Muc4/sialomucin complex in the mammary gland and breast cancer.

MUC4 is a one of the membrane mucins of the mucin gene (MUC) family, characterized by mucin tandem repeat domains and a transmembrane domain which associates it with the cell plasma membrane. Although MUC4 is encoded by a single gene, it is produced by epithelial cells as a heterodimer through a proteolytic cleavage mechanism. This heterodimer is found in both membrane and soluble forms associated with epithelia. Functionally, MUC4 is proposed to provide a protective mechanism for vulnerable epithelia, such as those of the airway, eye, female reproductive tract and mammary gland. The protective mechanism(s) may be highjacked by some carcinomas, such as those of the breast, to increase tumor progression. Two mechanisms are proposed to contribute to the MUC4 functions. First, MUC4 acts as an anti-adhesive or anti-recognition barrier at epithelial or tumor cell surfaces. Second, MUC4 can bind the receptor tyrosine kinase ErbB2 and alter its cellular signaling. Expression of MUC4 in mammary gland is repressed by posttranscriptional mechanisms involving basement membrane and TGF-beta, which are relieved during pregnancy to permit secretion of MUC4 into milk. These mechanisms are also abrogated in some breast cancers, providing a scenario for promotion of tumor progression. These observations imply important functions for MUC4 in both normal mammary function and in breast cancer.

Sialomucin complex (rat Muc4) transmembrane subunit binds the differentiation marker peanut lectin in the normal rat mammary gland.

Sialomucin complex (SMC, rat Muc4) is a heterodimeric glycoprotein composed of two subunits, the mucin component ASGP-1 and the transmembrane subunit ASGP-2. SMC/Muc4 is highly expressed on the surface of 13762 rat mammary adenocarcinoma cells at approximately 100 times the level found in the lactating mammary gland. Immunocytochemical staining of SMC/Muc4 in the developing rat mammary gland is localized to the apical membrane of the ductal epithelium. This staining pattern is similar to that for peanut lectin, a differentiation marker, which binds to cells expressing the disaccharide Thomsen-Friedenreich or TF antigen. Blotting of glycoproteins expressing the TF antigen from mammary tissues with peanut lectin detects a protein matching the migration of ASGP-2. Analysis of immunoprecipitated SMC/Muc4 by peanut lectin blotting shows that the TF antigen is abundantly present on the ASGP-2 subunit, hence the similarity of staining pattern with SMC/Muc4 antisera and peroxidase-conjugated lectin in mammary tissues. The TF antigen is also present on ASGP-2 of SMC/Muc4 produced by confluent cultures of Rama 37 rat mammary epithelial stem cells after their induction to an alveolar-like phenotype with prolactin. These results indicate that the TF antigen is present on the ASGP-2 transmembrane subunit of SMC/Muc4 from phenotypically normal tissues and cells, in contrast to malignant cells whose peanut lectin-binding disaccharide is located on ASGP-1.

Extracellular regulated kinase (ERK)-dependent regulation of sialomucin complex/rat Muc4 in mammary epithelial cells.

Sialomucin complex (SMC, rat Muc4) is a membrane mucin implicated in the protection of epithelia and the metastasis of some tumors. It is a heterodimeric complex, containing a mucin subunit with anti-adhesive activity and a transmembrane subunit with epidermal growth factor-like domains, one of which acts as an intramembrane ligand for ErbB2. Serum, insulin and insulin-like growth factor, but not epidermal growth factor, induce the expression of sialomucin complex in mammary epithelial cells. Induction correlates with sustained, but not transient, activation of extracellular-regulated protein kinase (ERK). MEK inhibitor U0126 blocked the induction, while activated MEK-1 transfected into a rat mammary adenocarcinoma cell line induced a sustained activation of ERK and up-regulated SMC/Muc4 expression. Northern and Western blotting indicated that up-regulation occurred concomitantly at the transcript and protein levels, both of which could be blocked by U0126. These results suggest that expression of SMC/Muc4 in mammary epithelial cells is regulated by selected growth factors through an ERK-dependent pathway at the transcript level.

Cloning and characterization of the 5' flanking region of the sialomucin complex/rat Muc4 gene: promoter activity in cultured cells.

Sialomucin complex (SMC/Muc4) is a heterodimeric glycoprotein complex consisting of a mucin subunit ascites sialoglycoprotein-1 (ASGP-1) and a transmembrane subunit (ASGP-2), which is aberrantly expressed on the surfaces of a variety of tumour cells. SMC is transcribed from a single gene, translated into a large polypeptide precursor, and further processed to yield the mature ASGP-1/ASGP-2 complex. SMC has complex spatial and temporal expression patterns in the normal rat, suggesting that it has complex regulatory mechanisms. A crude exon/intron map of the 5' regions of the SMC/Muc4 gene generated from clones isolated from a normal rat liver genomic DNA library reveals that this gene has a small first exon comprising the 5' untranslated region and signal peptide, followed by a large intron. The second exon appears to be large, comprising the 5' unique region and a large part (probably all) of the tandem repeat domain. This structure is strikingly similar to that reported for the human MUC4 gene. Using PCR-based DNA walking, 2.4 kb of the 5'-flanking region of the SMC/Muc4 gene was cloned and characterized. Promoter-pattern searches yielded multiple motifs commonly found in tissue-specific promoters. Reporter constructs generated from this 2.4 kb fragment demonstrate promoter activity in primary rat mammary epithelial cells (MEC), the human colon tumour cell line HCT-116, and the human lung carcinoma cell line NCI-H292, but not in COS-7 cells, suggesting epithelial cell specificity. Deletion constructs of this sequence transfected into rat MEC or HCT-116 cells demonstrate greatly varying levels of activity, suggesting that there are positive and negative, as well as tissue-specific, regulatory elements in this sequence. Taken together, these data suggest that the rat SMC/Muc4 promoter has been identified, that it is tissue- (epithelial cell-) specific, and that there are both positive and negative, as well as tissue-specific, regulatory elements in the sequence.

Sialomucin complex (rat Muc4) is regulated by transforming growth factor beta in mammary gland by a novel post-translational mechanism.

Sialomucin complex (SMC, rat Muc4) is a heterodimeric glycoprotein complex consisting of a mucin subunit ASGP-1 (for ascites sialoglycoprotein-1) and a transmembrane subunit ASGP-2, produced from a single gene and precursor. SMC expression is tightly regulated in mammary gland; the level in lactating mammary gland is about 100-fold that in virgin gland. In rat mammary epithelial cells, SMC is post-transcriptionally regulated by Matrigel by inhibition of SMC precursor synthesis. SMC is also post-transcriptionally regulated by transforming growth factor-beta (TGFbeta). The repression of SMC expression by TGFbeta is rapid, is independent of TGFbeta-induced cell cycle arrest, and does not require new protein synthesis. Unlike Matrigel, TGFbeta does not reduce SMC protein synthesis, as SMC precursor accumulation is equivalent in TGFbeta-treated and untreated cells. Instead, SMC precursor in TGFbeta-treated cells is more persistent and does not become processed as rapidly into mature ASGP-1 and ASGP-2, indicating that TGFbeta disrupts processing of SMC precursor. These results indicate that SMC, a product of normal mammary gland and milk, is regulated by TGFbeta by a novel post-translational mechanism. Thus, SMC is regulated by multiple post-transcriptional mechanisms, which serve to repress potential deleterious effects of overexpression.

Detection of sialomucin complex (MUC4) in human ocular surface epithelium and tear fluid.

PURPOSE: To evaluate human ocular surface epithelium and tear fluid for the presence of sialomucin complex (MUC4), a high-molecular-weight heterodimeric glycoprotein composed of mucin (ASGP-1) and transmembrane (ASGP-2) subunits. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) and Northern blot analysis assays were used to identify sialomucin complex RNA in ocular surface epithelia. Immunoprecipitation and immunoblot analysis were used to identify immunoreactive species in human tears and in the corneal and conjunctival epithelia using antibodies specific for carbohydrate and peptide epitopes on the sialomucin complex subunits. Immunofluorescence staining was used to detect sialomucin complex in frozen sections and impression cytology specimens of human cornea and conjunctival epithelia. RESULTS: ASGP-1- and ASGP-2-specific sequences were amplified from RNA extracted from both conjunctival and corneal epithelial biopsies by RT-PCR. Sialomucin complex transcripts were also detected in these tissues by Northern blot analysis, with a greater level of RNA detected in the peripheral than the central corneal epithelium. Sialomucin complex was immunoprecipitated from tear fluid samples and both corneal and conjunctival epithelia and detected by immunoblot analysis with specific anti-ASGP-1 and anti-ASGP-2 antibodies. The ASGP-1 peptide antibody HA-1 stained the full thickness of the corneal and conjunctival epithelia. In contrast, antibody 15H10, which reacts against a carbohydrate epitope on ASGP-1, stained only the superficial epithelial layers of these tissues. No staining was observed in the conjunctival goblet cells. CONCLUSIONS: Sialomucin complex was originally identified in rat mammary adenocarcinoma cells and has recently been shown to be produced by the ocular surface epithelia of rats. Furthermore, it has been identified as the rat homologue of human MUC4 mucin. The present studies show that it is expressed in the stratified epithelium covering the surface of the human eye and is present in human tear fluid. Expression of a carbohydrate-dependent epitope on the mucin subunit (ASGP-1) of sialomucin complex occurs in a differentiation-dependent fashion. Sialomucin complex joins MUC1 as another membrane mucin produced by the human ocular surface epithelia but is also found in the tear fluid, presumably in a soluble form, as found on the rat ocular surface.

Multiple facets of sialomucin complex/MUC4, a membrane mucin and erbb2 ligand, in tumors and tissues (Y2K update).

Sialomucin complex (SMC, MUC4) is a high Mr glycoprotein heterodimer, composed of mucin (ASGP-1) and transmembrane (ASGP-2) subunits. ASGP-2 contains two EGF-like domains and acts as an intramembrane ligand for the receptor tyrosine kinase ErbB2. Transfection studies with SMC DNAs showed that SMC expression could markedly reduce both cell-cell and cell-matrix interactions in vitro and increase the growth of primary tumors and the formation of metastatic foci of human A375 melanoma cells as xenotransplants in nude mice, possibly through the ability to suppress apoptosis. SMC is expressed in most vulnerable epithelia as a protective agent, which is found in both membrane and soluble forms at luminal surfaces and secreted into fluids such as milk and tears. SMC appears to be constitutively expressed by most accessible epithelia, notable exceptions being the mammary gland and uterine luminal epithelium, in which it is tightly regulated during pregnancy. Down-regulation at the luminal uterine surface appears necessary for blastocyst implantation. TGF-b is a potent repressor of SMC expression in the mammary gland and uterus, though by different mechanisms. These combined results suggest that SMC has multiple functions in epithelia and is tightly regulated in those tissues where its special functions are required.

An intramembrane modulator of the ErbB2 receptor tyrosine kinase that potentiates neuregulin signaling.

The ErbB2 receptor tyrosine kinase plays a critical role in a variety of developmental processes, and its aberrant activation may contribute to the progression of some breast and ovarian tumors. ASGP2, a transmembrane glycoprotein found on the surface of the highly metastatic ascites 13762 rat mammary adenocarcinoma cell line, is constitutively associated with ErbB2 in these cells and in mammary tissue from pregnant rats. Expression studies indicate that ASGP2 interacts directly and specifically with ErbB2 through one of its epidermal growth factor-like domains and that the co-expression of the two proteins in the same cell dramatically facilitates their direct stable interaction. Ectopic expression of ASGP2 in human melanoma tumor cells potentiates the response of endogenous ErbB2 to the neuregulin-1 growth factor. These observations point to a novel intramembrane mechanism for the modulation of receptor tyrosine kinase activity.

Post-transcriptional regulation of a milk membrane protein, the sialomucin complex (Ascites sialoglycoprotein (ASGP)-1/ASGP-2, rat muc4), by transforming growth factor beta.

Sialomucin complex (SMC, Rat Muc4) is a heterodimeric glycoprotein complex consisting of a mucin subunit ASGP-1 (ascites sialoglycoprotein-1) and a transmembrane subunit ASGP-2, which can act as a ligand for the receptor tyrosine kinase ErbB2. SMC is highly expressed on the surface of ascites 13762 rat mammary adenocarcinoma cells, approximately 100 times the level in lactating mammary gland and 10(4) times that in virgin mammary gland. SMC is sharply increased at mid-pregnancy in a manner similar to beta-casein. Unlike beta-casein, SMC appears to be regulated post-transcriptionally. Its transcript is present in both virgin and pregnant mammary tissue, and SMC synthesis is induced rapidly in cultured primary mammary epithelial cells from either normal pregnant or virgin rats. SMC protein, but not transcript, levels are significantly reduced when mammary cells are cultured in Matrigel, a reconstituted basement membrane which stimulates casein expression. SMC precursor is synthesized in Matrigel at a 10-fold lower rate. Matrigel has no effect on either the level of SMC or its transcript in cultured 13762 mammary tumor cells. The Matrigel effect on primary mammary and 13762 cells is mimicked by transforming growth factor beta, a component associated with this complex matrix. These results indicate that SMC is a novel product of normal mammary gland and milk, which is post-transcriptionally regulated by transforming growth factor beta in normal mammary gland, but not in 13762 mammary adenocarcinoma cells.

Sialomucin complex at the rat ocular surface: a new model for ocular surface protection.

The ocular surface, which is among the most accessible and vulnerable tissues in mammals, is protected by a complex tear film composed of lipid, aqueous and mucin layers. In spite of its importance, the molecular nature of the mucin contribution remains uncertain. Since membrane mucins have been implicated in the protection of other epithelia, we have analysed rat corneal and conjunctival tissues for sialomucin complex (SMC), a membrane mucin found at the apical epithelial cell surfaces in the airway and uterus. Using Northern and Western blot analyses, SMC expression was found in both ocular tissues, being particularly abundant in the cornea. In contrast with the other known membrane mucin, MUC1, SMC was localized more heavily towards the apical surface of the epithelial cells. SMC in ocular surface epithelia was produced in both soluble and membrane forms, the latter being found predominantly in the most superficial cells and at apical surfaces. The soluble form was found loosely adsorbed to apical cell surfaces, particularly of the cornea, as indicated by a mild rinsing protocol. Finally, the tear fluid contained substantial amounts of SMC. From these results, we propose a new model for tear mucin components in which SMC is expressed at the apical ocular surface in both membrane-bound and adsorbed soluble forms to provide a direct protective barrier. SMC secreted into the tear fluid may also participate in maintaining the stability of the preocular tear film by acting with other secreted mucins to determine the physical properties and protective behaviour of the tear film.

Sialomucin complex, a heterodimeric glycoprotein complex. Expression as a soluble, secretable form in lactating mammary gland and colon.

Ascites 13762 rat mammary adenocarcinoma cells express abundantly on their cell surfaces a heterodimeric glycoprotein complex composed of a sialomucin ascites sialoglycoprotein (ASGP)-1 and a transmembrane subunit ASGP-2. The latter, which contains two epidermal growth factor-like domains, binds the receptor tyrosine kinase p185(neu), suggesting that the complex is bifunctional as well as heterodimeric. Immunoblot analyses using monoclonal antibodies prepared against the complex demonstrate high levels of expression in rat lactating mammary gland and colon. Immunolocalization studies with anti-ASGP-2 indicate that ASGP-2 is present in these two tissues in the apical regions of secretory epithelial cells. Both mammary gland and colon contain a soluble, secretable form of ASGP-2, which is not found in the ascites cells; milk and mammary gland also have the membrane form. Immunoblot analyses using a COOH-terminal-specific polyclonal antibody indicate that the soluble form of ASGP-2 is missing its COOH-terminal domains. Both the soluble and membrane forms of ASGP-2 are similar to the membrane-associated form from the 13762 adenocarcinoma with respect to Mr, antigenicity, and association with ASGP-1. The presence of ASGP-1 in milk suggests that it is a candidate for the uncharacterized high Mr milk mucin, MUCX. ASGP-2 expression is up-regulated in mammary gland during pregnancy, because it is undetectable in virgin and early pregnant rats but abundant in the gland from late pregnant and lactating animals. However, compared with the lactating mammary gland, the 13762 ascites cells overexpress ASGP-2 by more than 100-fold, which may contribute to their malignancy. These combined results indicate that sialomucin complex is a unique secreted product in the mammary gland and colon, whose behavior is different from that in the mammary ascites tumors, and which may play important roles in mammary and intestinal physiology.