Báo cáo khoa học: Ets-1/ Elk-1 is a critical mediator of dipeptidyl-peptidase III transcription in human glioblastoma cells pdf

Results PCR amplification, sequence analysis and demonstration of promoter activity in the 5¢ upstream region of the human DPP-III gene Using primer designs based upon the DPP-III gene l

transcription in human glioblastoma cells

Abhay A Shukla, Misti Jain and Shyam S Chauhan

Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, India

Introduction

Dipeptidyl-peptidase III (DPP-III), a cytosolic

amino-peptidase has been purified and characterized from

dif-ferent tissues of various animal species such as rat and

human skin [1,2], bovine and human cataractous lens

[3], rabbit and human erythrocytes [4], rat brain [5]

and pancreas [6], monkey brain [7], human placenta

[8], neutrophils [9], Saccharomyces cerevisiae [10] and

Drosophila melanogaster[11] All mammalian DPP-IIIs

require zinc ions for their maximum activity and have

therefore been termed metalloaminopeptidases The

crystal structure of yeast DPP-III has been described

by Baral et al [12], providing an insight into its cata-lytic mechanism and mode of substrate binding No endogenous substrate for this enzyme has yet been identified However, it has broad specificity for a num-ber of polypeptides, suggesting its involvement in the terminal stage of intracellular protein catabolism Interestingly, DPP-III activity has been reported to increase in retroplacental serum [8] suggesting that it is synthesized in placental cells and released into the maternal circulation In view of its high affinity for angiotensin II and III [13], the potential role of this

Keywords

5¢-RACE; electrophoretic mobility shift

assays; promoter; site-directed

mutagenesis; transcription factors

Correspondence

S S Chauhan, Room No -3009,

Department of Biochemistry, All India

Institute of Medical Sciences, New Delhi

110029, India

Fax: +91 11 2658 8663

Tel: +91 11 2659 3272

E-mail: s_s_chauhan@hotmail.com

Database

The nucleotide sequence of the human

DPP-III promoter has been submitted to the

GenBank database under the accession

number FJ793449

(Received 9 November 2009, revised 25

December 2009, accepted 1 February 2010)

doi:10.1111/j.1742-4658.2010.07603.x

Dipetidyl-petidase III is a metallopeptidase involved in a number of physi-ological processes and its expression has been reported to increase with the histological aggressiveness of human ovarian primary carcinomas Because

no information regarding the regulation of its expression was available, experiments were designed to clone, define and characterize the promoter region of the human dipeptidyl-peptidase III (DPP-III) gene In this study,

we cloned a 1038 bp 5¢-flanking DNA fragment of the human DPP-III gene for the first time and demonstrated strong promoter activity in this region Deletion analysis revealed that as few as 45 nucleotides proximal to the transcription start site retained 40% of the activity of the full-length promoter This promoter lacked the TATA box but contained multiple GC boxes and a single CAAT box Similarly, two Ets-1⁄ Elk-1-binding motifs are present in the first 25 nucleotides from the transcription start site Bind-ing of Ets-1⁄ Elk-1 proteins to these motifs was visualized by electropho-retic mobility shift and chromatin immunoprecipitation assays Mutations

of these binding sites abolished not only binding of the Ets protein, but also the intrinsic promoter activity Increased DNA-binding activity of Ets-1⁄ Elk-1 by v-Ha-ras also augmented the mRNA level and promoter activity of this gene Similarly, co-transfection of DPP-III promoter–repor-ter constructs with Ets-1 expression vector led to a significant increase in promoter activity From these results, we conclude that Ets-1⁄ Elk-1 plays a critical role in transcription of the human DPP-III gene

Abbreviations

ChIP, chromatin immunoprecipitation; CLR, Chang liver Ras cells; CLDR, Chang liver DRas cells; DPP-III, dipeptidyl-peptidase III; EMSA, electrophoretic mobility shift assay; ERK, extracellular regulated kinase; Inr, initiator element; MEK, mitogen-activated protein kinase kinase.

peptidase in elevating the level of plasma angiotensin

hydrolysing activity during pregnancy has been

described Similarly, it exhibits high affinity for

Leu-enkephalins [5] These features suggest a potential role

for DPP-III in the regulation of blood pressure [10]

and in pain modulation [14] Human DPP-III has been

shown to increase with the histological aggressiveness

of human ovarian primary carcinomas [15] Because

levels of DPP-III alter in several physiological and

pathological conditions it must necessarily be

amena-ble to regulated expression However, no systematic

study has been carried out to elucidate the regulatory

molecular mechanisms associated with its expression

Therefore, this study was designed to clone and

char-acterize the human DPP-III promoter in order to

eluci-date the transcriptional regulation of the gene In this

regard, we identified the region that plays an

impor-tant role in determining the basal promoter activity of the gene Furthermore, with the help of binding assays and site-directed mutagenesis, we established that Ets-1⁄ Elk-1 play a key role in the regulation of DPP-III transcription in human glioblastoma cells

Results

PCR amplification, sequence analysis and demonstration of promoter activity in the 5¢ upstream region of the human DPP-III gene Using primer designs based upon the DPP-III gene located on chromosome 11q 12fi q13.1 of the human genome sequence (accession number NT_033903.7), we were able to amplify a single 1038 bp DNA fragment

by PCR (data not shown) This fragment was cloned

Fig 1 Nucleotide sequence of the 5¢-flank-ing region of the human DPP-III gene The transcriptional initiation site determined by 5¢-RACE in U87MG cells is denoted as +1 Different primers were used for amplifica-tion of the 1038 bp full-length promoter and its deletion fragments, 5¢-RACE and ChIP assays are shown by arrows The most 5¢-end base of the full-length promoter and the different deletion constructs are shown in bold and indicated by arrows ( fi ) with respect to the transcription initiation site (; +1) The translation initiation codon ATG is underlined Potential cis-element regulatory motifs are in italics and marked by dashed arrows Two AT-rich sequences present at positions )24 and )29 are written in bold The intronic sequences are written in lower case Primers used for the amplification

of DPP-III promoter sequence are also underlined.

into a TA cloning vector and sequenced Analysis of

its nucleotide sequence showed 100% homology to the

upstream region and part of the reported 5¢-end of

DPP-III mRNA These results indicated that the

amplified region is physically linked to exon 1 of the

DPP-III gene Nucleotide sequence analysis of

the cloned 1038 bp human DPP-III promoter revealed

that it contained 63.19% G + C nucleotides, no

detectable TATA box, a single CAAT box and several

GC boxes (Sp1-binding sites) Multiple putative

tran-scription factor binding sites were identified in this

region using the motif finder program (http://motif

genome.jp/) with high-stringency parameters for core

similarity of 0.85 and matrix similarity of 0.85 As

shown in Fig 1, these motifs include NF-jB, USF,

C⁄ EBP, CREB, NF-1 and multiple binding sites for

the Sp1 and Ets family of transcription factors,

sug-gesting that the amplified region is a potential

promoter To demonstrate promoter activity in the

amplified fragment, we cloned it upstream of the

lucif-erase reporter gene in the pGL3-Basic vector

Trans-fection of the resulting construct (pAAS-1) into

U87MG, Caov-2, Chang liver, Panc1 and NIH 3T3

cells yielded  800-,  200-,  100-,  80- and

25-fold higher luciferase activity respectively,

com-pared with the pGL3-Basic transfected cells (Fig 2)

These results established that the cloned fragment is a functional DPP-III promoter

Mapping of the transcriptional start site

As a first step towards characterization of the human DPP-III promoter, the transcriptional start site in U87MG cells was mapped using 5¢-RACE Resolution

of RACE products on agarose gel revealed the amplifi-cation of a single  200 bp fragment (Fig 3) This fragment was cloned into a TA cloning vector and six representative clones were subjected to double-stranded DNA sequencing All clones exhibited 100% homology

to DPP-III mRNA and contained the same nucleotide (G) corresponding to the 33rd nucleotide upstream of the translation initiation codon in the reported mRNA sequence (accession number NM_005700) These results suggested that this nucleotide is the transcrip-tion initiatranscrip-tion site (marked +1 in Fig 1)

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Fig 2 Demonstration of promoter activity in the 5¢ flanking region

of the human DPP-III gene in different cell lines The PCR-amplified

5¢ flanking region of human DPP-III gene (1038 bp) was cloned

upstream of the luciferase reporter gene in promoter-less plasmid

pGL3-Basic The resulting construct (pAAS-1) was transfected into

U87MG (human glioblastoma grade III), Caov-2 (ovarian carcinoma),

Chang liver (human liver), Panc1 (human pancreatic carcinoma) or

NIH 3T3 (mouse fibroblast) cells After 48 h of transfection, cells

were washed three times with ice-cold NaCl⁄ P i , lysed and

lucifer-ase activity was assayed in the cell lysates Cells transfected with

pGL3-Basic were processed in an identical way and served as the

negative control Values are the mean ± SE of at least three

inde-pendent experiments performed in triplicate Other details are

given in Materials and methods.

(T)nTTTV (A)nAAA

Oligo dT-anchor primer A

B

PCR anchor Primer

AAS2

AAS1

DL100 PCR negative control RACE product

~200 bp

1 2 3

500 bp

100 bp

Fig 3 Mapping of the transcription initiation site of the DPP-III gene (A) Schematic diagram showing the location of different primers used for 5¢-RACE on U87MG cDNA (B) Total cellular RNA isolated from U87MG cells was reverse transcribed using a gene-specific primer (AAS-2) and used in 5¢-RACE to map the transcrip-tion initiatranscrip-tion site PCR performed without a template served as the negative control RACE products were resolved on 2% agarose gel and stained with ethidium bromide The prominent 200 bp frag-ment (indicated on the right-hand side) was excised, cloned and subjected to double-strand DNA sequencing DL100 corresponds to

a 100 bp DNA ladder (MBI Fermentas, Vilnius, Lithuania).

Deletion analysis of human DPP-III promoter

In order to define the minimal promoter region and

identify the functional transcription factor binding

motif(s) in this region of the DPP-III gene, a series of

promoter–reporter constructs with varying lengths for

the 5¢-region were generated The 5¢-ends of these

constructs are marked ( ) in Fig 1 These constructs

were transfected into human glioblastoma cells

(U87MG) followed by estimation of the luciferase

activity The construct pAAS-2 ()781 ⁄ +5), which

lacks first 252 bases from the 5¢-end of the full-length

promoter, retained 92% of the promoter activity

( 740-fold promoter activity over pGL3-Basic vector)

(Fig 4A) Further deletion of 548 or more bases

from the 5¢-end resulted in a significant reduction in promoter activity Constructs which lacked 548 bp ()485 ⁄ +5, pAAS-3), 803 bp ()230 ⁄ +5; pAAS-4),

909 bp ()123 ⁄ +5; 5), 940 bp ()93 ⁄ +5; pAAS-6), 980 bp ()53 ⁄ +5; pAAS-7) and 993 bp ()40 ⁄ +5; pAAS-8) from the 5¢-end, retained 51, 48, 39, 42, 44 and 40% promoter activity, respectively, compared with the full-length promoter (Fig 4A) All these con-structs exhibited > 300-fold promoter activity com-pared with pGL3-Basic Thus, 45 nucleotides (minimal promoter) from the 3¢-end of the DPP-III promoter retained 40% of the full-length promoter (pAAS-1) activity (320-fold over pGL3-Basic) Sequence analysis

of the minimal promoter region ()40 ⁄ +5) revealed no obvious TATA or CAAT boxes However, two perfect

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Fig 4 Functional analysis of deletion constructs of the human DPP-III gene (A) A series of DNA fragments were PCR amplified using full-length promoter fragment as the template Seven fragments with different 5¢-ends (nucleotides )781, )485, )230, )124, )93, )53 and )40) and a common 3¢-end (nucleotide +5) were cloned upstream of the luciferase reporter gene in promoter-less plasmid pGL3-Basic to generate constructs pAAS-2, pAAS-3, pAAS-4, pAAS-5, pAAS–6, pAAS-7 and pAAS-8, respectively U87MG cells were transiently co-trans-fected with test plasmid and pRL-TK internal control plasmid Values significantly different from pAAS-1 are marked by * (B) A DNA frag-ment lacking 25 bases from the 3¢ end of pAAS-5 was amplified using DPP-III F-124 and DPP-III R-21 as the sense and antisense primers pAAS-1 was used as a template for the PCR The 99 bp amplified fragment was digested with XhoI and HindIII and cloned into the pGL3-Basic to generate pAAS-9 ( )124 ⁄ )21) U87MG cells were transiently transfected with pAAS-9 pAAS-1 and pAAS-5 were also transfected in separate experiments Luciferase activity in the cell lysates was measured 48 h after transfection Each transfection was performed in tripli-cate and the results are expressed as the mean ± SE of three independent experiments a Significantly higher compared with pAAS-9 (P < 0.001); b significantly higher compared with pAAS-9 (P < 0.001) Statistical analysis was performed using a paired two-tailed Student’s t-test.

consensus Ets-1⁄ Elk-1 core binding motifs (GGAA

GCAGGAA) separated by three bases were present in

this region (at positions)6 and )13) To elucidate the

role of these motifs, we deleted 25 bases from the

3¢-end of the construct pAAS-5, thus generating pAAS-9

()124 ⁄ )21) This construct, which lacked 5 bases of

exon 1 and 20 bases of the promoter region, including

Ets-1⁄ Elk-1-binding motifs, exhibited no promoter

activity (Fig 4B) These results established that

nucleo-tides between )20 and +5 are essential for DPP-III

promoter activity

Site-directed mutagenesis

To further corroborate our results, we mutated these

two Ets-1⁄ Elk-1 binding motifs sequentially in pAAS-1

using site-directed mutagenesis and assessed the

pro-moter activities of the resulting constructs (Fig 5)

pAAS-1Mut1 (harbouring a mutated Ets-1⁄ Elk-1 motif

at position )6) and pAAS-1Mut2 (having Ets-1 ⁄ Elk-1

mutated motifs at both position)6 and position )13),

were transfected in U87MG cells Mutations in the

motif at)6 (pAAS-1Mut1) resulted in an 81% loss of

basal promoter activity compared with the full-length

construct (pAAS-1) Whereas mutations in both Ets-1⁄ Elk-1-binding motifs (pAAS-1Mut2) resulted in

a 96% loss of basal promoter activity compared with the full-length construct (pAAS-1, Fig 5) Abolition of the promoter activity confirmed that Ets-1⁄ Elk-1 bind-ing motifs are essential for transcription of the human DPP-III gene Because mutation of the motif present

at the )6 position alone resulted in an 81% loss of promoter activity, we conclude that Ets-1⁄ Elk-1-bind-ing motifs are critical for DPP-III gene expression

Binding of transcription factors to the minimal promoter region (-40⁄ +5)

To show the in vivo binding of Ets-1 and Elk-1 tran-scription factors with the human DPP-III minimal promoter region, we performed chromatin immunopre-cipitation (ChIP) assays using Ets-1 and Elk-1 antibod-ies The immunoprecipitated chromatin was subjected

to PCR using primers flanking the binding motifs pres-ent at positions )6 and )13 in the DPP-III promoter Amplification of a specific 81 bp DNA fragment was observed when the DNA was immunoprecipitated using Ets-1 or Elk-1 antibodies However, no amplifi-cation of any DNA fragment was evident when DNA was precipitated using mouse IgG (negative control) (Fig 6) The identity of the amplified products as part

+ Anti mouse IgG

81 bp

200 bp

Fig 6 In vivo analysis of binding of Ets-1 and Elk-1 to the DPP-III promoter by ChIP assay U87MG cells were fixed with 1% formal-dehyde to cross-link the existing in vivo proteins–DNA complex Nuclei of the cross-linked cells were isolated and subjected to soni-cation to shear the DNA Anti-Ets-1 and anti-Ek-1 IgG were used to immunoprecipitate DNA bound to these proteins PCR was performed using DPP-III F-45 and ChIP R as the sense and antisense primers to specifically amplify 81 bp DPP-III promoter region including the Ets-1 ⁄ Elk-1-binding motifs present at positions )6 and )13 PCR using the same primers was also performed with DNA immunoprecipitated using mouse IgG as template and served as a negative control DL100 corresponds to a

100 bp DNA ladder and a single fragment of 200 bp is indicated by

an arrow.

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Mut1

pAAS-1 Mut2

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Fig 5 Functional relevance of Ets-1 ⁄ Elk-1-binding motifs in DPP-III

promoter activity U87MG cells were transiently transfected with

either wild-type promoter, construct pAAS-1 or promoter constructs

containing one ( )6; 1Mut1) or both ()6 and )13;

pAAS-1Mut2) mutated Ets-1 ⁄ Elk-1-binding motifs Luciferase activity was

measured 48 h after transfection and is plotted in the left-hand

panel Each transfection was performed in triplicate and the results

are expressed as the mean ± SE of three independent

experi-ments (a) Significantly higher compared with pAAS-1Mut1

(P < 0.001); (b) significantly higher compared with pAAS-1Mut2

(P < 0.001) Statistical analysis was performed using a paired

two-tailed Student’s t-test.

of the DPP-III promoter was confirmed by DNA

sequencing

An electrophoretic mobility shift assay (EMSA) was

performed to assess the specific binding of Ets-1⁄ Elk-1

to the DPP-III minimal promoter region EMSA was

performed using nuclear extracts obtained from

U87MG cells Nucleotide fragments (23 bp)

harbour-ing wild-type ()18 ⁄ +5; Ets-Wt) or mutated (Ets-M1

and Ets-M2) Ets-1⁄ Elk-1 motifs were used as

radiola-belled probes for this purpose (Table 1) Incubation of

radiolabelled Ets-Wt with the nuclear extract resulted

in the formation of two DNA–protein complexes

which migrated more slowly than the free radiolabelled

probe (Fig 7A,B; lanes 2) In the first complex

(Fig 7A; shift 1), nuclear proteins showed strong

bind-ing to the probe compared with the second complex

(Fig 7A; shift 2), which migrated more slowly than

the first Formation of these complexes was abrogated

in the presence of a 100 molar excess of unlabelled

Ets-Wt (Fig 7B, lane 3) Incubation of either

radiola-belled Ets-M1 or Ets-M2 with the nuclear lysate did

not result in the formation of any such complex

(Fig 7A; lanes 3 and 4) Consistent with these results,

in the presence of a 100 molar excess of unlabelled

Ets-M2, no change in the binding of nuclear proteins

to radiolabelled Ets-Wt was observed (Fig 7B; lane 4)

Incubation of the mixture of radiolabelled probe and

nuclear lysate with antibodies specific for Ets-1⁄ Elk-1

resulted in ‘supershifts’ of the complex (shift 1)

(Fig 7B; lanes 6 and 8) However, the formation of

such complexes was not observed when antibodies

were added to the nuclear lysate prior to addition of

the labelled probe (Fig 7B; lanes 5 and 7),

Upregulation of DPP-III expression and promoter

activity by v-Ha-ras

Promoter deletion analysis, site-directed mutagenesis,

EMSA and ChIP assays demonstrated that Ets-1

and Elk-1 play a critical role in the transcription of

Table 1 Sequence of double-stranded oligonucleotides used in the

gel-shift assay The two Ets-1 ⁄ Elk-1-binding motifs present

(posi-tions )6 and )13 in the dipeptidyl-peptidase III promoter sequence)

in the wild-type oligonucleotide (Ets-Wt; )18 ⁄ +5 with respect to

transcription initiation site) are shown in bold Mutated nucleotides

are shown in lowercase.

Ets-M2 Ets-M1 Ets-Wt Nuclear extract A

B

4 3 2 1

+ + + –

– – + +

– + – –

+ – – –

Shift 1 Shift 2

– – + +*

– – – –

Ets-1 antibody

+ +*

– – – – – –

Elk-1 antibody

– – – – + – – –

Unlabeled Ets-M2

– – – – – + – –

Unlabeled Ets-Wt

+ + + + + + + +

Radiolabeled Ets-Wt

+ + + + + + + –

Nuclear extract

8 7 6 5 4 3 2 1

Shift 1

Shift 2 Super shift

Fig 7 Binding profile of nuclear proteins from U87MG cells to the 5¢-flanking region of the human DPP-III gene (A) Radiolabelled 23-mer double-stranded wild-type (Ets-Wt) and mutated (Ets-M1 and Ets-M2) oligonucleotides were incubated with nuclear extracts (15 lg of protein) prepared from U87MG cells in the binding assay The DNA–protein complexes (black arrow) were resolved on a non-denaturing gel and subjected to autoradiography (B) The binding reactions were carried out in the absence or presence of a 100 molar excess of unlabelled wild-type or mutant double-stranded oli-gonucleotides In some of the reactions, 4 lg of antibody against c-Ets-1 and pElk-1 were incubated with nuclear lysate before (lanes

5 and 7) or after (lanes 6 and 8) adding labelled probe The shifts produced are shown by black arrows and supershifted complexes are shown by white arrows.

DPP-III gene Most Ets family proteins are nuclear

targets for phosphorylation by the RAS⁄

mitogen-activated protein kinase (MAPK) signalling pathway

These nuclear phospho-proteins in turn influence cell

proliferation, differentiation and oncogenic

transfor-mation Our laboratory has developed a human liver

cell line (Chang liver) stably expressing v-Ha-ras

(M Jain et al., unpublished results) These Chang liver

ras (CLR) cells exhibited approximately twofold higher

levels of pElk-1 (P£ 0.002) compared with cells stably

transfected with the empty vector (Chang liver DRas;

CLDR) (Fig 8A) Transfection of pAAS-1 in CLR

and CLDR cells demonstrated a twofold higher

lucifer-ase activity in CLR cells compared with CLDR cells,

(P < 0.001; Fig 8B) To demonstrate that the higher

DPP-III promoter activity was because of increased

levels of pElk-1 in v-Ha-ras-expressing cells (CLR), we

treated CLR and CLDR cells with mitogen-activated

protein kinase kinase⁄ extracellular regulated kinase

(MEK⁄ ERK) inhibitor U0126 (Sigma-Aldrich, Urbana,

IL, USA) Immunoblot analysis using antibodies

spe-cific for pERK confirmed a significant reduction in the

levels of its phosphorylated form (P£ 0.02; Fig 8D,E)

Similarly, levels of pElk-1 were found to be decreased

in U0126-treated CLR cells compared with untreated

cells (P£ 0.003) However, CLDR cells did not exhibit

any significant difference in pElk-1 levels on treatment

with the inhibitor (Fig 8F,G) Our efforts to compare

the levels of pEts-1 failed because the antibody did not

work in western blots

The effect of inhibition of the MAPK pathway by

the U0126 inhibitor and the concomitant influence of

pElk-1 levels on transcription of the DPP-III gene was

assessed In this regard, mRNA levels specific for the

DPP-III gene were estimated in both cell lines before

and after treatment with inhibitor We observed that

expression of DPP-III mRNA in CLR cells was

two-fold higher than in CLDR cells (P£ 0.002) Treatment

of CLR cells with U0126 decreased the DPP-III

mRNA level by threefold (P£ 0.03), although there

was no significant effect on DPP-III mRNA levels in

treated CLDR cells (Fig 8H) Thus we conclude that

upregulation of DPP-III expression by v-Ha-ras is

mediated by increased Elk-1 phosphorylation

Induction of DPP-III promoter activity by Ets-1

It is evident from the results presented in Fig 2 that the

DPP-III promoter exhibits minimal activity in human

cell lines like Panc1 and Chang liver Therefore, to

further establish the role of Ets-1 in the transcription

of DPP-III, we co-transfected Chang liver cells with

DPP-III promoter–reporter construct containing

wild-type (pAAS-1) or mutant (pAAS-1Mut2) Ets-1⁄ Elk-1-binding motifs with the Ets-1 expression vector (pEts-1)

in a molar ratio of 1 : 1 Simultaneously, pAAS-1 and pAAS-1Mut2 were also co-transfected along with empty Ets-1 expression vector (pcDNA3.1) and luciferase activity was assayed in all transfected cells Co-transfec-tion of pAAS-1 with pEts-1 resulted in a significant (P = 0.03) increase in promoter activity compared with co-transfection with empty vector (Fig 9) However, no such difference in promoter activity was observed when pAAS-1Mut2 was co-transfected with pEts-1 or empty vector (Fig 9) These results clearly establish that over-expression of Ets-1 induces the DPP-III promoter activity

Discussion

Human DPP-III, a metalloaminopeptidase, purified and characterized from a number of tissues, has been implicated in several physiological and pathological processes To understand the regulation of its expres-sion, the promoter of this peptidase has been cloned and characterized for the first time in this study

We amplified a 1038 bp genomic fragment located upstream of the previously published DPP-III tran-script (accession number NM_005700) This fragment exhibited maximal promoter activity in human glio-blastoma cells compared with other human and murine cells, as assessed by luciferase reporter assays (Fig 2) These results are in agreement with the high DPP-III activity reported in brain homogenates of rat, monkey and guinea pig [5,7,16] The presence of DPP-III in the central nervous system facilitates the degradation of angiotensins and encephalin, suggesting its role in blood pressure regulation and in pain modulation [5] Analysis of the amplified fragment (DPP-III pro-moter) revealed the absence of a consensus TATA box, a high GC content (63.16%), and the presence of multiple binding motifs for Sp1, suggesting that the human DPP-III gene is a housekeeping gene (Fig 1) [17–19] This is corroborated by reports demonstrating its expression in a wide range of tissues from different animal species [5,9,11,20] Our results demonstrate increased DPP-III promoter activity in v-Ha-ras trans-formed cells Consistent with these results, TATA-less promoters of human lysosomal cysteine cathepsins are also upregulated by malignant transformation [21] Most of the TATA-less promoters are known to initiate transcription from multiple sites However, TATA-less promoters with multiple GC boxes have been shown to initiate transcription from a single site,

as in the case of nerve growth factor receptor [22], the cellular retinol-binding protein [23], endothelial nitric

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oxide [24] and genes coding for DPP-I [18] Similarly,

we observed a single transcriptional start site located

33 nucleotides upstream of the translation initiation codon for DPP-III in U87MG cells (Fig 1) Analysis

of the DPP-III promoter sequence revealed the pres-ence of an initiator element (Inr)-like sequpres-ence sur-rounding the transcription initiation site and two

A⁄ T-rich sequences at positions )29 and )24 (Fig 1) The A⁄ T content of the )30 sequence in an Inr-containing synthetic promoter has been shown to have

a profound positive influence on the strength of the promoter, despite having minimal resemblance to the TATA consensus sequence [25,26] The stretch of DNA between the Inr sequence and the A⁄ T-rich region in the DPP-III promoter contains a GC box (Sp1-binding motif) and two Ets-1⁄ Elk-1-binding motifs Both of these transcription factors are known

to help in recruitment of the transcription initiation assembly [27–29] and therefore this arrangement of nu-cleotides in the DPP-III promoter is probably respon-sible for the strong promoter activity of DPP-III in U87MG cells

Deletion analysis of the DPP-III promoter revealed that the region between )781 and )485 bp contains several transcription factor binding motifs such as USF, C⁄ EBP and Sp1 (Fig 1), and removal of this region results in a 40% decrease in promoter activity

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Fig 9 Overexpression of pEts-1 results in increased DPP-IIII

promoter activity in human liver cells DPP-III promoter–reporter

construct harbouring wild-type (pAAS1) or mutant (pAAS1Mut2)

Ets-binding motifs were co-transfected with Ets-1 expression

vector (pEts-1) or empty vector (pcDNA3.1) at a molar ratio of 1 : 1

in Chang liver cells Forty-eight hours post transfection, cells

were lysed and luciferase activity was assayed Values are

the mean ± SE of four independent experiments performed in

triplicate Results were statistically analysed using the Student’s

t-test and luciferase activity significantly different from the

promoter–reporter construct transfected with empty vector is

denoted by ‘a’.

Fig 8 Elevation of DPP-III mRNA, promoter activity and pElk-1 levels by H-ras in human liver cells (A) An equal amount of total protein from Chang liver cells expressing v-Ha-ras (CLR) and control cells (CLDR) was resolved on SDS ⁄ PAGE and subjected to western blotting using mAb raised against phosphorylated form of Elk-1 (pElk-1) protein or a-tubulin (B) The individual pELK-1 bands of the blots given in (A) have been quantitated densitometrically and normalized with the a-tubulin levels Values are the mean ± SE of three independent experi-ments Results were statistically analysed using the Student’s t-test Values significantly different from CLDR cells are denoted by ‘a’ (C) To measure DPP-III promoter activity, CLR and CLDR cells were transiently transfected with DPP-III full-length promoter construct (pAAS-1) and luciferase activity was measured 48 h post transfection Other details are given in Material and methods Values are the mean ± SE of at least three independent experiments a Significantly higher compared with CLDR (P < 0.05) (D) Equal amounts of total protein from Chang liver cells expressing v-Ha-ras (CLR) and control cells (CLDR) treated with either dimethylsulfoxide or U0126 were resolved on SDS ⁄ PAGE and subjected to western blotting using an antibody raised against the phosphorylated form of Erk-1 or a-tubulin protein (E) Densitometric quantitation of the phosphorylated form of Erk-1 The bands of the blot given in (D) were subjected to densitometry and the values obtained were normalized with the levels of a-tubulin Values are the mean ± SE of three independent experiments Results were statistically analy-sed using the Student’s t-test Values significantly different from CLDR cells are denoted by ‘a’ (F) Equal amount of total protein from Chang liver cells expressing v-Ha-ras (CLR) and control cells (CLDR) treated with either dimethylsulfoxide or U0126 were resolved on SDS ⁄ PAGE and subjected to western blotting using an antibody raised against the phosphorylated form of Elk-1 or a-tubulin protein (G) Densitometric quantitation of phosphorylated form of Elk-1 Bands of the blot given in (F) were subjected to densitometry and the values obtained normal-ized with the levels of a-tubulin Values are the mean ± SE of three independent experiments Results were statistically analysed using the Student’s t-test Values significantly different from CLDR cells are denoted by ‘a’ (H) An equal amount of total RNA from CLR and CLDR cells before and after treating cells with either U0126 (10 lgÆmL)1) or dimethylsulfoxide was reverse transcribed and subjected to real time PCR using DPP-III (DPP F20 and AAS1) and b-actin (b-actin-F and b-actin-R) specific primers The DPP-III mRNA levels were calculated using b-actin as the internal control Cycle threshold (Ct) values were calculated for each PCR and relative fold change was calculated using 2)DD

Ct method [16] Each set of observations was compared with the other set using a paired two-tailed t-test, assuming unequal variances among the sample means A P-value of £ 0.05 was considered statistically significant a Significantly higher compared with Chang liver Ras treated (P < 0.05), b significantly higher compared with Chang liver DRas untreated (P < 0.05), c significantly higher as compared to Chang liver DRas untreated (P < 0.05).

(Fig 4A) These results suggest that some or all of

these motifs are important for DPP-III transcription

This analysis also suggested that the 25 nucleotides at

the 3¢-end of the DPP-III promoter are essential for its

activity (Fig 4B) Nucleotide sequence analysis of this

region revealed the presence of two Ets-1⁄

Elk-1-bind-ing motifs (GGAAGCAGGAA) at positions )6 and

)13 EMSA and ChIP assays established the binding

of the transcription factor(s) to these motifs Ets-1 has

been shown to positively regulate urokinase

plasmino-gen activator (uPA) expression in breast cancer,

gli-oma, astrocytoma and meningioma cells [30,31]

Similarly, Ets transcription factor(s) regulate the

expression of several other human genes such as

stromelysin [32], prolactin and growth hormone [33]

and chemokine [34] Mutations in the Ets-1⁄

Elk-1-binding motifs abolished promoter activity (Fig 5)

This was in agreement with the EMSA results using

the 23 bp DNA fragment, wherein no shift in mobility

was observed, (Fig 7) These results led us to conclude

that the mutations which abolish the binding of Ets-1

and Elk-1 to DPP-III promoter result in a concomitant

loss of promoter activity

Several studies have demonstrated that Ets-binding

motifs when present in a pair and in close proximity

with each other, induce gene expression to a higher

level [35–37] Consistent with these reports, the

DPP-III promoter containing two Ets-binding motifs

(at positions )6 and )13) separated by three

nucleo-tides exhibits robust activity (800-fold over

pGL3-Basic) in U87MG cells (Fig 2) Mutagenesis of just

one of these motifs ()6 position) resulted in a

> 80% decrease in promoter activity, suggesting that

both motifs are essential for strong promoter activity

(Fig 5) The cooperative binding of Ets proteins as a

homodimer or a heterodimer to form a ternary

com-plex with the promoter region of several genes has

been demonstrated [32,36] Stromelysin-1 promoter is

transactivated by a homo-dimer of Ets-1 through

head-to-head Ets-binding sites [36] The DPP-III

pro-moter contains two Ets-binding motifs arranged in a

head-to-tail orientation within the first 25 bp

upstream of the transcription start site However,

Ets-1 binds as a homodimer when the two motifs are

present in a head-to-head orientation [36] In

super-shift assays, the mobility of the DNA–protein

com-plex was further shifted to a similar extent by both

Ets-1 and Elk-1 antibodies (Fig 7B), suggesting the

binding of both these transcription factors to their

cognate motifs in this region The formation of two

DNA–protein complexes also suggests that both these

transcription factors bind individually, as well as in

the form of a heterodimer to the DPP-III promoter

The N-terminal domain of Ets-1 is involved in the formation of a complex with other proteins, whereas its C-terminal domain is involved in DNA binding [38] By contrast, the DNA-binding domain of Elk-1 is present at its N- terminal end and C-termini allows it

to form homo- or heterodimers In our supershift experiments, we used an antibody against Ets-1 raised against its N-terminal region and an antibody against Elk-1 raised against phosphorylated Ser383 present at the C-terminus of this protein Preincubation of nuclear proteins with antibodies completely abolished the formation of shift 2 (Fig 7B; lanes 5 and 7) This result suggests that shift 2 was created by binding a heterodimer of Ets-1 and Elk-1 to the probe, and the N-terminus of Ets-1 and the C-terminus of Elk-1 were involved in the formation of the heterodimer Preincu-bation of nuclear proteins with antibodies against Ets-1 or Elk-1 prevented the formation of any such com-plex, confirming the binding of a heterodimer of Ets-1 and Elk-1 to the probe Likewise, incubation of antibodies with the DNA–protein complexes did not produce any supershift of shift 2 because the N-termi-nus of Ets-1 and the C-termiN-termi-nus of Elk-1 were involved in the formation of the heterodimer and were not therefore available for binding to their respective antibodies However, these antibodies allowed us to identify binding of both Ets-1 and Elk-1 separately, as well as in the form of a complex to the DPP-III pro-moter The slower moving complex (Fig 7A, shift 2) showed strong binding of nuclear proteins to the probe only when antibodies were added after incubation of probe with the nuclear proteins (Fig 7B, lanes 6 and 8) suggesting stabilization of the DNA–protein complex

From these results it is apparent that Ets-1 and Elk-1 are involved in the formation of a ternary com-plex with the DPP-III promoter and in regulating its expression In addition, Ras has been shown to medi-ate the phosphorylation of Ets proteins thereby increasing their transactivation ability [39,40] In this regard, we observed significantly higher mRNA expres-sion and promoter activity of DPP-III associated with elevated levels of phosphorylated Elk-1 in CLR cells This further reiterates the role of Ets proteins in DPP-III expression Finally, co-transfection of Ets-1 expression vector (pEts-1) with promoter–reporter con-struct harbouring wild-type (pAAS-1), but not mutated (pAAS-1Mut2), Ets-1⁄ Elk-1-binding motifs exhibited a significant increase in promoter in Chang liver cells (Fig 9) These results confirm that Ets-1 is necessary for DPP-III transcription and convincingly demon-strate the critical role of Ets-1⁄ Elk-1 in the expression

of this peptidase