MUC1 is a substrate for γ‐secretase

Understanding the underlying mechanisms by which a normal cell avoids the oncogenic potential of MUC1 signaling requires further definition of the pathways by which the MUC1 cytoplasmic tail is processed in both normal and tumor‐derived cells. In the present study we describe the processing pathway initiated by TACE/ADAM17 cleavage of MUC1. Utilizing the human uterine epithelial cell line, HES, derived from normal endometrium, we show that endogenous full length MUC1 undergoes regulated intramembranous proteolysis mediated by presenillin‐dependent γ‐secretase. Cytokine‐stimulated HES cells exposed to γ‐secretase inhibitors accumulated a membrane‐associated 15 kDa fragment of the MUC1 C‐terminal subunit (CTF15). Inhibitors of TACE/ADAM17‐mediated shedding inhibited accumulation of MUC1‐CTF15 and MUC1 ectodomain release to a similar extent consistent with MUC1‐CTF15 being a product of TACE/ADAM17 action. Reduction of catalytically active γ‐secretase complex by nicastrin siRNA treatment also resulted in CTF15 accumulation. Furthermore, mature nicastrin, the substrate receptor for γ‐secretase, co‐immunoprecipitated with CTF15 in the presence of γ‐secretase inhibitors indicating the formation of CTF15: nicastrin complexes. MUC1‐CTF15 accumulation in response to γ‐secretase inhibition was demonstrated in both normal and tumor‐derived cells from humans and mice indicating that this processing pathway exists in many cell contexts. We did not detect products of MUC1 cleavage by γ‐secretase in the presence of various proteasomal inhibitors indicating that subsequent degradation is either non‐proteasomal or extremely efficient. We suggest that this efficient pathway attenuates potential signaling mediated by cytoplasmic tail fragments. J. Cell. Biochem. 108: 802–815, 2009. © 2009 Wiley‐Liss, Inc.     

Transmembrane and truncated (SEC) isoforms of MUC1 in the human endometrium and Fallopian tube

The cell surface mucin MUC1 is expressed by endometrial epithelial cells with increased abundance in the secretory phase of the menstrual cycle, when it is found both at the apical cell surface and in secretions. This suggests the presence of a maternal cell surface glycoprotein barrier to embryo implantation, arising from the anti-adhesive property of MUC1. In previous work, we demonstrated alternatively spliced MUC1 variant forms in tumour cells. The variant MUC1/SEC lacks the transmembrane and cytoplasmic sequences found in the full-length variant. We now show that MUC1/SEC mRNA is present in endometrial carcinoma cell lines, endometrial tissue and primary cultured endometrial epithelial cells. The protein can be detected using isoform-specific antibodies in uterine flushings, suggesting release from endometrium in vivo. However, on the basis of immunolocalisation studies, MUC1/SEC also remains associated with the apical epithelial surface both in tissue and in cultured cells. Transmembrane MUC1 and MUC1/SEC are both strikingly localised to the apical surface of tubal epithelium. Thus MUC1 may contribute to the anti-adhesive character of the tubal surface, inhibiting ectopic implantation. The mechanism by which this barrier is overcome in endometrium at implantation is the subject of ongoing investigation.     

CA125/MUC16 interacts with Src family kinases,and over-expression of its C-terminal fragment in human epithelial cancer cells reduces cell–cell adhesion

MUC16/CA125 is over-expressed in human epithelial tumors including ovarian, breast and some other carcinomas. The purpose of this study is to investigate how cell surface MUC16 is functionally involved in tumor progression, with a special focus on the role of its cytoplasmic tail. Forced expression of C-terminal MUC16 fragment (MUC16C) in epithelial cancer cells increased cell migration. We found that MUC16C directly interacted with Src family kinases (SFKs). Notably, localizations of E-cadherin and β-catenin at the cell–cell contacts were more diffuse in MUC16C transfectants compared with mock transfectants. Furthermore, MUC16C transfectants showed reduced Ca²⁺-dependent cell–cell adhesion, but the treatment of cells with PP2, a SFKs inhibitor, restored this. Because cell surface MUC16 is also associated with the E-cadherin/β-catenin complex, the over-expression of MUC16 and its interaction with SFKs may enhance SFKs-induced deregulation of E-cadherin. Thus, our results suggest a role for cell surface MUC16 in cell–cell adhesion of epithelial cancer cells.     

Human mucin MUC1 RNA undergoes different types of alternative splicing resulting in multiple isoforms

MUC1 is a transmembrane mucin with important functions in normal and transformed cells, carried out by the extracellular domain or the cytoplasmic tail. A characteristic feature of the MUC1 extracellular domain is the variable number of tandem repeats (VNTR) region. Alternative splicing may regulate MUC1 expression and possibly function. We developed an RT-PCR method for efficient isolation of MUC1 mRNA isoforms that allowed us to evaluate the extent of alternative splicing of MUC1 and elucidate some of the rules that govern this process. We cloned and analyzed 21, 24, and 36 isoforms from human tumor cell lines HeLa, MCF7, and Jurkat, respectively, and 16 from normal activated human T cells. Among the 78 MUC1 isoforms we isolated, 76 are new and different cells showed varied MUC1 expression patterns. The VNTR region of exon 2 was recognized as an intron with a fixed 5′ splice site but variable 3′ splice sites. We also report that the 3506 A/G SNP in exon 2 can regulate 3′ splice sites selection in intron 1 and produce different MUC1 short isoform proteins. Furthermore, the SNP A to G mutation was also observed in vivo, during de novo tumor formation in MUC1^+/?Kras^G12D/+Pten^loxP/loxP mice. No specific functions have been associated with previously reported short isoforms. We now report that one new G SNP-associated isoform MUC1/Y-LSP, but not the A SNP-associated isoform MUC1/Y, inhibits tumor growth in immunocompetent but not immunocompromised mice.     

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.     

Human corin isoforms with different cytoplasmic tails that alter cell surface targeting

Corin is a cardiac serine protease that activates natriuretic peptides. It consists of an N-terminal cytoplasmic tail, a transmembrane domain, and an extracellular region with a C-terminal trypsin-like protease domain. The transmembrane domain anchors corin on the surface of cardiomyocytes. To date, the function of the corin cytoplasmic tail remains unknown. By examining the difference between human and mouse corin cytoplasmic tails, analyzing their gene sequences, and verifying mRNA expression in hearts, we show that both human and mouse corin genes have alternative exons encoding different cytoplasmic tails. Human corin isoforms E1 and E1a have 45 and 15 amino acids, respectively, in their cytoplasmic tails. In transfected HEK 293 cells and HL-1 cardiomyocytes, corin isoforms E1 and E1a were expressed at similar levels. Compared with isoform E1a, however, isoform E1 was more active in processing natriuretic peptides. By cell surface labeling, glycosidase digestion, Western blotting, and flow cytometry, we found that corin isoform E1 was activated more readily as a result of more efficient cell surface targeting. By mutagenesis, we identified a DDNN motif in the cytoplasmic tail of isoform E1 (which is absent in isoform E1a) that promotes corin surface targeting in both HEK 293 and HL-1 cells. Our data indicate that the sequence in the cytoplasmic tail plays an important role in corin cell surface targeting and zymogen activation.     

The N-terminal region of neuregulin isoforms determines the accumulation of cell surface and released neuregulin ectodomain

Two neuregulin-1 isoforms highly expressed in the nervous system are the type III neuregulin III-beta1a and the type I neuregulin I-beta1a. The sequence of these two isoforms differs only in the region that is N-terminal of the bioactive epidermal growth factor-like domain. While the biosynthetic processing of the I-beta1a isoform has been well characterized, the processing of III-beta1a has not been reported. In this study, we compared III-beta1a and I-beta1a processing. Both III-beta1a and I-beta1a were synthesized as transmembrane proproteins that were proteolytically cleaved to produce an N-terminal fragment containing the bioactive epidermal growth factor-like domain. For I-beta1a, this product was released into the medium. However, for III-beta1a, this product was a transmembrane protein. In cultures of cells expressing III-beta1a, the amount of neuregulin at the cell surface was much greater, and the amount in the medium was much less than in cultures expressing I-beta1a. Phorbol ester treatment and truncation of the cytoplasmic tail had markedly different effects on III-beta1a and I-beta1a processing. These results demonstrate an important role for the N-terminal region in determining neuregulin biosynthetic processing and show that a major product of III-beta1a processing is a tethered ligand that may act as a cell surface signaling molecule.     

Differential expression of the human CD8beta splice variants and regulation of the M-2 isoform by ubiquitination

The CD8alphabeta heterodimer functions as a coreceptor with the TCR, influencing the outcome of CD8(+) T cell responses to pathogen-infected and tumor cells. In contrast to the murine CD8B gene, the human gene encodes alternatively spliced variants with different cytoplasmic tails (M-1, M-2, M-3, and M-4). At present, little is known about the expression patterns and functional significance of such variants. We used quantitative RT-PCR to demonstrate differential mRNA expression patterns of these splice variants in thymocytes and in resting, memory, and activated primary human CD8(+) T cells. In total CD8(+) T cells, mRNA levels of the M-1 variant were the most predominant and levels of M-3 were the least detected. The M-4 isoform was predominant in effector memory CD8(+) T cells. Upon stimulation of CD8(+) T cells, the M-2 variant mRNA levels were elevated 10-20-fold relative to resting cells in contrast to the other isoforms. Curiously, the M-2 isoform was not expressed on the cell surface in transfected cell lines. Using fluorescent chimeras of the extracellular domain of mouse CD8beta fused to the cytoplasmic tails of each isoform, the M-2 isoform was localized in a lysosomal compartment regulated by ubiquitination of a lysine residue (K215) in its cytoplasmic tail. In contrast, upon short-term stimulation, the M-2 protein localized to the cell surface with the TCR complex. The relatively recent evolution of CD8B gene splice variants in the chimpanzee/human lineage is most likely important for fine-tuning the CD8(+) T cell responses.     

Phosphoinositide 3-kinase is activated by MUC1 but not responsible for MUC1-induced suppression of Toll-like receptor 5 signaling

MUC1 is a membrane-tethered mucin-like glycoprotein expressed on the surface of various mucosal epithelial cells as well as hematopoietic cells. Recently, we showed that MUC1 suppresses flagellin-induced Toll-like receptor (TLR) 5 signaling both in vivo and in vitro through cross talk with TLR5. In this study, we determined whether phosphoinositide 3-kinase (PI3K), a negative regulator of TLR5 signaling, is involved in the cross talk between MUC1 and TLR5 using various genetically modified epithelial cell lines. Our results showed 1) activation of MUC1 induced recruitment of the PI3K regulatory subunit p85 to the MUC1 cytoplasmic tail (CT) as well as Akt phosphorylation, 2) MUC1-induced Akt phosphorylation required the presence of Tyr(20) within the PI3K binding motif of the MUC1 CT, and 3) mutation of Tyr(20) or pharmacological inhibition of PI3K activation failed to block MUC1-induced suppression of TLR5 signaling. We conclude that whereas PI3K is downstream of MUC1 activation and negatively regulates TLR5 signaling, it is not responsible for MUC1-induced suppression of TLR5 signaling.     

MUC1 initiates a calcium signal after ligation by intercellular adhesion molecule-1

The MUC1 mucin is normally restricted to the apical surface of breast epithelial cells. In tumors, it is frequently overexpressed and underglycosylated. The MUC1 peptide core mediates firm adhesion of tumor cells to adjacent cells via binding to intercellular adhesion molecule-1 (ICAM-1). There is increasing evidence that MUC1 is involved in signaling, with current reports focusing on phosphorylation of the MUC1 cytoplasmic tail after indirect or artificial modes of stimulation. ICAM-1 is the only known direct ligand of the MUC1 extracellular domain. The data presented herein show that MUC1 expressed on the surface of breast cancer cell lines or transfected 293T cells can initiate a calcium-based oscillatory signal on contact with ICAM-1-transfected NIH 3T3 cells, and we present a novel method of quantifying and comparing calcium oscillations. The MUC1-induced signal appears to be distinct from those previously described, and may involve a Src family kinase, phosphoinositol 3-kinase, phospholipase C, and lipid rafts, but not mitogen-activated protein kinase. As calcium signaling has been associated with cytoskeletal change and motility, it is possible that the functions of MUC1 include heterotypic cell-cell adhesion followed by a calcium-based promigratory signal within tumor cells, thus facilitating metastasis.     

Nuclear association of the cytoplasmic tail of MUC1 and beta-catenin

MUC1, an integral membrane mucin associated with the metastatic phenotype, is overexpressed by most human carcinoma cells. The MUC1 cytoplasmic tail (CT) is postulated to function in morphogenetic signal transduction via interactions with Grb2/Sos, c-Src, and beta-catenin. We investigated intracellular trafficking of the MUC1 CT, using epitope-tagged constructs that were overexpressed in human pancreatic cancer cell lines S2-013 and Panc-1. The MUC1 CT was detected at the inner cell surface, in the cytosol, and in the nucleus of cells overexpressing MUC1. Fragments of the MUC1 CT were associated with beta-catenin in both cytoplasm and nuclei. Overexpression of MUC1 increased steady state levels of nuclear beta-catenin but decreased nuclear levels of plakoglobin (gamma-catenin). There was no detectable association between plakoglobin and the MUC1 CT. Coimmunoprecipitation experiments revealed that the cytoplasmic and nuclear association of MUC1 CT and beta-catenin was not affected by disruption of Ca2+-dependent intercellular cadherin interactions. These results demonstrate nuclear localization of fragments of MUC1 CT in association with beta-catenin and raise the possibility that overexpression of the MUC1 CT stabilizes beta-catenin and enhances levels of nuclear beta-catenin during disruption of cadherin-mediated cell-cell adhesion.     

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.     

Cutting edge: enhanced pulmonary clearance of Pseudomonas aeruginosa by Muc1 knockout mice

MUC1 (MUC1 in human and Muc1 in nonhumans) is a membrane-tethered mucin that interacts with Pseudomonas aeruginosa (PA) through flagellin. In this study, we compared PA pulmonary clearance and proinflammatory responses by Muc1(-/-) mice with Muc1(+/+) littermates following intranasal instillation of PA or flagellin. Compared with Muc1(+/+) mice, Muc1(-/-) mice showed increased PA clearance, greater airway recruitment of neutrophils, higher levels of TNF-alpha and KC in bronchoalveolar lavage fluid, higher levels of TNF-alpha in media of flagellin-stimulated alveolar macrophages, and higher levels of KC in media of tracheal epithelial cells. Knockdown of MUC1 enhanced flagellin-induced IL-8 production by primary human bronchial epithelial cells. Expression of MUC1 in HEK293T cells attenuated TLR5-dependent IL-8 release in response to flagellin, which was completely ablated when its cytoplasmic tail was deleted. We conclude that MUC1/Muc1 suppresses pulmonary innate immunity and speculate its anti-inflammatory activity may play an important modulatory role during microbial infection.     

Cell Surface-Associated Anti-MUC1-Derived Signal Peptide Antibodies: Implications for Cancer Diagnostics and Therapy

The MUC1 tumor associated antigen is highly expressed on a range of tumors. Its broad distribution on primary tumors and metastases renders it an attractive target for immunotherapy. After synthesis MUC1 is cleaved, yielding a large soluble extracellular alpha subunit containing the tandem repeats array (TRA) domain specifically bound, via non-covalent interaction, to a smaller beta subunit containing the transmembrane and cytoplasmic domains. Thus far, inconclusive efficacy has been reported for anti-MUC1 antibodies directed against the soluble alpha subunit. Targeting the cell bound beta subunit, may bypass limitations posed by circulating TRA domains. MUC1’s signal peptide (SP) domain promiscuously binds multiple MHC class II and Class I alleles, which upon vaccination, generated robust T-cell immunity against MUC1-positive tumors. This is a first demonstration of non-MHC associated, MUC1 specific, cell surfaces presence for MUC1 SP domain. Polyclonal and monoclonal antibodies generated against MUC1 SP domain specifically bind a large variety of MUC1-positive human solid and haematological tumor cell lines; MUC1-positive bone marrow derived plasma cells obtained from multiple myeloma (MM)-patients, but not MUC1 negative tumors cells, and normal naive primary blood and epithelial cells. Membranal MUC1 SP appears mainly as an independent entity but also co-localized with the full MUC1 molecule. MUC1-SP specific binding in BM-derived plasma cells can assist in selecting patients to be treated with anti-MUC1 SP therapeutic vaccine, ImMucin. A therapeutic potential of the anti-MUC1 SP antibodies was suggested by their ability to support of complement-mediated lysis of MUC1-positive tumor cells but not MUC1 negative tumor cells and normal naive primary epithelial cells. These findings suggest a novel cell surface presence of MUC1 SP domain, a potential therapeutic benefit for anti-MUC1 SP antibodies in MUC1-positive tumors and a selection tool for MM patients to be treated with the anti-MUC1 SP vaccine, ImMucin.     

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.