepared using pzfp <dig> as a template.
the following forward primers with restriction site for saci or nhei were used in pcr reactions:
f1: 5' gctagccagtttccttctacaagcctcag 3'
f <dig>  r5: 5' gctagccgcgtccgggaag 3'
f5: 5' gagctcgcgtccgggaagc 3'
f6: 5' gagctcggccccggccccgg 3
the set of promoter constructs lacking different 3'-terminal sequences were generated with the following reverse primers:
r01: 5' tcagatctggaactagagcttgccag 3'
r <dig>  r5: 5' tcagatctagagttggaggttctgag 3'
r1: 5' cttaagcacgcgtcgggatctc 3'
r2: 5' cttaagtttgagcgaagagccgggtg 3'
r <dig>  f <dig>  f5: 5' gctagcctcgtagatggcagtcag 3'
the constructs containing mutations in selected transcription factors' binding sites were generated using quikchange xl site-directed mutagenesis kit  according to the manufacturer's procedure.
the sequences were changed as follows:
ebs2: cccccacccc to cccccagctg
ebs3: ccgggggcg to ccatgggcg
ap-1: caagcctcag to caagccatgg
ets3: atccggaa to tgcagcca
ets4: acccatttcc to acccatggcc
ebs6: gggtggggcg to gggtaccgcg
the point mutant ets <dig> was created by means of deletion mutation after a pcr reaction performed with a reverse primer 5' tcagatcttgcggaaatgggtcggt 3'.
reporter gene assay
transient transfection experiments were carried out using lipofectamine  <dig> reagent  in 12-well plate.
total amount of  <dig>  μg of dna per each well was used, including  <dig>  μg of reporter vectors with zfp <dig> promoter fragments and  <dig> ng of pef1/myc-his/lacz.
for some experiments indicated amounts of pelk-vp <dig> or pelk-en were used.
the amount of dna per well was equalized using mock dna .
luciferase assays were carried out using the dual light reporter gene assay system  according to the manufacturer's procedure.
luciferase activity was measured  <dig> hrs after transfection or at indicated time point after stimulation.
β-galactosidase activity was measured to normalize the efficiency of transfection.
all experiments were repeated at least three times in duplicates.
western blot
western blot was carried out using immobilon western chemiluminescent hrp substrate  and anti-phospho erk1/ <dig> , anti-elk- <dig> , anti- phospho p <dig> , anti-phospho jnk , anti-c-fos , anti-egr- <dig> , anti-gapdh , anti-phospho ser <dig> elk- <dig> , anti-erk, anti-p <dig>  anti-jnk .
sirna/dna co-transfection
was carried out using lipofectamine  <dig> reagent  in 12-well plate.
total amount of  <dig> ng plasmid dna and  <dig> pmol sirna oligonucleotides per each well was used.
chromatin immunoprecipitation assay
chromatin immunoprecipitation was carried out as described before  <cit>  using anti-elk- <dig>  anti-egr- <dig>  and nonspecific igg .
following primers forward: acctccaactctgggttcct and reverse: gactcagtccctccatggtc for fragment containing ets4/ <dig> sites and forward: cggaagggaaccagtccag and reverse: agagtgggagcgctgaagt for fragment containing ebs <dig> binding site were used.
authors' contributions
mf, pt, ab, as constructs generation, reporter gene assays.
as qpcr.
pt, jk, mw, ks western blots.
ak chromatin immunoprecipitation, conception and interpretation of data.
mf, pt, ak writing of manuscript.
all authors drafted, read and approved the manuscript.

 gene zfp encodes for tristetraprolin. the gene product is the prototype of the tandem ccch zinc finger protein family, called tis which includes four structurally and sequentially related proteins - ttp, brf- brf- and zfp36l. tis family members feature a conserved non-typical tandem zinc finger domain that mediates its interaction with target rna species. ttp is the most thoroughly described member of the tis family, and has been identified as a nucleo-cytoplasmic protein that specifically binds mrnas containing adenine/uridine-rich elements in their 3'-utrs and directs them to exosome- or p-body-mediated degradation. genome-wide experiments have revealed many potential targets for ttp-mediated degradation. a more direct approach confirmed that ttp interacts with transcripts encoding for a number of cytokines, pro-inflammatory factors, proteins which have important roles in breast cancer invasion and metastasis, immediate-early response proteins like c-fos and tristetraprolin itself. the activity of ttp is regulated predominantly via its phosphorylation by map kinases, kinases downstream of mapks or akt in response to pro-inflammatory cytokines, lps and anti-inflammatory factors. the ttp protein sequence contains several key serine/threonine residues, the modification of which was previously shown to determine the ability of ttp to recruit the mrna-degradation machinery, bind transcripts, be shuttled to p-bodies or stress granules and its nuclear/cytoplasmic localization and protein stability. less is known about the regulation of ttp expression. both mrna and protein are induced rapidly after stimulation of the cells with pro- and anti-inflammatory factors. the structure of the murine zfp gene promoter has been characterized, with the identification of a conserved proximal egr- transcription factor binding site, ap sp ttp promoter element stat and smad biding sites and a functional gas element. the first intron has also been shown to play a key role in mitogen-induced expression of zfp. the elk- transcription factor is a representative member of ets protein family characterized by the presence of the evolutionary conserved ets domain stabilized by three key tryptophan residues and responsible for the interaction with dna. the elk- domain structure includes an ets domain at the n-terminus, box b domain in the middle part of the sequence and c-terminal transactivation domain. box b is also found in other members of tcf subfamily and is relevant for the formation of ternary complex with srf on sres. box a is a site of recruitment of the msin3a/hdac complex, which confers the repressor function of elk-. hdac- is recruited to elk- through the repressive r-motif in sumo-dependent manner. the phosphorylation of the tad serine/threonine residues is crucial for switching from repression to activation of transcription. the phosphorylation is catalyzed mainly by mapks such as erk1/ and ser phosphorylation serves as a hallmark of elk- activation. only a handful of the genes directly targeted by elk- are known. among them egr- and fos seem to have the most important function in the regulation of the immediate-early cell response and widen the spectrum of elk-1-regulated genes. the role of elk- in the regulation of immunological response has also been emphasized.  egf regulates tristetraprolin expression in erk1/2-dependent manner stimulation of the human breast cancer mcf- cell line with egf resulted in a rapid induction of ttp expression, the maximal effect being observed minutes after egf treatment. the erk1/ pathway inhibitor, u inhibited this process. in order to confirm the involvement of egf in the activation of erk1/ in the mcf- cell line we performed western blot analysis using anti-phospho erk1/ anti-phospho p and anti-phospho jnk antibodies. we found that egf was a specific activator of erk1/ phosphorylation in this system. we were unable to detect the phosphorylation of jnk or p after egf treatment whereas pma treatment induced phosphorylation of all tested map kinases. we concluded that egf activates the erk1/ pathway in the mcf- cell line and that activation of this pathway resulted in increase of ttp mrna. regulation of zfp promoter by egf we decided to test the hypothesis that erk-mediated expression of ttp is regulated at the promoter level. we therefore generated a reporter construct containing the zfp promoter fragment, further abbreviated as zfp the promoter fragment included the first exon, intron and the upstream promoter sequence of zfp gene. this promoter fragment was activated after egf treatment, and this activation was blocked by the erk1/ pathway inhibitor, u. elk- a well-characterized substrate of erk1/ is phosphorylated on ser after erk1/ activation. we therefore investigated elk- ser phosphorylation status upon egf treatment, and observed an increase after minutes of egf stimulation. as all canonical mapk are capable of phosphorylating elk- on ser we found that inhibition of erk1/ using u abrogated the observed phosphorylation. we also confirmed the expression of elk- in mcf- cells at the protein level, and found that elk- level was relatively high in comparison to other tested cell lines. having shown that elk- is activated by erk1/ in response to egf stimulation, we checked whether the promoter of zfp is the target of elk- regulation. for this purpose we used the zfp reporter vector and expression vectors for constitutively active and dominant-negative forms of elk- elk-vp and elk-en, respectively. due to the presence of strong and non-facultative activation or repression domains fused in frame with c-terminus of elk- the transcription factor acts independently of mapk activation while preserving the dna binding specificity. zfp is activated by elk-vp and repressed by elk-en in a dose-dependent manner. the intensity of activation of the promoter by elk-vp is much stronger than the intensity of activation observed after egf treatment. one possible explanation of this phenomenon may be that vp domain conjugated to elk- is more powerful on zfp promoter than phosphorylated form of elk- dose-dependent regulation of activation/repression of zfp promoter by elk-vp or elk-en suggests that observed regulation is specific. in the mock-transfected mcf- cells, egf stimulation caused an increase in promoter activity, which was completely abolished by the inhibition of erk1/. the transient overexpression of native elk- did not change the fold of stimulation by egf in mcf- cell line, which already expresses high levels of endogenous elk-. in hek cell line with very low level of elk- the overexpression of this transcription factor restored the responsiveness of zfp promoter to egf. in mcf- as well as elk- transfected hek cells the egf-dependent activation of the promoter was blocked by u this made us conclude, that elk- can be engaged in the regulation of zfp promoter after egf stimulation. these findings were further supported by the results obtained by qpcr. in elk-1-limited hek cells the stimulation of ttp expression by egf is not observed. sequences in zfp promoter responsible for elk- regulation in order to find sequences involved in the observed regulation of zfp promoter by elk- we designed a series of truncation mutants. deletion of two regions, - to - bp and + to + bp, has resulted in a substantial decrease in promoter activation by elk-vp. the region - to - bp contains two ebs sites, namely ebs and ebs and one ap- binding site. we introduced point-mutations into each of these sites in the full-length zfp promoter. results of these experiments exclude the role of ap- and ebs binding sites in the investigated regulation, despite the fact that elk- can stimulate the expression of both c-fos and egr- in mcf- cell line after egf treatment. only the mutation of ebs sequence resulted in 30% decrease in zfp promoter activation by elk-vp. comparison of the sequence of ttp gene in different species revealed the presence of conservative elements in this region. importance of murine homologue of human ebs in serum responsiveness was already shown earlier. we have confirmed the involvement of egr- in the regulation of zfp promoter by experiments with sirna against egr- the knockdown of egr- in mcf- cells caused the lack of activation of zfp promoter by egf. taken together, we conclude that egr- by possible interaction with ebs site can upregulate the activity of zfp promoter. the region + to + bp contains three ets sequences which potentially can bind transcription factors from the ets family and ebs sequence which can potentially interact with egr-. we have generated point-mutations of ets ets or ebs and deletion mutation of ets in the full-length zfp promoter. despite high degree of conservation of ebs sequence among analyzed species, its mutation did not influence the activation of zfp promoter by elk-vp also mutation of ets did not result in decrease of promoter activation. mutations of ets and ets sequences leaded to about 50% reduction of elk-vp16-induced up regulation of zfp promoter activity. these results suggest that ets and ets can participate in the regulation of zfp promoter activity by elk- since deletions of the regions containing ebs or ets4/ets did not result in a loss of dose-dependent responsiveness to elk-vp we decided to check whether deletion of both regions will abolish this regulation. the results indicate that both investigated regions are jointly needed for the regulation of zfp removing of both of them resulted in a loss of dose-dependent regulation of zfp promoter by elk-vp. to confirm the binding of egr- to the sequence located - to - bp and the binding of elk- to the sequence located + to + bp chromatin immunoprecipitation was performed. the lysates from mcf- cells were immunoprecipitated with anti-egr- anti-elk- or nonspecific antibody. by pcr with primers flanking the investigated sequences, the levels of immunoprecipitated promoter sequences was analyzed. we have observed increased level of - to - bp amplicon after immunoprecipitation with anti-egr- antibody, in comparison to the level of template immunoprecipitated with anti-elk- or nonspecific igg. when the primers flanking the region + to + bp were used, we have observed a higher amplification in samples immunoprecipitated with anti-elk- antibody. these results made us conclude that in vivo egr- interacts with promoter sequence at the region - to - bp and elk- interacts with the region + to + bp. discussion our research focused on the mechanisms of ttp transcript induction by egf in a cellular model of human breast cancer, using mcf- cell line. our data indicate that the expression is under a stringent control of erk1/2-dependent pathway. the activation of zfp promoter by egf is abolished when erk1/ pathway inhibitor, u is present. dose-dependent regulation of investigated promoter by constitutively active and dominant-negative forms of elk- suggests involvement of this transcription factor in the regulation of ttp transcription. the dose-dependent activation of the investigated promoter by elk-vp is lost when both regions of the promoter are removed. the region + to + bp contains two ets sites important for the activation of the zfp promoter by elk-vp the sequence of ets is gcggaa, whereas the most frequent motif recognized by elk- is ccggaa. such motif was characterized in egr- tr pip mcl- and srf promoters. however in other known elk- target genes modifications of this canonical sequence are present. in c-fos promoter elk- binds to caggat, in nur promoter to gaggaa, in mcpip- and pai- to caggaa. in the sequence of ets, the gga core is preserved but the rest of the sequence is changed in respect to the canonical one. the ets and ets sequences are located in close proximity and the disruption of any of them results in the similar effect, which may suggest that ets and ets are in a functional relationship. a cooperation between proteins from ets family was already described for promoters of stromelisine- and p where two ets- proteins have to bind to the promoters to obtain their full activation. recognition of ets sequence through first ets- molecule and its interaction with the second ets- molecule results in conformational changes and formation of complex with dna. this type of interaction between proteins and dna enhances the affinity of the second transcription factor, even to the sequence which is not canonical. it could not be excluded that elk- forms a complex with other protein from ets family on the zfp promoter, especially that such complexes were described on other promoters. for instance, the formation of heterodimer between ets- and elk- is a key step in the regulation of dpp-iii gene expression. the c-terminal domain of elk- and the n-terminal domain of ets- are engaged in this interaction so that ets sequences participating in this process have to be arranged in the orientation "head to tail". such orientation of ets and ets is present in zfp promoter which rises the possibility of such heterodimer formation on the investigated promoter. ebs located at - to - bp turned out to be another sequence important for the regulation of human zfp promoter by elk-. murine ebs homologue was already shown to play a role in the regulation of zfp promoter after serum stimulation. elk- activates zfp promoter through ebs indirectly by stimulation of egr- transcription which in turn binds to ebs knockdown of egr- in mcf- cells abrogates the activation of zfp promoter by egf. two other investigated ebs sequences do not take part in the regulation of zfp promoter by egf. also ap- binding site, despite activation of c-fos by egf in mcf- cell line, is not important for the activation of ttp promoter by egf. lai et al described the contribution of ebs ap and tpe to the serum induction of murine zfp promoter. despite very high degree of conservation of all these elements in human and murine promoter we have detected only the importance of ebs in the regulation of human zfp promoter by egf. we hypothesize that the regions containing ebs and ets4/ets are equally important for the stimulation of ttp expression by egf. removing of both regions resulted in a complete loss of dose-dependent regulation of the promoter by elk-vp and point mutations of any of these sites abrogated the egf-dependent promoter activation. elimination of egr- from the cells causes the same effect. neither ebs nor ets4/ site is sufficient enough to drive the activation of zfp promoter alone. the binding of elk- and egr- to zfp promoter detected by means of chromatin immunoprecipitation confirmed involvement of these transcription factors in the regulation of ttp expression.  egf regulates zfp expression through activation of transcription factor elk- elk- binds directly to the zfp promoter through the sequences localized at + to + bp. elk- induces also the expression of another transcription factor egr- which as well binds to the zfp promoter to the sequence at - to - bp. ttp was shown to negatively modulate a number of factors connected with mammary gland tumor progression. among them il- cox- c-fos, urokinase, urokinase receptor, metalloproteinase- can be pointed out and notably, all of them are down-modulated at their mrna level by tristetraprolin. our results demonstrate that the expression of zfp is stimulated by egf. the results show complex influence of egf on the development of breast cancer. egf is well-known as a factor which promotes tumor growth and survival. this growth factor is able to induce heterodimerization between her-2/neu and its exclusive receptor c-erbb elevated levels of c-erbb and her- in breast cancer is correlated with high disease recurrence rates and decreased patient survival. upregulation of ttp expression by egf described in this paper reveals unexpected influence of egf on breast cancer development. ttp expression is diminished in many cancers and overexpression of ttp in tumors delayed tumor growth and vascularization. thus induction of ttp expression by egf can be classified as a anti-tumor activity of this growth factor. materials and methods cell culture adenocarcinoma cell line mcf-, human embryonic kidney epithelium cell line hek, human epithelium cell line hela and human hepatoma cell line were cultured at 37°c and 5% co mcf- cells in minimal eagle's medium supplemented with 10% fbs and bovine insuline, hek hela and hepg in dulbecco's modified eagle's minimum essential medium with g/l d-glucose supplemented with 10% fbs. reagents and cell stimulation cells were stimulated with egf or pma. when applied, the inhibitor of mek1/ u was added to the medium min prior stimulation. pre-designed oligonucleotide silencer sirna targeted to egr- and control one were supplied by ambion. rna preparation and northern blot analysis total rna isolation and northern blot analysis was performed as described previously. plasmid constructs pef1/myc-his/lacz is a control vector containing the gene for β-galactosidase. pelk-vp pelk-en were described before. pzfp containing human zfp promoter fragment was generated by two step pcr, using total dna isolated from mcf- cells. the first round pcr was carried out with the primer forward: 5' gtcttccctcccttcctcac 3' and reverse 5' gtcagggctcagcgacag 3'. then the second round was performed with nested primers introducing saci and bglii restriction sites: forward: 5' tcgagctcttcctcaccctgtctatc 3' and reverse: 5' tcagatctcaggaggctctgcggaaatg 3'. the introduced restriction sites were used during cloning of pcr product to pgl2-basic reporter vector. the set of deletion mutants was prepared using pzfp as a template. the following forward primers with restriction site for saci or nhei were used in pcr reactions: f1: 5' gctagccagtttccttctacaagcctcag 3' f r5: 5' gctagccgcgtccgggaag 3' f5: 5' gagctcgcgtccgggaagc 3' f6: 5' gagctcggccccggccccgg 3 the set of promoter constructs lacking different 3'-terminal sequences were generated with the following reverse primers: r01: 5' tcagatctggaactagagcttgccag 3' r r5: 5' tcagatctagagttggaggttctgag 3' r1: 5' cttaagcacgcgtcgggatctc 3' r2: 5' cttaagtttgagcgaagagccgggtg 3' r f f5: 5' gctagcctcgtagatggcagtcag 3' the constructs containing mutations in selected transcription factors' binding sites were generated using quikchange xl site-directed mutagenesis kit according to the manufacturer's procedure. the sequences were changed as follows: ebs2: cccccacccc to cccccagctg ebs3: ccgggggcg to ccatgggcg ap-1: caagcctcag to caagccatgg ets3: atccggaa to tgcagcca ets4: acccatttcc to acccatggcc ebs6: gggtggggcg to gggtaccgcg the point mutant ets was created by means of deletion mutation after a pcr reaction performed with a reverse primer 5' tcagatcttgcggaaatgggtcggt 3'. reporter gene assay transient transfection experiments were carried out using lipofectamine reagent in 12-well plate. total amount of μg of dna per each well was used, including μg of reporter vectors with zfp promoter fragments and ng of pef1/myc-his/lacz. for some experiments indicated amounts of pelk-vp or pelk-en were used. the amount of dna per well was equalized using mock dna. luciferase assays were carried out using the dual light reporter gene assay system according to the manufacturer's procedure. luciferase activity was measured hrs after transfection or at indicated time point after stimulation. β-galactosidase activity was measured to normalize the efficiency of transfection. all experiments were repeated at least three times in duplicates. western blot western blot was carried out using immobilon western chemiluminescent hrp substrate and anti-phospho erk1/, anti-elk-, anti- phospho p, anti-phospho jnk, anti-c-fos, anti-egr-, anti-gapdh, anti-phospho ser elk-, anti-erk, anti-p anti-jnk. sirna/dna co-transfection was carried out using lipofectamine reagent in 12-well plate. total amount of ng plasmid dna and pmol sirna oligonucleotides per each well was used. chromatin immunoprecipitation assay chromatin immunoprecipitation was carried out as described before using anti-elk- anti-egr- and nonspecific igg. following primers forward: acctccaactctgggttcct and reverse: gactcagtccctccatggtc for fragment containing ets4/ sites and forward: cggaagggaaccagtccag and reverse: agagtgggagcgctgaagt for fragment containing ebs binding site were used. authors' contributions mf, pt, ab, as constructs generation, reporter gene assays. as qpcr. pt, jk, mw, ks western blots. ak chromatin immunoprecipitation, conception and interpretation of data. mf, pt, ak writing of manuscript. all authors drafted, read and approved the manuscript. 
so far the contribution of p mapk to the regulation of ttp expression and function has been best described. egf activates ttp expression via elk- and egr- transcription factors. transcripts modulated by ttp encode proteins such as cytokines, pro-inflammatory agents and immediate-early response proteins. our results demonstrate the induction of the gene coding ttp by egf through the erk1/2-dependent pathway and implicates the transcription factor elk- in this process.
 tristetraprolin is a key mediator of processes such as inflammation resolution, the inhibition of autoimmunity and in cancer. it carries out this role by the binding and degradation of mrna transcripts, thereby decreasing their half-life. transcripts modulated by ttp encode proteins such as cytokines, pro-inflammatory agents and immediate-early response proteins. ttp can also modulate neoplastic phenotypes in many cancers. ttp is induced and functionally regulated by a spectrum of both pro- and anti-inflammatory cytokines, mitogens and drugs in a mapk-dependent manner. so far the contribution of p mapk to the regulation of ttp expression and function has been best described.  our results demonstrate the induction of the gene coding ttp by egf through the erk1/2-dependent pathway and implicates the transcription factor elk- in this process. we show that elk- regulates zfp expression by two mechanisms: by binding the zfp promoter directly through ets-binding site and by inducing expression of egr- which in turn increases zfp expression through sequences located between - and - bp.  egf activates ttp expression via elk- and egr- transcription factors. 