From INSERM U 531, Institut Louis Bugnard, 31403, Toulouse, France
and the Department of Medicine, Tulane University and
Veterans Affairs Medical Center School of Medicine, New Orleans,
Louisiana 70112
Received for publication, December 6, 2000, and in revised form, January 16, 2001
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ABSTRACT |
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The sst2 somatostatin receptor is an inhibitory G
protein-coupled receptor, which exhibits anti-tumor properties.
Expression of sst2 is lost in most human pancreatic cancers. We
have cloned 2090 base pairs corresponding to the genomic DNA region
upstream of the mouse sst2 (msst2) translation
initiation codon (ATG). Deletion reporter analyses in mouse pituitary
AtT-20 and human pancreatic cancer PANC-1, BxPC-3, and Capan-1 cells
identify a region from nucleotide Among the wide spectrum of biological functions exerted by the
ubiquitous regulatory neurohormone somatostatin, its action as an
inhibitor of cell proliferation and endocrine and exocrine secretory
processes, together with its immunomodulatory and antiangiogenic properties have lately raised great interest in cancer research. Cellular mechanisms that are induced by somatostatin include inhibition of adenylate cyclase, modulation of K+ and Ca2+
channels, and protein dephosphorylation. The biological effects of
somatostatin are mediated through its specific binding to a family of G
protein-coupled receptors
(ssts)1 encoded by five
different genes (sst1-sst5) (1-2).
Studies with CHO cells expressing each sst subtype reveal a predominant
role of sst2 in mediating the antiproliferative effect of somatostatin
analogs (3). Upon ligand stimulation, sst2 induces transient
association and activation of the tyrosine phosphatase SHP-1, which in
turn, associates with and dephosphorylates activated insulin receptor
and its substrates leading to inhibition of the insulin mitogenic
signal (4, 5). sst2 is expressed in a variety of human tissues such as
brain, kidney, pituitary, adrenals, stomach, colon, and pancreas (1).
Interestingly, it has recently been shown that sst2 expression
disappears in human pancreatic adenocarcinomas and their metastases and
is also absent in most human pancreatic adenocarcinoma-derived cell
lines (6). The molecular mechanism responsible for this loss is
currently unknown. In non-sst2-expressing human pancreatic cancer cells
BxPC-3 and Capan-1, heterologous expression of sst2 triggers an
increase in somatostatin expression and secretion that induces an
autocrine inhibition of cell proliferation (7). Furthermore, expression of sst2 reverses the malignant properties of these cells and induces an
anti-tumor bystander effect highlighting the tumor suppressor properties and the potential therapeutic interest of sst2 in pancreatic cancer (7-8).
In recent years, considerable effort has been directed at understanding
the molecular alterations that occur in pancreatic carcinoma. The loss
of negative growth constraints as a result of dysregulation in
TGF- Very recently, an alternative promoter usage of the mouse sst2 gene
(msst2) has been reported (19). The authors identified two
short exons separated by introns larger than 25 kbp located upstream
from the first coding exon, and three tissue and cell specific
promoters located each at the 5' border to the referred exons. In
addition, sequence information corresponding to a 3.8 kbp region
upstream from the hsst2 start codon (20), and a novel initiator element (SSTR2inr) (21) have recently been
reported. Whereas several studies have pointed out important modulators of sst2 expression, very little information concerning the potential involvement of transcriptional mechanisms has been brought up. Thus,
growth factors, somatostatin, glucocorticoids, and estrogens have been
demonstrated to regulate sst2 mRNA expression levels (22-25).
Given the tumor suppressor properties displayed by sst2, the
understanding of the molecular mechanisms underlying its
transcriptional regulation is of great interest. The aims of this work
were: (1) to describe a novel msst2 promoter sequence and to
identify potentially relevant transcriptional regulatory elements, (2)
to characterize the transcriptional activity of 5' deleted promoter
fragments in different cellular contexts, and (3) in view of the
critical role of TGF- Genomic Library Screening and DNA Sequencing--
A Plasmid Constructions for Luciferase Assays--
Appropriate
promoter 5'-deleted fragments from p9K were cleaved at their 3'-end
with NcoI enzyme and at their 5'-end with KpnI
(Luc21: 2090 bp), SacI (partial digestion) (Luc17: 1704 bp), HindII (Luc12: 1180 bp), SacI (Luc6: 635 bp), or
NheI (Luc3: 325 bp) enzymes. Following, subcloning into pGL3
basic (pGL3) (Promega) was carried out using the same enzyme pairs with
the exception of Luc12 where PGL3 was digested with SmaI and
NcoI enzymes. To assemble Luc9 (898 bp) and Luc10 (1037 bp),
PCR amplification of DNA fragments from nt Cell Culture--
Mouse pituitary AtT-20 (ATCC ref. CCL-89) and
human pancreatic adenocarcinoma PANC-1 (ATCC ref. CRL-1469), BxPC-3
(ATCC ref. CRL-1687), and Capan-1 (ATCC ref. HTB-79) cells were grown
in Dulbecco's modified Eagle's medium (AtT-20, PANC-1, and BxPC3 cells) or RPMI 1640 (Capan-1 cells) supplemented with 10% FCS, 5%
streptomycin/penicillin, 1% fungizon, and 2 mM
L-glutamine (growth medium, Life Technologies, Inc.).
Transient Transfection and Luciferase Assays--
AtT20
(5 × 104 cells/ml), PANC-1 (7 × 104
cells/ml), BxPC-3 (105 cells/ml), and Capan-1
(105 cells/ml) cells were seeded in 35-mm dishes and left
to attach and grow overnight. Subsequently, they were co-transfected
with a mixture of luciferase plasmid (either pGL3 or a test construct, 2 µg) and pCMV
Cells were solubilized in 200 µl of cell culture lysis reagent
(Promega). Luciferase activity was measured after addition of 100 µl
of luciferin solution (Promega) to 50 µl of cell lysate using a
Labsystem Luminoskan. Cell Growth Assay--
BxPC-3 cells were grown and plated in
35-mm dishes at 60 × 103 cells/ml (2 ml/dish).
Following an overnight attachment phase, cells were transfected with 2 µg of either pSmad4 or pCMV5 (empty vector), and subsequently allowed
to recover for 8 h as described above. Cells were cultured in
basic medium overnight (time 0) and then in basic medium containing or
not 10% FCS with or without 1 nM RC-160. Cell growth was
measured at the indicated times by counting cells with a Coulter
counter Z1 (Coulter Electronics) as described previously (28).
Reverse Transcription (RT-PCR)--
Total RNA was extracted
using RNAble (Eurobio). 1 µg of total RNA was denatured at 94 °C
for 10 min, immediately chilled on ice, and treated with RNase-free
DNase I. First-strand cDNA synthesis was undertaken as formerly
described (6). sst2 and Nuclear Extract Preparation--
Cells were washed, scraped, and
harvested in phosphate-buffered saline, then lysed in buffer HNB (0.5 M sucrose, 15 mM Tris, pH 7.5, 60 mM KCl, 0.25 mM EDTA, 0.125 mM
EGTA, 0.5 mM spermidine, 0.15 mM spermine, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 2 mM benzamidine, 5 µg/ml aprotinin, and 5 µg/ml leupeptin with 0.1% Nonidet P-40). The nuclei were pelleted
and resuspended in buffer HNB without Nonidet P-40 but containing 0.31 M NaCl. After four freeze-thaw cycles, nuclei were
separated by centrifugation at 100,000 × g for 15 min,
and the supernatant was stored at Solubilization and Immunoblotting--
Cells were washed twice
and collected in phosphate-buffered saline. After centrifugation at
1000 × g for 5 min at 4 °C, cells were solubilized
in 50 mM Tris-HCl buffer, pH 7.5 containing 140 mM NaCl, 1 mM EDTA, 0.1 mg/ml soybean trypsin
inhibitor, 0.1 mM phenylmethylsulfonyl fluoride and 1.5%
CHAPS. After gently shaking for 30 min at 4 °C, the mixture was
centrifuged at 13 000 × g for 20 min. Solubilized or
nuclear proteins (50 µg) were resolved in SDS-polyacrylamide gels,
transferred to a nitrocellulose membrane, and immunoblotted with
anti-Smad 4 (Santa-Cruz Biotechnology) or anti-sst2 antibodies (4).
Immunoreactive proteins were visualized by the ECL immunodetection system.
msst2 Gene Cloning and Sequence Analysis of Its Proximal
Promoter--
Following the screening of a
The presence of typical promoter features and consensus sequences for
transcription factor binding sites comprised within the
msst2 5'- flanking region available was examined by the use of the transcription factor binding site database MATRIX SEARCH (29).
This analysis failed to identify TATA- and CAAT-like promoter sequences, likewise all ssts promoters described to date and
the majority of G protein-coupled receptors. This region of the
msst2 gene contains consensus sequences for AP1 (nt
Sequence comparison between the msst2 5'- flanking region
available and that of hsst2 (20) revealed several stretches
of significant similarity in terms of both sequence homology and spatial distribution, therefore suggesting shared transcriptional regulatory pathways between both mouse and human sst2 genes
(Fig. 1).
Functional Organization of the msst2 Proximal Promoter--
To
functionally characterize the msst2 promoter, transient
transfection assays were undertaken with expression constructs containing different deletions of the 2090 bp 5'-flanking sequence available inserted upstream from the translation initiation (ATG start)
codon of the luciferase reporter gene. Thus, vector expression constructs assembled included Luc21, Luc17, Luc12, Luc6, and Luc3 (Fig.
2A). To this end, human
pancreatic cancer cells PANC-1 and mouse pituitary cells AtT-20, both
endogenously expressing sst2, were used (28, 34).
As shown in Fig. 2B, the cloned sst2 promoter
sequence was able to induce transcription of the reporter gene both in
AtT-20 and PANC-1 cells. In both cell lines, deletion from nt Transcriptional Activation of the msst2 Proximal Promoter by
TGF-
To identify the msst2 promoter region(s) involved in the
up-regulation elicited by TGF-
To determine whether the region between nt
To determine whether Smad3 and Smad4 were involved in the
transcriptional induction of msst2 by TGF- Restoration of TGF- Restoration of Endogenous sst2 Expression by Overexpression of
Smad4 in BxPC-3 Cells--
Because Smad4 was found to be essential for
msst2 transcriptional activation in Smad4-deficient BxPC-3
cells, we sought to determine whether overexpression of Smad4 could
up-regulate sst2 expression in these cells, which lack endogenous sst2
receptors. After transient expression of Smad4 in BxPC-3 cells, Smad4
was localized in the nucleus of BxPC-3 cells as revealed by
immunoblotting of nuclear extracts with anti-Smad4 antibodies, compared
with no expression of Smad4 in control vector-transfected cells (Fig. 7A). Analysis of sst2 mRNA
expression by RT-PCR revealed the presence of sst2 transcripts in
BxPC-3 cells expressing Smad4, but not in cells expressing control
vector, suggesting that overexpression of Smad4 induced endogenous
sst2 gene transcription (Fig. 7B). Smad4-induced
up-regulation of sst2 mRNA levels was associated with an increase
of sst2 protein as observed by immunoblotting experiments using
anti-sst2 antibodies (Fig. 7C).
To determine whether transient overexpression of Smad4 could
re-establish somatostatin-inducible growth inhibition, cellular proliferation assays were performed in the presence or absence of
somatostatin analogue, RC-160 (1) in BxPC-3 cells overexpressing Smad4.
RC-160 has previously been shown to bind sst2 with high affinity (1, 3)
and to inhibit proliferation of cells expressing sst2 (3, 5, 28). As
shown in Fig. 8, RC-160 treatment for
96 h resulted in a significant reduction of cell growth (-32 ± 5%; mean ± S.E., n = 3; p < 0.05) in cells overexpressing Smad4, whereas it did not affect control
vector-transfected cell proliferation. This result was in agreement
with the view that Smad4 induces the expression of functional sst2
receptors in BxPC-3 cells.
This work provides novel information on the msst2
somatostatin receptor promoter sequence and functional organization.
Promoter sequence-based analysis has further enabled the identification of potentially important regulatory regions. Moreover, we demonstrate that msst2 is transcriptionally induced by TGF- The cloning and sequencing of 2090 nucleotides upstream of the
msst2 start codon has allowed the identification of
sequences of potential regulatory interest as they may participate in
both constitutive and induced sst2 promoter activities.
Thus, the presence of a Sp1 and a USF/MLTF site suggests the
possibility of a cell cycle-dependent regulation of msst2
(38, 39).
In addition, the presence of the consensus sequence for TEF, a member
of the leucine zipper transcription factor family that exhibits
thyrotroph-restricted expression during embryogenesis (40), and for the
adult pituitary restricted factor Pit-1/GHF-1, may suggest an important
role for sst2 during embryonic development and maintenance of a
differentiated phenotype of pituitaries (41). Furthermore, cis-acting
elements such as C/EBP and H-APF-1, known to mediate transcriptional
activation of acute phase response genes in response to diverse
cytokines (42) might be involved in msst2 transcriptional
activation by proinflammatory cytokines (43). Finally, the presence of
a cluster of three half-sites of the consensus ERE could be related to
the reported estrogen-dependent sst2 up-regulation
(24).
Reporter assays performed in mouse pituitary AtT-20 and human
pancreatic cancer PANC-1, BxPC-3, and Capan-1 cells reveal that the
msst2 promoter is organized in distinct functional
regulatory regions. A proximal region comprising 325 nucleotides
upstream of the ATG includes the minimal promoter and displays maximal transcriptional activity. Because a consensus AP1 site is located within this region, members of the c-Jun and c-Fos transcription factor
family might account for some of the activity observed. A distal region
consisting of the following upstream ~1700 nt is likely to comprise
silencers or transcriptional repressor elements, because
transcriptional activity of reporter constructs comprising different 5'
length tracts of this region decreases as the length of the 5'-end
portion increases. Despite differences in basal transcription levels,
reporter assays performed with analogous regions of the
hsst2 promoter and cell lines of breast and neuronal origin
reveal a similar activity profile (21, 25). This result, together with
the following additional features shared by hsst2 and msst2: (i) high
sequence homology within the ~120 nucleotides upstream from their
corresponding ATG start site, including the hsst2 initiator element
(SSTR2inr, Refs. 20, 25), (ii) transcriptional start site
position and splicing acceptor site consensus sequence found to be
functional in msst2 (19), and (iii) additional stretches of
significant sequence homology, would suggest that both promoters have
similar functional organization and might share regulatory features.
A growing body of evidence indicates that loss of responsiveness to the
antiproliferative effects of TGF- The present work demonstrates the transcriptional activation of
msst2 by TGF- Recent evidence indicates that Smad3 and Smad4 can directly bind to
specific DNA sequences in either artificial or natural TGF- In addition, our results demonstrate that overexpression of Smad4 in
human Smad4-deficient BxPC-3 cells devoid of endogenous sst2, restores
both endogenous expression of functional sst2 receptors as demonstrated
by mRNA, immunoblotting and binding studies, and somatostatin-induced cell growth inhibition. This suggests that Smad4
can activate the human sst2 promoter in pancreatic cells. This
hypothesis is strengthened by the observation that Sp1 and CAGA-box
like sequences are also present in the hsst2 promoter. In
addition, these results demonstrate that Smad4 is a key transactivator that regulates sst2 gene expression and suggest that the lack of Smad4
may be responsible in part for the loss of sst2 expression in human
pancreatic cancers, which in turn may contribute to a gain of tumor
growth advantage. Strikingly in this respect is the fact that
aberration of the Smad4 gene observed in human pancreatic cell lines or orthotopic xenografts of human pancreatic carcinoma correlates with the loss of sst2 (3, 12,
46).2
In conclusion, the results obtained recognize msst2 as a
TGF-260 to the ATG codon (325 base
pairs) showing maximal activity, and a region between nucleotides
2025 and
260 likely to comprise silencer or transcriptional
suppressor elements. In PANC-1 and AtT-20 cells, transforming growth
factor (TGF)-
up-regulates msst2 transcription.
Transactivation is mediated by Smad4 and Smad3. The cis-acting region
responsible for such regulation is comprised between nucleotides
1115
and
972 and includes Sp1 and CAGA-box sequences. Expression of Smad4
in Smad4-deficient Capan-1 and BxPC-3 cells restores
TGF-
-dependent and -independent msst2
transactivation. Expression of Smad4 in BxPC-3 cells reestablishes both
endogenous sst2 expression and somatostatin-mediated inhibition of cell
growth. These findings demonstrate that msst2 is a
new target gene for TGF-
transcription regulation and underlie the possibility that loss of Smad4 contributes to the lack of sst2 expression in human pancreatic cancer, which in turn may contribute to
a stimulation of tumor growth.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-dependent pathways may significantly contribute to
the malignancy of this tumor type (9). Upon ligand binding, type II
TGF-
receptor (TGF-
RII) heterodimerizes with and phosphorylates
type I TGF-
receptor, which in turn initiates signaling by
phosphorylating intracellular targets, the most important of which are
Smad proteins (10, 11). Thus, phosphorylation of Smad2 and Smad3 leads
to heterodimerization with Smad4 and translocation of the
hetero-oligomeric complex into the nucleus, where it affects
transcription of specific genes by either binding directly to DNA or by
complex formation with other components (11). Smad4, also known as
DPC4 is mutated or deleted in ~50% of pancreatic cancers
(12), and it has been clearly established that Smad4 mutations
interfere with the signaling pathway that mediates TGF-
induced
growth suppression in pancreatic cancer cells (13). However, until now,
very few Smad4-regulated genes have been identified. They include
plasminogen activator inhibitor-1 (14), p21Waf1 (15), Jun-B
(16), human type VII collagen gene (17), and platelet-derived growth
factor B-chain (18).
transduction pathways in the negative control
of cell growth, to explore the possibility that TGF-
and associated signaling molecules Smad3 and Smad4 contribute to the transcriptional regulation of msst2.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
DASH II
mouse 129 genomic DNA library (a gift from A. Begue, Institut Pasteur,
Lille, France) was screened with a 32P-randomly labeled
probe (26) corresponding to the full-length msst2A cDNA sequence
(1.2-kbp XbaI-XbaI fragment isolated from pCMV6c,
kindly donated by G. I. Bell (Howard Hughes Medical Institute, The
University of Chicago)). Positively hybridizing phagemid clones
6
and
9 were isolated following standard protocols (27). Subsequently, a KpnI-KpnI fragment comprising sst2 N-terminal
coding and 5'-non-coding sequence (~0.7 and ~2.1 kbp fragments,
respectively), and a BamHI-BamHI fragment
comprising the full-length sst2 coding sequence and 5'- and 3'-flanking
non-coding sequence (~1.7 and ~1.5 kbp fragments, respectively)
from
9 were subcloned into pUC19 (p9K and p9B, respectively). p9K
and p9B inserts were sequenced on both strands using ABI PRISM Dye
Terminator cycle Sequencing Ready Reaction Kit (PerkinElmer Life
Sciences) and ABI 373A sequencer apparatus. Sequencing was initiated
using the standard M13 forward and reverse primers. Analysis was
performed using PCGene and Matrix Search programs.
972 to
557 and from
833 to
557 of the reported msst2 promoter sequence was
undertaken using Luc12 construct as a template and primer pairs 5'Kpn10
(ATCGCGGTACCAGCATAGAGTTGTTCTTGG) and 3'Sac (CTAGCCTGGAGCTCACTATG), and
5'Kpn9 (ATCGCGGTACCATAGCCGTCTGCCACATG) and 3'Sac, respectively.
Amplified fragments were then subcloned into pLuc21 backbone
using KpnI and SacI restriction enzymes. Construction of the TGF-
responsive element-containing vectors (RE
and (RE)2, single and tandem repeats, respectively) involved the
initial insertion of HSV-thymidine kinase promoter from pRL-TK (Promega) into pGL3 using BglII and HindIII as
cloning restriction sites to obtain TK-pGL3. Subsequently, one or two
RE fragments (nt
115 to
972) previously PCR amplified using primer
pair 5'-GACAAAGGAGAGTTACAGCAG-3' and 5'-GCTTGATGTCTGCCACCATC-3' and
subcloned into Topo TA cloning vector (Invitrogen) were inserted into
TK-pGL3 vector to obtain RE-TK and (RE)2-TK. Integrity and orientation
of the inserts were checked by restriction enzyme analysis and
sequencing. Control plasmids included pGL3 and pCMV
gal (a gift from
H. Paris, INSERM U388, France).
gal (1 or 2 µg) with or without 0.5 µg each of the following expression vectors or vector combinations:
pCMV5-FLAG-Smad4 (pSmad4), pCMV5-Smad4-(1-514)
(pSmad4-(1-514)), pCDNA3-myc-Smad3 (pSmad3), or pSmad3 and pSmad4.
All pSmad constructs were kindly given by Dr. Ten Dijke (Ludwig
Institute for Cancer Research, Uppsala, Sweden). Transfection mixtures
where pSmad vectors or combinations of them were absent were completed
with an equivalent amount of the parental pSmad empty vector(s).
Similarly, 2 µg of RE-TK or (RE)2-TK vectors and 1 µg pCMV
gal
were used in the corresponding transfection assays. Cells were
transfected using FuGENE6 transfection reagent (Roche Molecular
Biochemicals) and subsequently allowed to recover for 8 h. In
basal transcriptional activity experiments, cells were starved in basic
medium (growth medium without FCS) for 16 h and then allowed to
grow in fresh basic medium supplemented with 0.5% FCS for 24 h.
Experiments of recombinant human TGF-
(R&D Systems)-treated cells
were carried out after a starvation period of either 16 h (BxPC3
and Capan-1) or 40 h (AtT-20 and PANC-1) in basic medium.
Treatment was carried out in fresh basic medium supplemented with 4 ng/ml TGF-
1 and 0.5% FCS for all cell lines with the exception of
AtT-20 cells where medium was supplemented with 2 ng/ml TGF-
1, and
no FCS was added. A parallel set of mock-treated cells was always
included in each experiment as a control.
galactosidase was measured by spectrophotometry (MRX Dynex technologies) on 90-µl cell lysate aliquots after addition of 200 µl of O-nitrophenyl
-D-galactopyranoside (1 mg/ml) solution.
-actin PCR fragment amplification were
carried out for 35 (sst2) and 25 (
-actin) cycles using,
respectively, primer pairs: (5'-ATGGACATGGCGGATGAGCCA-3', sense) and
(5'-TACTGGTTTGGAGGTCTCCAT-3', antisense), and
(5'-TCACGCCATCCTGCGTCTGGACT-3', sense) and (5'-CCGGACTCATCGTACTCCT-3',
antisense) as described previously (6). Amplified fragments were
separated on 7% SDS-PAGE and stained with ethidium bromide.
80 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
DASH II mouse genomic
DNA library (5 × 105 pfu) with a homologous probe
corresponding to the full-length msst2A cDNA (1.2 kbp), hybridizing
clones
6 and
9 were isolated. Southern blot analysis with a panel
of restriction enzymes indicated that they putatively contained the
full msst2 coding sequence and ~4.5 and 11.5 kbp, and 7.5 and 8.5 kbp
5'- and 3'-flanking regions, respectively. Subcloning and subsequent
sequencing analysis of overlapping ~4.5 kbp
BamHI-BamHI and ~2.7 kbp
KpnI-KpnI fragments from clone
9 allowed the
description of a 4730 nt genomic DNA tract comprising 2090 nt
corresponding to the 5'-region upstream of the msst2
translation initiation codon (ATG) and 2640 nt including coding and
3'-non-coding sequence of the msst2 gene
(GenBankTM/EBI accession number AF008914).
231 to
225), Sp1 (nt
1009 to
999), USF/MLTF (nt
1077 to
1070), TEF
(nt
598 to
593) and HAPF-1 (nt
1676 to
1670 and
32 to
26).
A putative Pit-1/GH-1 binding site identical to that identified in the
rat sst1 promoter region (ATGAATA, Ref. 30) is also found
between nt
151 and
145. We have identified two sequences: TTTGCAC
(nt
1161 and
1155) and CTTGCAA (nt
388 to
381) that show
homology with a natural C/EBP
and C/EBP
inducible binding
sequence in the mouse hepatocyte growth factor gene (31). Moreover, the second half of the reported C/EBP consensus sequence (32) is totally
conserved between nt positions
321 and
312 (TATCTGTAAT). Additionally, a cluster of three sequences corresponding to half of the
consensus motif for the estrogen responsive element (GGTCA) is located
between positions 1143 and 1174. Finally, three E-boxes, (nt
1992 to
1987,
1954 to
1949, and
28 to
23), which constitute the
binding site for members of the helix-loop-helix transcription factor
family, one GA-box (nt
1550 to
1523), capable of binding to SP1 and
related factors, and four putative CAGA-like boxes (nt
1302 to
1296,
986 to
978,
935 to
927, and
297 to
289), recently
described as TGF-
responsive elements (15, 33) were identified (Fig.
1).
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Fig. 1.
2090-Nucleotide sequence fragment of the
msst2 5'-flanking region. The ATG start codon is
shown in bold. The transcription start site (+1) (19) is
indicated by an asterisk. Open boxes indicate
consensus sequences for several transcription factors. The tract
underlined corresponds to an A-T rich region. The region
found to be responsive to TGF- is double underlined.
Gray-shaded regions outline those tracts where significant
homology between msst2 and its cognate hsst2
sequences have been found (identical and distinct nucleotides in
black and white characters, respectively).
Positions corresponding to hsst2 boundary nucleotides at
each tract (20) are indicated under gray-shaded regions.
Percentage of nucleotide identity shared between msst2 and
hsst2 sequences at each tract are (from top to
bottom): 83.1%; 71.4%; 75.5%; 88.9%; and 70.2%.
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Fig. 2.
Transcriptional activity of the
msst2 promoter in AtT-20 and PANC-1 cells.
A, schematic view of reporter constructs used in the
functional characterization of the msst2 proximal promoter.
Relative position of transcription factor consensus sequences
identified. B, transcriptional activity is indicated as
relative luciferase units (RLU) (luciferase activity
corrected for transfection efficiency and standardized as -fold changes
relative to empty vector (PGL3, basic = 1)). RLU values shown are
the mean ± S.E. of three experiments performed in
duplicate.
2025 to
1639 upstream from the msst2 transcription start site (+1)
(19) did not change promoter activity. However, successive deletions of
the promoter from nt
1639 to
260 corresponding to Luc17, Luc12,
Luc6, and Luc3 constructs resulted in an increase of promoter activity.
Maximal promoter activity was observed with Luc3, the shorter reporter
construct tested. This indicates that a minimal promoter was comprised
within the 325 nucleotides upstream of the msst2 ATG start
codon. This result was in agreement with the location of a
transcription start site 65 base pairs upstream from the start codon
(19).
, Role of Smad3 and Smad4--
To investigate the effect of
TGF-
on the transcriptional activity of the msst2
promoter, reporter gene assays were performed using the Luc17
construct, because of the presence of Sp1, AP1, and CAGA box-like
sequences recently recognized as cis-acting elements for
TGF-
-mediated transcriptional induction (13, 33, 35) within ~1400
base pairs upstream of the msst2 start codon. Our results
showed that treatment of Luc17-transfected AtT-20 and PANC-1 cells with
TGF-
for 24 h, induced a 2- and 1.8-fold transcriptional
activation of Luc17, respectively (Fig.
3) suggesting that the msst2
gene promoter is up-regulated by TGF-
.
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Fig. 3.
Transcriptional up-regulation of the
msst2 proximal promoter by TGF-
in AtT-20 and PANC-1 cells. TGF-
-responsiveness of
reporter constructs in AtT-20 and PANC-1 cells following a 24-h
treatment with 2 and 4 ng/ml TGF-
, respectively (filled
bars). Results are expressed as percent of control value obtained
with untreated cells (set arbitrarily to 100%, open bars).
Values are the mean ± S.E. of three experiments performed in
duplicate. *, p < 0.01 versus the
corresponding value of untreated cells.
, shorter reporter constructs were used in similar expression analyses. Whereas Luc12 retained TGF-
responsiveness comparable with that of Luc17, neither Luc10, Luc9, Luc6, or Luc3 constructs were responsive to TGF-
(Fig. 3). These results suggested the presence of a cis-acting TGF-
responsive element comprised between nt
1115 and
972 of the msst2
promoter sequence.
1115 and
972 (RE) could
function as an enhancer in the context of a heterologous minimal
promoter, one or two copies of the RE were cloned upstream of the
TK promoter in the pGL3 basic vector and were used in
transient PANC-1 cell transfection experiments. As shown in Fig.
4, a tandem repeat but not a single copy,
of the RE conferred TGF-
responsiveness of the TK promoter, the
promoter activity being increased 2-fold after treatment with TGF-
.
These data indicate that the region between nt 911 and 1053 of the
msst2 promoter can function as an enhancer element driving
the transcriptional activation of a heterologous promoter when present
as a dimer and suggest that this region is responsible for TGF-
activation of msst2 promoter activity.
View larger version (12K):
[in a new window]
Fig. 4.
msst2 promoter region between nt
1115 and
972 confers TGF-
responsiveness
to a heterologous promoter in PANC-1 cells. Constructs containing
one (RE-TK) or two copies ((RE)2-TK) of the
msst2 promoter region between nt
1115 and
972 cloned
upstream from the thymidine kinase promoter were transiently
transfected in PANC-1 cells. Cells were treated (filled
bars) or not (open bars) with 4 ng/ml TGF-
for
24 h. Transcriptional activation in each case is expressed as
percent of control value obtained with untreated transfected cells (set
arbitrarily to 100%). Values are the mean ± S.E. of four
experiments performed in duplicate. *, p < 0.05 versus the corresponding value of untreated cells.
, PANC-1 cells
were transiently co-transfected with Luc12 reporter construct and
expression vectors (individually or in combination) coding for Smad3,
Smad4, and the truncated form of Smad4, Smad4-(1-514), previously
shown to inhibit Smad4-dependent signaling pathways (36).
Expression of Smad3, Smad4, or Smad4 (1) alone did not affect
basal Luc12 transcriptional activity and neither Smad3 nor Smad4
enhanced the response to TGF-
(Fig.
5). However, overexpression of truncated Smad4 abolished TGF-
induction of Luc12 activity demonstrating the
involvement of Smad4 in TGF-
mediated up-regulation of
msst2 promoter activity in these cells. Furthermore,
co-expression of Smad3 and Smad4 in the absence of TGF-
increased
Luc12 transcription to levels comparable with those previously reached
with TGF-
, and addition of TGF-
did not further increase promoter
activity (Fig. 5). These results therefore identified Smad3 and Smad4
as important components of TGF-
-mediated msst2 transcriptional
activation in PANC-1 cells.
View larger version (23K):
[in a new window]
Fig. 5.
Regulation of the msst2
proximal promoter activity by Smad proteins and
TGF- in PANC-1 cells. Luc12 and single or
pair combinations of expression vectors corresponding to Smad3, Smad4,
and the Smad4 dominant-negative mutant Smad4-(1-514) were transiently
expressed in PANC-1 cells, and cells were treated (filled
bars) or not (open bars) with 4 ng/ml TGF-
for
24 h. Transcriptional activation in each case is expressed as
percent of control value obtained with untreated Luc12 transfected
cells (set arbitrarily to 100%). Values experiments performed in
duplicate. *, p < 0.05 versus the
corresponding value of untreated cells. **, p < 0.01 versus Luc12-transfected control cells.
-mediated msst2 Transcriptional
Activation in Smad4-deficient Pancreatic Cancer Cells by Expression of
Smad4--
Additional evidence for the role of Smad4 in
TGF-
-dependent msst2 up-regulation was pursued by
performing similar transient expression assays with human pancreatic
cancer cell lines Capan-1 and BxPC-3 lacking endogenous Smad4 activity
as a result of homologous deletion or mutation at the Smad4
corresponding locus (DPC4) and unresponsive to TGF-
(12,
37). Furthermore, these cells have lost sst2 endogenous expression and
hence, sst2-induced negative regulation of cell growth (6, 28). The
transcriptional activity of the msst2 promoter was first
verified in both cell lines. As observed in PANC-1 and AtT-20 cells,
Luc3 displayed maximal transcriptional activity, and activity of
constructs Luc6 to Luc21 decreased progressively both in Capan-1 and
BxPC-3 cells (not shown). Furthermore, Luc12 construct was not
responsive to TGF-
in either Capan-1 or BxPC-3 cells, and expression
of Smad3 did not affect Luc12 transcriptional levels (Fig.
6). Moreover, in Capan-1 cells, transient
transfection of Smad4 induced TGF-
-dependent Luc12
activation, and expression of Smad4-(1-514) abrogated this
up-regulation. Co-expression of Smad3 and Smad4 in Capan-1 cells in the
absence of TGF-
increased Luc12 transcription to a level equivalent
to that obtained in TGF-
treated cells with Smad4 alone, and this
increase was not further enhanced by TGF-
(Fig. 6). In BxPC-3 cells,
the same results were found except that Luc12 transcriptional
activation was maximally induced by expression of Smad4 alone. This
activation was abrogated in the presence of Smad4-(1-514). Luc12
transcriptional activation by Smad4 was not further increased by
co-expression with Smad3 and/or TGF-
treatment (Fig. 6). These
results indicated that expression of Smad4 conferred
TGF-
-dependent and -independent transcriptional
activation of the msst2 promoter in Capan-1 and BxPC-3
cells, respectively.
View larger version (18K):
[in a new window]
Fig. 6.
msst2 proximal promoter
transactivation by Smad proteins and TGF- in
Capan-1 and BxPC-3 cells. Luc12 and single or pair combinations of
expression vectors corresponding to Smad3, Smad4, and the Smad4
dominant-negative mutant Smad4-(1-514) were transiently expressed, and
cells were treated (filled bars) or not (open
bars) with 4 ng/ml TGF-
for 24 h. Transcriptional
activation in each case is expressed as percent of control value
obtained with untreated Luc12-transfected cells (set arbitrarily to
100%). Values are the mean ± S.E. of four experiments performed
in duplicate. *, p < 0.01 versus the
corresponding value of untreated cells. **, p < 0.01 versus Luc12-transfected control cells.
View larger version (34K):
[in a new window]
Fig. 7.
Smad4 induction of sst2 expression in BxPC-3
cells. BxPC-3 cells were transfected with either the Smad4
expression vector (Smad4) or the PCMV5 empty vector and
cultured in Dulbecco's modified Eagle's medium supplemented with 10%
FCS for 48 h. A, detection of nuclear Smad4. Nuclear
extracts were prepared from BxPC-3 cells transfected with Smad4
expression vector or PCMV5 vector and fractionated by 10% SDS-PAGE
before immunoblotting with anti-Smad4 antibodies. B,
induction of sst2 mRNA by Smad4. RT-PCR amplifications of sst2
mRNA (sst2: 1105 bp) or -actin (actin: 517 bp) were performed on
BxPC-3 cells transfected with Smad4 expression vector or PCMV5 vector
as described under "Materials and Methods." The resulting PCR
products were analyzed by polyacrylamide gel electrophoresis and
ethidium bromide staining. C, induction of sst2 protein by
Smad4. BxPC-3 cells transfected with Smad4 expression vector or PCMV5
vector were solubilized, and proteins were resolved by SDS-PAGE on a
7.5% acrylamide gel, transferred to a nitrocellulose membrane, and
blotted with anti-sst2 antibodies.
View larger version (25K):
[in a new window]
Fig. 8.
Restoration of RC-160-mediated growth
inhibition by expression of Smad4 in BxPC-3 cells. BxPC-3 cells
were transfected with Smad4 expression vector (hatched bar)
or the PCMV5 empty vector (open bar). After an 8-h
expression, cells were cultured in basic medium overnight and then
cultured in basic medium containing 10% FCS with or without
(untreated) 1 nM RC-160 for 96 h. Cell growth was
evaluated by cell counting. Results are expressed as a percentage of
control values obtained in untreated cells measured at the indicated
times by counting. Values are mean ± S.E. of three experiments
performed in triplicate. *, p < 0.05 versus
the corresponding value of untreated cells.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and identify the
region that includes the cis-acting element(s) required for this
up-regulation. In addition, we provide evidence that the tumor
suppressor Smad4 (DPC4) plays a central role in
TGF-
-dependent msst2 transcriptional induction. Finally,
we demonstrate that expression of Smad4 is sufficient to restore sst2
expression and re-establish somatostatin-induced growth inhibition in
Smad4-deficient human pancreatic cancer cells, which lack endogenous
sst2 receptors.
may enhance the ability of tumor
cells to invade surrounding tissue structures during malignant
progression through stimulation of angiogenesis, immunosupression, and
synthesis of extracellular matrix. In pancreatic cancer, a defect in
TGF-
signaling appears to be crucial to the aggressiveness and
growth advantage of this tumor type (44-47). Noteworthy, transgenic
mice expressing a dominant-negative TGF-
RII mutant in several
targeted epithelial tissues, reveal increased proliferation and severe
perturbed differentiation restricted to pancreatic acinar cells (48).
Additionally, somatic inactivation of the tumor suppressor Smad4,
whether as a result of homozygous deletion or mutation, has been
observed in ~50% of human pancreatic carcinomas (11). Finally, a
correlation between the lack of Smad4 activity and unresponsiveness to
TGF-
-mediated growth inhibition has been established in a number of
human pancreatic cancer cell lines (12).
. Indeed, assays carried out in AtT-20 and
PANC-1 cells reveal a 2-fold increase of Luc12 construct transcription levels after treatment with TGF-
. Analogous discrete
TGF-
-mediated transactivation has been reported for other reporter
gene constructs (17, 49-51). Therefore, msst2 is recognized
as the first G-protein-coupled receptor-encoding gene transcriptionally
up-regulated by TGF-
. On the other hand, we provide evidence that
Smad4 plays a crucial role in this activation. Overexpression of the
dominant-negative mutant Smad4-(1-514) in PANC-1 cells abrogates
TGF-
-dependent msst2 promoter activation.
Moreover, whereas transient expression of Luc12 construct in human
pancreatic cancer BxPC-3 and Capan-1 cells, which lack Smad4
expression, results in lack of TGF-
induction; restoration of Smad4
expression reestablishes TGF-
-mediated msst2 promoter
induction to a level comparable with that observed in PANC-1 cells
containing functional Smad4. These results underscore a central role
for Smad4 in msst2 up-regulation. In addition, our results suggest that
Smad3 alone does not activate transcription but acts in concert with
Smad4 to induce TGF-
-independent msst2 transcription.
This is in agreement with the required cooperation of both Smad
proteins in a heteromeric complex and their capacity to act as
ligand-independent activators when transiently overexpressed (36, 52).
Transcriptional activation of the msst2 promoter by Smad4 in
the absence of Smad3 and/or TGF-
in BxPC3 cells and not in Capan 1 cells could reflect a different expression level of endogenous Smad3
molecules in these cells as reported for other cell types (53). It is
noteworthy that, for a given cell line, transcriptional activation of
Luc12 construct reaches similar maximal levels in each of the referred
situations. This might suggest that the response is at a saturating
level and/or that other nuclear factors are required for greater
induction. Supporting the former hypothesis would be the enhanced
expression of TGF-
isoforms in several human pancreatic cancer cell
lines (54).
-inducible promoters and thus activate gene transcription. To
date, such sequences (CAGA-box) have been identified in the promoter of
the human PAI-1 and PDGF-B genes (33, 18), the mouse junB (16), and human COL1A2 genes (55).
However, it is not clear whether direct binding of Smad proteins to
these closely related DNA sequences is sufficient to confer maximal TGF-
-induced transcription. It is interesting that Sp1 binding activity has been attributed to TGF-
responsive elements identified in the promoter of p15INK4B (56) and
p21WAF1/Cip1 (35) genes, and that
Smad proteins have been shown to activate transcription through
TGF-
-responsive elements, which display Sp1 binding activity within
the same or adjacent DNA regions in the COL7A1 (16), the
COL1A2 (55), and the p21WAF1/Cip1
(57) genes. Moreover, Sp1 and Smad proteins can functionally interact
to activate gene transcription (57). Deletion analysis of the
msst2 promoter reveals that the region between nt
1115 and
972 is required for msst2 TGF-
-dependent
up-regulation. Furthermore, this region functions as an enhancer in the
context of a heterologous promoter. It is tempting to propose the
neighboring Sp1 site and CAGA-box like sequence identified in this
region (nt
1009 to
999 and
986 to-978, respectively) as the
cis-acting elements responsible for the transcriptional activation
induced by TGF-
.
-regulated gene and identify Smad4 as a critical element in such
regulation. The ability of Smad4 to restore the expression of sst2, one
of the key negative control elements of cell proliferation in
pancreatic cancer cells raises the possibility that functional Smad4 is
required for TGF-
-mediated induction of sst2 and subsequent somatostatin-mediated growth inhibition. In consequence, the lack of
sst2 induction as a result of Smad4 inactivation may represent a new
possible molecular mechanism contributing to the growth advantage of
pancreatic tumors.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Dr G. I. Bell for providing msst2 cDNA and Dr Ten Dijke for providing Smad3, Smad4, and Smad4-(1-514) constructs.
![]() |
FOOTNOTES |
---|
*
This work was supported by Grant 5576 from the Association
pour la Recherche contre le Cancer, Grant 2ACFH0113C from the Conseil Rgional Midi-Pyr
n
es, and Grant 2578DB06D from the Ligue
Nationale contre le Cancer.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§ To whom correspondence should be addressed: INSERM U 531, Institut Louis Bugnard, IFR 311, Av. Jean Poulhès, CHU Rangueil Bât. L3 31403, Toulouse, France. Tel.: 33 5 61 32 24 07; Fax: 33 5 61 32 24 03; E-mail: susinich@rangueil.inserm.fr.
Published, JBC Papers in Press, January 22, 2001, DOI 10.1074/jbc.M010981200
2 J. Torrisani, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: sst2, somatostatin receptor; hsst2, human sst2 gene; msst2, mouse sst2 gene; kbp, kilobase pairs; FCS, fetal calf serum; PAGE, polyacrylamide gel electrophoresis; pfu, plaque forming unit; PCR, polymerase chain reaction; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; nt, nucleotide.
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