Characterization of human mucin gene MUC4 promoter: importance of growth factors and proinflammatory cytokines for its regulation in pancreatic cancer cells.

The human mucin gene MUC4 encodes a large transmembrane mucin that is thought to play important roles in tumor cell biology and that is overexpressed in human pancreatic carcinomas. In this report, we describe the structure and functional activity of the 5'-flanking region, including 1.0 kilobase of the promoter. The long 5'-untranslated region (2.7 kilobases) is characterized by a high content of GC in its 3'-end. The first TATA box was located at -2672/-2668. Multiple transcription start sites and a high density of putative binding sites for Sp1 (GC and CACCC boxes), AP-1/-2/-4, cAMP-responsive element-binding protein, GATA, GR, and STAT transcription factors were found within the 5'-flanking region. Transcriptional activity of the promoter was assessed using pGL3-luciferase deletion mutants in two MUC4-expressing (CAPAN-1 and CAPAN-2) and one nonexpressing (PANC-1) pancreatic cancer cell line. Two highly active fragments (-219/-1 and -2781/-2572) that drive MUC4 transcription in CAPAN-1 and CAPAN-2 cells were identified. Gel retardation assays indicated that Sp1 and Sp3 bind to cognate cis-elements found in the 5'-flanking region and that Sp1 transactivates, whereas Sp3 inhibits the GC-rich region (-464/-1) in CAPAN-2 cells. Activation of protein kinase C with phorbol ester and treatment of cells with epidermal growth factor and transforming growth factor-alpha resulted in up-regulation of the promoter. Tumor necrosis factor-alpha and interferon (IFN)-gamma inflammatory cytokines had no or mild effect on MUC4 transcriptional activity when used alone. However, a very strong synergistic effect (10-12-fold activation) between IFN-gamma and tumor necrosis factor-alpha or IFN-gamma and transforming growth factor-alpha was obtained in CAPAN-2 cells. Altogether these results demonstrate that the 5'-flanking region of MUC4 contains epithelial cell-specific, positive, and negative regulatory cis-elements, that Sp1/Sp3 are important regulators of MUC4 basal expression, and that its regulation in pancreatic cancer cells involves complex interplay between several signaling pathways.

Mucins are large, highly O-glycosylated proteins that are encoded by at least 10 genes in humans (1,2).The common structural feature among all mucins is the presence of tandemly repeated amino acid regions that serve as a docking site for O-glycans (1).Mucins are now classified in two main categories: secreted mucins including gel-forming (MUC2, MUC5AC, MUC5B, and MUC6) and non-gel-forming (MUC7) and membrane-bound mucins (2).This last group includes MUC1, MUC3, MUC4, and MUC12 (3).
Human MUC4 has been extensively studied in our laboratory.Its first partial cDNA was isolated from a human tracheobronchial library (4), and the gene was located on chromosome 3q29 (5).Recently its complete genomic organization has been established.The 5Ј-region of the gene is characterized by a single exon (exon 2) made of a 48-bp1 minimal unit repeated in tandem that encodes a large Ser/Thr-rich domain (6).The tandem repeat varies in size between 7 and 19 kb because of a variable number of tandem repeat polymorphism.The 3Ј-end region of MUC4 mainly consists of two EGF-like domains, a transmembrane domain, and a short cytoplasmic tail (7) and shows extensive alternative splicing downstream of the tandem repeat that generates a family of putative secreted and membrane-associated MUC4 isoforms (8).
The human MUC4 displays extensive similarities in the Nt and Ct regions with the rat sialomucin complex (SMC) previously isolated from mammary adenocarcinoma ascites cells (9).SMC is a heterodimer glycoprotein composed of an O-glycosylated mucin subunit ASGP-1 tightly bound to a N-glycosylated transmembrane subunit ASGP-2 (10).Interestingly, ASGP-2 contains two EGF-like domains in its extracellular part, one of which acts as a ligand for ErbB2/Neu oncogene (11).The whole SMC molecule is transcribed from a single gene as a 9-kb mRNA, which is translated into a single polypeptide that is secondary cleaved to yield the heterodimer complex ASGP-1/ ASGP-2 (12).In the same way, MUC4 may also be cleaved in two subunits: the mucin subunit MUC4␣ and a transmembrane subunit with two EGF-like domains MUC4␤ (7).MUC4 is therefore considered to be the human homologue of rat SMC.
In situ hybridization studies have shown that MUC4 is expressed in numerous normal tissues such as trachea, lung, stomach, colon, uterus, and prostate (13,14) as well as very early during the development of the primitive gut (6.5 weeks of gestation) (15,16).No MUC4 mRNAs are detected in normal pancreas (17), gall bladder, liver, biliary epithelial cells, or intrahepatic bile ducts (18).Interestingly, abnormal expression of MUC4 was demonstrated to occur in several human epithelial cancers such as lung and pancreas carcinomas (17,19,20).Furthermore high levels of MUC4 mRNAs have been found in several differentiated pancreatic tumor cell lines (21)(22)(23).The dysregulation of MUC4 expression, often dramatic, together with the homology to SMC/rMuc4, which is considered to promote tumor cell metastasis (24), points out an important role for MUC4 in human tumor biology, especially in pancreatic tumors.Knowledge of the molecular mechanisms underlying the dysregulation of MUC4 observed in pancreatic cancer is thus necessary to understand the role and contribution of MUC4 altered expression during carcinogenesis.However, very few data concerning the factors involved in the control of MUC4 expression have been published so far.Gollub et al. (25) have shown that estrogen and dexamethasone up-regulate MUC4 mRNA levels in the endometrial Ishikawa epithelial cell line.In bronchial cells, Bernacki et al. (26) showed that MUC4 is regulated at the transcriptional level by retinoic acid.More recently, Choudhury et al. (27) have demonstrated that alltrans-retinoic acid induces MUC4 expression in CD18/HPAF pancreatic carcinoma cells and is mediated by TGF-␤ 2 .
Recently, the 5Ј-flanking region as well as 2.4 kb of the promoter of the rat Muc4 was described (10,28).The promoter is TATA-less and is characterized by the presence of an initiator consensus sequence located 27 bp upstream of the unique transcription start site.The cell-specific activity of the promoter is thought to be under the control of cis-elements present upstream from the initiator (28).
The purpose of our study was to isolate and characterize the promoter of the human mucin gene MUC4 and identify ciselements as well as define the molecular mechanisms involved in the regulation of its expression in pancreas cancer cells.To this end, three human well differentiated pancreatic duct cancer cell lines CAPAN-1 (MUC4 positive), CAPAN-2 (MUC4 positive), and PANC-1 (MUC4 negative) were chosen (21, 29 -31).We have identified regulatory cis-elements and trans-acting factors involved in the control of MUC4 expression.Moreover, this study shows that MUC4 promoter regulation is complex and involves many signaling pathways that may be responsible for the overexpression of MUC4 expression in carcinoma of the pancreas and hepatobiliary system.

EXPERIMENTAL PROCEDURES
Cloning-Inserts were prepared using the restriction map of a cosmid clone called LEA 51 (6), which covers the 5Ј-proximal region of MUC4.Gel-purified fragments (Qiaquick gel extraction kit, Qiagen) were subcloned into the promoterless pGL3 Basic vector (Promega).Internal deletion mutants were generated by PCR using pairs of primers bearing specific restriction sites at their 5Ј-and 3Ј-ends (Table I).PCR products were digested, gel-purified, and subcloned into the pGL3 vector that had been previously cut with the same restriction enzymes.Fragments 1488 (1.43 kb, KpnI-SacI) and 1489 (1.32 kb, KpnI-SacI) were generated using the restriction sites present within the LEA 51 genomic clone.Fragment 1744 was generated using the Genome walker kit (CLONTECH).The EcoRV 650-bp PCR fragment was first subcloned into pCR 2.1 vector (Invitrogen) before being subcloned into pGL3 basic vector.All clones were sequenced on both strands on an automatic LI-COR sequencer (ScienceTec, France) using infrared labeled RV3 and GL2 primers (Promega).Plasmids used for transfection studies were prepared using the Endofree plasmid Mega kit (Qiagen).
Cell Culture-Pancreatic cancer cell lines CAPAN-1 and CAPAN-2 were purchased from American Type Culture Collection, and PANC-1 cells were from European Collection of Cell Cultures.CAPAN-1 and CAPAN-2 cells were cultured in RPMI 1640 medium supplemented with 2 mM glutamine and 15 and 10% fetal calf serum (Roche Diagnostics), respectively.PANC-1 cells were grown in Dulbecco's modified Eagle's medium supplemented with 2 mM glutamine and 10% fetal calf serum.The cells were maintained in a 37 °C incubator with 5% CO 2 .
RT-PCR-Total RNAs from pancreatic cancer cells were prepared using the RNeasy midi-kit from Qiagen.The cells were harvested at confluence, and 1.5 g of total RNA was used to prepare cDNA (Advantage TM RT-for-PCR kit, CLONTECH).PCR was then performed on 5 l of cDNA as described by Van Seuningen et al. (32) using specific pairs of primers for MUC4 mucin gene (MUC4 forward primer, 5Ј-CGCGGT-GGTGGAGGCGTTCTT-3Ј; MUC4 reverse primer, 5Ј-GAAGAATCCT-GACAGCCTTCA-3Ј; positions 3094 -3114 and 3670 -3690; accession number AJ242549) (7).The PCR product expected size is 596 bp.Singlestranded oligonucleotides were synthesized by MWG-Biotech.Glyceraldehyde-3-phosphate dehydrogenase was used as an internal control.PCR reactions and PCR product analyses were carried out as described previously (32,33).
Oligonucleotide Probes-The oligonucleotides used for gel shift assays are indicated in Table II.They were synthesized by MWG-Biotech.Equimolar amounts of single-stranded oligonucleotides were annealed and radiolabeled using T4 polynucleotide kinase (Promega) and [␥-32 P]dATP.The radiolabeled probes were then separated from free

TABLE I
Oligonucleotides used in PCR Sequences of the pairs of oligonucleotides used in PCR to produce deletion mutants covering MUC4 5Ј-flanking region (see Fig. 5).KpnI (GGTACC), SacI (GAGCTC), MluI (ACGCGT), and BglII (AGATCT) sites were added at the end of the primers to direct subcloning and are italicized and underlined.Positions of the DNA fragments relative to the first ATG are indicated.S, sense; AS, antisense.Primer Extension-Primer extension reactions were performed using 20 g of total RNAs prepared from CAPAN-1 and CAPAN-2 cells as above.Annealing and labeling of the primers and extension reactions were performed as described previously (32).The reverse primer NAU 979 (5Ј-CCTGTTGTGGAGGACACCTGTG-3Ј) is located downstream of the TATA box, and the reverse primer NAU 1035 (5Ј-CAGCAGCTG-CAGTGTGAGGAG-3Ј) is located in the 3Ј-end of the 5Ј-UTR.X174 DNA/HinfI dephosphorylated markers (Promega) were radiolabeled with [␥-32 P]dATP just before use.Manual sequencing of M13mp18 control DNA was performed using the T7 Sequenase version 2.0 kit (Amersham Pharmacia Biotech).The samples were denatured for 10 min at 90 °C before loading on a 8% sequencing gel (Sequagel-8; National Diagnostic, Prolabo, France).The gel was then vacuum-dried and autoradiographed for 3-4 days at Ϫ80 °C.
Transfections-The transfections were performed using Effectene® reagent (Qiagen) as described previously (33).Briefly, the cells were passed at 0.5 ϫ 10 6 cells/well the day before the transfection.Total cell extracts were prepared after 48 h of incubation at 37 °C using 1ϫ reagent lysis buffer (Promega) as described in the manufacturer's instruction manual.The results were corrected for transfection efficiency by co-transfecting 0.1 g of pSV-␤Gal vector (Promega).␤-Galactosidase and luciferase activities were measured as described by Perrais et al. (33).The luciferase activity is expressed as fold of induction of the test plasmid activity compared with that of the corresponding control vector (pGL3 control vector, Promega) after correction for transfection efficiency by dividing by ␤-galactosidase activity.Each plasmid was assayed in triplicate in at least three separate experiments.Co-transfection studies with pCMV-Sp1 and pCMV-Sp3 expression vectors were performed as described previously (32).PMA (100 nM, 24 h), cholera toxin (1 g/ml, 24 h), and calcium ionophore A23187 (250 nM, 30 min) were added to the transfected cells as indicated, and the cells were then harvested to measure luciferase activity.The cells were also treated with EGF (25 ng/ml, 24 h) and TGF-␣ (25 ng/ml, 24 h) growth factors and with TNF-␣ (40 ng/ml, 24 h) and IFN-␥ (25 ng/ml, 24 h) inflammatory cytokines.All reagents were from Sigma unless otherwise indicated.In some experiments, the cells were pretreated for 30 min with tyrosine-kinase inhibitor tyrphostin AG 1478 (10 M) (Calbiochem) before adding the growth factor of interest.
Nuclear Extract Preparation-Nuclear extracts from cell lines of interest were prepared as described by Van Seuningen et al. (34) and kept at Ϫ80 °C until use.Protein content (5 l of cell extracts) was measured using the bicinchoninic acid method in 96-well plates as described in the manufacturer's instruction manual (Pierce).
Electrophoretic Mobility Shift Assays (EMSA)-Nuclear proteins (5 g) were preincubated for 20 min on ice in 20 l of binding buffer with 2 g of poly(dI-dC) (Sigma) and 1 g of sonicated salmon sperm DNA.Radiolabeled DNA probe was added (120,000 cpm/reaction), and the reaction was left for another 20 min on ice.For supershift analyses, 1 l of the antibody of interest (anti-Sp1 and anti-Sp3, Santa Cruz Laboratories, Tebu, France) was added to the proteins and left for 1 h on ice before adding the radiolabeled probe.The reaction was stopped by adding 2 l of loading buffer and loaded onto a 4% nondenaturing polyacrylamide gel, and electrophoresis conditions were as described by Van Seuningen et al. (32).The gels were vacuum-dried and autoradiographed overnight at Ϫ80 °C.
DNA Sequence and Transcription Factor Binding Site Analyses-DNA sequences were analyzed using PC-GENE software, and the Transfac 4.0 data base was used to localize putative transcription factor binding sites within the 5Ј-flanking region of MUC4.The search was conducted using MatInspector V2.2 software (35).

RESULTS
Cloning and Characterization of the MUC4 Promoter-To isolate the promoter of the human mucin gene MUC4, a human genomic pWE15 library was screened with RAC3 probe (183 bp), which spans 106 bp of the 5Ј-UTR and 77 bp of the first exon of MUC4 (6).One positive clone, called LEA 51, was isolated and contains an insert of 40 kb (Fig. 1).EcoRI digestion of the insert generated a 2.2-kb fragment that was sequenced and showed 100% homology in its 3Ј-end region with the previously published 5Ј-flanking cDNA sequence of MUC4 (6).To obtain more clones containing the promoter region, a PCRbased DNA walking technique was used.A 2.8-kb fragment was obtained from the PvuII library using the two primers NAU 376 and NAU 357 that are located in the 5Ј-end of the 5Ј-UTR of MUC4.The sequence of the 2.8-kb fragment perfectly aligned with the 3Ј-end of the cosmid clone LEA51, which indicated that we had most likely isolated the promoter of MUC4.A second round of PCR was then carried out using two primers (NAU 598 and NAU 599) located in the 5Ј-end of the 2.8-kb fragment.A 650-bp-long PCR product was isolated from the EcoRV library.The 3Ј-end of that fragment matched the 5Ј-end of the 2.8 kb.Altogether 3.7 kb of the MUC4 5Ј-flanking region upstream of the first ATG was isolated using this strategy.
The sequence of the promoter and of the 5Ј-UTR is presented in Fig. 2. The first typical TATA box (TATAA) was found at Ϫ2672/Ϫ2668 upstream of the translational start site.The 5Ј-UTR is mainly composed of GC-rich domains that are potential binding sites for Sp1 but also for the CACCC box-binding protein.This region is also characterized by numerous putative binding sites for transcription factors of the AP family (AP-1, AP-2, and AP-4), PEA3 transcription factor (Ets family), glucocorticoid receptor element, and two sites for the progesterone receptor.A very high density of binding sites for factors known to initiate transcription in TATA-less promoters (Sp1, CACCC box, glucocorticoid receptor element, AP-2, PEA3, and Med-1) (36 -40) was found in the sequence encompassing the Ϫ330/ Ϫ29 nucleotides.Finally, two putative STAT-binding sites, a transcription factor activated by IFN-␥ (41), were found in the 3Ј-end of the 5Ј-UTR.Upstream of the first TATA box, numerous putative binding sites for Sp1, glucocorticoid receptor element, AP-1, AP-4, GATA, and cAMP-responsive element-binding protein transcription factors were found.
Characterization of the Transcription Start Sites-The expression of MUC4 in CAPAN-1 (lane 4) and CAPAN-2 (lane 5) and its absence in PANC-1 (lane 6) cells was confirmed by RT-PCR (Fig. 3).One can note that MUC4 expression is higher in CAPAN-1 (lane 4) cells compared with CAPAN-2 (lane 5).To localize transcription initiation sites, primer extension experiments were then carried out with total RNAs from the two cell lines expressing MUC4 (CAPAN-1 and CAPAN-2) (Fig. 4).Three extension products of 89, 90, and 91 bp were produced when using NAU 979 reverse primer located downstream of the TATA box (Fig. 4B), indicating the existence of three consecutive transcription initiation sites at positions Ϫ2603, Ϫ2604, and Ϫ2605, respectively (Fig. 4A).Intensity of the extension products was much more important in CAPAN-1 cells (lane 2) compared with CAPAN-2 (lane 1), which indicates that cellspecific transcription factors are involved in the transcriptional activity of that region of the promoter and for the higher expression of MUC4 in these cells as shown by RT-PCR (Fig. 3, compare lanes 4 and 5).Because it is widely known that transcription initiation sites are found in GC-rich regions of mammalian promoters, several oligonucleotides localized in the 3Јend of the 5Ј-UTR were used.One additional extension product of 153 bp was obtained when using NAU 1035 (Fig. 4C) and corresponds to a thymidine residue located at position Ϫ199 from the translational start site (Fig. 4A).One can note that the intensity of the extension product is the same in both cell TAGCGTGGGGCCCCGCCCTCTTTT T31 (CACCC box), Ϫ214/Ϫ188 GGTGGGGTAGTGGGGTGGGGCTGAGGA lines (lanes 2 and 3).RT-PCR experiments performed with pairs of primers covering the region stretching from the TATA box down to the first ATG confirmed the fact that this region is transcribed in CAPAN-1 and CAPAN-2 but not in PANC-1 cells (not shown).Because previous results from our laboratory determined the first ATG to be located 2.67 kb downstream of the TATA box described in this report (6), one can conclude that a rather long 5Ј-UTR lays between the first TATA box and the translation initiation site of MUC4.
Transcriptional Activity of the Promoter and of the 5Ј-UTR of MUC4 in Pancreatic Cancer Cells-From the above experiments, two main zones can be distinguished in the MUC4 5Ј-flanking region located upstream of the first ATG: a long 5Ј-UTR rich in GC and the region containing the promoter upstream of the TATA box.A series of deletion mutants covering 3712 nucleotides upstream of the first ATG were made in pGL3 basic vector to characterize the DNA sequences involved in MUC4 promoter activity (Fig. 5A).
The luciferase diagram shown on Fig. 5B clearly indicates that two highly active regions (Ϫ219/Ϫ1 and Ϫ2781/Ϫ2572) are responsible for MUC4 transcription in CAPAN-1 and CAPAN-2 cell lines.On the contrary, the activity of these two regions was kept to a minimal level in the MUC4 negative PANC-1 cells.Fragments covering the Ϫ1187/Ϫ1 region of the 5Ј-UTR (fragments 1555, 2038, and 2150) have about the same range of activity in the three cell lines, indicating that cell-specific elements are not present in that region of the 5Ј-UTR.The minimal sequence with the highest luciferase activity covers the first 219 nucleotides upstream of the ATG.When the bases Ϫ219/Ϫ145 are deleted the luciferase activity dramatically drops (fragment 1919).This result indicates that region Ϫ219/ Ϫ145 contains cis-elements that are essential to drive transcription of MUC4.Fragment 1489 (Ϫ1708/Ϫ387), which does not contain the first 386 nucleotides, does not have any luciferase activity.This points out again, in accordance with the primer extension data (Fig. 4C), that initiation of transcription in the GC-rich region occurs within the first 386 nucleotides upstream of the ATG.
Interestingly, the region covering the Ϫ3135/Ϫ1703 nucleotides (1488), which is 1.43 kb long and which contains the first TATA box, is also inactive.This may indicate that inhibitory cis-elements are present in that region of the 5Ј-UTR downstream of the TATA box.This hypothesis was confirmed when cells were transfected with the next set of fragments in which a large part of the sequence downstream of the TATA box was deleted.The most active fragment covering the Ϫ2781/Ϫ2572 nucleotides (fragment 1959) is 2-and 4-fold more active in CAPAN-1 cells than in CAPAN-2 and PANC-1 cells, respec-tively.This results indicates that this domain contains regulatory elements that confer cell specificity to the expression pattern of MUC4.This is in good agreement with the results obtained by primer extension in which transcription was shown to be more important in that region of the promoter in CA-PAN-1 cells (Fig. 4B, lane 2).The other two fragments, covering the Ϫ3135/Ϫ2572 (fragment 1641) and Ϫ3135/Ϫ2614 (fragment 2095) nucleotides, although much less active, showed also a higher activity in CAPAN-1 cells.The removal of the TATA box sequence to obtain a fragment covering the Ϫ3135/Ϫ2837 region (1718) resulted in a total loss of the luciferase activity, confirming the fact that this TATA box is essential and drives transcription of MUC4 promoter.A fragment covering the Ϫ3713/Ϫ3059 nucleotides (1744) was generated because our computational studies indicated that it contained two putative TATA boxes.This region, however, does not have any luciferase activity in any cell line tested.One can conclude from these results that the Ϫ219/Ϫ145 and Ϫ2781/Ϫ2572 DNA regions contain essential cell-specific cis-elements that drive MUC4 transcription in CAPAN-1 and CAPAN-2 cells.
Binding of Sp1 and Sp3 to the Promoter and the 5Ј-UTR of MUC4 -The region adjacent to the first ATG is characterized by a high amount of GC-rich domains as well as numerous putative binding sites for Sp1.Two Sp1 binding sites were also found right upstream of the TATA box.To confirm the binding of Sp1 in these regions, EMSA studies were carried out with nuclear proteins from the three cell lines and radiolabeled double-stranded oligonucleotide probes each containing a Sp1binding site present either in the Ϫ219/Ϫ1 (T30 and T31) or Ϫ2781/Ϫ2572 (T16) region (Fig. 6).A very faint binding occurs with the T16 probe (Ϫ2723/Ϫ2703) representative of a Sp1binding site located upstream of the TATA box (lane 2).Supershift with anti-Sp1 antibody was only visualized in CAPAN-1 and CAPAN-2 cells (lane 3).The addition of Sp3 antibody did not produce any supershift (lane 4).The strongest binding was obtained with the T30 probe (Sp1 binding site at Ϫ173/Ϫ150).Three main shifted bands were visualized (complexes 1, 2, and 3) in the three cell lines (lane 6).Complex 3 corresponds to Sp1 binding because a supershift occurred upon addition of anti-Sp1 antibody in the mixture to produce complex 4 (lane 7).Complexes 1 and 2 correspond to the two forms of Sp3 (70 and 105 kDa) (lane 6) because these two bands were supershifted upon addition of anti-Sp3 antibody to produce complex 5 (lane 8).The same profile was obtained with T31 probe representative of another Sp1/Sp3 binding site at Ϫ214/Ϫ188 in the 5Ј-UTR (lanes 10 -12), although binding was not as intense.Altogether, it can be stressed from these data that Sp1 and Sp3 engage with two of their cognate cis-elements (Ϫ214/Ϫ188 and ing Pathways in MUC4 Promoter Activity in Pancreatic Cancer Cells-The proximal and the distal regions of the promoter of MUC4 are characterized by the presence of numerous potential binding sites for transcription factors of the AP family (AP-1, AP-2 and AP-4) as well as the PEA3 transcription factor of the Ets family.These factors can be activated by PKC signaling pathway and heterodimerize with c-Jun or c-Fos transcription factors to induce transcription of the target gene (42,43).To study PKC signaling pathway, the transfected cells were treated with PMA for 24 h before harvesting cells.As expected, the luciferase diagram (Fig. 8A) shows that PMA strongly induces the activity of all the fragments tested whether they are located in the 5Ј-UTR region (fragments 1809, 1555, and 2150) or in the region containing the TATA box (fragments 1641 and 1959).The activation is, however, more important in the fragments containing the TATA box (3.5-4-fold activation compared with 2.0 -2.5-fold, respectively).From these data, one can conclude that PKC is a strong activator of MUC4 transcription and that PMA-sensitive cis-elements are present throughout the 5Ј-flanking region of MUC4.
A few putative binding sites for cAMP-responsive elementbinding protein/activating transcription factor transcription factors were found in the promoter.Because these factors are activated by PKA (44), involvement of PKA signaling pathway was tested with cholera toxin.At the most, cholera toxin induced a 2-fold activation of the Ϫ219/Ϫ1 region (fragment 1809) in CAPAN-1 cells (Fig. 8B).Thus, it seems that MUC4 up-regulation by PKA is not very strong and is confined to the Ϫ219/Ϫ1 region in CAPAN-1 cells.
MUC4 is one of the major mucins expressed in the respiratory tract.Moreover, in bronchial epithelial cells, mucin secretion is influenced by inositol 1,4,5-triphosphate-sensitive intracellular calcium release (45).When we studied the effect of intracellular calcium increase in the three cell lines using calcium ionophore A23187, the diagram profile of luciferase activity mimicked that of cholera toxin with a 2-fold activation of the Ϫ219/Ϫ1 region (fragment 1809) in CAPAN-1 cells (Fig. 8C).
From these results, it can be postulated that PKC-sensitive cis-elements present throughout the region upstream of the first ATG are responsible for the up-regulation of MUC4 and that PKA and calcium signaling pathways induce a mild activation of the Ϫ219/Ϫ1 region of the 5Ј-UTR.

Involvement of Growth Factors (EGF and TGF-␣) in the Regulation of MUC4 Promoter Activity in Pancreatic Cancer
Cells-The high density of binding sites for transcription factors activated by growth factors in the 5Ј-flanking region of MUC4 is in favor of regulatory mechanisms involving these molecules.Moreover, mucin secretion and/or expression has already been shown to be regulated by growth factors (46,47), and PKC, which was shown in this report to up-regulate MUC4 transcription (Fig. 8), is also known to be activated by EGF.In our study, biological effect of EGF as well as TGF-␣, an EGFrelated growth factor, were tested on MUC4 promoter activity.As shown in Fig. 9, the activating effect of these two growth factors in CAPAN-1 cells was directed toward the Ϫ219/Ϫ1 region (fragment 1809) of the 5Ј-UTR (2-fold activation).In CAPAN-2 cells, EGF and TGF-␣ strongly induced the activity of Ϫ3135/Ϫ2572 region (fragment 1641) (3-fold activation) and to a lesser extent that of fragments 1809, 2150, and 1959 (approximately 2-fold activation).Because EGF receptor is known to be up-regulated by the proinflammatory cytokine TNF-␣, which is increased in hypersecretory diseases (48), the synergistic effect between TNF-␣ and EGF or TGF-␣ was tested.In our studies, TNF-␣ alone had no effect on MUC4 promoter in either cell line.Cell treatment with EGF and TNF-␣ did not result in a synergistic up-regulation of MUC4 transcriptional activity.Finally, when cells were pretreated with the tyrosine kinase inhibitor tyrphostin AG1478 before adding EGF or TGF-␣, the luciferase activity dramatically dropped (Fig. 9), indicating that EGF-and TGF-␣-mediated up-regulation of MUC4 transcription involves downstream intracellular tyrosine kinases.As expected, EGF and TGF-␣ had no significant effect on MUC4 transcriptional activity in PANC-1 cells.
Involvement of Interferon-␥ in the Regulation of MUC4 Promoter Activity in Pancreatic Cancer Cells-INF-␥, a proinflammatory cytokine, is a potent antitumoral, antiviral, and antibacterial molecule that exerts its effects through the stimulation of a signaling cascade that activates transcription factors of the STAT family (41).Because two putative STATbinding sites were found in the 3Ј-end of MUC4 5Ј-UTR (Fig. 2), the biological effect of IFN-␥ was tested on the MUC4 5Јflanking region.Opposite effects were seen when cells were treated with IFN-␥ (Fig. 10).In CAPAN-1 cells, IFN-␥ led to a slight decrease of the activity of all fragments.In CAPAN-2 cells, a 2-fold activation of the fragments covering the GC-rich (lane 2) cells.Lane 3, no RNA added.Three extension products of 89, 90, and 91 bp (lanes 1 and 2) were produced when using NAU 979 reverse primer located downstream of the TATA box.C, primer extension on 20 g of total RNA from CAPAN-1 (lane 2) and CAPAN-2 (lane 3) cells.Lane 1, no RNA added.One extension product of 153 bp was produced (lanes 2 and 3) when using NAU 1035 reverse primer located in the 3Ј-end of the 5Ј-UTR.X174 DNA/HinfI dephosphorylated markers previously radiolabeled and denatured are indicated on the left side of the gels.The sequence of the M13mp18 plasmid control is shown.FIG. 6. Binding of Sp1 and Sp3 to three different binding sites in MUC4 promoter using EMSA.Autoradiograms of EMSAs performed with 5 g of nuclear proteins isolated from CAPAN-1, CAPAN-2, and PANC-1 cell lines.Nuclear proteins were incubated with the radiolabeled DNA probes as indicated.Lanes 1-4, T16, Sp1-binding site at Ϫ2723/Ϫ2703; lanes 5-8, T30, Sp1-binding site at Ϫ173/Ϫ150; lanes 9 -12, T31, CACCC box at Ϫ214/Ϫ188.Supershift experiments were carried out by adding 1 l of the antibodies of interest (anti-Sp1, lanes 3, 7, and 11; anti-Sp3, lanes 4, 8, and 12).Radiolabeled probe alone were loaded in the first lane of each series (lanes 1, 5, and 9).DNA-protein complexes with Sp1 (complex 3) and Sp3 (complexes 1 and 2) and supershifted complexes with anti-Sp1 (complex 4) and anti-Sp3 (complex 5) are indicated on the right side of the figure with arrows.region of the 5Ј-UTR (fragments 1809 and 1555) was observed, whereas it had no significant effect on the two fragments containing the TATA box (fragments 1641 and 1959).In PANC-1 cells, no effect was observed.
Because IFN-␥ effects on MUC4 transcription activity were not dramatic, we hypothesized that it could act in synergy with TGF-␣ or TNF-␣, as it has already been shown for numerous other genes involved in cell differentiation and proliferation during inflammatory processes (49,50) or carcinogenesis (51).As shown in Fig. 10, IFN-␥ and TNF-␣ did not synergize to induce MUC4 transcription in CAPAN-1 cells.On the contrary, in CAPAN-2 cells, a strong synergistic effect was visualized on the region covering the first 1.2 kb of the 5Ј-UTR (fragments 1809, 1555, and 2150; 4-, 10-, and 6-fold activation, respectively).No synergistic effect was seen on the two fragments containing the TATA box (fragments 1641 and 1959).In CAPAN-1 cells, IFN-␥ and TGF-␣ led to a decrease of the activity of all the fragments but 1809.In CAPAN-2 cells, the results were opposite because IFN-␥ strongly potentiated the effect of TGF-␣ (from a 3-fold activation on fragments 1555 and 1959 and a 4-fold activation on fragment 1809 to a 8-fold activation on fragment 2150) on all fragments.In PANC-1 cells, IFN-␥, TNF-␣, and TGF-␣ did not have any regulatory effects on MUC4 promoter activity, nor did they have synergistic effects.

DISCUSSION
Human mucin gene MUC4 belongs to the subfamily of mucin genes encoding transmembrane mucins like MUC1, MUC3, and MUC12 (1-3).Several reports show that the cytoplasmic tail of these transmembrane mucins is involved in cell signaling events.MUC1 possesses multiple tyrosine phosphorylation sites in its C-terminal part that interact with either the adaptor protein Grb2 and then the Sos-Ras pathway (52) or ␤-catenin (53).The frequent aberrant expression of MUC1 in cancer cells over the entire cell surface also may promote metastasis via the anti-adhesive effects of the extracellular mucin domain (54,55).Studies on rMuc4 also demonstrated its anti-adhesive and anti-immune recognition effects when overexpressed (10) and its link to ErbB2 signaling pathway (11), pointing out an important role for MUC4 in tumor progression.In this paper, we describe the structure of the 5Ј-flanking region of MUC4 and its transcriptional regulation in pancreatic cancer cells.This model was chosen because MUC4 is not expressed in normal pancreas (17), whereas it is aberrantly expressed in pancreatic cancer (17,21).The identification of regulatory regions and responsive elements to growth factors and proinflammatory cytokines involved in tumorigenesis provides new insights as to how MUC4 is regulated and its potential role as a tumor marker in pancreatic cancer.
Sequencing of the 5Ј-flanking region of MUC4 shows that a long 5Ј-UTR (2.5 kb) lays between the first ATG and the first active TATA box (Ϫ2672/Ϫ2668).When compared with the recently published promoter sequence of the rMuc4 (28), the human sequence shows ϳ70% homology over the first 464 nucleotides upstream of the first ATG, and then both sequences diverge.The absence of TATA box and the presence of a transcription start site in the close vicinity of the first ATG was also found in the rat (28).Functional studies of the 5Ј-flanking region of MUC4 in pancreatic cancer cells show that MUC4 transcription is controlled by two regulatory regions (Ϫ219/ Ϫ145 and Ϫ2781/Ϫ2572).Cell-specific elements are present in these two transcription units because their activity was kept to a minimum in PANC-1 cells, which do not express MUC4.The residual activity found in these cells, however, indicates that other cis-elements or mechanisms are necessary to completely MUC4 described previously in inflammatory diseases of the lung ( 16), gastrointestinal tract (57), or epithelial tumors (17,19,20).Growth factors are thought to be involved in mucussecreting cell production, because hypersecretory diseases are associated with abnormal epithelial cell growth and proliferation (48).Among growth factors, EGF seems a good candidate because it has already been shown to activate MUC5AC production in the human lung mucoepidermoid carcinoma NCI-H292 cell line (47).In this report, we now show that MUC4 transcription is activated by EGF and TGF-␣ via the activation of downstream tyrosine kinases.PKC-mediated activation of MUC4 shown in this report could also be another pathway used by growth factors to increase MUC4 expression in cancer cells.
Mucins have long been shown to be target molecules during inflammatory reactions.Inflammatory diseases of the epithelium are often characterized by mucin up-regulation and mucus hypersecretion in response to the aggression (63)(64)(65)(66)(67).In previous reports, TNF-␣ was shown to activate MUC2 (68) and MUC5AC (47).In this report, MUC4 does not seem to respond to that proinflammatory cytokine, nor does it respond to IFN-␥.Up-regulation of MUC4 was thus hypothesized to be the result of synergistic mechanisms between TNF-␣ and IFN-␥.Redundancy and synergy are fundamental and ubiquitous phenomena in the immune system are needed to elicit specific and efficient response of the body toward infection (69).Synergy between IFN-␥ and TNF-␣ or TGF-␣ has been extensively studied and occurs in the regulation of numerous genes involved in cell differentiation and proliferation during inflammatory processes (49,50) as well as in carcinogenesis (51).In this report, we show that MUC4 is strongly up-regulated by INF␥/TGF␣ or IFN␥/TNF␣ in CAPAN-2 cells.Synergy between IFN-␥ and TNF-␣ is mediated through the simultaneous activation of STATs (IFN-␥) and NF-B (TNF-␣) transcription factors.Interaction between STAT and NF-B once bound to their cognate cis-elements then induces the promoter of the target gene (69,70).In the 5Ј-UTR of MUC4 such a proximity between NF-B and STAT binding sites was found.Regarding IFN-␥ and TGF-␣, the mechanism is different and goes through an autocrine loop in which IFN-␥ induces the transcription and expression of TGF-␣ that will then bind to the EGF receptor and consequently activates the gene of interest (71).
From our results, it is clear that MUC4 promoter is regulated by growth factors that affect cell growth and proliferation and by proinflammatory cytokines, which besides their important role in inflammation have anti-tumoral activities.Thus, it may be hypothesized that MUC4, which is a transmembrane mucin aberrantly expressed in pancreas and gall bladder adenocarcinomas or overexpressed in mucoepidermoid cancers (lung, esophagus, and gall bladder) and thought to participate in ErbB2/Neu signaling, plays important roles in cell proliferation, tumorigenesis, and metastasis.Moreover, being up-regulated by molecules involved in chronic inflammatory diseases, MUC4 could be considered to be a molecule participating to the host defense against pathogens like other adhesion molecules such as ␤-catenin and ICAM-1 (72)(73)(74).
In conclusion, we have isolated the promoter of the human mucin gene MUC4 and showed that it is characterized by a typical TATA box flanked by a very long 5Ј-UTR.The 3Ј-end of the 5Ј-UTR is characterized by a GC-rich region that acts as a second transcription unit.From our results was drawn a schematic representation of MUC4 5Ј-flanking region in which signaling pathways that mediate MUC4 regulation in pancreatic cancer cells identified in this paper are shown (Fig. 11).Regulation by transcription factors involves Sp1, Sp3, and probably factors from the AP family as well as NF-B and STATs.MUC4 transcription is up-regulated by PKC, and EGF-and TGF-␣mediated up-regulation involves activation of downstream tyrosine kinase cascades.The most dramatic effect was the cellspecific up-regulation because of a synergistic effect between IFN-␥ and TNF-␣ and IFN-␥ and TGF-␣.Investigations are now in progress to demonstrate whether the inactivation of these pathways represses MUC4 expression and affects the behavior of human pancreatic cancer cells.

FIG. 1 .FIG. 2 .
FIG. 1. Schematic representation of the genomic clones covering the 5flanking region of human mucin gene MUC4 upstream of the ATG.The 3Јend of the cosmid LEA 51 is shown and aligned with the PvuII 2.8-kb clone and the EcoRV 0.65-kb clone obtained with the Genome walker kit.

FIG. 5 .
FIG. 5. Position and transcriptional activity of MUC4 pGL3 deletion mutants in pancreatic cancer cell lines.A, schematic representation of the localization of the different pGL3 deletion mutants covering 3721 nucleotides upstream of the first ATG.The numbering refers to the translational start site designated ϩ1.The TATA box locations are indicated as well as some restriction sites found in the 5Ј-flanking region of the gene.B, transcriptional activity of the deletion mutants was studied in CAPAN-1 (black bars), CAPAN-2 (white bars), and PANC-1 (hatched bars) cell lines.The background activity of pGL3 Basic promoterless vector used to subclone MUC4 fragments is shown.The results are the means Ϯ S.D. and represent more than three different experiments in triplicate for each fragment.

TABLE II
Sequences of the sense oligonucleotides used for gel shift assay experimentsAntisense complementary oligonucleotides were also synthesized and annealed to the sense oligonucleotide to produce double-stranded DNA.Positions of the DNA fragments relative to the first ATG are indicated.