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.     

Expression, location, and interactions of ErbB2 and its intramembrane ligand Muc4 (sialomucin complex) in rat mammary gland during pregnancy

Muc4 (also called Sialomucin complex) is a heterodimeric glycoprotein complex consisting of a peripheral O-glycosylated subunit ASGP-1 (ascites sialoglycoprotein-1) tightly but non-covalently bound to an N-glycosylated transmembrane subunit ASGP-2. Muc4/SMC can act as an intramembrane ligand for ErbB2 via an EGF-like domain present in the transmembrane subunit. The complex is developmentally regulated in normal rat mammary gland and overexpressed in a number of mammary tumors. Overexpression of Muc4/SMC has been shown to block cell-cell and cell-matrix interactions, protect tumor cells from immune surveillance, promote metastasis, and protect from apoptosis. We have investigated whether Muc4/SMC and ErbB2 are co-expressed and co-localized in normal rat mammary gland and whether Muc4/SMC-ErbB2 complex formation is developmentally regulated in this tissue. Muc4/SMC and ErbB2 have different expression patterns and regulatory mechanisms in the developing rat mammary gland, but both are maximally expressed during late pregnancy and lactation. The two proteins form a complex in lactating mammary gland which is not detected in the virgin gland. Moreover, this complex does not contain ErbB3. ErbB2 is co-localized with Muc4/SMC at the apical surfaces of ductal and alveolar cells in lactating gland; however, another form of ErbB2, recognized by a different antibody, localizes to the basolateral surfaces of these cells. ErbB2 phosphorylated on Tyr 1248 co-localized with Muc4/SMC at the apical surface but not at the basolateral surfaces of these cells. To investigate the function of Muc4 in the mammary gland, transgenic mice were derived using an MMTV-Muc4 construct. Interestingly, mammary gland development in the transgenic mice was aberrant, exhibiting a bifurcated pattern, including invasion down the blood vessel, similar to that exhibited by transgenic mice inappropriately expressing activated ErbB2 in the mammary gland. These data provide further evidence of the ability of Muc4/SMC to interact with ErbB2 and influence its behavior in normal epithelia.     

Enzymatic cleavage as a processing step in the maturation of Muc4/sialomucin complex

Cleavage of Muc4/SMC precursor into two subunits is an essential processing step for maturation and occurs within a GD-PH sequence. Recent evidence indicates that cleavage of the precursor of gel-forming mucin MUC2 within the same tetrapeptide sequence occurs by a non-enzymatic, autocatalytic cleavage at low pH, and in cells in the late secretory pathway. Here we provide evidence that the cleavage step of Muc4/SMC processing occurs by a proteolytic mechanism. First, processing of Muc4/SMC precursor to ASGP-2 was inhibited in the presence of the mechanism-based serine protease inhibitor, Pefabloc SC, under conditions that did not block synthesis of other proteins. This inhibition led to an increased level of the precursor. Second, neutralization of the acidic environment of the late secretory pathway with NH4Cl did not inhibit cleavage of Muc4/SMC precursor. These results indicate that the two mucins can be processed by cleavage at the same peptide site by different mechanisms.     

Non-apoptotic desquamation of cells from corneal epithelium: putative role for Muc4/sialomucin complex in cell release and survival

Muc4/sialomucin complex (SMC), a large heterodimeric mucin composed of an extracellular mucin subunit ASGP-1 and a transmembrane subunit ASGP-2, is present at the rat ocular surface localized mainly to the most superficial layers of the epithelia. To investigate corneal homeostasis and the functions of Muc4/SMC at the ocular surface, we developed a corneal epithelial cell culture system from corneal explants, from which migrating cells formed an epithelial sheet resembling the native epithelium with regard to microanatomy, expression of characteristic markers, cell migration, and Muc4/SMC expression. Cells migrating from the explants expressed smooth muscle actin. Proliferation was detected only on the edge of epithelial sheet in the immature epithelium and throughout the sheet in confluent cultures. Microscopy revealed that the epithelial sheet was formed from four to six layers of cells expressing keratin 3 and Muc4/SMC in forms identical to those expressed at ocular surface in vivo. Electron microscopy showed cells in various morphological states in the process of releasing from the surface of the multilayer (desquamating). Surprisingly, few of these cells showed evidence of apoptosis, either by morphological or DNA fragmentation analyses. These results suggest a new model for desquamation from stratified epithelia, in which desquamation and apoptosis are independent and sequential processes. Desquamating cells also exhibit a high level of Muc4/SMC. Since Muc4/SMC has been shown to be a potent anti-adhesive and a repressor of apoptosis, we propose that it plays a role in the non-apoptotic desquamation process.     

Sialomucin complex (Muc4) expression in the rat female reproductive tract

Previous studies in our laboratory demonstrated the presence of sialomucin complex (SMC)/Muc4 covering the rat uterine luminal epithelium. SMC/Muc4 expression in the uterus is regulated by estrogen and progesterone and lost at the time of receptivity. In contrast to this hormonal regulation at the uterine luminal surface, SMC/Muc4 in the uterine glandular epithelium, oviduct, cervix, and vagina was constitutively expressed at all stages of the estrous cycle. Furthermore, SMC was expressed in the cervix and vagina of the ovariectomized rat, even though it is not found in the uterine luminal epithelium. Both soluble and membrane-bound forms of SMC were present in these tissues. Immunohistochemical analyses showed distinctive localization patterns of SMC in the various tissues during the estrous cycle. Moreover, the previously unreported expression of SMC/Muc4 in the isthmus, ampulla, and infundibulum of the oviduct suggests potential functions in gamete development. These results indicate that SMC/Muc4 is expressed in most tissues of the female reproductive tract, in which it may have multiple functions. However, hormonal regulation appears to be restricted to the uterine luminal epithelium.     

Muc4/sialomucin complex,the intramembrane ErbB2 ligand,translocates ErbB2 to the apical surface in polarized epithelial cells

Muc4/Sialomucin complex (SMC) acts as an intramembrane ligand for the receptor tyrosine kinase ErbB2, inducing a limited phosphorylation of the receptor. Because Muc4/SMC is found at the apical surfaces of polarized epithelial cells and ErbB2 is often basolateral, the question arises as to whether these components become associated in polarized cells. To address this question, we examined the localization of these proteins in polarized human colon carcinoma CACO-2 cells. Dual color immunofluorescence analysis by confocal microscopy demonstrated the basolateral localization of the ErbB2 in these cells; it is primarily co-localized with E-cadherin at adherens junctions. Expression of apical Muc4/SMC in these cells by transient transfection results in the localization of the ErbB2 at the apical surface. Two-color confocal microscopy indicated that ErbB2 is colocalized with Muc4/SMC in the transfected cells but not in untransfected cells in the same culture. The change of localization of ErbB2 was confirmed by cell surface biotinylation of apical and basolateral proteins, followed by streptavidin precipitation and the subsequent detection of ErbB2 by immunoblotting. In contrast, Na+/K+-ATPase maintains its basolateral localization in Muc4/SMC-transfected cells, indicating that the translocation of ErbB2 is not the result of depolarization of the cells. A potential physiological role for the apical localization of ErbB2 is indicated by the fact that ErbB2 phosphorylated at tyrosine 1248 is found predominantly in Muc4/SMC-transfected cells, but not in untransfected cells, and is co-localized with the apical Muc4/SMC. The ability of Muc4/SMC to alter the localization of ErbB2, particularly a phosphorylated form of it, in epithelial cells, suggests that it has an important role in regulating ErbB2 signaling.     

Enzymes of orithine metabolism in adult developing rat intestine

The levels of 11 enzymes, most of them involved in the metabolism of orithine, were measured in whole upper intestine, or in duodenum, small intestine and colon of adult rats. The developmental formations in small intestine of arginase, orithine aminotransferase, and orithine transcarbamylase were compared with those in liver. Changes with age (late gestation to adult) of the intestinal activities of pyrroline-5-carboxylate reductase, proline oxidase and glutamyl transpeptidase are also described.The results suggests that the proximal part of the intestine is well endowed with enzymes involved in the conversion of ornithine to proline as well as to citrulline. Fetal intestine is rich in proline oxidase and pyrroline-5-carboxylate reductase. The peak levels of ornithine aminotraferase found in intestine in the first 3 postnatal weeks were higher than seen in any other rat tissue.Some of the properties of arginase, ornithine aminotransferase and pyrroline-5-carboxylate reductase in small intestine were compared with those in liver. Isozymes of arginase in small intestine differed from those in liver; the kinetic properties of ornithine aminotransferase were similar in the two tissues. In intestine of 14-day-old rats, the orithine aminotransferase reaction was reversible, forming ornithine from pyrroline-5-carboxylate. The intestinal pyrroline-5-carboxylate reductase was cold-labile as was the hepatic enzyme in rat.     

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.     

Enzymes of ornithine metabolism in adult and developing rat intestine.

The levels of 11 enzymes, most of them involved in the metabolism of ornithine, were measured in whole upper intestine, or in duodenum, small intestine and colon of adult rats. The developmental formations in small intestine of arginase, ornithine aminotransferase, and ornithine transcarbamylase were compared with those in liver. Changes with age (late gestation of adult) of the intestinal activities of pyrroline-5-carboxylate reductase, proline oxidase and glutamyl transpeptidase are also described. The results suggest that the proximal part of the intestine is well endowed with enzymes involved in the conversion of ornithine to proline as well as to citrulline. Fetal intestine is rich in proline oxidase and pyrroline-5-carboxylate reductase. The peak levels of ornithine aminotransferase found in intestine in the first 3 postnatal weeks were higher than seen in any other rat tissue. Some of the properties of arginase, ornithine aminotransferase and pyrroline-5-carboxylate reductase in small intestine were compared with those in liver. Isozymes of arginase in small intestine differed from those in liver; the kinetic properties of ornithine aminotransferase were similar in the two tissues. In intestine of 14-day-old rats, the ornithine aminotransferase reaction was reversible, forming ornithine from pyrroline-5-carboxylate. The intestinal pyrroline-5-carboxylate reductase was cold-labile as was the hepatic enzyme in rat.     

Expression and localization of Tmie in adult rat cochlea

Loss-of function mutations in transmembrane inner ear expressed (Tmie/TMIE) gene have been shown to cause deafness in mice and humans (DFNB6). However, the functional roles of TMIE in the cochlea remain unclear. A primary step toward the understanding of the role of TMIE in hearing and its dysfunction is the documentation of its cellular and sub-cellular location within the cochlea, the auditory organ. In this study, we located and determined the cellular expression of Tmie within the rat cochlea using a polyclonal anti-Tmie antibody. The anti-Tmie antibody identified a specific band of 17 kDa in a variety of rat tissues by using Western blot analyses. The expression products of Tmie were also detected in the spiral limbus, spiral ligament, organ of Corti, and stria vascularis by immunohistochemistry analysis and RT-PCR. Our results point out the presence and localization of Tmie products in the cochlea of rat. Knowledge of spatial distribution of Tmie will provide important insight into the mechanisms that lead to deafness due to mutations in the TMIE gene.     

Tenascin in the developing and adult human intestine

The tenascins are a family of multifunctional extracellular matrix glycoproteins subject to complex spatial and temporal patterns of expression in the course of various organogenetic processes, namely those involving epithelial-mesenchymal interactions. In the intestine, the tenascins, in particular tenascin-C, have been found to be differentially expressed in the developing and adult small intestinal and colonic mucosa as well as in neoplasm. While tenascin-C emerges as a key player likely to be involved in intestinal mucosa development, maintenance and disease, its exact role in the regulation of fundamental intestinal cell function(s) such as proliferation, migration and tissue-specific gene expression remains however to be established.     

Creatine transporter localization in developing and adult retina: importance of creatine to retinal function

Creatine and phosphocreatine are required to maintain ATP needed for normal retinal function and development. The aim of the present study was to determine the distribution of the creatine transporter (CRT) to gain insight to how creatine is transported into the retina. An affinity-purified antibody raised against the CRT was applied to adult vertebrate retinas and to mouse retina during development. Confocal microscopy was used to identify the localization pattern as well as co-localization patterns with a range of retinal neurochemical markers. Strong labeling of the CRT was seen in the photoreceptor inner segments in all species studied and labeling of a variety of inner neuronal cells (amacrine, bipolar, and ganglion cells), the retinal nerve fibers and sites of creatine transport into the retina (retinal pigment epithelium, inner retinal blood vessels, and perivascular astrocytes). The CRT was not expressed in Müller cells of any of the species studied. The lack of labeling of Müller cells suggests that neurons are independent of this glial cell in accumulating creatine. During mouse retinal development, expression of the CRT progressively increased throughout the retina until approximately postnatal day 10, with a subsequent decrease. Comparison of the distribution patterns of the CRT in vascular and avascular vertebrate retinas and studies of the mouse retina during development indicate that creatine and phosphocreatine are important for ATP homeostasis. photoreceptor; development; glutamine synthetase; neurochemistry     

Expression and localization of cathepsin k in adult rat sertoli cells

The cathepsins are a family of cysteine proteases that have been broadly implicated in proteolytic processes during cell growth, cell development, and normal adult cellular function. Cathepsin L is a major secretory product of rat and mouse Sertoli cells, the absence of which in furless mice is associated with atrophy of some seminiferous tubules. However, furless mice produce viable sperm, suggesting the possibility that other members of the cathepsin family of proteases may complement cathepsin L action in the testis. Our objective herein was to begin to test this hypothesis. To this end, we first utilized cDNA microarray technology to identify the members of the cathepsin gene family expressed by freshly isolated adult rat Sertoli cells. This approach, complemented by Northern blot analyses, showed that in addition to cathepsin L, cathepsin K is highly and specifically expressed in Sertoli cells. As is also true of cathepsin L, cathepsin K mRNA was found to be expressed by Sertoli cells at specific stages of the cycle of the seminiferous epithelium, with maximal expression at stages VI-VII. The use of immunocytochemical methods revealed that cathepsin K protein localizes to the cytoplasm of Sertoli cells at stages VI-VIII, to small punctuate lysosomes at stages I-VIII and XIII-XIV, and to early and late residual bodies at stages IX-XII. This localization was found to be similar to that of cathepsin L. The similarity in the expression and localization of cathepsin K and cathepsin L suggest that the two proteases may have similar functions. If true, this might explain the fertility of furless mice. Further, the results suggest that cathepsin K, in both its secreted and lysosomal forms, may play a role in the degradation of Sertoli cell residual bodies.     

Expression and localization of the multidrug resistance-associated protein 3 in rat small and large intestine

Multidrug resistance-associated protein 3 (MRP3; symbol ABCC3), has been shown to mediate ATP-dependent transport of organic anions including 17beta-glucuronosyl estradiol, glucuronosyl bilirubin, monovalent, and sulfated bile salts. MRP3 mRNA expression was reported in rat intestine suggesting a role of MRP3 in the intestinal transport. We examined the expression and localization of MRP3 in rat small and large intestine by RT-PCR, immunofluorescence, and immunoblot analysis. MRP3 was identified in all intestinal segments by RT-PCR. MRP3 expression was low in duodenum and jejunum but markedly increased in ileum and colon. With the use of a rat MRP3 specific antibody, MRP3 was localized to the basolateral domains of enterocytes. Immunofluorescence analysis and immunoblot analysis confirmed a strong expression of rat MRP3 in ileum and colon. In contrast, MRP2 was predominantly expressed in the proximal segments of rat small intestine. Our findings demonstrate a high expression of rat MRP3 in ileum and colon and provide evidence for an involvement of MRP3 in the ATP-dependent transport of organic anions, including bile salts from the enterocyte into blood.