Production and localization of Muc4/sialomucin complex and its receptor tyrosine kinase ErbB2 in the rat lacrimal gland.

PURPOSE: To show the presence and forms of sialomucin complex (rat Muc4) and receptor tyrosine kinase ErbBs in the rat lacrimal gland and analyze for complexes of ErbB2 and its ligand Muc4. METHODS: Northern blot analyses were used to identify sialomucin complex/Muc4 (SMC/Muc4) mRNA in rat lacrimal gland. Immunoblot analyses were performed to detect SMC/Muc4 and ErbBs. Sequential immunoprecipitation and immunoblot analyses were used to differentiate membrane and soluble forms of the SMC/Muc4 transmembrane subunit ASGP-2. Methacarn-fixed, paraffin-embedded sections of lacrimal glands from female adult rats were immunocytochemically stained using antisera to SMC/Muc4 and ErbBs to determine their relative locations in the gland. Colocalization of SMC/Muc4 and ErbB2 was confirmed by confocal immunofluorescence. Sequential immunoprecipitation and immunoblot were performed to analyze complexes of the SMC/Muc4 and ErbB2 in the lacrimal tissue. RESULTS: Northern blot analyses of rat lacrimal glands, with a probe for SMC/Muc4, demonstrated the presence of a approximately 9-kb transcript, consistent with observations in other tissues. Similarly, immunoblot analyses with antibodies against both the transmembrane (ASGP-2) and mucin (ASGP-1) subunits showed the presence of the two SMC/Muc4 subunits in lysates from rat lacrimal gland. Significantly, two different forms of ASGP-2 were observed, a high-molecular-weight ( approximately 200-kDa) form and the more common 120- to 140-kDa form. Sequential immunoprecipitation and immunoblot analyses to differentiate membrane and soluble forms of SMC/Muc4 indicated that the high-molecular-weight form of ASGP-2 was predominantly associated with membranes, whereas the 120- to 140-kDa form was both membrane-associated and soluble. The lacrimal gland consists of acini connected by intercalated and interlobular ducts. Both acini and some intercalated ducts were stained by anti-ASGP-2 monoclonal antisera. Two patterns of acinar staining were observed: membrane staining at the borders of the epithelial cells and a granular staining within the cells. Staining of ductal surfaces with antibody to the cytoplasmic domain of ASGP-2 suggests that membrane SMC/Muc4 is being produced by the ductal cells and is not simply an adsorbed soluble product from the acinar cells. Immunoblot and immunocytochemical analyses demonstrated the presence of all four ErbBs, with ErbB2 showing the most widespread distribution, similar to that of SMC/Muc4. Immunofluorescence colocalization of membrane SMC/Muc4 and ErbB2 and coimmunoprecipitation of a complex of the two provided evidence of their association in membranes of lacrimal gland acinar cells. CONCLUSIONS: SMC/Muc4 is produced by the rat lacrimal gland as both membrane and soluble forms, specifically associated with both acinar and ductal cells. Because sialomucin complex is also present in the ocular tear film, the rat lacrimal gland represents a second source of this mucin for the tear film, in addition to the corneal and conjunctival epithelia. Moreover, the presence of a complex of SMC/Muc4 and the receptor tyrosine kinase ErbB2 in lacrimal tissue suggests that SMC/Muc4 acts as a ligand for the receptor and has functions in the lacrimal gland other than that of a mucin.

Muc4/sialomucin complex, an intramembrane modulator of ErbB2/HER2/Neu, potentiates primary tumor growth and suppresses apoptosis in a xenotransplanted tumor.

Overexpression of the membrane mucin MUC4/Sialomucin complex (SMC) has been observed during malignant progression of mammary tumors in both humans and rats, suggesting that deregulation of MUC4/SMC expression might facilitate development of these malignancies. As previously reported, overexpression of SMC results in suppression of both cell adhesion and immune killing of tumor cells. SMC also acts as a ligand for ErbB2/Neu, modulating phosphorylation of the receptor tyrosine kinase in the presence and absence of heregulin. The present studies investigated the effect of Muc4/SMC up-regulation on primary tumor growth using a tetracycline-inducible SMC expression system in a xenotransplanted tumor model. SMC up-regulation provoked rapid growth of transfected A375 melanoma in nude mice. Up-regulation of SMC, however, did not significantly increase proliferation of A375 cells in vitro. Instead, a strong suppression of apoptosis was observed in situ in SMC-overexpressing tumors. These data suggest that Muc4/SMC expression promotes tumor growth in vivo at least in part via suppression of tumor cell apoptosis. Importantly, reduction of apoptosis was also observed in vitro, indicating that anti-apoptotic effect of SMC is independent of tumor-host interactions. These findings strongly suggest that SMC up-regulation alters intracellular signaling to favor cell survival, providing for the first time evidence for the regulation of programmed cell death by a gene of the MUC family.

Expression and localization of immunoreactive-sialomucin complex (Muc4) in salivary glands.

Sialomucin Complex (SMC; Muc4) is a heterodimeric glycoprotein consisting of two subunits, the mucin component ASGP-1 and the transmembrane subunit ASGP-2. Northern blot and immunoblot analyses demonstrated the presence of SMC/Muc4 in submaxillary, sublingual and parotid salivary glands of the rat. Immunocytochemical staining of SMC using monoclonal antisera raised against ASGP-2 and glycosylated ASGP-1 on paraffin-embedded sections of parotid, submaxillary and sublingual tissues was performed to examine the localization of the mucin in the major rat salivary glands. Histological and immunocytochemical staining of cell markers showed that the salivary glands consisted of varying numbers of serous and mucous acini which are drained by ducts. Parotid glands were composed almost entirely of serous acini, sublingual glands were mainly mucous in composition and a mixture of serous and mucous acini were present in submaxillary glands. Since immunoreactive (ir)-SMC was specifically localized to the serous cells, staining was most abundant in parotid glands, intermediate levels in submaxillary glands and least in sublingual glands. Ir-SMC in sublingual glands was localized to caps of cells around mucous acini, known as serous demilunes, which are also present in submaxillary glands. Immunocytochemical staining of SMC in human parotid glands was localized to epithelial cells of serous acini and ducts. However, the staining pattern of epithelial cells was heterogeneous, with ir-SMC present in some acinar and ductal epithelial cells but not in others. This report provides a map of normal ir-SMC/Muc4 distribution in parotid, submaxillary and sublingual glands which can be used for the study of SMC/Muc4 expression in salivary gland tumors.