| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 282, Issue 31, 22638-22650, August 3, 2007
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1213
4
From the
INSERM, U560, Place de Verdun, Lille, F-59045, France, the Department of Pediatrics, Divisions of Gastroenterology and Nutrition and **Neonatology, Erasmus MC and Sophia Children's Hospital, Rotterdam, 3015GE The Netherlands, the ¶Department of Cell Biology and Anatomy, University of Miami School of Medicine, Miami, Florida 33136, and the ||INSERM, U682, 3 avenue Molière, Strasbourg F-67200, France
Received for publication, January 31, 2007 , and in revised form, June 5, 2007.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
and -1
, FOXA1/A2, HNF-4 and -4
, and GATA-4, -5, and -6 factors in a cell-specific manner. Binding of TFs was assessed by chromatin immunoprecipitation, and gel-shift assays. Altogether, these results demonstrate that MUC4 is a target gene of endodermal TFs and thus point out an important role for these TFs in regulating MUC4 expression during epithelial differentiation during development, cancer, and repair. |
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
Recently, we characterized the promoter region of MUC4, which is composed of a proximal and a distal promoter (13) and identified binding sites for transcription factors (TFs)5 of the hepatocyte nuclear factor (HNF), GATA, and caudal-related homeobox (CDX) families in the distal promoter (14). We also recently showed that MUC4 endogenous expression is regulated by HNF-1 and HNF-4 TFs in esophageal cancer cells (15). Altogether, these findings suggest that MUC4 expression may be regulated by TFs responsible for cell differentiation programs leading to the formation of organs derived from the primitive gut.
HNF, GATA, and CDX TFs regulate regional cell differentiation of the endoderm and its derivatives during embryonic development (16–18). Their participation in maintaining a functional epithelium is the result of a tight spatio-temporal regulation of cell- or tissue-specific genes in the lung (19–21) and intestine (22–25). Among these genes, we recently identified the secretory mucin MUC2 as a target of CDX-1/CDX-2 (26) and GATA-4 (27).
The GATA family consists of six members, GATA-1 to -6, that bind to the 5'-(A/T)GATA(A/G)-3' nucleotide motif via their zinc finger domains. They are grouped into two subfamilies based on structural features and expression patterns. GATA-1, -2, and -3 are involved in hematopoiesis and neurogenesis. GATA-4, -5, and -6 are found mainly in heart and endoderm-derived tissues, including liver, lung, pancreas, stomach, and intestine, and are involved in regulation of cardiogenesis and gut development (28).
Hepatocyte nuclear factors belong to a heterogeneous family of TFs involved in a wide variety of biological pathways. Although important in liver development and function, they are also involved in visceral endoderm differentiation and found in kidney, pancreas, stomach, small intestine, and colon (29). HNF-1 and -1 are homeodomain proteins that form homo- or heterodimers and bind the consensus sequence 5'-GTTAATGATTAAC-3'. HNF-3 and -3 belong to the forkhead/winged helix DNA binding domain family. They bind the consensus sequence 5'-GATTATTGACTT-3' as monomers (30) and are expressed in embryonic endoderm and in the adult intestine (31). In this paper we will use the new nomenclature FOXA1 (HNF-3) and FOXA2 (HNF-3). HNF-4 and -4 are members of the steroid hormone receptor superfamily. They are zinc finger TFs and bind the consensus sequence 5'-TGGACTTAG-3'. HNF-4 has been implicated in early endodermal development and differentiation of the liver, kidney, pancreas, stomach, and intestine (32).
Homeobox CDX-1 and -2 are intestine-specific genes that bind the AT-rich consensus sequence (T/C)ATAAA(T/G) either as homo- or heterodimers (33). In vivo and in vitro studies suggest that these TFs are important in intestinal development, intestinal cell proliferation and differentiation, and in the control of intestinal identity (33–35).
In this paper we report that Muc4 mucin is expressed before and after cytodifferentiation in the lung and gastrointestinal tract of the developing mouse and that the apical surface expression observed prior to cytodifferentiation in the gastrointestinal tract is also observed in mucus-secreting goblet cells after differentiation. Furthermore, we report, by deciphering the molecular mechanisms of transcriptional regulation, that MUC4 is a target gene of CDX-1/-2, HNF-1/-4, FOXA1/A2, and GATA-4, -5, and -6 TFs.
|
EXPERIMENTAL PROCEDURES |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
Immunohistochemistry—Immunohistochemistry was carried out as in Ref. 38. Muc4 expression at different stages of mouse development and in Cdx-1-/- and Cdx-2+/- mice was carried out using rabbit polyclonal HA-1 antibody (1:2000 dilution) that recognizes a C-terminal peptidic region of the ASGP-1 (MUC4) subunit (39). Negative controls included staining sections without primary HA-1 antibody or by incubating with rabbit preimmune serum (supplemental data Figs. S1 and S7).
Cell Culture—Human pancreatic CAPAN-1 and CAPAN-2, colonic HT-29 STD, HT-29 5F12, LS174T, and Caco-2, gastric KATO-III, and respiratory NCI-H292 cancer cell lines were cultured as previously described (13, 40–43). All cell lines were supplemented with penicillin (100 units/ml), streptomycin (100 µg/ml) and cultured at 37 °C in a 5 (CAPAN-1, CAPAN-2, KATO-III, NCI-H292) or 10% (HT-29 STD, HT-29 5F12, LS174T, Caco-2) CO2-jacketed incubator.
RT-PCR—Total RNAs from cultured cells were prepared using the RNeasy mini-kit from Qiagen (Courtaboeuf, France). Total RNA (1.5 µg) was used to synthesize cDNA (AdvantageTM RT-for-PCR kit, Clontech) as described (43). PCR was performed on 5 µl of cDNA using specific pairs of primers (MWG-Biotech, Ebersberg, Germany) for MUC4 (44) and 28S rRNA as the internal control. PCR were carried out in 50-µl final solutions (5 µl of 10x PCR buffer containing 15 mM MgCl2, 4 µl of 2.5 mM dNTPs, 10 pmol of each primer, and 1 unit of Taq polymerase (Roche Diagnostics). Cycling conditions were as follows: 1) denaturation: 94 °C, 2 min for one cycle; 2) denaturation: 94 °C, 45 s; annealing: 60 °C, 1 min; and extension: 72 °C, 1 min for 27 cycles; and 3) final extension: 72 °C, 10 min. PCR products were analyzed on 1.5% agarose gels containing ethidium bromide run in 1x Tris borate-EDTA buffer. PCR primers and annealing temperature to analyze TF expression are described in supplemental Table S1. A 100-bp DNA ladder was purchased from GE Healthcare.
Small Interfering RNA (siRNA) Assays—Cell seeding and cell transfection were performed as described before (15) with 100 nM human GATA-4, GATA-6, CDX-1, CDX-2, HNF-1, HNF-1, FOXA1, FOXA2, HNF-4, or combinations of SMARTpool® siRNA, using 1 µl of DharmaFECTTM 1 transfection reagent (Dharmacon, Perbio Science, Brebières, France). Controls included mock-transfected cells, and cells transfected with siCONTROLTM GAPD siRNA or siCONTROLTM Non-Targeting siRNA Pool. Total RNA isolation and RT-PCR were as described above. siRNAs were assayed in triplicate in at least two separate experiments. MUC4/glyceraldehyde-3-phosphate dehydrogenase ratio was calculated by densitometric analysis using the GelAnalyst-GelSmart software (Clara Vision, Orsay, France).
pGL3-MUC4 Promoter Constructs and Site-directed Mutagenesis—The four pGL3-MUC4 deletion mutants used in this study and that cover the distal promoter of MUC4 were previously described (13). Plasmids used for transfection studies were prepared using the Endofree plasmid Maxi kit (Qiagen). QuikChange site-directed mutagenesis kit (Stratagene) was used to generate site-specific mutations. Oligonucleotides containing the desired mutations are shown in supplemental Table S2.
Transient Transfection Assays—Transfection experiments were performed using Effectene® reagent (Qiagen) as previously described (42). Luciferase activity was corrected for transfection efficiency by co-transfecting cells with pRL-TK vector (Promega). Total cell extracts were prepared after a 48-h incubation at 37 °C using 1x Passive Lysis Buffer (Promega). Co-transfection experiments were carried out in the presence of 1 µg of each pGL3-MUC4 promoter fragment with either 0.5 µg of the expression vector encoding the TF of interest or 0.5 µg of empty control vectors as the reference. pCMV-FOXA1 and pCMV-FOXA2 were a kind gift of Dr R. Costa (University of Illinois, Chicago, IL). pCB6-HNF-1, pCB6-HNF-1, pMT2-HNF-4, and pMT2-GATA-4 were a kind gift of Dr. S. Cereghini (UMR7622 CNRS, Paris, France). pSG5-HNF-4, pSG5-GATA-5, and pSG5-GATA-6 were a kind gift of Dr. J. K. Divine (Washington University, St. Louis, MO). pCMV-Cdx-1 and pCMV-Cdx-2 were previously described (45). Relative luciferase activity was expressed as -fold activation in samples transfected with vector expressing the TF of interest compared with empty vector. Each construct or combination was assayed in triplicate in three separate experiments. To study the effects of the TFs on endogenous expression of MUC4 mRNA, cells were transfected with 4 µg of the expression vector of interest or empty control as previously described (27). Three independent experiments were carried out. MUC4/28S ratio was calculated by densitometric analysis as above.
Electrophoretic Mobility Shift Assay (EMSA)—Putative binding sites were identified using MatInspector (www.genomatix.de) software (46). Oligonucleotides used for EMSA (supplemental Table S3) were synthesized by MWG-Biotech. Annealed oligonucleotides were radiolabeled using T4 polynucleotide kinase (Promega) and [-32P]dATP (GE Healthcare) and purified by chromatography on a Bio-Gel P-6 column (Bio-Rad). Nuclear extracts were prepared as described before (47), and quantified using the bicinchoninic acid method (Pierce). Nuclear protein incubation with radiolabeled probes and competitions with unlabeled probes were as described in Ref. 26. For supershift analyses, 2 µl of the antibody of interest (anti-GATA-4, anti-GATA-6, anti-HNF-1, anti-HNF-1, anti-FOXA1, anti-FOXA2, anti-HNF-4, and anti-HNF-4, 10x solutions (Santa Cruz Laboratories) and anti-CDX-2 (Biogenex, Alphelys, Plaisir, France) were added to the proteins and left for 1 h at room temperature before adding the radiolabeled probe. Electrophoresis conditions and gel processing were as described (42).
Chromatin Immunoprecipitation (ChIP)—Cells (3 x 106) were treated with 1% (v/v) formaldehyde for 10 min at room temperature and cross-links were quenched with glycine at a final concentration of 0.125 M for 5 min. ChIP experiments were then carried out as previously described (15). All antibodies were from Santa Cruz except GATA-4, GATA-5, and GATA-6, which were from R&D Systems. Primer information is given in supplemental Table S4.
Statistics—All values in this article are mean ± S.D. When indicated, Student's t test was used for statistical evaluations; a p < 0.05 was considered statistically significant.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
MUC4 Expression in Epithelial Cancer Cell Lines—Because MUC4 has a cell-specific pattern of expression in respiratory and gastrointestinal tracts, we studied its mRNA expression and promoter regulation in a panel of epithelial cell lines of different origins (respiratory, gastric, colonic, and pancreatic) and phenotypes (enterocyte, mucus-secreting, and undifferentiated colonic cancer cells). MUC4 mRNA was strongly expressed in respiratory NCI-H292, pancreatic CAPAN-1 and CAPAN-2, and to a lower extent in gastric KATO-III and intestinal HT-29 5F12 cell lines (Fig. 2A). The basal level of the TFs in the different cell lines is shown in Fig. 2B.
Regulation of MUC4 mRNA Expression by Endodermal Transcription Factors—To investigate whether endodermal CDX-1 and -2, HNF-1 and -4, FOXA1/A2, and GATA-4, -5, and -6 TFs regulate MUC4 endogenous expression, knockdown assays were carried out with specific siRNA in CAPAN-1, HT-29 5F12, and KATO-III cell lines that expressed the TFs of interest (supplemental data Fig. S2). The strongest inhibitions of MUC4 mRNA expression were observed with FOXA2, GATA-6, CDX-2, and HNF-1 siRNAs (75–80% inhibition, Fig. 2C). siRNA for HNF-1 and FOXA1 led to a 60% decrease of MUC4 expression, whereas CDX-1, HNF-4, and GATA-4 siRNA effects were milder (20–30% inhibition). Regulation of endogenous MUC4 expression by HNF-4 and GATA-5 could not be studied by the siRNA approach because none of the cell lines studied expressed these TFs or expression was extremely low (HNF-4 in KATO-III). Forced expression of HNF-4 and GATA-5 in NCI-H292 and CAPAN-2 MUC4-expressing cells led to a 5.5–7.5- and 1.5–2.0-fold increase of MUC4 mRNA, respectively (Fig. 2D). Together, these results demonstrate that CDX, HNF, FOXA, and GATA TFs regulate MUC4 mRNA expression in cancer cell lines of endodermal origin.
|
Regulation of MUC4 Distal Promoter by CDX-1 and CDX-2 Transcription Factors—As expected, activation of the MUC4 promoter by intestine-specific CDX-1 (Fig. 3A) and CDX-2 (Fig. 3B) TFs was the strongest in intestinal cells. This activation occurred in a promoter region -3135/-2837 (black bars) that does not contain any consensus CDX binding site. Non-negligible activation of the promoter region containing either the T60 (gray bar, CDX-1/HT-29 STD) or the T90 (CDX-2/LS174T, hatched bar) CDX binding sites was also observed.
Regulation of MUC4 Distal Promoter by HNF-1 and -1, FOXA1/A2, and HNF-4 and -4 Transcription Factors—The strongest MUC4 promoter activation by HNF-1 (Fig. 3C) and HNF-1 (Fig. 3D) was observed in HT-29 STD and KATO-III gastrointestinal cell lines in -3135/-2837 (T144 HNF binding site) and -3713/-3059 (T91 and T106 HNF binding sites) promoter regions. When HNF-4 and HNF-4 were overexpressed, the effects were the most potent in the respiratory NCI-H292 cells (Fig. 3, E and F) in a region containing the T91 and T106 binding sites for HNF-4 (hatched bar) and a region devoid of HNF binding site (white bar) for HNF-4. Milder effects of HNF-4 on different regions of the promoter were also observed in the other cell lines studied (supplemental data Fig. S4). Activation of MUC4 promoter by FOXA1 and FOXA2 were the strongest in colonic cancer cell lines and implied different regions of the promoter for FOXA1 (Fig. 3G) and the -3135/-2837 region (T144 HNF binding site) for FOXA2 (Fig. 3H).
Regulation of MUC4 Distal Promoter by GATA-4, -5, and -6 Transcription Factors—Overexpression of GATA-4 led to the transactivation of the MUC4 distal promoter in all the cell lines studied except for pancreatic CAPAN-2 (Fig. 3I and supplemental data Fig. S4). All the promoter fragments were stimulated by GATA-4 although there were cell-specific differences. For instance, the region -3135/-2572 (T60 GATA binding site) was strongly activated in pancreatic CAPAN-1 but not in LS174T (Fig. 3I). Transactivation of the MUC4 promoter by GATA-5 (Fig. 3J) was the strongest in pancreatic CAPAN-2 and enterocyte Caco-2 but like GATA-4 activations could be seen in all the cell lines studied and implied different regions of the promoter (supplemental data Fig. S4). The same conclusions can be drawn for GATA-6 (supplemental data Fig. S4). Interestingly, in respiratory NCI-H292 cells a strong activation of the promoter region -2781/-2572 that does not contain any putative GATA binding site was observed (Fig. 3K).
|
|
|
Mutation of the T60 cis-element significantly abrogated (80% loss, p < 0.05) the transactivation of the MUC4 promoter by both CDX-1 and CDX-2 in colonic HT-29 STD cells (Fig. 4C). Single mutations in the T90 region (mut#1 and mut#2) led to a significant (p < 0.05) 20–40% reduction of the transactivating effect of CDX-2 in LS174T cells and the double mutation (mut#1–2) led to 65% loss of transactivation, indicating an additive effect (Fig. 4D). Consistent with the absence of DNA binding, mutation of the T164 site (mut#3), alone or in combination with mut#1 and/or mut#2 did not alter MUC4 promoter transactivation by CDX-2.
|
|
cis-elements (15). Here, we provide evidence by EMSA that T144 not only engages with HNF-1 (Fig. 5A, lane 5) but also with HNF-1 (lane 6) and that both TFs interact with the chromatin region encompassing T144 by ChIP (Fig. 5B). Incubation of the radiolabeled probe T91 with nuclear extract from gastric KATO-III cells, which express HNF TFs gave one retarded band (Fig. 5C, lane 2), which disappeared by competition with a x50 excess of the corresponding unlabeled probe (lane 3). Use of the T91 radiolabeled probe in which the putative HNF binding site was mutated (Mut.T91) prevented DNA-protein complex formation (lane 11), confirming the specificity of the interaction. Inhibition of the shifted complex upon addition of anti-FOXA1 antibody in the reaction mixture confirmed the involvement of FOXA1 in the complex formation (lane 6). In vivo binding of FOXA1 to the chromatin region encompassing the T91 probe was then confirmed by ChIP (Fig. 5D).
Mutation of the T144 HNF-1 cis-element in the construct -3135/-2837 resulted in a 86 and 78% decrease of promoter transactivation by HNF-1 and HNF-1, respectively (Fig. 5E). Mutation of that same cis-element did not alter the MUC4 promoter transactivation by FOXA2 (supplemental data Fig. S5). The T144 cis-element appears thus essential in MUC4 promoter regulation by HNF-1 and HNF-1.
Mutation of the T106 HNF-1 element led to a 80% loss of transactivation by HNF-1, HNF-1, and FOXA2 but not FOXA1 (Fig. 5E). Mutation of the T91 FOXA element had a lower impact, alone (40–50% inhibition) or in combination with the T106 mutation (equivalent to T106 mutation), on the regulation by HNF-1, HNF-1, and FOXA2. On the other hand, the T91 mutation abrogated MUC4 transactivation by FOXA1 and the double mutation T91/T106 was equivalent to the single T91 mutant. Altogether these results indicate that T106 participates in MUC4 regulation by HNF-1, HNF-1, and FOXA2. T91 is more important in conveying MUC4 activation by FOXA1. The T91 and T106 mutations partly affected promoter activation by HNF-4 or HNF-4 (supplemental data Fig. S5). Absence of binding and partial loss of transactivation of the MUC4 promoter by these two TFs indicate that their regulation mode is most likely indirect.
Identification and Functionality of the GATA cis-Elements Present in MUC4 Distal Promoter—Two putative GATA binding sites are present in the MUC4 distal promoter (supplemental data Fig. S3). EMSA with T59 and T60 radiolabeled probes and nuclear extracts from CAPAN-1 cells (Fig. 6A), which express GATA TFs, revealed a retarded complex with both probes (lanes 2 and 7), that disappeared by competition with a x50 excess of unlabeled probe (lanes 3 and 8). T59 binds GATA-4 because anti-GATA-4 antibody totally inhibited the shifted band (lane 9), whereas partial inhibition with antibodies suggested the binding of GATA-4 (lane 4) and GATA-6 (lane 5) to T60. In vivo binding of GATA-4 and GATA-6 to chromatin encompassing T59 and T60 binding sites was confirmed by ChIP (Fig. 6B).
Single mutations of T59 and T60, respectively, led to 87 and 80% decrease of GATA-4 transactivating effect in CAPAN-1 cells and the double mutation increased that loss up to 95% (Fig. 6C). Single mutations had more moderate effects on GATA-6 activity in KATO-III cells (20 and 60% loss for T59 and T60, respectively), whereas the double mutation led to a complete loss of transactivation by GATA-6. Together, these data demonstrate that GATA-4 and -6 regulate MUC4 transcription by binding to two promoter elements.
Synergistic Activity of CDX-2, GATA-4, and HNF-1 TFs on MUC4 Promoter Activity—HNF-1, GATA-4, and CDX-2 are known to cooperate synergistically to regulate intestine-specific gene expression. As MUC4 is expressed in the intestine and regulated by these TFs (this report), we tested whether such a synergistic mechanism would exist for MUC4. Cell specificity was assessed by performing co-transfections in enterocytes (Caco-2), mucus-secreting (LS174T), and undifferentiated (HT-29 STD) colonic cancer cells on the construct -3135/-2837 (Fig. 7). A strong synergistic activation by HNF-1 and GATA-4 was observed in the three cell lines, whereas HNF-1 and CDX-2 specifically synergized in enterocyte Caco-2 cells. When combination of the three factors HNF-1, GATA-4, and CDX-2 was tested, a significant synergistic effect was visualized in mucus-secreting LS174T cells (p < 0.01), whereas addition of CDX-2 to HNF-1 and GATA-4 had the opposite effect both in undifferentiated HT-29 STD and enterocyte Caco-2 cells.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
In this paper we also demonstrate that the human mucin gene MUC4 is regulated at the transcriptional level by transcription factors (HNF-1/-4, FOXA1/A2, GATA-4, -5, and -6, and CDX-1 and -2) involved in cell differentiation programs during embryonic development. HNF, GATA, and CDX TFs have a specific spatio-temporal pattern of expression during embryonic development and in adults and participate in regional cell differentiation by regulating the expression of tissue-specific genes. It is known that they hierarchically regulate each other in a cell- and tissue-specific manner during development (49, 50) and that they act in various combinations to direct cell-specific transcription during cell differentiation. This is most likely why we did not always get complete inhibition of MUC4 expression when we knocked down their expression one by one. We confirmed that hypothesis by using combinations of TFs in siRNA experiments that led to increased repression of MUC4 (supplemental data Fig. S6). This is also in agreement with data obtained from targeted disruption of these TFs in mice that only resulted in moderate reduction of target gene expression (49).
|
MUC4 transactivation by HNF-1 and HNF-1 is mediated by their direct binding to two HNF cis-elements (T106, T144) in the MUC4 promoter (Fig. 8). In the small intestine, both TFs are highly expressed in the crypts (55), where MUC4 is also found, which suggests a role in the early steps of intestinal epithelial differentiation. We also showed that both TFs were able to regulate MUC4 in all the cell lines tested regardless of their tissue origin, phenotype, or differentiation status suggesting that they may regulate MUC4 in other organs in which they are expressed, such as stomach, colon, and pancreas (56). This is in agreement with recent data in which we showed that HNF-1 is a key regulator of MUC4 expression in esophageal cancer cells (15).
Activation of MUC4 by FOXA1 and FOXA2 was very strong in colonic cancer cell lines, suggesting intestinal specificity. Activation by FOXA1 was mediated by its direct binding to one HNF element (T91) in the MUC4 promoter, whereas FOXA2 acts indirectly (Fig. 8). Indirect regulation by FOXA1 may also occur as we showed in HT-29 STD cells that it could induce strong activation of the -2781/-2572 promoter region, which does not contain any FOXA binding site. From that, it can be hypothesized that FOXA1 is able to recruit and/or activate a cell-specific TF that then interacts with the MUC4 promoter. During embryonic development, FOXA TFs are expressed in the early steps of endoderm differentiation (30). In the adult intestine, FOXA1 is strongly expressed in intestinal crypts and decreases along the crypt-villus axis, whereas FOXA2 is found in the crypts (57) and in goblet cells.6 Thus, MUC4 as a target gene of these TFs expressed both in the crypts and in the villi may play a role in both intestinal differentiation induction and terminal differentiation. In the lung, the same role could be played in differentiation of goblet cells, as FOXA2 was shown to be a major actor in lung epithelial cell differentiation and in regulating lung-specific genes (20, 21).
|
and -4 showed strong transactivating activity in the respiratory cancer cell line and a non-negligible effect in pancreatic, gastric, and colonic cell lines. Our data suggest that their effects are indirect (transactivation of promoter regions devoid of HNF binding sites) and involve cooperation with co-factors (HNF-4/HNF-1 and HNF-4/GATA-6) (supplemental data Fig. S6). This is in agreement with previous results showing cooperation between these TFs to induce transcription of their target genes (58). In adults, HNF-4 is expressed in kidney, pancreas, stomach, and intestine (29). In the intestine, both TFs are expressed in the villi, whereas HNF-4 is also found in the crypts (49). They may thus participate to the complex regulatory system controlling MUC4 expression along the crypt-villus axis. GATA factors play essential roles in the development and function of endoderm-derived tissues (28). GATA-4 is required for the development of visceral endoderm and for proper differentiation of glandular gastric epithelial cells, whereas GATA-6 is essential for pulmonary development (59). In this paper we show that both TFs are important regulators of MUC4 expression by directly mediating their effects via two cis-elements (T59, T60) (Fig. 8). Activation of a promoter region devoid of the GATA binding site by GATA-6 in NCI-H292 cells also suggests indirect regulation via the recruitment and/or activation of a cell-specific TF able to then activate the MUC4 promoter. GATA-5 activation of MUC4 transcription is indirect or requires other elements not present in the distal promoter as it did not bind to DNA. In the small intestine, GATA-4 expression increases along the crypt-villus axis, whereas GATA-6 expression is highly expressed in progenitor cells of the crypts (60), which suggests that the role of GATA-4 in intestinal differentiation is to activate terminal differentiation-specific genes, whereas GATA-6 participates in maintaining the pool of proliferating cells. GATA TFs are thus most likely important in determining the spatio-temporal pattern of expression of MUC4 during development and later on in adult respiratory and gastrointestinal tracts.
Until now, extensive work has been published regarding the regulation of enterocyte-specific markers, such as sucrase isomaltase, lactase, and fatty acid-binding protein by HNF, GATA, and CDX TFs (18, 22–25). In this work we showed that, like sucrase isomaltase, MUC4 is synergistically activated by HNF-1 and GATA-4 in Caco-2 and HT-29 STD colonic cancer cells. The synergy is even stronger in the presence of CDX-2 in mucus-secreting LS174T cells. This could represent a combinatorial system able to drive MUC4 expression in the small intestine where the three TFs are co-expressed (61). This suggests that the MUC4 mucin can be added to the list of intestine-specific genes regulated by these TFs and as such plays a role in gastrointestinal differentiation.
Embryonic cells share common biological properties with tumor cells. HNF, GATA, and CDX TFs are known to participate in carcinogenesis, thus they may be responsible for some aberrant patterns of MUC4 expression in epithelial cancers. In general, MUC4 overexpression in epithelial cancers is associated with poor prognosis and tumor progression (2, 5, 62). Interestingly, among HNF, GATA, and CDX TFs, some have tumor suppressor functions (FOXA2 (63)), whereas others are thought to participate in tumor progression (GATA-6 (64, 65), HNF-1 (66), and FOXA1 (67)). Others like CDX-2 or GATA-4 have antagonist functions. CDX-2 is a tumor suppressor gene in colorectal cancer (68, 69), whereas its ectopic expression in intestinal metaplasia in the stomach and esophagus is considered as a preneoplastic condition (70). GATA-4 is overexpressed in pancreatic intraepithelial neoplasia (65) and down-regulated in gastric, colorectal, and lung tumors (64). Taken together, these data suggest that, depending on the function attributed to the TF in tumor progression and to the active oncogenic pathway(s) the TF is facing in a given cancer cell, activation of tumor-associated MUC4 mucin expression will have profound consequences on the tumor behavior. As we saw, multiple TFs may be able to activate MUC4 in the same cell/organ, so the level of expression of these TFs will be an important factor in determining the level of MUC4 expression and consequently cancer cell fate toward either proliferation or differentiation.
In conclusion, we have shown in this report that MUC4 mucin has a spatio-temporal pattern of expression during development and is a target gene of CDX-1 and -2, HNF-1 and -1, FOXA1/A2, HNF-4 and -4, and GATA-4, -5, and -6 transcription factors. The complexity of the mechanisms described in this report points out that MUC4 is tightly regulated by these TFs and suggests that these TFs are responsible for the MUC4 spatio-temporal pattern of expression during embryonic development of gut endoderm-derived tissues. The regulatory mechanisms described in this paper will also help in a better understanding of MUC4 regulation in epithelial cancers and epithelial repair during which cell differentiation is altered and recapitulates molecular mechanisms observed during development.
|
FOOTNOTES |
|---|
The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S7 and Tables S1–S4.
1 Both authors participated equally to this work.
2 Recipient of a Centre Hospitalier Régional et Universitaire de Lille-Région Nord-Pas de Calais Ph.D. fellowship.
3 Recipient of an INSERM-Région Nord-Pas de Calais Ph.D. fellowship.
4 To whom correspondence should be addressed. Tel.: 33-320-29-88-67; Fax: 33-320-53-85-62; E-mail: isabelvs{at}lille.inserm.fr.
5 The abbreviations used are: TF, transcription factor; HNF, hepatocyte nuclear factor; CDX, caudal-related homeobox; E, embryonic day; P, postnatal day; RT, reverse transcriptase; siRNA, small interfering RNA; EMSA, electrophoretic mobility shift assay; ChIP, chromatin immunoprecipitation.
6 I. B. Renes and I. Van Seuningen, unpublished data.
|
ACKNOWLEDGMENTS |
|---|
vectors; Dr. R. Costa (University of Illinois, Chicago, IL) for the kind gift of pCMV-FOXA1 and pCMV-FOXA2 vectors; and Dr. S. Cereghini (UMR7622 CNRS, Paris, France) for the kind gift of pMT2-GATA-4, pMT2-HNF-4, pCB6-HNF-1, and pCB6-HNF-1 expression vectors. We thank Pr. F. Beck (University of Leicester, Leicester, UK) for providing the Cdx-2+/- mice and Pr. J. Deschamps (Hubrecht laboratory, Utrecht, The Netherlands) for the Cdx-1-/- mice. |
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
