Involvement of the MAP kinase ERK2 in MUC1 mucin signaling

MUC1 mucin is a receptor-like glycoprotein expressed abundantly in various cancer cell lines as well as in glandular secretory epithelial cells, including airway surface epithelial cells. The role of this cell surface mucin in the airway is not known. In an attempt to understand the signaling mechanism of MUC1 mucin, we established a stable cell line from COS-7 cells expressing a chimeric receptor consisting of the extracellular and transmembrane domains of CD8 and the cytoplasmic (CT) domain of MUC1 mucin (CD8/MUC1 cells). We previously observed that treatment of these cells with anti-CD8 antibody resulted in tyrosine phosphorylation of the CT domain of the chimera. Here we report that treatment of CD8/MUC1 cells with anti-CD8 resulted in activation of extracellular signal-regulated kinase (ERK) 2 as assessed by immunoblotting, kinase assay, and immunocytochemistry. The activation of ERK2 was completely blocked either by a dominant negative Ras mutant or in the presence of a mitogen-activated protein kinase kinase (MEK) inhibitor. We conclude that tyrosine phosphorylation of the CT domain of MUC1 mucin leads to activation of a mitogen-activated protein kinase pathway through the Ras-MEK-ERK2 pathway. Combined with the existing data by others, it is suggested that one of the roles of MUC1 mucin may be regulation of cell growth and differentiation via a common signaling pathway, namely the Grb2-Sos-Ras-MEK-ERK2 pathway.     

MUC17, a novel membrane-tethered mucin

Membrane mucins have several functions in epithelial cells including cytoprotection, extravasation during metastases, maintenance of luminal structure, and signal transduction. In this paper we describe a large membrane mucin expressed in the normal intestine. This novel mucin, designated MUC17, contains an extended, repetitive extracellular glycosylation domain and a carboxyl terminus with two EGF-like domains, a SEA module domain, a transmembrane domain, and a cytoplasmic domain with potential serine and tyrosine phosphorylation sites. RNA blot analysis and in situ hybridization indicates that MUC17 is expressed in select pancreatic and colon cancer cell lines and in intestinal absorptive cells. Radiation hybrid mapping localized MUC17 to chromosome 7q22 where it resides in close proximity with three other membrane mucin genes, MUC3A, MUC3B, and MUC12. Thus, these membrane mucins reside together in a gene cluster, but are expressed in different tissues and are likely to have different functions as well.     

Membrane-tethered MUC1 mucin is phosphorylated by epidermal growth factor receptor in airway epithelial cells and associates with TLR5 to inhibit recruitment of MyD88

MUC1 is a membrane-tethered mucin glycoprotein expressed on the apical surface of mucosal epithelial cells. Previous in vivo and in vitro studies established that MUC1 counterregulates airway inflammation by suppressing TLR signaling. In this article, we elucidate the mechanism by which MUC1 inhibits TLR5 signaling. Overexpression of MUC1 in HEK293 cells dramatically reduced Pseudomonas aeruginosa-stimulated IL-8 expression and decreased the activation of NF-κB and MAPK compared with cells not expressing MUC1. However, overexpression of MUC1 in HEK293 cells did not affect NF-κB or MAPK activation in response to TNF-α. Overexpression of MyD88 abrogated the ability of MUC1 to inhibit NF-κB activation, and MUC1 overexpression inhibited flagellin-induced association of TLR5/MyD88 compared with controls. The MUC1 cytoplasmic tail associated with TLR5 in all cells tested, including HEK293T cells, human lung adenocarcinoma cell line A549 cells, and human and mouse primary airway epithelial cells. Activation of epidermal growth factor receptor tyrosine kinase with TGF-α induced phosphorylation of the MUC1 cytoplasmic tail at the Y46EKV sequence and increased association of MUC1/TLR5. Finally, in vivo experiments demonstrated increased immunofluorescence colocalization of Muc1/TLR5 and Muc1/phosphotyrosine staining patterns in mouse airway epithelium and increased Muc1 tyrosine phosphorylation in mouse lung homogenates following P. aeruginosa infection. In conclusion, epidermal growth factor receptor tyrosine phosphorylates MUC1, leading to an increase in its association with TLR5, thereby competitively and reversibly inhibiting recruitment of MyD88 to TLR5 and downstream signaling events. This unique ability of MUC1 to control TLR5 signaling suggests its potential role in the pathogenesis of chronic inflammatory lung diseases.     

MUC1 tyrosine phosphorylation activates the extracellular signal-regulated kinase

MUC1 is a transmembrane glycoprotein expressed on the apical surface of epithelial cells and exhibiting structural features characteristic of receptors for cytokines and growth factors. Its intracellular cytoplasmic tail (CT) contains multiple amino acid sequence motifs that, once phosphorylated, serve as docking sites for SH2 domain-containing proteins mediating signal transduction. Most studies examining MUC1 signaling have focused on cancer cells where MUC1 is overexpressed, aberrantly glycosylated, and constitutively phosphorylated. No studies have determined the signaling pathways activated in response to stimulation of its ectodomain. To better understand the signaling mechanisms of MUC1, we stably transfected HEK293 cells with an expression plasmid encoding a chimeric protein consisting of the extracellular and transmembrane domains of CD8 and the MUC1 CT (CD8/MUC1). Extracellular treatment of HEK293-CD8/MUC1 cells with CD8 antibody induced intracellular Tyr phosphorylation of the MUC1 CT and activated ERK1/2, but not the p38, SAPK/JNK, or ERK5 MAP kinases. Moreover, phosphorylation of ERK1/2 was completely blocked using a CT deletion mutant or a mutant construct in which all Tyr residues in the CT were changed to Phe. These results establish that Tyr phosphorylation of the MUC1 CT is required for activation of a downstream ERK1/2 pathway.     

Role of phosphorylation of the cytoplasmic domain of the alpha(2)-macroglobulin receptor (LRP)

The low density lipoprotein receptor-related protein (alpha(2)MR/LRP) is a cell surface receptor which is present on most cells and tissues. We show that the 85 kDa subunit, containing the transmembrane region and cytoplasmic domain is phosphorylated in vivo. Comparison of the phosphorylation of the low density lipoprotein receptor (LDLR) with a chimeric receptor containing the cytoplasmic domain of the alpha(2)MR/LRP (LDLR/LRP) showed that phosphorylation is exclusive to the cytoplasmic domain. Staurosporine, a general kinase inhibitor, resulted in a 40% lowering of phosphorylation of LDLR/LRP, but did not give rise to measurable changes in its membrane traffic in MDCK cells. The role of phosphorylation on degradation of the receptor was studied using inhibitors of lysosomal and proteasomal degradation. These studies showed that LDLR/LRP was rapidly turned over by proteasomal degradation but that this turnover was also not a consequence of phosphorylation.     

Identification of four sites of stimulated tyrosine phosphorylation in the MUC1 cytoplasmic tail

MUC1 is an integral membrane protein expressed on the apical surface of epithelial cells where it acts as a signaling receptor. Its cytoplasmic tail (CT) contains seven, highly conserved tyrosine residues, some of which are constitutively phosphorylated and serve as recognition sites for SH2 domain proteins involved in intracellular signal transduction. However, no studies have determined which MUC1 tyrosines are phosphorylated or which signaling pathways are activated in response to stimulation of its ectodomain. In this report, we used our previously characterized CD8/MUC1 chimeric protein that is tyrosine phosphorylated on the MUC1 CT in response to extracellular treatment with CD8 antibody and performed site-directed mutagenesis of all seven tyrosines, both individually and in multiple combinations, to identify the particular sites of stimulated phosphorylation. We observed four phosphorylation sites, three present in sequence motifs with known signaling potential (Y(20), Y(46), and Y(60)) and one previously uncharacterized (Y(29)). These results are discussed in the context of the role of MUC1 in signal transduction.     

MUC4 involvement in ErbB2/ErbB3 phosphorylation and signaling in response to airway cell mechanical injury

The receptor tyrosine kinases ErbB2 and ErbB3 are phosphorylated in response to injury of the airway epithelium. Since we have shown that the membrane mucin MUC4 can act as a ligand/modulator for ErbB2, affecting its localization in polarized epithelial cells and its phosphorylation, we questioned whether Muc4 was involved, along with ErbB2 and ErbB3, in the damage response of airway epithelia. To test this hypothesis, we first examined the localization of MUC4 in human airway samples. Both immunocytochemistry and immunofluorescence showed a co‐localization of MUC4 and ErbB2 at the airway luminal surface. Sequential immunoprecipitation and immunoblotting from airway cells demonstrated that the MUC4 and ErbB2 are present as a complex in airway epithelial cells. To assess the participation of MUC4 in the damage response, cultures of NCI‐H292 or airway cells were scratch‐wounded, then analyzed for association of phospho‐ErbB2 and ‐ErbB3 with MUC4 by sequential immunoprecipitation and immunoblotting. Wounded cultures exhibited increased phosphorylation of both receptors in complex with MUC4. Scratch wounding also increased activation of the downstream pathway through Akt, as predicted from our previous studies on Muc4 effects on ErbB2 and ErbB3. The participation of MUC4 in the phosphorylation response was also indicated by siRNA repression of MUC4 expression, which resulted in diminution of the phosphorylation of ErbB2 and ErbB3. These studies provide a new model for the airway epithelial damage response, in which the MUC4–ErbB2 complex is a key element in the sensor mechanism and phosphorylation of the receptors. J. Cell. Biochem. 107: 112–122, 2009. © 2009 Wiley‐Liss, Inc.     

Distinct roles for the NK cell-activating receptors in mediating interactions with dendritic cells and tumor cells

Hyperproduction of goblet cells and mucin in the airway epithelium is an important feature of airway inflammatory diseases. We investigated the involvement of Notch signaling in MUC5AC expression in NCI-H292 cells, a human lung carcinoma cell line. Epidermal growth factor (EGF) stimulated generation of the Notch intracellular domain (NICD) in a RBP-Jκ-dependent manner. Treatment with γ-secretase inhibitors L-685,458 or DAPT or introduction of small interfering RNA directed against Notch1 reduced EGF-induced MUC5AC expression. The inhibitory effect of L-685,458 on EGF-induced MUC5AC mRNA and protein expression was also observed in primary human bronchial epithelial cells. Blockage of Notch signaling with L-685,458 or Notch siRNA resulted in a decrease in EGF-induced phosphorylation of ERK. These results suggested that ERK activation is necessary for the regulation of EGF receptor (EGFR)-mediated MUC5AC expression by Notch signaling. Conversely, forced expression of NICD induced both EGFR and ERK phosphorylation with MUC5AC expression even in the absence of EGF. Treatment of the NICD-expressing cells with EGF further augmented ERK phosphorylation in an additive manner. The ERK phosphorylation induced by exogenous NICD was inhibited by treatment with an Ab that antagonizes EGFR activity as well as by inhibitors of EGFR and ERK, implying that Notch signaling induces MUC5AC expression by activating the EGFR pathway. Collectively, these results suggest that MUC5AC expression is regulated by a bidirectional circuit between Notch and EGFR signaling pathways.     

The epidermal growth factor receptor regulates interaction of the human DF3/MUC1 carcinoma antigen with c-Src and beta-catenin

The DF3/MUC1 mucin-like, transmembrane glycoprotein is aberrantly overexpressed in most human carcinomas. The MUC1 cytoplasmic domain interacts with the c-Src tyrosine kinase and thereby increases binding of MUC1 and beta-catenin. In the present work, coimmunoprecipitation studies demonstrate that MUC1 associates constitutively with the epidermal growth factor receptor (EGF-R) in human ZR-75-1 breast carcinoma cells. Immunofluorescence studies show that EGF-R and MUC1 associate at the cell membrane. We also show that the activated EGF-R phosphorylates the MUC1 cytoplasmic tail on tyrosine at a YEKV motif that functions as a binding site for the c-Src SH2 domain. The results demonstrate that EGF-R-mediated phosphorylation of MUC1 induces binding of MUC1 to c-Src in cells. Moreover, in vitro and in vivo studies demonstrate that EGF-R increases binding of MUC1 and beta-catenin. These findings support a novel role for EGF-R in regulating interactions of MUC1 with c-Src and beta-catenin.     

Transmembrane and secreted MUC1 probes show trafficking-dependent changes in O-glycan core profiles

The human mucin MUC1 is expressed both as a transmembrane heterodimeric protein complex that recycles via the trans-Golgi network (TGN) and as a secreted isoform. To determine whether differences in cellular trafficking might influence the O-glycosylation profiles on these isoforms, we developed a model system consisting of membrane-bound and secretory-recombinant glycosylation probes. Secretory MUC1-S contains only a truncated repeat domain, whereas in MUC1-M constructs this domain is attached to the native transmembrane and cytoplasmic domains of MUC1 either directly (M0) or via an intermitting nonfunctional (M1) or functional sperm protein-enterokinase-agrin (SEA) module (M2); the SEA module contains a putative proteolytic cleavage site and is associated with proteins receiving extensive O-glycosylation. We showed that MUC1-M2 simulates endogenous MUC1 by recycling from the cell surface of Chinese hamster ovary (CHO) mutant ldlD14 cells through intracellular compartments where its glycosylation continues. The profiles of O-linked glycans on MUC1-S secreted by epithelial EBNA-293 and MCF-7 breast cancer cells revealed patterns dominated by core 2-based oligosaccharides. In contrast, the respective membrane-shed probes expressed in the same cells showed a complete shift to patterns dominated by sialyl core 1. In conclusion, glycan core profiles reflected the subcellular trafficking pathways of the secretory or membranous probes and the modifying activities of the resident glycosyltransferases.     

Inhibition of E-Cadherin Dependent Cell-Cell Contact Promotes MUC5AC Mucin Production through the Activation of Epidermal Growth Factor Receptors

Mucin production by epithelial cells is modulated by many soluble factors, including epidermal growth factor (EGF). E-Cadherin promotes EGF receptor (EGFR)-mediated MUC5AC mucin production in airway epithelial cells in dense cultures, suggesting the involvement of E-cadherin in activating EGFRs and mucin production. However, the role of E-cadherin in modulating mucin production is not completely understood. We examined its role in MUC5AC production in a human lung epithelial cell line, NCI-H292. Treatment of low density NCI-H292 cells with an anti-E-cadherin monoclonal antibody (SHE78-7) inhibited cell-cell contact in the dispersed colonies, but promoted MUC5AC production. Furthermore, treatment of the NCI-H292 cells with anti-E-cadherin antibody stimulated phosphorylation of extracellular signal-regulated kinase (ERK). The enhanced production of MUC5AC was inhibited with an EGFR inhibitor and with a MEK inhibitor, but not with a Src family kinase inhibitor. These results suggest that inhibition of E-cadherin activates EGFRs independently of Src and promotes MUC5AC production through the ERK signaling pathway in sparsely cultured NCI-H292 cells.     

Inhibition of E-cadherin dependent cell-cell contact promotes MUC5AC mucin production through the activation of epidermal growth factor receptors

Mucin production by epithelial cells is modulated by many soluble factors, including epidermal growth factor (EGF). E-Cadherin promotes EGF receptor (EGFR)-mediated MUC5AC mucin production in airway epithelial cells in dense cultures, suggesting the involvement of E-cadherin in activating EGFRs and mucin production. However, the role of E-cadherin in modulating mucin production is not completely understood. We examined its role in MUC5AC production in a human lung epithelial cell line, NCI-H292. Treatment of low density NCI-H292 cells with an anti-E-cadherin monoclonal antibody (SHE78-7) inhibited cell-cell contact in the dispersed colonies, but promoted MUC5AC production. Furthermore, treatment of the NCI-H292 cells with anti-E-cadherin antibody stimulated phosphorylation of extracellular signal-regulated kinase (ERK). The enhanced production of MUC5AC was inhibited with an EGFR inhibitor and with a MEK inhibitor, but not with a Src family kinase inhibitor. These results suggest that inhibition of E-cadherin activates EGFRs independently of Src and promotes MUC5AC production through the ERK signaling pathway in sparsely cultured NCI-H292 cells.     

Prostaglandin E2 induces MUC8 gene expression via a mechanism involving ERK MAPK/RSK1/cAMP response element binding protein activation in human airway epithelial cells

MUC8 gene expression is overexpressed in nasal polyp epithelium and is also increased by treatment with inflammatory mediators in nasal epithelial cells. These data suggest that MUC8 may be one of important mucin genes expressed in human airway. However, the mechanisms of various inflammatory mediator-induced MUC8 gene expression in normal nasal epithelial cells remain unclear. We examined the mechanism by which prostaglandin E(2) (PGE2), an arachidonic acid metabolite, increases MUC8 gene expression levels. Here, we show that ERK mitogen-activated protein kinase is essential for PGE2-induced MUC8 gene expression in normal human nasal epithelial cells and that p90 ribosomal S 6 protein kinase 1 (RSK1) mediates the PGE2-induced phosphorylation of cAMP-response element binding protein. Our results also indicate that cAMP-response element at the -803 region of the MUC8 promoter is an important site of PGE2-induced MUC8 gene expression. In conclusion, this study gives insights into the molecular mechanism of PGE2-induced MUC8 gene expression in human airway epithelial cells.     

Cytoplasmic domain of P-selectin (CD62) contains the signal for sorting into the regulated secretory pathway.

P-selectin (CD62), formerly called GMP-140 or PADGEM, is a membrane protein located in secretory storage granules of platelets and endothelial cells. To study the mechanisms responsible for the targeting of P-selectin to storage granules, we transfected its cDNA into COS-7 and CHO-K1 cells, which lack a regulated exocytic pathway, or into AtT20 cells, which are capable of regulated secretion. P-selectin was expressed on the plasma membrane of COS-7 and CHO-K1 cells but was concentrated in storage granules of AtT20 cells. Immunogold electron microscopy indicated that the electron-dense granules containing P-selectin in AtT20 cells also stored the endogenous soluble hormone ACTH. Activation of AtT20 cells with 8-Br-cAMP increased the surface expression of P-selectin, consistent with agonist-induced fusion of granule membranes with the plasma membrane. Deletion of the last 23 amino acids of the 35-residue cytoplasmic domain resulted in delivery of P-selectin to the plasma membrane of AtT20 cells. Replacement of the cytoplasmic tail of tissue factor, a plasma membrane protein, with the cytoplasmic domain of P-selectin redirected the chimeric molecule to granules. We conclude that the cytoplasmic domain of P-selectin is both necessary and sufficient for sorting of membrane proteins into the regulated pathway of secretion.     

MUC16 is produced in tracheal surface epithelium and submucosal glands and is present in secretions from normal human airway and cultured bronchial epithelial cells

The gel-forming MUC5AC and MUC5B mucins have been identified as major components of human airway mucus but it is not known whether additional mucin species, possibly with other functions, are also present. MUC16 mucin is a well-known serum marker for ovarian cancer, but the molecule has also been found on the ocular surface and in cervical secretions suggesting that it may play a role on the normal mucosal surface. In this investigation, the LUM16-2 antiserum (raised against a sequence in the N-terminal repeat domain) recognized MUC16 in goblet and submucosal gland mucous cells as well as on the epithelial surface of human tracheal tissue suggesting that the mucin originates from secretory cells. MUC16 mucin was present in 'normal' respiratory tract mucus as well as in secretions from normal human bronchial epithelial (NHBE) cells. MUC16 from NHBE cells was a high-molecular-mass, monomeric mucin which gave rise to large glycopeptides after proteolysis. N- and C-terminal fragments of the molecule were separated on gel electrophoresis showing that the MUC16 apoprotein undergoes a cleavage between these domains, possibly in the SEA domain as demonstrated for other transmembrane mucins; MUC1 and MUC3. After metabolic labeling of NHBE cells, most of the secreted monomeric, high-molecular-mass [(35)S]sulphate-labelled molecules were immunoprecipitated with the OC125 antibody indicating that MUC16 is the major [(35)S]sulphate-labelled mucin in NHBE cell secretions.     

Neutrophil elastase induces MUC5AC mucin production in human airway epithelial cells via a cascade involving protein kinase C, reactive oxygen species, and TNF-alpha-converting enzyme

Mucus hypersecretion is a prominent manifestation in patients with chronic inflammatory airway diseases and contributes to their morbidity and mortality by plugging airways and causing recurrent infections. Human neutrophil elastase (HNE) exists in high concentrations (1-20 microM) in airway secretions of these patients and induces overproduction of MUC5AC mucin, a major component of airway mucus. Previous studies showed that HNE induces MUC5AC mucin production involving reactive oxygen species (ROS) generation and TGF-alpha-dependent epidermal growth factor receptor (EGFR) activation in human airway epithelial cells. However, the molecular mechanisms involved in these responses are not defined. TNF-alpha-converting enzyme (TACE) cleaves pro-TGF-alpha into soluble TGF-alpha and can be activated by ROS. We hypothesize that HNE activates TACE via ROS generation, resulting in cleavage of pro-TGF-alpha, EGFR activation, and MUC5AC mucin expression in airway epithelial cells. Here we show that in human airway epithelial cells HNE increases TGF-alpha release, EGFR phosphorylation, and MUC5AC mucin expression, effects that were attenuated by TACE inhibitor TAPI-1 and by specific knockdown of TACE expression with small interfering RNA, implicating TACE in HNE-induced responses. These responses to HNE were also reduced by pretreatment with ROS scavengers, implicating ROS. Furthermore, we show that HNE causes protein kinase C (PKC) activation and translocation from cytosol to plasma membrane; blockade of this effect by PKC inhibitors reduced HNE-induced ROS generation and other responses, implicating PKC. We conclude that HNE induces MUC5AC mucin expression via a cascade involving PKC-ROS-TACE in human airway epithelial cells.     

MCP-1/CCR2B-dependent loop upregulates MUC5AC and MUC5B in human airway epithelium

Cigarette smoke represents a major risk factor for the development of chronic obstructive pulmonary disease (COPD), a respiratory condition associated with airflow obstruction, mucus hypersecretion, chronic inflammation, and upregulation of inflammatory mediators such as the monocyte chemotactic protein-1 (MCP-1). MCP-1 through its receptor CCR2 induces chemotaxis and activates (44/42)MAPK, a kinase known to play a key role in mucin regulation in bronchial epithelium. In the present study we used differentiated primary cultures of normal human bronchial epithelial (NHBE) cells to test whether MCP-1 through its receptor CCR2 induces mucin upregulation. We have provided evidence that NHBE cells release MCP-1 to the epithelial surface and express the CCR2B isoform of the receptor mainly at the apical pole. In addition, we found that MCP-1 has a novel function in airway epithelium, increasing the two major airway mucins MUC5AC and MUC5B, an effect mediated, at least in part, by a cascade of events initiated by interaction of its receptor CCR2B with G(q) subunits in caveolae, followed by PLCβ, PKC, and (44/42)MAPK activation. We also have shown that MCP-1 is able to induce its own expression using the same receptor but through a different pathway that involves RhoA GTPase. Furthermore, we found that a single exposure to MCP-1 is enough to induce MCP-1 secretion and sustained mucin upregulation up to 7 days after initial exposure, an effect mediated by CCR2B as confirmed using short hairpin RNA. These results agree with our data in smoker's airway epithelium, where CCR2B is present in MUC5AC- and MUC5B-expressing cells and augmented MCP-1 expression is associated with increased MUC5AC and MUC5B immunolabeling, suggesting that the mechanisms described in primary cell cultures in the present study are operative in vivo. Therefore, therapeutic approaches targeting MCP-1/CCR2B may be useful in preventing not only influx of inflammatory cells to the airways but also mucus hypersecretion and goblet cell hyperplasia.