miR-219-1-3p is a negative regulator of the mucin MUC4 expression and is a tumor suppressor in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is among the most lethal cancers in the world with one of the worst outcome. The oncogenic mucin MUC4 has been identified as an actor of pancreatic carcinogenesis as it is involved in many processes regulating pancreatic cancer cell biology. MUC4 is not expressed in healthy pancreas whereas it is expressed very early in pancreatic carcinogenesis. Targeting MUC4 in these early steps may thus appear as a promising strategy to slow-down pancreatic tumorigenesis. miRNA negative regulation of MUC4 could be one mechanism to efficiently downregulate MUC4 gene expression in early pancreatic neoplastic lesions. Using in silico studies, we found two putative binding sites for miR-219-1-3p in the 3'-UTR of MUC4 and showed that miR-219-1-3p expression is downregulated both in PDAC-derived cell lines and human PDAC tissues compared with their normal counterparts. We then showed that miR-219-1-3p negatively regulates MUC4 mucin expression via its direct binding to MUC4 3'-UTR. MiR-219-1-3p overexpression (transient and stable) in pancreatic cancer cell lines induced a decrease of cell proliferation associated with a decrease of cyclin D1 and a decrease of Akt and Erk pathway activation. MiR-219-1-3p overexpression also decreased cell migration. Furthermore, miR-219-1-3p expression was found to be conversely correlated with Muc4 expression in early pancreatic intraepithelial neoplasia lesions of Pdx1-Cre;LSL-Kras(G12D) mice. Most interestingly, in vivo studies showed that miR-219-1-3p injection in xenografted pancreatic tumors in mice decreased both tumor growth and MUC4 mucin expression. Altogether, these results identify miR-219-1-3p as a new negative regulator of MUC4 oncomucin that possesses tumor-suppressor activity in PDAC.

The MUC4 mucin mediates gemcitabine resistance of human pancreatic cancer cells via the Concentrative Nucleoside Transporter family.

The fluorinated analog of deoxycytidine, Gemcitabine (Gemzar), is the main chemotherapeutic drug in pancreatic cancer, but survival remains weak mainly because of the high resistance of tumors to the drug. Recent works have shown that the mucin MUC4 may confer an advantage to pancreatic tumor cells by modifying their susceptibility to drugs. However, the cellular mechanism(s) responsible for this MUC4-mediated resistance is unknown. The aim of this work was to identify the cellular mechanisms responsible for gemcitabine resistance linked to MUC4 expression. CAPAN-2 and CAPAN-1 adenocarcinomatous pancreatic cancer (PC) cell lines were used to establish stable MUC4-deficient clones (MUC4-KD) by shRNA interference. Measurement of the IC50 index using tetrazolium salt test indicated that MUC4-deficient cells were more sensitive to gemcitabine. This was correlated with increased Bax/BclXL ratio and apoptotic cell number. Expression of Equilibrative/Concentrative Nucleoside Transporter (hENT1, hCNT1/3), deoxycytidine kinase (dCK), ribonucleotide reductase (RRM1/2) and Multidrug-Resistance Protein (MRP3/4/5) was evaluated by quantitative RT-PCR (qRT-PCR) and western blotting. Alteration of MRP3, MRP4, hCNT1 and hCNT3 expression was observed in MUC4-KD cells, but only hCNT1 alteration was correlated to MUC4 expression and sensitivity to gemcitabine. Decreased activation of MAPK, JNK and NF-κB pathways was observed in MUC4-deficient cells, in which the NF-κB pathway was found to have an important role in both sensitivity to gemcitabine and hCNT1 regulation. Finally, and in accordance with our in vitro data, we found that MUC4 expression was conversely correlated to that of hCNT1 in tissues from patients with pancreatic adenocarcinoma. This work describes a new mechanism of PC cell resistance to gemcitabine, in which the MUC4 mucin negatively regulates the hCNT1 transporter expression via the NF-κB pathway. Altogether, these data point out to MUC4 and hCNT1 as potential targets to ameliorate the response of pancreatic tumors to gemcitabine treatment.

Galectin-3 regulates MUC1 and EGFR cellular distribution and EGFR downstream pathways in pancreatic cancer cells.

MUC1 is a transmembrane glycoprotein which is typically expressed at the apical membrane of normal epithelial cells. In cancer cells, the over-expression of MUC1 and its aberrant localization around the cell membrane and in the cytoplasm favours its interaction with different protein partners such as epidermal growth factor receptor (EGFR) and can promote tumour proliferation through the activation of oncogenic signalling pathways. Our aims were to study the mechanisms inducing MUC1 cytoplasmic localization in pancreatic cancer cells, and to decipher their impact on EGFR cellular localization and activation. Our results showed that galectin-3, an endogenous lectin, is co-expressed with MUC1 in human pancreatic ductal adenocarcinoma, and that it favours the endocytosis of MUC1 and EGFR. Depletion of galectin-3 by RNA interference increased the interaction between MUC1 and EGFR, EGFR and ERK-1,2 phosphorylation, and translocation of EGFR to the nucleus. On the contrary, silencing of galectin-3 led to a decrease of cyclin-D1 levels and of cell proliferation. The galectin-3-dependent regulation of MUC1/EGFR functions may represent an interesting mechanism modulating the EGFR-stimulated cell growth of pancreatic cancer cells.

Clinical impact of MUC1 and MUC4 expression in Barrett-associated oesophageal adenocarcinoma.

AIMS: To study the expression of MUC1 and MUC4 mucins in Barrett-associated oesophageal adenocarcinoma and coexisting lesions of the carcinogenic sequence (normal mucosa, metaplasia, dysplasia) if present, and to investigate their prognostic significance. METHODS: The expression profiles of MUC1 and MUC4 were investigated by immunohistochemistry in tissue samples obtained from consecutive patients with primary surgically resected lower third oesophageal adenocarcinoma (OA) between 1997 and 2002. Histopathological parameters, recurrence and long-term survival were correlated with the number of cells stained. RESULTS: All 52 patients exhibited OA, with 25 patients (48.1%) having associated Barrett oesophagus lesions (metaplasia or/and dysplasia). MUC1 and MUC4 were expressed in 52 and 41 of the 52 patients with adenocarcinoma (100% and 78%), respectively. All samples expressed MUC1 strongly. The prevalence of MUC4 staining was significantly decreased in metaplasia compared with normal mucosa (53% versus 92%, p<0.001). No correlation was found between the level of MUC1 or MUC4 expression in OA and histopathological variables, recurrence or survival. CONCLUSIONS: MUC1 and MUC4 are strongly expressed in OA. The results do not support a role for these two membrane-bound mucins as either a phenotypic or a prognostic marker for the development of Barrett OA. There are several other membrane-bound mucins that have not yet been evaluated in this situation.

Tumour growth and resistance to gemcitabine of pancreatic cancer cells are decreased by AP-2alpha overexpression.

BACKGROUND: Activator protein-2alpha (AP-2alpha) is a transcription factor that belongs to the family of AP-2 proteins that have essential roles in tumorigenesis. Indeed, AP-2alpha is considered as a tumour-suppressor gene in different tissues such as colonic, prostatic or breast epithelial cells. Moreover, AP-2alpha also participates in the control of colon and breast cancer cells sensitivity towards chemotherapeutic drugs. Despite its potential interest, very few data are available regarding the roles of AP-2alpha in pancreatic cancer. METHODS: We have developed a stable pancreatic CAPAN-1 cell line overexpressing AP-2alpha. Consequences of overexpression were studied in terms of in vivo cell growth, gene expression, migration capacity and chemosensitivity. RESULTS: In vivo tumour growth of CAPAN-1 cells overexpressing AP-2alpha was significantly decreased by comparison to control cells. An altered expression pattern of cell cycle-controlling factors (CDK-4, CDK-6, cyclin-G1, p27(kip1) and p57(kip2)) was observed in AP-2alpha-overexpressing clones by microarrays and western blot analysis. Promoter activity and ChIP analysis indicated that AP-2alpha induces p27(kip1) protein levels by direct binding to and transactivation of its promoter. Moreover, AP-2alpha overexpression increased the chemosensitivity of CAPAN-1 cells to low doses of gemcitabine and reduced their in vitro migration capacity. CONCLUSION: Our data suggested that AP-2alpha overexpression could be exploited to decrease in vivo tumour growth of pancreatic cancer cells and to increase their sensitivity to gemcitabine.

Key elements of the BMP/SMAD pathway co-localize with CDX2 in intestinal metaplasia and regulate CDX2 expression in human gastric cell lines.

Helicobacter pylori infection induces intestinal metaplasia of the stomach, a preneoplastic lesion associated with an increased risk for gastric cancer development. Intestinal metaplasia is induced by the intestine-specific transcription factor CDX2 but the mechanisms responsible for this ectopic expression have never been described. We hypothesized that the BMP/SMAD pathway has a role in CDX2 regulation, in this context, for the following reasons: (1) the BMP pathway is crucial for normal intestinal differentiation and (2) there is an influx of BMP2 and BMP4-producing cells to the stomach upon Helicobacter pylori infection. We evaluated the expression of key elements of the BMP pathway in human stomach specimens with IM. Growth factor treatments, with BMP2 and BMP4, were performed in cultured cells and a knock-down experiment of SMAD4 was done using RNAi. We showed overexpression in IM of BMP2/4, BMPR1A, and SMAD4 in 56% of IM foci, and pSMAD1/5/8 in 100% of IM foci as compared to adjacent mucosa. In vitro, treatment of AGS cells with BMP2 and BMP4 increased endogenous CDX2 expression as well as the intestinal differentiation markers MUC2 and LI-cadherin. On the other hand, SMAD4 knock-down led to decreased endogenous CDX2, MUC2, and LI-cadherin in AGS. Treatment of the SMAD4 knock-down cells had no influence on CDX2 expression as opposed to wild-type cells. A 9.3 kb CDX2 promoter could be transactivated by SMAD4 and SMAD1 in a cell-dependent manner. In conclusion, we identified for the first time that the BMP pathway is active in intestinal metaplasia and that BMP2 and BMP4 regulate CDX2 expression and promote intestinal differentiation through the canonical signal transducers.

Epigenetic regulation (DNA methylation, histone modifications) of the 11p15 mucin genes (MUC2, MUC5AC, MUC5B, MUC6) in epithelial cancer cells.

The human genes MUC2, MUC5AC, MUC5B and MUC6 are clustered on chromosome 11 and encode large secreted gel-forming mucins. The frequent occurrence of their silencing in cancers and the GC-rich structure of their promoters led us to study the influence of epigenetics on their expression. Pre- and post-confluent cells were treated with demethylating agent 5-aza-2'-deoxycytidine and histone deacetylase (HDAC) inhibitor, trichostatin A. Mapping of methylated cytosines was performed by bisulfite-treated genomic DNA sequencing. Histone modification status at the promoters was assessed by chromatin immunoprecipitation assays. Our results indicate that MUC2 was regulated by site-specific DNA methylation associated with establishment of a repressive histone code, whereas hypermethylation of MUC5B promoter was the major mechanism responsible for its silencing. DNA methyltransferase 1 was identified by small interfering RNA approach as a regulator of MUC2 and MUC5B endogenous expression that was potentiated by HDAC2. MUC2 and MUC5B epigenetic regulation was cell-specific, depended on cell differentiation status and inhibited their activation by Sp1. The expression of MUC5AC was rarely influenced by epigenetic mechanisms and methylation of MUC6 promoter was not correlated to its silencing. In conclusion, this study demonstrates the important role for methylation and/or histone modifications in regulating the 11p15 mucin genes in epithelial cancer cells.

MUC4 expression is regulated by cystic fibrosis transmembrane conductance regulator in pancreatic adenocarcinoma cells via transcriptional and post-translational mechanisms.

MUC4 mucin is a high molecular weight transmembrane glycoprotein that plays important roles in tumour biology. It is aberrantly expressed in pancreatic adenocarcinoma, while not being detectable in the normal pancreas. Previous studies have demonstrated that the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel that is defective in CF, is implicated in multiple cellular functions, including gene regulation. In the present study, using a CFTR-defective pancreatic cancer cell line and its derived subline expressing functional CFTR, we report that MUC4 expression is negatively regulated by CFTR. Short-interfering RNA (siRNA)-mediated silencing of CFTR also leads to an increased expression of MUC4. Additionally, our results suggest that CFTR-mediated regulation of MUC4 is cell density-dependent and is achieved by transcriptional and posttranslational mechanisms. Moreover, in a panel of pancreatic cancer cell lines and normal pancreas, we observed that CFTR was downregulated in pancreatic cancer cells and negatively correlated with MUC4 in most cases. An aberrant expression of MUC4 was also detected in the CF pancreas. Downregulation of CFTR in pancreatic adenocarcinoma and its inverse association with the tumour-linked mucin, MUC4, indicate novel function(s) of CFTR in pancreatic tumour biology and suggest the implication of new signalling pathway(s) in MUC4 regulation.

Surgery alone in the curative treatment of localised oesophageal carcinoma.

AIM: To document the results of surgery alone in patients with localised oesophageal carcinoma. METHODS: Between January 1982 and 2002, 179 consecutive patients who underwent curative oesophagectomy for stage 0, I or II oesophageal carcinoma, without neo-adjuvant treatment, were analysed retrospectively. RESULTS: Postoperative mortality and morbidity rates were 2.8 and 30.7%, respectively. The overall actuarial survival rate at 5 years was 59%. No patients with more than four invaded lymph nodes survived 5 years. A lymph node ratio more than 0.2, more than four invaded lymph nodes and an advanced pTNM stage were predictors of poor prognosis. CONCLUSION: Long-term survival after surgery alone for localised oesophageal carcinoma can be achieved in some 2/3rds of patients. These results should be considered before designing neo-adjuvant therapeutic regimen or embarking into exclusive treatment alternate to oesophagectomy.

Value of endoscopic ultrasonography as a predictor of long-term survival in oesophageal carcinoma.

BACKGROUND: : Endoscopic ultrasonography (EUS) is considered to be the best locoregional staging technique for cancer of the oesophagus. This study evaluated the relationship between preoperative EUS findings, completeness of surgical resection and survival. METHODS: : Between January 1995 and July 2002, 150 patients who underwent EUS for staging of tumours of the oesophagus were analysed prospectively. All underwent surgical resection with curative intent without neoadjuvant treatment. RESULTS: : Patient survival was significantly related to tumour (EUS T), node (EUS N) and Union Internacional Contra la Cancrum classification (EUS UICC) stage according to sonographic findings (P = 0.003, P = 0.009 and P = 0.004 respectively), and the presence of stenosis determined by EUS (P = 0.004). EUS T stage was a prognostic factor for survival (relative risk 1.7 (95 per cent confidence interval (c.i.) 1.1 to 3.0); P = 0.046). Complete surgical resection (R0) was also significantly related to EUS T, N and UICC classification (P < 0.001). EUS UICC stage was a factor predictive of R0 resection (relative risk 2.6 (95 per cent c.i. 1.4 to 4.8); P = 0.003). CONCLUSION: : R0 resection and survival of patients with tumours of the oesophagus were strongly related to endosonographic findings. These results support the proposal that EUS should be performed in all patients with oesophageal cancer, not only for staging patients before therapy but also to determine prognosis.

Extent of oesophageal resection for adenocarcinoma of the oesophagogastric junction.

AIMS: The optimal extent of oesophageal resection and surgical approach in patients treated for adenocarcinomas of the oesophagogastric junction (OGJ) are still uncertain. We report the correlations between resection margin involvement and outcome. METHODS: Patients with positive proximal resection margin (PPRM) and those with negative proximal resection margin (NPRM) were compared. RESULTS: Of 94 patients with macroscopically complete resection, eight were PPRM. There was no difference between the two groups in postoperative mortality or morbidity rates, in anastomotic leakage or in recurrence rates. The median survival in the PPRM group was 11.1 months compared with 36.3 months in the NPRM group (P=0.02). No infiltration was observed in patients whose proximal margin exceeded 7 cm. The extended transthoracic approach was the only prognostic factor for tumours type II (P=0.03, RR=1.4, 95% CI: 1.1-1.8). CONCLUSION: Histologic infiltration of oesophageal resection margin influences 5-year survival rate. In adenocarcinomas of the OGJ that can be treated curatively, a transection with a 8 cm oesophagectomy above the tumour in fresh specimen should be performed, and by thoracoabdominal approach for tumours type I and II.

Transcriptional regulation of the 11p15 mucin genes. Towards new biological tools in human therapy, in inflammatory diseases and cancer?

Mucin production and secretion by specialized epithelial cells is a common mechanism used by mammals to protect the underlying mucosae against various injuries (pollutants, pathogens, pH). The expression of mucin genes is cell- and tissue-specific but is submitted to variations during cell differentiation, inflammatory process, and is altered during carcinogenesis. The molecular mechanisms responsible for the control of mucin transcription and expression are beginning to be understood as mucin gene promoters and regulatory regions are characterized. The four gel-forming mucin genes, MUC2-MUC5AC-MUC5B-MUC6, are clustered on the p15 arm of chromosome 11. Common regulatory mechanisms (PKA, PKC, PKG and Ca2+ signaling, Sp1/Sp3) may account for the capability of mucous-secreting cells to express several mucin genes simultaneously. In response to an insult or during carcinogenesis, the normal pattern of expression is altered and results from specific answers of the cell by activating different intracellular signaling pathways. 11p15 mucin genes are regulated at the transcriptional level by pro-inflammatory cytokines (IL-1beta, IL-6, TNF-alpha), pleiotropic cytokines (IL-4, IL-13, IL-9), bacterial exoproduct (LPS), growth factors (EGF, TGF-alpha), lipid mediator (PAF), retinoids and hormones. To date, the only downstream cascade known to activate mucin gene transcription is the Src/Ras/MAPK/pp90rsk cascade, which leads to the activation of the transcription factor NF-kappaB. Mucin gene transcription is also regulated by ATF-1, CREB and RAR-alpha transcription factors. Finally, repression of mucin transcription in cancer cells is under the control of the epigenetic mechanism of methylation. As transcriptional regulation of mucin genes begins to be unraveled, it becomes clear that many signaling pathways are involved. Our understanding of mucin gene transcriptional regulation, which awaits more data (identification of the signaling cascades and active cis-elements within promoters and introns), will most certainly lead to the use of mucin genes as molecular markers in cancer and molecular tools in human gene therapy, and to the synthesis of new therapeutic agents in inflammatory diseases of the epithelium.

Influence of culture conditions on the alpha 1,2-fucosyltransferase and MUC gene expression of a transformed cell line MM-39 derived from human tracheal gland cells.

Human tracheal glands cells (HTGC) in culture are able to respond to adrenergic, cholinergic and purinergic agonists by increasing their serous and mucin secretions. These secretagogues are also able to maintain an optimal responsiveness of serous cells to stimulation when they are regularly and briefly delivered to the cells, making the HTGC a suitable model to study the serous secretion (Merten, in press). Our interest has been focused on the effects of cholinergic and purinergic secretagogues associated to histamine, on the mucous function of the transformed human tracheal gland cell line MM-39, which has a mixed, both serous and mucous, phenotype. When the cells were exposed to short stimulation every 2 days for 3 weeks with 10 or 100 microM carbachol, UTP and histamine, modifications of their mucous phenotype were observed. The expression of MUC genes appeared dependent on the culture conditions. Transcripts of MUC1, MUC4, and MUC5B genes were observed when the cells were regularly exposed to the mixture of secretagogues at a concentration of 10 microM, in contrast to the unstimulated expression of MUC1 and MUC4 in control cells. MUC1, MUC4, MUC7, MUC6 and MUC11 transcripts were observed when the cells were regularly exposed to the mixture of secretagogues at a concentration of 100 microM. These culture conditions were also able to induce an alpha 1,2-fucosyltransferase activity absent in the MM-39 cells cultivated with standard conditions. There was no marked effect on the alpha 2,3-sialyltransferase activity although the expression pattern of the sialyltransferase genes was reduced to the unique presence of ST3Gal III. In conclusion, MM-39 cells exposed to repeated stimulation by secretagogues at different concentrations express different sero-mucous phenotypes.

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.

Influence of TNFalpha on the sialylation of mucins produced by a transformed cell line MM-39 derived from human tracheal gland cells.

In order to investigate the influence of inflammation on the peripheral glycosylation of airway mucins, a human respiratory glandular cell line (MM-39) was treated by TNFalpha. The expression and the activity of sialyl- and fucosyl-transferases, involved in the biosynthesis of peripheral carbohydrate determinants like sialyl-Lewis x, were investigated by RT-PCR and by HPAEC respectively. The mRNA steady-state level of sialyl- (ST3Gal III) and of fucosyl- (FUT3) transferases was moderately up-regulated by TNFalpha; a 52% increase of alpha2,3-sialyltransferase activity was also observed in TNFalpha-stimulated MM-39 cells. After metabolic radio-labelling with [(3)H]glucosamine and [(3)H]fucose, the mucins released in the culture supernatant were purified by Sepharose CL-4B, density-gradient centrifugation and treatment with glycosaminoglycans-degrading enzymes. The mucins, released in the culture supernatant from control MM-39 cells, were constituted by two populations of molecules having the same 1.39-1.44 mg/ml density but carrying either high or low amounts of sialic acid residues at their periphery. TNFalpha was able to increase the sialylation of the weakly sialylated mucins. This effect and the enhancement of the alpha2,3-sialyltransferase activity by TNFalpha argue in favour of a regulation of the mucin sialylation by this pro-inflammatory cytokine. Despite the moderate overexpression of FUT3, no fucosylation of mucins produced by MM-39 cells was induced by TNFalpha. In conclusion, the influence of TNFalpha on the sialylation of mucins could explain why the mucins from infected patients suffering either from cystic fibrosis or from chronic bronchitis are more sialylated.

Sequence of the 5'-flanking region and promoter activity of the human mucin gene MUC5B in different phenotypes of colon cancer cells.

Control of gene expression in intestinal cells is poorly understood. Molecular mechanisms that regulate transcription of cellular genes are the foundation for understanding developmental and differentiation events. Mucin gene expression has been shown to be altered in many intestinal diseases and especially cancers of the gastrointestinal tract. Towards understanding the transcriptional regulation of a member of the 11p15.5 human mucin gene cluster, we have characterized 3.55 kb of the 5'-flanking region of the human mucin gene MUC5B, including the promoter, the first two exons and the first intron. We report here the promoter activity of successively 5'-truncated sections of 956 bases of this region by fusing it to the coding region of a luciferase reporter gene. The transcription start site was determined by primer-extension analysis. The region upstream of the transcription start site is characterized by the presence of a TATA box at bases -32/-26, DNA-binding elements for transcription factors c-Myc, N-Myc, Sp1 and nuclear factor kappaB as well as putative activator protein (AP)-1-, cAMP-response-element-binding protein (CREB)-, hepatocyte nuclear factor (HNF)-1-, HNF-3-, TGT3-, gut-enriched Krüppel factor (GKLF)-, thyroid transcription factor (TTF)-1- and glucocorticoid receptor element (GRE)-binding sites. Intron 1 of MUC5B was also characterized, it is 2511 nucleotides long and contains a DNA segment of 259 bp in which are clustered eight tandemly repeated GA boxes and a CACCC box that bind Sp1. AP-2alpha and GATA-1 nuclear factors were also shown to bind to their respective cognate elements in intron 1. In transfection studies the MUC5B promoter showed a cell-specific activity as it is very active in mucus-secreting LS174T cells, whereas it is inactive in Caco-2 enterocytes and HT-29 STD (standard) undifferentiated cells. Within the promoter, maximal transcription activity was found in a segment covering the first 223 bp upstream of the transcription start site. Finally, in co-transfection experiments a transactivating effect of Sp1 on to MUC5B promoter was seen in LS174T and Caco-2 cells.

[MUC genes: a superfamily of genes? Towards a functional classification of human apomucins]. / Gènes MUC: une superfamille de gènes? Vers une classification fonctionnelle des apomucines humaines.

The MUC genes encode epithelial mucins. Eight different human genes have been well characterized, and two others identified more recently. Among them, a family of four genes, expressed in the respiratory and digestive tracts, is clustered to chromosome 11p15.5; and these genes encode gel-forming mucins which are structurally related to the superfamily of cystine-knot growth factors. A second group is composed of three independent genes encoding various isoforms of mucins including membrane-bound mucins associated to carcinomas. In this second group, MUC3 and MUC4 encode large apomucins containing EGF-like domains.

Genomic organization of the 3' region of the human mucin gene MUC5B.

MUC5B, mapped clustered with MUC6, MUC2, and MUC5AC to chromosome 11p15.5, is a human mucin gene of which the genomic organization is being elucidated. We have recently published the sequence and the peptide organization of its huge central exon, 10,713 base pairs (bp) in length. We present here the genomic organization of its 3' region, which encompasses 10,690 bp. The genomic sequence has been completely determined. The 3' region of MUC5B is composed of 18 exons ranging in size from 32 to 781 bp, contrasting thus with the very large central exon. The sizes of the 18 introns range from 114 to 1118 bp. Some repetitive sequences were identified in four introns. The peptide deduced from the sequence of the 18 exons consists of an 808-amino acid peptide. This carboxyl-terminal region exhibits extensive sequence similarity to MUC2, MUC5AC, and von Willebrand factor, particularly the number and the positions of the cysteine residues, suggesting that this domain may be derived from a common ancestral gene. The presence in these components of a cystine knot also found in growth factors such as transforming growth factor-beta is of particular interest. Moreover, one part of this peptide is identical to the 196-amino acid sequence deduced from the cDNA clone pSM2-1, which codes for a part of the high molecular weight mucin MG1 isolated from human sublingual gland. Considering the expression pattern of MUC5B and the origin of MG1, we can thus conclude that MUC5B encodes MG1.

Diverse molecular interactions of the hnRNP K protein.

The hnRNP K protein is a versatile molecule that interacts with RNA, DNA, the proto-oncoprotein VaV, Src-like tyrosine and inducible serine/threonine kinases, the transcription factor TBP and a number of zinc-finger transcriptional repressors. The interaction of K protein with some of its protein partners is modulated by nucleic acids and K protein can alter the in vivo and in vitro rate of transcription. K protein can simultaneously engage several proteins and may facilitate molecular cross-talk. Taken together these diverse interactions suggest that K protein may act as a nucleic acid-regulated docking platform.

Human mucin genes assigned to 11p15.5: identification and organization of a cluster of genes.

Four distinct genes that encode mucins have previously been mapped to chromosome 11p15.5. Three of these genes (MUC2, MUC5AC, and MUC6) show a high level of genetically determined polymorphism and were analyzed in the CEPH families. Linkage analysis placed all three genes on the genetic map in a cluster between HRAS and INS, and more detailed analysis of recombinant breakpoints revealed that MUC6 is telomeric to MUC2. Using these recombinants D11S150 was mapped close to MUC2. Ten of the 11 recombinant chromosomes studied in detail were paternal, and the recombinant events were distributed throughout the 11p15 region, suggesting that the high level of recombination observed in 11p15.5 is not due to a particular recombinational hot spot. Pulsed-field gel electrophoresis was used to make a detailed physical map of the MUC cluster and to integrate the physical and genetical maps. The gene order was determined to be HRAS-MUC6-MUC2-MUC5AC-MUC5B-IGF2. The MUC genes span a region of some 400 kb and the map extends 770 kb and contains 15 putative CpG islands. The order of the MUC genes on the map corresponds to the relative order of their expression along the anterior-posterior axis of the body, suggesting a possible functional significance to the gene order.