cell culture  tissue culture reagents were purchased from 
  commercial sources ( invitrogen ) .
the human pancreatic epithelial cancer cell 
  lines , panc-1 , aspc-1 , capan-1 , capan-2 , bxpc-3 , l3.6 , miapaca-2 , and cfpac-1 , 
  were obtained from american type culture collection and maintained according 
  to the supplier 's suggestions .
all cells were grown at 37 c in a 
  humidified incubator under 5% co2 .
plasmid construction  standard molecular biology techniques 
  were used to clone klf4 , klf5 , klf7 , klf9 , klf11 , klf14 , and 
  klf15 into the pcdna3.1/his ( invitrogen ) and pcmvtag2 ( stratagene ) 
  vectors for expression as his - tagged or flag - tagged proteins , respectively , as 
  well as the truncated ( 263 bp ) tgfrii promoter into 
  the pgl3-lux vector ( promega ) . the p3tp - lux reporter plasmid containing 
  tgf-responsive elements
anita roberts 
  ( national institutes of health ) as a positive control for tgf1 
  stimulation experiments ( data not shown ) .
semi - quantitative rt - pcr  total rna was extracted from cells 
  according to the manufacturer 's instructions using an rneasy kit ( qiagen ) , and 
  5 g was used for cdna synthesis using oligo(dt ) primer using the 
  superscript iii first - strand synthesis system for rt - pcr ( invitrogen ) 
  per the manufacturer 's protocol .
rt - pcr was performed using la taqdna 
  polymerase with a gc buffers kit ( takara ) per the manufacturer protocols . 
 
semiquantitative rt - pcr analysis was performed with the primer sets provided 
  in supplemental table s1 .
amplification of four human housekeeping genes , gapdh ( unigene 
  hs.544577 ) , 2-microglobulin ( b2 m , hs.709313 ) , 
  2-tubulin ( tubb2 , hs.300701 ) , and hypoxanthine 
  phosphoribosyltransferase 1 ( hprt1 , hs.412707 ) was used for all 
  samples as an internal control .
densitometric values were obtained and 
  normalized to the average of the housekeeping cdnas for each individual sample 
  using scion image beta 4.02 software ( scion corp . ) . to represent the tgf inducibility of individual klf 
  transcripts ,
each tgf-treated sample was compared with an untreated 
  control that was originally plated and ultimately collected at the same time 
  to minimize compounding factors , which can often influence gene expression 
  ( i.e. cell cycle stage , cell density , potential unknown paracrine , 
  and/or autocrine stimuli ) . at each time point , values were determined by first 
  normalizing the tgf-treated sample ( t , treated ) to the average of the 
  four aforementioned housekeeping genes in the same sample ( tc , treated 
  housekeeping gene control ) .
this resultant value was divided by the 
  corresponding untreated sample ( ut , untreated ) normalized in the same manner 
  ( utc , untreated housekeeping gene control ) , to express the fold of tgf 
  induction ( [ t / tc]/[ut / utc ] = fold tgf-induction ) . another housekeeping 
  gene , -actin ( actb , hs.520640 ) , was not used in our analyses , 
  because it showed significant differences with tgf treatment over 
  control values ( data not shown ) .
total protein extracts were prepared by lysing 
  cells in radioimmune precipitation assay buffer supplemented with complete 
  protease inhibitor mixture ( roche applied science ) .
cellular lysates were 
  subjected to 10% sds - page and then separated proteins are transferred to 
  polyvinylidene difluoride membranes ( millipore ) .
membranes were incubated 
  overnight at 4 c in blocking solution ( tris - buffered saline solution 
  containing 5% nonfat dried milk and 0.1% tween 20 ) .
immune 
  complexes were visualized by enhanced chemiluminescence ( pierce ) and exposed 
  to x - ray film .
transcriptional reporter assays  cells were transfected with 
  specified reporter constructs along with expression constructs and/or empty 
  vector using electroporation ( 2  10 cells/0.4-cm 
  microcuvette , 360 v , and 10 ms ) and subsequently serum - starved overnight . 
 
transfection was performed with equimolar concentrations of dna , and 
  expression was quantified with western blotting directed against 
  epitope - tagged proteins as described .
cells were stimulated with tgf1 
  ( r&d systems ) as specified and assayed at various specified time points . 
  at 24 or 48 h after transfection and treatment as noted ,
cells were lysed , and 
  luciferase measurements were performed using a 20/20 luminometer ( turner 
  designs ) according to manufacturer 's suggestions ( promega ) .
data were 
  normalized as relative light units and normalized to the protein concentration 
  as the mean  s.d .
cells were transfected with flag - tagged 
  constructs . at 24 h post - transfection , cells were washed and lysed in lysis 
  buffer ( 150 mm nacl , 0.5% nonidet p-40 , 50 mm tris - hcl , 
  ph 7.5 , 20 mm mgcl2 ) supplemented with complete protease 
  inhibitor tablets ( roche applied science ) for 30 min at 4 c . 
 
immunoprecipitations were performed using anti - flag m2 agarose - conjugated 
  antibodies ( sigma ) for 2 h at 4 c . to detect interaction with endogenous 
  co - repressors ,
immunocomplexes were collected by centrifugation , washed with 
  lysis buffer , and analyzed by western blot as described above using 
  anti - msin3a and hdac2 antibodies ( santa cruz biotechnology ) .
the following primer set for the 263-bp tgfrii 
  promoter was used for pcr : 5-gca gat gtt ctg atc tac ta-3 
  ( forward ) ; 5-agc tgg gca gga cct ctc tc-3 ( reverse ) using takara 
  la taq according to the manufacturer 's protocol ( mirus ) .
site - directed mutagenesis was 
  performed with quikchange ii site - directed mutagenesis kits per the 
  manufacturer 's protocol ( stratagene ) .
all constructs were sequenced by the 
  mayo clinic molecular biology core facility . gst fusion 
the klf14 cdna fragment encoding amino 
  acids 191323 corresponding to the dna - binding zinc finger region was 
  cloned into the gst fusion vector pgex 5x-1 ( amersham biosciences ) using 
  standard techniques .
gst fusion protein expression was induced in bl21 cells 
  ( stratagene ) by the addition of 1 mm 
  isopropyl - d - thiogalactopyranoside and incubation for 2 h. cells 
  were lysed and subsequently purified by using glutathione - sepharose 4b 
  affinity chromatography as previously described 
  ( 15 ) .
briefly , 1.75 pmol of double - stranded oligonucleotides were end - labeled with 
  [ -p]atp using 10 units of t4 polynucleotide kinase and 
  appropriate buffer ( 700 mm tris - hcl , ph 7.6 , 100 mm 
  mgcl2 , 50 mm dithiothreitol ) according to the 
  manufacturer 's instructions ( promega ) .
the reaction was incubated at 37 c 
  for 10 min and halted with addition of te plus edta ( 0.5 m edta , 1 
  m tris , ph 8.0 , h2o ) .
protein lysates included 0.5 g 
  of purified gst - klf14/zf fusion protein and in some experiments rhsp1 
  ( promega ) at the indicated dilutions .
a 5 zncl2 buffer was 
  used in this reaction ( 100 mm hepes , ph 7.5 , 250 mm kcl , 
  25 mm mgcl2 , 50 m zncl2 , 30% 
  glycerol , 1 mg / ml bovine serum albumin , 250 g / ml poly(di - dc ) , 
  h2o ) for 10 min at room temperature .
the 
  -p - labeled oligonucleotides were added for 20 min . in some 
  cases ,
an excess of cold probe , at the indicated dilutions , was added 
  concomitant with the addition of radiolabeled probe in addition to anti - sp1 
  polyclonal rabbit antibody purchased from commercial sources ( millipore ) .
the 
  mixtures were electrophoresed in a 4% nondenaturing polyacrylamide gel in a 
  hoeffer midi - gel using 0.5 tris borate - edta for 4 h at 160 v. gels 
  were then transferred to blotting paper ( whatman 3 mm ) , covered in plastic 
  wrap , and vacuum dried for 1.5 h at 65 c .
dried gels were then analyzed 
  using a storm scanner 860 phosphorimager ( amersham biosciences ) .
sp1 consensus 
  and sp1 mutant double - stranded oligonucleotides were obtained from commercial 
  sources ( santa cruz biotechnology ) with the following sequences : sp1 
  consensus , 5-att cga tcg ggg cgg ggc gag c-3 ( forward ) ; 
  5-gct cgc ccc gcc ccg atc gaa t-3 ( reverse ) and sp1 mutant , 
  5-att cga tcg gtt cgg ggc gag c-3 ( forward ) ; 5-gct cgc 
  ccc gaa ccg atc gaa t-3 ( reverse ) .
figure 1.a , repression of the tgfrii promoter 
  occurs after 24 h of tgf stimulation in panc1 cells .
panc1 cells were 
  transfected with a full - length and a truncated 263-bp 
  tgfrii promoter luciferase reporter and then 
  stimulated with tgf1 after overnight serum starvation .
luciferase levels 
  were obtained at specified time points after treatment and compared with 
  untreated controls .
data are the mean  s.d . from three 
  independent experiments , with triplicates for each experiment .
 
  binding sites . the diagram represents a schematic of the human 
  tgfrii proximal promoter containing five gc - rich 
  sp1-like elements .
a , repression of the tgfrii promoter 
  occurs after 24 h of tgf stimulation in panc1 cells .
panc1 cells were 
  transfected with a full - length and a truncated 263-bp 
  tgfrii promoter luciferase reporter and then 
  stimulated with tgf1 after overnight serum starvation .
luciferase levels 
  were obtained at specified time points after treatment and compared with 
  untreated controls .
data are the mean  s.d . from three 
  independent experiments , with triplicates for each experiment .
the diagram represents a schematic of the human 
  tgfrii proximal promoter containing five gc - rich 
  sp1-like elements .
a yet undefined sp / klf repressor protein plays a role in silencing of 
  the type ii tgf-receptor promoter  previous studies 
  have described the regulation of the tgfrii by 
  tgf ligands , primarily showing a bimodal response consisting of an 
  sp1-dependent up - regulation 
  ( 14 , 
  17 ) and a subsequent 
  down - regulation of receptor transcript levels upon this stimulation 
  ( 1820 ) . 
 
interestingly , although the activation of tgfrii promoter , in particular 
  by sp1 , has received precise attention , how this receptor is repressed remains 
  poorly understood .
consequently , the major goal of the current study has been 
  to characterize the role of a specific family of non - smad proteins ( sp / klf 
  transcription factors ) , which may functionally explain the down - regulation 
  tgfrii through gc - rich sp1-like sequences .
our studies 
  began with examination of the transcriptional activity of the 
  tgfrii gene promoter in panc1 epithelial cells , a 
  widely used model for studying tgf signaling .
we have performed an 
  initial series of reporter assays using full - length 
  tgfrii and the previously described , 
  tgf-sensitive , 263-bp core promoter , which is located 5 of the 
  transcriptional start site 
  ( 14 ) .
treatment of panc1 cells 
  with exogenous tgf1 leads to a marked reduction in activity of the 
  full - length tgfrii reporter when compared with 
  untreated control cells ( fig . 
 
this silencing effect is recapitulated in the 263-bp 
  tgfrii core promoter , suggesting that both the 
  previously described activation pathway 
  ( 14 ) and the negative 
  transcriptional regulatory mechanism characterized further here 
  ( fig .
1a ) , are 
  operational at this core promoter level . using bioinformatics analyses 
  ( transfac public )
, we have identified five putative sp / klf binding sites 
  within this tgf-sensitive , 263-bp core promoter region 
  ( fig .
four of 
  these sites ( # 14 ) have been previously identified , although an 
  additional site ( # 5 ) has not been previously reported 
  ( 14 , 
  21 ) .
some of these previously 
  identified sites have been shown to be activated by sp1 . however
, whether 
  novel sp / klf silencer proteins can also bind to this sequence to reverse the 
  activation by sp1 remained unknown .
therefore , this analysis has led us to the 
  hypothesis that a yet undefined sp / klf repressor protein plays a role in the 
  silencing of this promoter .
novel tgf-inducible non - smad , sp / klf proteins are 
  identified as candidate regulators of the type ii 
  tgf-receptor to test our hypothesis , we have 
  developed a four - tier screening approach .
this approach includes first , 
  testing which of the 24 known sp / klf proteins are expressed in 
  tgf-sensitive epithelial cells and , thus , can be considered initial 
  candidates to target the tgfrii promoter .
our 
  experimental cell model , panc1 , a human epithelial cell line , is an optimal 
  model for our studies , because they have adequate expression of 
  tgfrii mrna and display growth inhibition to exogenous 
  tgf1 stimulation ( 22 ) . 
 
in addition , as shown in fig . 
  2a , each of the 24 known
sp / klf transcription 
  factors are consistently expressed in these cells , which made it ideal for 
  performing a comprehensive screen .
second , we determine whether any 
  sp / klf genes are tgf-inducible with a kinetic that is 
  consistent with playing a role in the down - regulation of the 
  tgfrii .
third , by utilizing transfection studies 
  combined with reporter assays , we test the potential of distinct members of 
  this family to repress tgfrii promoter activity and 
  then , by electromobility shift assays , determine which sites on the promoter 
  are utilized by our candidate klf protein .
finally , we examine whether the 
  repressor that is isolated according to these criteria binds to the endogenous 
  tgfrii gene and can remodel chromatin on this target , 
  suggesting the bona fide target status of the candidate klf protein .
thus , by 
  applying this comprehensive screening , our study has been robust in evaluating 
  the klf protein family in the tgf response and regulation of the 
  tgfrii . the genomic axiom that functionally related genes follow a similar pattern 
  of expression suggested that , hypothetically , the protein that represses the 
  tgfrii may be expressed in a similar manner after 
  tgf treatment , in particular , during the repression response to this 
  cytokine .
consequently , we have evaluated which , if any , of these 
  sp / klf members are inducible by tgf1 treatment .
thus , using rna 
  from panc1 cells either untreated or treated with tgf1 , we have 
  performed rt - pcr at various time points 
  ( fig . 2b ) . as a 
  positive control for tgf-mediated transcriptional induction
, we monitor 
  the expression of p21 , a known tgf-inducible gene within the 
  pathway . of the 24 known sp / klf transcription factors ,
we have 
  identified 7 that were markedly induced with exogenous tgf1 treatment , 
  suggesting that these could be potential candidate transcriptional repressors 
  of the tgfrii promoter 
  ( fig .
these 
  results are not only consistent with our previous work , which identified 
  klf11 as a tgf-inducible gene 
  ( 16 , 
  23 ) , but , more importantly , it 
  characterizes previously unidentified klf targets for this cascade , 
  namely klf4 , -5 , -7 , -9 , -14 , and -15 .
thus , based upon 
  their expression patterns , these seven genes are good candidates to further 
  investigate their regulation of the tgfrii 
  promoter .
figure 2.a , eight pancreatic cancer cell lines were screened by rt - pcr for 
  expression of 24 members of the sp / klf family of transcription 
  factors .
the figure shows that all 24 sp / klf members are expressed in 
  the tgf-sensitive panc1 cell line .
panc1 cells were 
  serum - starved overnight , treated with 10 ng / ml tgf1 , and screened for 
  the expression of each klf gene by semi - quantitative rt - pcr .
the 
  expression levels for each klf gene from tgf1-treated samples 
  were compared with untreated controls and normalized to housekeeping genes as 
  described under  material and methods . 
the expression of the 
  p21 gene was used as a positive control for tgf1 treatment . 
 
four different housekeeping genes , namely gapdh , b2 m , tubb2 , and 
  hprt1 , were used as internal standards for normalization .
c , 
  quantification of tgf induction of transcript levels over time . at each 
  time point , values were determined by first normalizing the densitometric 
  measurement of the tgf-treated sample to the average of four 
  housekeeping genes ( gapdh , b2 m , tubb2 , and hprt1 ) in the 
  same sample .
this resulting value was divided by the corresponding value of 
  the untreated sample normalized in the same manner in order to express the 
  -fold of tgf induction .
a , eight pancreatic cancer cell lines were screened by rt - pcr for 
  expression of 24 members of the sp / klf family of transcription 
  factors .
the figure shows that all 24 sp / klf members are expressed in 
  the tgf-sensitive panc1 cell line .
panc1 cells were 
  serum - starved overnight , treated with 10 ng / ml tgf1 , and screened for 
  the expression of each klf gene by semi - quantitative rt - pcr .
the 
  expression levels for each klf gene from tgf1-treated samples 
  were compared with untreated controls and normalized to housekeeping genes as 
  described under  material and methods . 
the expression of the 
  p21 gene was used as a positive control for tgf1 treatment . 
 
four different housekeeping genes , namely gapdh , b2 m , tubb2 , and 
  hprt1 , were used as internal standards for normalization .
c , 
  quantification of tgf induction of transcript levels over time . at each 
  time point , values were determined by first normalizing the densitometric 
  measurement of the tgf-treated sample to the average of four 
  housekeeping genes ( gapdh , b2 m , tubb2 , and hprt1 ) in the 
  same sample .
this resulting value was divided by the corresponding value of 
  the untreated sample normalized in the same manner in order to express the 
  -fold of tgf induction .
figure 3.a , panc1 cells were transfected with klf(flag / his ) epitope - tagged 
  expression constructs or control empty vector to test 
  tgfrii promoter activity .
b , panc1 cells were 
  transfected with indicated klf(flag / his ) epitope - tagged expression constructs 
  or control vector and then stimulated with 10 ng / ml tgf1 after overnight 
  serum starvation to test tgfrii promoter activity . 
 
c , panc1 cells were transfected with various concentrations of 
  epitope - tagged klf14(flag ) expression construct or control vector and then 
  stimulated with 10 ng / ml tgf1 after overnight serum starvation to 
  observe the effect on tgfrii promoter activity . 
 
western controls ( flag / his ) were shown for epitope - tagged klf expression in 
  all experiments .
expression of flag - tagged klf4 , klf7 , klf14 , and klf15 was 
  confirmed by an anti - flag antibody ( sigma ) , whereas expression of his - tagged 
  klf5 , klf9 , and klf11 was verified by the omni d8 antibody ( santa cruz 
  biotechnology ) .
data are the mean  s.d . from three independent 
  experiments , with triplicates for each experiment .
a , panc1 cells were transfected with klf(flag / his ) epitope - tagged 
  expression constructs or control empty vector to test 
  tgfrii promoter activity .
b , panc1 cells were 
  transfected with indicated klf(flag / his ) epitope - tagged expression constructs 
  or control vector and then stimulated with 10 ng / ml tgf1 after overnight 
  serum starvation to test tgfrii promoter activity . 
 
c , panc1 cells were transfected with various concentrations of 
  epitope - tagged klf14(flag ) expression construct or control vector and then 
  stimulated with 10 ng / ml tgf1 after overnight serum starvation to 
  observe the effect on tgfrii promoter activity . 
 
western controls ( flag / his ) were shown for epitope - tagged klf expression in 
  all experiments .
expression of flag - tagged klf4 , klf7 , klf14 , and klf15 was 
  confirmed by an anti - flag antibody ( sigma ) , whereas expression of his - tagged 
  klf5 , klf9 , and klf11 was verified by the omni d8 antibody ( santa cruz 
  biotechnology ) .
subsequently , we have tested the transcriptional activity of these 
  candidates on the tgfrii promoter using reporter 
  assays by co - transfecting the 263-bp core tgfrii 
  promoter - luciferase construct with cdnas encoding each of the seven 
  tgf-inducible klf candidates . out of these
, five candidates 
  induced a marked decrease in tgfrii promoter activity , 
  namely klf4 , -7 , -11 , -14 , and -15 ( fig . 
 
klf5 did not affect tgfrii 
  promoter activity above control levels , whereas klf9 appeared to slightly 
  activate this promoter , therefore these two proteins were not continued in 
  subsequent experiments due to our objective of identifying 
  tgfrii repressors . to confirm whether this observed 
  repression of tgfrii promoter regulation is consistent 
  with tgf pathway activation
, these five proteins were further tested in 
  panc1 cells treated with exogenous tgf1 treatment .
interestingly , upon 
  treatment , these klf proteins were capable of further repressing 
  tgfrii promoter activity , with the largest repression 
  achieved by klf14 ( fig . 
 
these data also indicate that a second 
  tgf-dependent mechanism , directly ( signal - induced klf expression or 
  post - translational modifications ) or indirectly ( induction of another 
  transcription factor ) , cooperates with klf proteins to additionally repress 
  this gene . upon increasing concentrations of klf14 cdna in 
  transfection studies
, we found a concentration - dependent repression of the 
  core tgfrii promoter ( fig . 
 
together , these results strongly identify klf14 as a 
  good candidate to be a regulator of tgfrii promoter 
  activity and expression .
klf14 , a novel non - smad protein , regulates the type ii 
  tgf-receptor  the gene encoding klf14 has been 
  previously identified by our group and named bteb5 due to its 
  sequence similarities to members of this subfamily of klf silencing 
  proteins.5 although , 
  recently , genetic studies have reported the genomic structure , intronless 
  nature , and potential imprinted status of this gene 
  ( 24 ) , a functional 
  characterization of this protein at the cellular and biochemical level has 
  never been performed . because our data indicate that klf14 appears to be a 
  potent regulator of the tgfrii in promoter assays 
  combined with this existing gap in knowledge on this klf family member and its 
  targets , the choice to further investigate the role of this particular klf in 
  tgfrii regulation would significantly expand the current knowledge on 
  the functional properties of members of this family .
noteworthy , we have validated these in vitro reporter results 
  in vivo by assessing whether this protein has a regulatory effect on 
  endogenous tgfrii levels in panc1 cells .
initial correlative experiments 
  demonstrate that the levels of tgfrii mrna levels 
  decrease at a time in which the amount of klf14 increases ( repression phase of 
  the bimodal expression pattern of tgfrii in response 
  to tgf ) , raising the possibility that klf14 is induced to 
  subsequently down - regulate the tgfrii 
  ( fig .
mechanistically support this correlation , we have performed rt - pcr on cells 
  overexpressing klf14 to determine tgfrii levels in comparison to mock 
  transfected cells in the presence or absence of exogenous tgf1 
  stimulation ( fig .
4b ) . 
  klf14 overexpression alone is sufficient to decrease tgfrii mrna , 
  interestingly to the same extent as tgf1 stimulation alone . furthermore
, 
  subsequent tgf1 treatment in cells transfected with klf14 leads 
  to further down - regulation of tgfrii transcripts . because klf14 
  is a tgf-inducible gene , the further decrease in 
  tgfrii mrna levels observed upon tgf1 treatment 
  likely results from the induction of endogenous sp / klf transcription 
  factors by this cytokine .
these results suggest that tgf down - regulates 
  tgfrii transcripts through klf14 expression 
  with subsequent negative regulation of promoter activity .
next , we have tested 
  whether the effect of klf14 on tgfrii expression 
  interfered with tgf-induced signals that target downstream genes , such 
  as p21 ( 25 ) .
indeed , 
  as expected , tgf1 treatment increases p21 promoter activity , as 
  observed via reporter assays , as well as its mrna levels 
  ( fig .
4 , c and 
  d ) , whereas klf14 reduces both p21 promoter 
  activity and mrna levels upon tgf1 treatment 
  ( fig .
these results suggest that klf14 interferes with the 
  activation of downstream tgfsignaling effects , at least in part , by its 
  ability to repress the tgfrii .
figure 4.a , tgfrii and klf14 transcript levels were 
  analyzed by determination of density , and relative transcript levels were 
  represented by the ratio tgfrii cdna / gapdh 
  cdna and klf14 cdna / gapdh cdna .
panc1 cells were transfected 
  with klf14 or control empty vector and then stimulated with 10 ng / ml 
  tgf1 for 24 h after overnight serum starvation .
densitometry was 
  performed after normalizing to the average of gapdh , b2 m , tubb2 , and hprt1 
  levels and untreated control cdna .
c , panc1 cells were transfected 
  with klf14 flag epitope - tagged constructs or control empty vector along with a 
  p21 promoter reporter construct and then stimulated with 10 ng / ml 
  tgf1 after overnight serum starvation .
panc1 cells were transfected with 
  flag - klf14 or control empty vector and then stimulated with 10 ng / ml 
  tgf1 for 24 h after overnight serum starvation .
densitometry was 
  obtained after normalizing to the average of gapdh , b2 m , tubb2 , and hprt1 
  levels and untreated control cdna .
a , tgfrii and klf14 transcript levels were 
  analyzed by determination of density , and relative transcript levels were 
  represented by the ratio tgfrii cdna / gapdh 
  cdna and klf14 cdna / gapdh cdna .
panc1 cells were transfected 
  with klf14 or control empty vector and then stimulated with 10 ng / ml 
  tgf1 for 24 h after overnight serum starvation .
densitometry was 
  performed after normalizing to the average of gapdh , b2 m , tubb2 , and hprt1 
  levels and untreated control cdna .
c , panc1 cells were transfected 
  with klf14 flag epitope - tagged constructs or control empty vector along with a 
  p21 promoter reporter construct and then stimulated with 10 ng / ml 
  tgf1 after overnight serum starvation .
panc1 cells were transfected with 
  flag - klf14 or control empty vector and then stimulated with 10 ng / ml 
  tgf1 for 24 h after overnight serum starvation .
densitometry was 
  obtained after normalizing to the average of gapdh , b2 m , tubb2 , and hprt1 
  levels and untreated control cdna .
klf14 represses the tgfrii promoter via distinct 
  sp1-like gc - rich sequences and competition with sp1repressor klf 
  proteins , such as klf14 , have been previously shown to compete with the 
  canonical sp1 protein for overall transcriptional activity of a promoter , such 
  as the cyp1a1 and ldlr promoters 
  ( 26 , 
  27 ) . therefore , herein we test 
  the ability of klf14 to repress transcription of the 
  tgfrii promoter in the presence of exogenous 
  sp1 expression . indeed , as shown in 
  fig .
5a , the 
  expression of exogenous sp1 relieves the repression mediated by klf14 
  in a dose - dependent manner . due to the direct , sp1-dependent up - regulation of 
  the tgfrii promoter as part of its bimodal response 
  during activation
, we dissect whether the repression of the 
  tgfrii promoter via klf14 is also acting through a 
  direct mechanism on the promoter rather than indirect due to klf14 mediating a 
  secondary effector .
panc1 cells were transfected 
  with klf14(flag ) and sp1(his ) epitope - tagged expression constructs or control 
  empty vector as indicated to evaluate in vivo competition on 
  tgfrii promoter activity .
western control ( flag / his ) 
  is shown for epitope - tagged expression .
b , klf14 
  binds to four of the putative sp1 sites and competes with sp1 on the 
  tgfrii promoter .
electromobility shift assay was 
  performed using klf14-zf gst fusion proteins and radiolabeled oligonucleotides 
  for each of the five putative sp1 sites .
lane 1 : control - sp1 
  consensus oligonucleotide ; lane 2 : control - sp1 mutant 
  oligonucleotide ; lane 3 : sp1 consensus oligonucleotide and rhsp1 
  protein ; lane 4 : wt oligonucleotide ; lane 5 : mut#1 
  oligonucleotide ; lane 6 : mut#2 oligonucleotide ; lane 7 : wt 
  oligonucleotide and 25 cold probe ; lane 8 : wt oligonucleotide 
  and 100 cold probe ; lane 9 : wt oligonucleotide and anti - gst ; 
  lane 10 : wt oligonucleotide and 1 rhsp1 protein ; lane 
  11 : wt oligonucleotide and 5 rhsp1 protein .
c , klf14 
  utilizes four sp / klf sites to repress the tgfrii 
  promoter in vivo .
panc1 cells were transfected with 
  tgfrii - luciferase with mutated sp1 sites as indicated 
  and stimulated with 10 ng / ml tgf1 after overnight serum starvation , and 
  transcriptional activity was measured and normalized to control empty vector . 
 
data are the mean  s.d . from three independent experiments , with 
  triplicates for each experiment .
panc1 cells were transfected 
  with klf14(flag ) and sp1(his ) epitope - tagged expression constructs or control 
  empty vector as indicated to evaluate in vivo competition on 
  tgfrii promoter activity .
western control ( flag / his ) 
  is shown for epitope - tagged expression .
b , klf14 
  binds to four of the putative sp1 sites and competes with sp1 on the 
  tgfrii promoter . electromobility shift assay was 
  performed using klf14-zf gst fusion proteins and radiolabeled oligonucleotides 
  for each of the five putative sp1 sites .
lane 1 : control - sp1 
  consensus oligonucleotide ; lane 2 : control - sp1 mutant 
  oligonucleotide ; lane 3 : sp1 consensus oligonucleotide and rhsp1 
  protein ; lane 4 : wt oligonucleotide ; lane 5 : mut#1 
  oligonucleotide ; lane 6 : mut#2 oligonucleotide ; lane 7 : wt 
  oligonucleotide and 25 cold probe ; lane 8 : wt oligonucleotide 
  and 100 cold probe ; lane 9 : wt oligonucleotide and anti - gst ; 
  lane 10 : wt oligonucleotide and 1 rhsp1 protein ; lane 
  11 : wt oligonucleotide and 5 rhsp1 protein .
c , klf14 
  utilizes four sp / klf sites to repress the tgfrii 
  promoter in vivo .
panc1 cells were transfected with 
  tgfrii - luciferase with mutated sp1 sites as indicated 
  and stimulated with 10 ng / ml tgf1 after overnight serum starvation , and 
  transcriptional activity was measured and normalized to control empty vector . 
 
data are the mean  s.d . from three independent experiments , with 
  triplicates for each experiment .
thus , to first delineate the dna binding properties of klf14 to the 
  promoter of tgfrii in vitro , we characterize the five 
  putative sp / klf sites ( shown in fig . 
 
radiolabeled oligonucleotides for each of the 5 sp1 sites have 
  been created as well as mutants for each site containing either 2- or 
  4-nucleotide substitutions ( mut#1 or mut#2 , respectively ) within the gc - rich 
  binding sequence .
sp1-consensus oligonucleotides and respective mutants have 
  been used as internal binding controls 
  ( fig .
the interaction between a recombinant klf14 zinc finger 
  dna - binding domain and four of the sp / klf site probes ( # 13 and 
  5 ) is specific as indicated by the fact that an excess of unlabeled 
  probe competes with the radiolabeled probes 
  ( fig .
furthermore , we were able to abolish this binding by 
  using antibodies against the recombinant klf protein ( anti - gst antibody ) , thus 
  confirming specificity of the complex ( fig . 
  5b , lane 9 ) .
further specificity is evidenced by 
  the impaired ability of recombinant klf14 protein to bind to mutated sp / klf 
  probes ( fig .
visible but reduced binding is noted with the 
  2-bp mutant probes ( mut#1 , lane 5 ) for sites # 13 , however a 
  significant loss of binding was observed with the 4-bp mutant probes ( mut#2 , 
  lane 6 ) at all sites .
moreover , through addition of recombinant sp1 
  protein to the reaction , an increase in the concentration of sp1 diminished 
  the binding of klf14 , demonstrating that klf14 and sp1 compete for these 
  gc - rich binding sites ( fig . 
 
5b , lanes 10 and 11 ) , complementing the 
  competition results we observed in vivo 
  ( fig .
5a ) . only a weak 
  complex was visualized with site # 4 , indicating that this site was less 
  specific for klf14 binding than the other four sites . to functionally complement these studies ,
we have performed 
  tgfrii reporter assays with both wild - type and mutant 
  promoter - luciferase constructs .
site - directed mutagenesis has been utilized to 
  create a 2-nucleotide substitution in each of the 5 gc - rich sp / klf sites 
  ( fig .
5c ) . these 
  assays resulted in a marked loss of the repression observed with the wild - type 
  tgfrii promoter upon klf14 overexpression with 
  mutation in sp / klf sites # 1 , 2 , 3 , and 5 , particularly with a significant gain 
  of transcriptional activity upon mutating site # 1 .
these findings demonstrate 
  that these four are operational to silence the transcriptional activity of the 
  tgfrii promoter by klf14 .
furthermore , we did not observe a significant 
  loss of repression with mutation of site # 4 with klf14 overexpression , which 
  supports our finding of negligible binding of klf14 to this specific sp / klf 
  site ( fig .
5b ) . all 
  together , klf14 appears to bind and act on four of the gc - rich sites of the 
  tgfrii promoter to repress its transcriptional 
  activity , and this occurs through a mechanism that includes , at least in part , 
  competition with sp1 .
repression of the tgfrii gene by klf14 occurs via 
  mechanisms involving chromatin - modifying enzymes 
careful 
  examination of klf14 sequence reveals a 26-amino acid domain that is highly 
  similar to repression domains of klf9 ( bteb1 ) , klf13 ( bteb3 ) , and klf16 
  ( bteb4 ) proteins ( fig . 
 
6a ) , which we have previously shown to repress 
  transcription by recruiting sin3a via its paired amphipathic helix 2 domain , 
  as well as hdac ( 15 , 
  28 ) .
interestingly , other 
  studies have found that hdac inhibition leads to transcriptional activation of 
  the tgfrii promoter in various cancer cell lines , 
  raising the possibility that a repressor of this type may be operational to 
  achieve this effect ( 29 , 
  30 ) .
thus , we have tested 
  whether klf14 does indeed interact with sin3a and hdac by immunoprecipitating 
  full - length flag - tagged klf14 from panc1 cells .
the presence of sin3a and hdac 
  complexed to klf14 is detected by western - blot analysis 
  ( fig .
thus , these 
  results indicate that klf14 interacts with sin3a and hdac in mammalian cells , 
  implying that these proteins are part of a repressor complex .
subsequently , we 
  have investigated whether this repressor complex occupies the endogenous 
  tgfrii promoter in vivo via chip assays . 
 
first , we find that klf14 indeed binds to the tgfrii 
  promoter ( fig .
6c ) , treatment 
  with tgf1 increases the amount of klf14 bound to the 
  tgfrii promoter .
in addition , the treatment with 
  tgf1 coincides with the appearance of sin3a on the same region of the 
  promoter as klf14 , further implicating this repressor complex in the mechanism 
  of klf14 repression of the tgfrii promoter 
  ( fig .
6d ) . to gain 
  insight into the type of chromatin remodeling that accompanies klf14 and sin3a 
  occupation , we also have performed chip using antibodies to various histone 
  marks .
interestingly , we find that , under native conditions , acetylated 
  histones h3 and h4 are present on the tgfrii promoter ; 
  however , upon tgf1 treatment , these fall to negligible levels , 
  indicating a change from a transcriptionally  active ,  acetylated 
  state to a relatively  inactive ,  non - acetylated state 
  ( fig .
moreover , 
  we observe that , although methylated k20 h4 , a mark of repression 
  ( 31 ) , is not present under 
  native conditions , it occupies the promoter upon tgf1 treatment along 
  with sin3a ( fig .
these types of experiments are in agreement with the  histone code 
  hypothesis  ( 32 ) , 
  revealing that the state of chromatin in the tgfrii 
  promoter appears to switch from  active  to 
   repressed  upon klf14 occupation after treatment with tgf1 , 
  all consistent with the idea that klf14 binds to the 
  tgfrii promoter to cause repression 
  ( fig .
therefore , both competition with sp1 and direct 
  repression via the n - terminal domain , are likely to behave as a dual mechanism 
  of repression that would make it more difficult to reverse than if one of them 
  was operational .
this redundancy would ensure that the promoter remains silent 
  even under circumstances that inactivate either of the single mechanisms 
  ( fig .
figure 6.a , alignment displaying the 26-amino acid sin3-interacting domain 
  of klf14 similar to other klf repressors , klf9 , klf13 , and klf16 .
panc1 cells were 
  transfected with flag - tagged klf14 or control empty vector constructs and 
  flag - immunoprecipitated .
panc1 cells were transfected with flag - tagged klf14 
  or control empty vector constructs and subject to chromatin 
  immunoprecipitation .
a 263-bp fragment of the tgfrii 
  promoter was amplified by pcr from anti - flag or mock immunoprecipitated dna 
  samples .
note that the tgfrii promoter was amplified 
  from anti - flag ( -flag ) but not mock immunoprecipitated samples 
  ( mock ) transfected with klf14 .
d , tgf1 leads to repressive chromatin modifications upon klf14 
  occupation of the tgfrii promoter .
panc1 cells were 
  transfected with flag - tagged klf14 and treated with tgf1 and chromatin 
  landscape chip assay was performed using specified antibodies . a , alignment displaying the 26-amino acid sin3-interacting domain 
  of klf14 similar to other klf repressors , klf9 , klf13 , and klf16 .
panc1 cells were 
  transfected with flag - tagged klf14 or control empty vector constructs and 
  flag - immunoprecipitated .
panc1 cells were transfected with flag - tagged klf14 
  or control empty vector constructs and subject to chromatin 
  immunoprecipitation .
a 263-bp fragment of the tgfrii 
  promoter was amplified by pcr from anti - flag or mock immunoprecipitated dna 
  samples .
note that the tgfrii promoter was amplified 
  from anti - flag ( -flag ) but not mock immunoprecipitated samples 
  ( mock ) transfected with klf14 .
d , tgf1 leads to repressive chromatin modifications upon klf14 
  occupation of the tgfrii promoter .
panc1 cells were 
  transfected with flag - tagged klf14 and treated with tgf1 and chromatin 
  landscape chip assay was performed using specified antibodies .
the current study provides us with several novel mechanistic insights on 
  the regulation of the tgf pathway . for instance
, the data reported here 
  represent the first functional characterization of the klf14 protein , outline 
  a novel pathway for the silencing of the tgfrii 
  promoter , provide insight into the molecular mechanisms by which these 
  phenomena are achieved , and report additional tgf-inducible , klf 
  proteins that are good candidates to regulate this promoter .
these findings 
  are of significant relevance to the areas of klf proteins , tgf 
  signaling , the maintenance of cell homeostasis , and their potential 
  contribution to disease states .
tgf1 ligand itself may play a role in regulating signaling by 
  down - regulating the cell surface receptor and thus abrogating downstream 
  messages .
others have also shown that the tgf receptors are under 
  autoregulation control by ligand stimulation 
  ( 18 ) .
it has been previously 
  shown that treatment with tgf1 leads to down - regulation of tgfrii 
  levels ( 19 ) .
derynck and 
  colleagues ( 20 ) have 
  previously described a process in osteoblastic differentiation in which at 
  first there is a marked up - regulation of tgf1 and sensitivity followed 
  by marked down - regulation of receptors and insensitivity to tgf1 . 
 
furthermore , recent in vitro studies on the biosynthesis of the 
  receptors indicate that the half - life of tgfrii is 60 min and is 
  further reduced to 45 min in the presence of exogenous tgf1 
  ( 33 ) .
therefore , understanding 
  how this down - regulation of the tgfrii promoter occurs 
  is of primary biological importance for better understanding tgf 
  signaling .
activation of the 
  tgfrii would occur via tgf1 ligand initiating an up - regulation of 
  the tgfrii promoter by sp1 . in our model ,
activation 
  of the tgf pathway also causes an induction of klf14 expression 
  ( a ) , which would , in turn , bind to the tgfrii 
  promoter through its gc - rich sites ( b ) , competing away sp1 
  ( c ) , recruiting the sin3a / hdac complex ( d ) , and remodeling 
  chromatin from a transcriptionally  active ,  acetylated state to 
  an  inactive  state with silencing methyl marks ( e ) . 
 
thus , klf14 mediates a negative feedback loop to down - regulate the type ii 
  tgf receptor upon tgf stimulation . model of klf14-mediated tgfrii silencing .
activation of the 
  tgfrii would occur via tgf1 ligand initiating an up - regulation of 
  the tgfrii promoter by sp1
. in our model , activation 
  of the tgf pathway also causes an induction of klf14 expression 
  ( a ) , which would , in turn , bind to the tgfrii 
  promoter through its gc - rich sites ( b ) , competing away sp1 
  ( c ) , recruiting the sin3a / hdac complex ( d ) , and remodeling 
  chromatin from a transcriptionally  active ,  acetylated state to 
  an  inactive  state with silencing methyl marks ( e ) . 
 
thus , klf14 mediates a negative feedback loop to down - regulate the type ii 
  tgf receptor upon tgf stimulation . in this regard ,
the current study reports several novel observations of 
  significant biochemical relevance for extending our knowledge of tgf 
  signaling regulation by non - smad proteins .
for instance , this study represents 
  the first functional characterization of klf14 as a novel tgf-inducible 
  repressor protein that mediates silencing of the tgfrii , and the 
  mechanisms by which this phenomenon occurs , in particular the role of 
  corepressors and chromatin modifications on the tgfrii 
  promoter .
previous promoter studies have primarily focused on the role of sp1 
  in the activation of this promoter .
however , recent studies have uncovered a 
  paradoxical behavior for this promoter , namely that histone deacetylase 
  inhibitors are needed to activate this promoter 
  ( 34 , 
  35 ) .
these data point to the 
  existence of a repressive state of this promoter , which is less likely to be 
  regulated by sp1 but rather by sp / klf repressors of the type previously 
  described by our laboratory 
  ( 11 ) . however , in the current 
  study , rather than looking for a candidate gene within the family of klf 
  repressors , we have performed an unbiased screening for the tgf 
  inducibility of each of the 24 klf transcription factors and find that 
  klf14 is up - regulated upon tgf treatment and also can 
  efficiently repress the tgfrii .
interestingly , we 
  demonstrate that there is a strong inverse correlation between expression 
  patterns in a temporal manner of tgfrii mrna and klf14 mrna in human 
  pancreatic cancer cells .
expression of exogenous klf14 decreases the level of 
  transcription from the tgfrii promoter , implying a 
  repressive role for klf14 in tgfrii expression .
further analysis reveals the mechanism by which this protein works , 
  indicating that klf14 is a member of the sin3-dependent klf repressors .
this 
  is important in light of there being two types of repressors in this family as 
  recently reviewed by us ( 11 ) , 
  the sin3a - dependent proteins , which include klf9 , -10 , -11 , -16 , and now -14 , 
  and the ctbp - dependent proteins , such as klf3 and -8 .
this knowledge can now 
  be very useful for performing rapid screening of the proteins that may be 
  acting as repressors of particular genes , besides the traditional hdac 
  inhibitor experiments .
this screening , we propose , can utilize sirna to either 
  target ctbp or sin3a .
an abolition of the silencing activity would then 
  rapidly focus the investigations on a reduced number of candidates belonging 
  to each of these groups .
in addition to the recruitment of a 
  klf14-msin3a - hdac2 repressor complex to the tgfrii 
  promoter , we observed a concurrent remodeling of chromatin , which involves 
  loss of transcriptionally  active  acetylated histone marks and an 
  increase in histone marks that associate with transcriptional silencing . 
 
finally , we defined binding sites involved in klf14-mediated repression of the 
  tgfrii promoter , namely sites -143/138 ( # 1 ) , -101/-95 
  ( # 2 ) , -59/52 ( # 3 ) and -11/-5 ( # 5 ) , which are consistent with previously 
  reported sp1 sites , and these same sites , when mutated , lose the repressive 
  effect of klf14 .
overall , however , the major relevance of this study is the fact that it 
  contributes to organize our thoughts on how tgf signaling is regulated . 
  it has recently been appropriately proposed to classify these mechanisms by 
  whether they are directly mediated by smads ( the best studied to date ) or 
  those less understood events regulated by non - smad proteins 
  ( 4 ) .
in fact , our laboratory 
  has previously described a role for klf repressors , namely klf10 ( tieg1 ) and 
  klf11 ( tieg2 ) , as non - smad effectors of the tgf response in cell growth 
  ( 9 , 
  36 ) .
now , due to the findings 
  reported in the current study , we can propose a model wherein tgf 
  cytokines activate and repress the tgfrii promoter 
  using the very same gc - rich sites utilized by this family of proteins . 
 
activation would occur in an initial phase via sp1 , whereas inhibition would 
  follow activation and require the action of klf14 , competition of sp1 , 
  recruitment of sin3a , hdac , and distinct chromatin modifications on the 
  promoter ( see model in fig .
therefore , klf14 becomes another important non - smad protein , in which at least 
  one of its functions is to silence the tgfrii .
this 
  novel transcriptional pathway thus becomes an important step for modulating 
  the activity of tgf signaling .
 the role of non - smad proteins in the regulation of transforming growth 
  factor- ( tgf ) signaling is an emerging line of active 
  investigation . here 
 , we characterize the role of klf14 , as a 
  tgf-inducible , non - smad protein that silences the tgf receptor ii 
  ( tgfrii ) promoter . 
 together with endocytosis , 
  transcriptional silencing is a critical mechanism for down - regulating 
  tgf receptors at the cell surface . 
 however , the mechanisms underlying 
  transcriptional repression of these receptors remain poorly understood . 
 klf14 
  has been chosen from a comprehensive screen of 24 members of the sp / klf family 
  due to its tgf inducibility , its ability to regulate the 
  tgfrii promoter , and the fact that this protein had 
  yet to be functionally characterized . 
 we find that klf14 represses the 
  tgfrii , a function that is augmented by tgf treatment . 
 mapping of 
  the tgfrii promoter , in combination with site - directed 
  mutagenesis , electromobility shift , and chromatin immunoprecipitation assays , 
  have identified distinct gc - rich sequences used by klf14 to regulate this 
  promoter . 
 mechanistically , klf14 represses the tgfrii 
  promoter via a co - repressor complex containing msin3a and hdac2 . 
 furthermore , 
  the tgf pathway activation leads to recruitment of a klf14-msin3a - hdac2 
  repressor complex to the tgfrii promoter , as well as 
  the remodeling of chromatin to increase histone marks that associate with 
  transcriptional silencing . 
 thus , these results describe a novel 
  negative - feedback mechanism by which tgfrii activation at the cell 
  surface induces the expression of klf14 to ultimately silence the tgfrii 
  and further expand the network of non - smad transcription factors that 
  participate in the tgf pathway . 