Transcriptional Activation of Mouse sst2 Somatostatin Receptor Promoter by Transforming Growth Factor-beta

INVOLVEMENT OF Smad4*

Elena Puente, Nathalie Saint-Laurent, Jérôme Torrisani, Christophe Furet, Andrew V. SchallyDagger , Nicole Vaysse, Louis Buscail, and Christiane Susini§

From INSERM U 531, Institut Louis Bugnard, 31403, Toulouse, France and the Dagger  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



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 -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)-beta 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-beta -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-beta 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

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-beta -dependent pathways may significantly contribute to the malignancy of this tumor type (9). Upon ligand binding, type II TGF-beta receptor (TGF-beta RII) heterodimerizes with and phosphorylates type I TGF-beta 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-beta -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).

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-beta transduction pathways in the negative control of cell growth, to explore the possibility that TGF-beta and associated signaling molecules Smad3 and Smad4 contribute to the transcriptional regulation of msst2.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Genomic Library Screening and DNA Sequencing-- A lambda 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 lambda 6 and lambda 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 lambda 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.

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 -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-beta 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 pCMVbeta gal (a gift from H. Paris, INSERM U388, France).

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 pCMVbeta 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 pCMVbeta 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-beta (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-beta 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-beta 1, and no FCS was added. A parallel set of mock-treated cells was always included in each experiment as a control.

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. beta -galactosidase was measured by spectrophotometry (MRX Dynex technologies) on 90-µl cell lysate aliquots after addition of 200 µl of O-nitrophenyl beta -D-galactopyranoside (1 mg/ml) solution.

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 beta -actin PCR fragment amplification were carried out for 35 (sst2) and 25 (beta -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.

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 -80 °C.

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.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

msst2 Gene Cloning and Sequence Analysis of Its Proximal Promoter-- Following the screening of a lambda 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 lambda 6 and lambda 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 lambda 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).

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 -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/EBPbeta and C/EBPgamma 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-beta 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-beta 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%.

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).


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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.

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 -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).

Transcriptional Activation of the msst2 Proximal Promoter by TGF-beta , Role of Smad3 and Smad4-- To investigate the effect of TGF-beta 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-beta -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-beta 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-beta .


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Fig. 3.   Transcriptional up-regulation of the msst2 proximal promoter by TGF-beta in AtT-20 and PANC-1 cells. TGF-beta -responsiveness of reporter constructs in AtT-20 and PANC-1 cells following a 24-h treatment with 2 and 4 ng/ml TGF-beta , 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.

To identify the msst2 promoter region(s) involved in the up-regulation elicited by TGF-beta , shorter reporter constructs were used in similar expression analyses. Whereas Luc12 retained TGF-beta responsiveness comparable with that of Luc17, neither Luc10, Luc9, Luc6, or Luc3 constructs were responsive to TGF-beta (Fig. 3). These results suggested the presence of a cis-acting TGF-beta -responsive element comprised between nt -1115 and -972 of the msst2 promoter sequence.

To determine whether the region between nt -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-beta responsiveness of the TK promoter, the promoter activity being increased 2-fold after treatment with TGF-beta . 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-beta activation of msst2 promoter activity.


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Fig. 4.   msst2 promoter region between nt -1115 and -972 confers TGF-beta 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-beta 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.

To determine whether Smad3 and Smad4 were involved in the transcriptional induction of msst2 by TGF-beta , 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-beta (Fig. 5). However, overexpression of truncated Smad4 abolished TGF-beta induction of Luc12 activity demonstrating the involvement of Smad4 in TGF-beta mediated up-regulation of msst2 promoter activity in these cells. Furthermore, co-expression of Smad3 and Smad4 in the absence of TGF-beta increased Luc12 transcription to levels comparable with those previously reached with TGF-beta , and addition of TGF-beta did not further increase promoter activity (Fig. 5). These results therefore identified Smad3 and Smad4 as important components of TGF-beta -mediated msst2 transcriptional activation in PANC-1 cells.


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Fig. 5.   Regulation of the msst2 proximal promoter activity by Smad proteins and TGF-beta 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-beta 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.

Restoration of TGF-beta -mediated msst2 Transcriptional Activation in Smad4-deficient Pancreatic Cancer Cells by Expression of Smad4-- Additional evidence for the role of Smad4 in TGF-beta -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-beta (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-beta 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-beta -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-beta increased Luc12 transcription to a level equivalent to that obtained in TGF-beta treated cells with Smad4 alone, and this increase was not further enhanced by TGF-beta (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-beta treatment (Fig. 6). These results indicated that expression of Smad4 conferred TGF-beta -dependent and -independent transcriptional activation of the msst2 promoter in Capan-1 and BxPC-3 cells, respectively.


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Fig. 6.   msst2 proximal promoter transactivation by Smad proteins and TGF-beta 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-beta 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.

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).


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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 beta -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.

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.


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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

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-beta 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-beta -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.

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-beta 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-beta 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-beta 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-beta -mediated growth inhibition has been established in a number of human pancreatic cancer cell lines (12).

The present work demonstrates the transcriptional activation of msst2 by TGF-beta . 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-beta . Analogous discrete TGF-beta -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-beta . 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-beta -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-beta induction; restoration of Smad4 expression reestablishes TGF-beta -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-beta -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-beta 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-beta isoforms in several human pancreatic cancer cell lines (54).

Recent evidence indicates that Smad3 and Smad4 can directly bind to specific DNA sequences in either artificial or natural TGF-beta -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-beta -induced transcription. It is interesting that Sp1 binding activity has been attributed to TGF-beta 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-beta -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-beta -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-beta .

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-beta -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-beta -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 Riota gional Midi-Pyriota niota 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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