IL-4 induced MUC4 enhancement in respiratory epithelial cells in vitro is mediated through JAK-3 selective signaling.
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BACKGROUND: Recent studies have identified MUC4 mucin as a ligand for activation of ErbB2, a receptor tyrosine kinase that modulates epithelial cell proliferation following epithelial damage in airways of asthmatics. In this study, we investigated the potential role of IL-4, one of the Th2 inflammatory cytokines persistent in asthmatic airways, in regulating MUC4 expression using a cell line NCI-H650. METHODS: Real time PCR analysis was performed to determine concentration and time dependent effects of IL-4 upon MUC4 expression. Nuclear run on experiments were carried out to explore potential transcriptional modulation. Western blotting experiments using a monoclonal antibody specific to ASGP-2 domain of MUC4 were performed to analyze MUC4 glycoprotein levels in plasma membrane fractions. To analyze potential signal transduction cascades, IL-4 treated confluent cultures were co-incubated, separately with a pan-JAK inhibitor, a JAK-3 selective inhibitor or a MEK-1, 2 (MAPK) inhibitor at various concentrations before MUC4 transcript analysis. Corresponding transcription factor activation was tested by western blotting using a monoclonal p-STAT-6 antibody. RESULTS: MUC4 levels increased in a concentration and time specific fashion reaching peak expression at 2.5 ng/ml and 8 h. Nuclear run on experiments revealed transcriptional enhancement. Corresponding increases in MUC4 glycoprotein levels were observed in plasma membrane fractions. Pan-JAK inhibitor revealed marked reduction in IL-4 stimulated MUC4 levels and JAK3 selective inhibitor down-regulated MUC4 mRNA expression in a concentration-dependent fashion. In accordance with the above observations, STAT-6 activation was detected within 5 minutes of IL-4 stimulus. No effect in MUC4 levels was observed on using a MAPK inhibitor. CONCLUSION: These observations signify a potential role for IL-4 in MUC4 up-regulation in airway epithelia. (+info)
Muc4-ErbB2 complex formation and signaling in polarized CACO-2 epithelial cells indicate that Muc4 acts as an unorthodox ligand for ErbB2.
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Muc4 serves as an intramembrane ligand for the receptor tyrosine kinase ErbB2. The time to complex formation and the stoichiometry of the complex were determined to be <15 min and 1:1 by analyses of Muc4 and ErbB2 coexpressed in insect cells and A375 tumor cells. In polarized CACO-2 cells, Muc4 expression causes relocalization of ErbB2, but not its heterodimerization partner ErbB3, to the apical cell surface, effectively segregating the two receptors. The apically located ErbB2 is phosphorylated on tyrosines 1139 and 1248. The phosphorylated ErbB2 in CACO-2 cells recruits the cytoplasmic adaptor protein Grb2, consistent with previous studies showing phosphotyrosine 1139 to be a Grb2 binding site. To address the issue of downstream signaling from apical ErbB2, we analyzed the three MAPK pathways of mammalian cells, Erk, p38, and JNK. Consistent with the more differentiated phenotype of the CACO-2 cells, p38 phosphorylation was robustly increased by Muc4 expression, with a consequent activation of Akt. In contrast, Erk and JNK phosphorylation was not changed. The ability of Muc4 to segregate ErbB2 and other ErbB receptors and to alter downstream signaling cascades in polarized epithelial cells suggests that it has a role in regulating ErbB2 in differentiated epithelia. (+info)
The mucin Muc4 potentiates neuregulin signaling by increasing the cell-surface populations of ErbB2 and ErbB3.
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Mucins provide a protective barrier for epithelial surfaces, and their overexpression in tumors has been implicated in malignancy. We have previously demonstrated that Muc4, a transmembrane mucin that promotes tumor growth and metastasis, physically interacts with the ErbB2 receptor tyrosine kinase and augments receptor tyrosine phosphorylation in response to the neuregulin-1beta (NRG1beta) growth factor. In the present study we demonstrate that Muc4 expression in A375 human melanoma cells, as well as MCF7 and T47D human breast cancer cells, enhances NRG1beta signaling through the phosphatidylinositol 3-kinase pathway. In examining the mechanism underlying Muc4-potentiated ErbB2 signaling, we found that Muc4 expression markedly augments NRG1beta binding to A375 cells without altering the total quantity of receptors expressed by the cells. Cell-surface protein biotinylation experiments and immunofluorescence studies suggest that Muc4 induces the relocalization of the ErbB2 and ErbB3 receptors from intracellular compartments to the plasma membrane. Moreover, Muc4 interferes with the accumulation of surface receptors within internal compartments following NRG1beta treatment by suppressing the efficiency of receptor internalization. These observations suggest that transmembrane mucins can modulate receptor tyrosine kinase signaling by influencing receptor localization and trafficking and contribute to our understanding of the mechanisms by which mucins contribute to tumor growth and progression. (+info)
IL-9 modulated MUC4 gene and glycoprotein expression in airway epithelial cells.
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Compromised epithelial cell integrity is a common feature associated with chronic lung inflammatory states such as asthma. While epithelial cell damage is largely due to sustained effects of inflammatory mediators localized to airways, the subsequent process of epithelial cell differentiation is attributed to members of the transmembrane receptor tyrosine kinase family called the ErbB's. MUC4, a large molecular weight membrane-bound glycoprotein, has recently been identified as a potential ligand for the ErbB-2 receptor. In this study, we investigated the possible role of interleukin-9 (IL-9), a Th2 cytokine, on MUC4 expression using a lung cancer cell line, NCI-H650. We determined that IL-9 up-regulates MUC4 expression in a time and concentration-dependent fashion. Nuclear run-on assays indicated transcriptional regulation of MUC4 while no post-transcriptional mRNA stabilization was observed by actinomycin D chase experiments. IL-9 also increased MUC4 glycoprotein expression as determined by Western blots using a monoclonal antibody specific for a non-tandem repeat region on ASGP-2 region of MUC4. Furthermore, a JAK3-selective inhibitor 4-(4'-hydroxyphenyl) amino-6, 7-dimethoxyquinazoline (WHI-P131), substantially reduced IL-9-induced MUC4 mRNA expression in a dose-dependent fashion. These results implicate a potential role for IL-9 upon MUC4 expression in human airway epithelial cells. (+info)
MUC4 gene polymorphism and expression in women with implantation failure.
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INTRODUCTION: The molecular mechanism of human embryo implantation is poorly understood. The role of MUC4 mucin, present in endometrial epithelium, has never been explored, and results obtained in animal studies strongly suggest a role in implantation. We investigated the role of MUC4 in human embryo implantation. METHODS AND RESULTS: We analysed the MUC4 variable number of tandem repeat (VNTR) polymorphism in three populations by Southern blot analysis: spontaneously fertile patients (C), infertile patients with repeated unexplained implantation failures after IVF (IF) and patients with a child after IVF (IVF-C). We found no differences in the size or allelic distribution of MUC4 VNTR between these three populations. We also examined, in IVF-C and IF groups, the endometrial expression of MUC4 mRNA as well as the expression of the MUC4 glycoprotein together with estrogen receptor (ER) and progesterone receptor (PR). No expression differences could be detected. However, we noticed a pattern of expression for MUC4 protein, which is limited to patches of cells in the luminal and glandular epithelium. CONCLUSIONS: We conclude that the different-sized MUC4 alleles do not interfere with implantation. The absence of coexpression of MUC4 and the steroid receptors suggests that MUC4 expression is not directly regulated by steroids. (+info)
MUC4 is a novel prognostic factor of extrahepatic bile duct carcinoma.
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PURPOSE: Many of the patients with extrahepatic bile duct carcinoma (EHBDC) show a poor outcome. We have reported that MUC4 is a novel prognostic factor of pancreatic adenocarcinoma and intrahepatic cholangiocarcinoma. The aim of this study is to evaluate the prognostic significance of MUC4 expression in EHBDC. EXPERIMENTAL DESIGN: We examined the expression profile of MUC4 in EHBDC tissues from 70 patients using immunohistochemistry. MUC4 is a membrane mucin like MUC1. In addition, MUC4 is an intramembrane ligand for receptor tyrosine kinase ErbB2 and is related with regulation of p27. We compared the MUC4 expression with MUC1, ErbB2, or p27 expression in EHBDC. RESULTS: MUC4 was expressed in 36 of the 70 patients with EHBDC. There was no significant correlation between the MUC4 expression and MUC1, ErbB2, or p27 expression. The survival of 19 patients with high MUC4 expression (>or=20% of carcinoma cells stained) was significantly worse than that of the 51 patients with low MUC4 expression (under 20% of carcinoma cells stained; P = 0.0072). The univariate analysis showed that high MUC4 expression (P = 0.0072), high MUC1 expression (P = 0.0092), histologic grading (P = 0.0029), surgical margin involvement (P = 0.0137), and nodal metastasis (P = 0.0036) were statistically significant risk factors. The backward stepwise multivariate analysis showed that high MUC4 expression (P = 0.0195) and surgical margin involvement (P = 0.0358) were statistically significant independent risk factors. CONCLUSIONS: MUC4 expression in EHBDC is a new independent factor for poor prognosis and predicts the outcome of patients with EHBDC. (+info)
Aberrant expression of MUC4 in ovarian carcinoma: diagnostic significance alone and in combination with MUC1 and MUC16 (CA125).
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Mucins are being implicated in diagnosis, prognosis, and as therapeutic targets due to their aberrant expression in a variety of carcinomas. Here, we have analyzed the expression of MUC4 and have compared its potential usefulness in early detection and prognosis of ovarian carcinoma alone and in combination with other mucin antigens, MUC1 and MUC16. Clinical significance of the differential mucin expression was evaluated by grouping the tumor samples in early (stage I and II) and advanced (stage III and IV) stage cases and histological subtypes (serous, mucinous, endometrioid and clear cell). Correlation of these mucins with patient's survival (n=63) was determined by Kaplan-Meier analysis in order to predict their prognostic value. MUC4 showed significant overexpression in tumor cases (P<0.0001) with highest incidence (92.0%) among all three mucins. A significant overexpression of MUC1 (P<0.018) and MUC16 (P<0.0001) was also observed in 83.0 and 79.0% of tumor samples, respectively. Notably, MUC4 expression was significantly higher (P+info)
Membrane mucin Muc4 induces density-dependent changes in ERK activation in mammary epithelial and tumor cells: role in reversal of contact inhibition.
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The membrane mucin Muc4 has been shown to alter cellular behavior through both anti-adhesive effects on cell-cell and cell-extracellular matrix interactions and its ability to act as an intramembrane ligand for the receptor tyrosine kinase ErbB2. The ERK pathway is regulated by both cell-matrix and cell-cell adhesion. An analysis of the effects of Muc4 expression on ERK phosphorylation in mammary tumor and epithelial cells, which exhibit both adhesion-dependent growth and contact inhibition of growth, showed that the effects are density dependent, with opposing effects on proliferating cells and contact-inhibited cells. In these cells, cell-matrix interactions through integrins are required for activation of the ERK mitogenesis pathway. However, cell-cell interactions via cadherins inhibit the ERK pathway. Expression of Muc4 reverses both of these effects. In contact-inhibited cells, Muc4 appears to activate the ERK pathway at the level of Raf-1; this activation does not depend on Ras activation. The increase in ERK activity correlates with an increase in cyclin D(1) expression in these cells. This abrogation of contact inhibition is dependent on the number of mucin repeats in the mucin subunit of Muc4, indicative of an anti-adhesive effect. The mechanism by which Muc4 disrupts contact inhibition involves a Muc4-induced relocalization of E-cadherin from adherens junctions at the lateral membrane of the cells to the apical membrane. Muc4-induced abrogation of contact inhibition may be an important mechanism by which tumors progress from an early, more benign state to invasiveness. (+info)