Glycoprotein contributions to mammary gland and mammary tumor structure and function: roles of adherens junctions, ErbBs and membrane MUCs

Mammary function is dependent on its three-dimensional organization, which is established and maintained by cell adhesive junctions linked through the membrane to the cell cytoskeleton. These junctions serve not only as structural elements, but also function as initiators and integrators of cell signals. In this review we discuss three types of glycoproteins whose interactions impinge on the function of mammary cell-cell junctions, cadherins, ErbB receptor tyrosine kinases and membrane mucins, as a microcosm of events regulating mammary cell behaviors. Actions of these components are integrated by the critical signaling element beta-catenin. When functioning properly, these glycoproteins, beta-catenin and associated signaling pathways mesh into a highly structured program for development and function of the gland. However, disruption or dysfunction of these glycoproteins or the signaling elements can lead to disorganization of the epithelia and ultimately to neoplasia.     

Increased level of linkage disequilibrium in rural compared with urban communities: a factor to consider in association-study design

Few studies have investigated genetic differentiation within nonisolate European populations, despite the initiation of large national sample collections such as U.K. Biobank. Here, we used short tandem repeat markers to explore fine-scale genetic structure and to examine the extent of linkage disequilibrium (LD) within national subpopulations. We studied 955 unrelated individuals of local ancestry from nine Scottish rural regions and the urban center of Edinburgh, as well as 96 unrelated individuals from the general U.K. population. Despite little overall differentiation on the basis of allele frequencies, there were clear differences among subpopulations in the extent of pairwise LD, measured between a subset of X-linked markers, that reflected presumed differences in the depths of the underlying genealogies within these subpopulations. Therefore, there are strategic advantages in studying rural subpopulations, in terms of increased power and reduced cost, that are lost by sampling across regions or within urban populations. Similar rural-urban contrasts are likely to exist in many other populations with stable rural subpopulations, which could influence the design of genetic association studies and national biobank data collections.     

Heterogeneity Analysis in 40 X-linked Retinitis Pigmentosa Families

Analysis of genetic heterogeneity in 40 kindreds with X-linked retinitis pigmentosa (XLRP), with 20 polymorphic markers, showed that significant heterogeneity is present (P=.001) and that 56% of kindreds are of RP3 type and that 26% are of RP2 type. The location of the RP3 locus was found to be 0.4 cM distal to OTC in the Xp21.1 region, and that of the RP2 locus was 6.5 cM proximal to DXS7 in Xp11.2-p11.3. Bayesian probabilities of linkage to RP2, RP3, or to neither locus were calculated. This showed that 20 of 40 kindreds could be assigned to one or the other locus, with a probability >.70 (14 kindreds with RP3 and 6 kindreds with RP2 disease). A further three kindreds were found to be unlinked to either locus, with a probability >.8. The remaining 17 kindreds could not be classified unambiguously. This highlights the difficulty of classifying families in the presence of genetic heterogeneity, where the two loci are separated by an estimated 16 cM.     

STUDIES OF SPERMATOGENESIS IN THE HEPATICAE : II. Blepharoplast Structure in the Spermatid of Marchantia

The blepharoplast in a young, developing spermatid of Marchantia polymorpha, is a composite structure consisting of two basal bodies and a subjacent narrow band of axonemal-type tubules that we have termed the "spline." For most of its length, the spline consists of six long parallel tubules that nearly encircle the cell. The spline anterior is asymmetrically widened for about 2 µ by shorter tubules of the same kind. The lateral displacement of three long, adjacent marginal tubules by three short intervening tubules at the spline tip produces a long narrow aperture. Distally, the aperture is closed by the convergence of the displaced tubules with another trio of long tubules. Together, these form the six-membered cell-encircling portion. The expanded spline anterior has, at this stage of development, the four-layered (Vierergruppe) structure, of which the aforementioned tubules constitute the uppermost layer. The lower three strata consist of diagonal fins, elongated chambers, and fine tubules, respectively. The two flagellar bases lie close above the spline tip—one slightly anterior to the other—and diverge unequally from the spline axis. A few triplets extend proximally from the basal bodies, but do not connect with the spline. The anterior basal body is longer than the posterior one.     

Mechanistic and signaling analysis of Muc4–ErbB2 signaling module: New insights into the mechanism of ligand‐independent ErbB2 activity

The membrane mucin Muc4 is aberrantly expressed in numerous epithelial carcinomas and is currently used as a cancer diagnostic and prognostic tool. Muc4 can also potentiate signal transduction by modulating differential ErbB2 phosphorylation in the absence and in the presence of the ErbB3 soluble ligand heregulin (HRG‐β1). These features of Muc4 suggest that Muc4 is not merely a cancer marker, but an oncogenic factor with a unique‐binding/activation relationship with the receptor ErbB2. In the present study, we examined the signaling mechanisms that are associated with the Muc4–ErbB2 module by analyzing ErbB2 differential signaling in response to Muc4 expression. Our study was carried out in the A375 human melanoma and BT‐474 breast cancer cell lines as our model systems. Quantitative and comparative signaling modulations were evaluated by immunoblot using phospho‐specific antibodies, and densitometry analysis. Signaling complex components were identified by chemical cross‐linking, fractionation by gel filtration, immunoprecipitation, and immunoblotting. Activated downstream signaling pathways were analyzed by an antibody microarray screen and immunoblot analyses. Our results indicate that Muc4 modulates ErbB2 signaling potential significantly by stabilizing and directly interacting with the ErbB2–ErbB3 heterodimer. Further analyses indicate that Muc4 promotes ErbB2 autocatalysis, but it has no effect on ErbB3 phosphorylation, although the chemical cross‐linking data indicated that the signaling module is composed of Muc4, ErbB2, and ErbB3. Our microarray analysis indicates that Muc4 expression promotes cell migration by increasing the phosphorylation of the focal adhesion kinase and also through an increase in the levels of β‐catenin. J. Cell. Physiol. 224: 649–657, 2010. © 2010 Wiley‐Liss, Inc.     

Plasma membrane-microfilament interaction in animal cells

Microfilament interactions with the plasma membranes of animal cells appear to vary with cell type and localization. In the erythrocyte, actin oligomers are associated with the membrane via spectrin and ankyrin. The ends of stress fibers in cultured cells, such as fibroblasts, are attached to the plasma membrane at focal adhesion sites and may involve the protein vinculin as a linking protein. In intestinal brush border microvilli a 110,000 dalton protein links the microfilament bundles to sites on the microvillus. A transmembrane complex containing actin stably associated with a cell surface glycoprotein can be isolated from ascites tumor cell microvilli and can be obtained still associated with microfilaments by gentle extraction and gradient centrifugation of the microvilli. These varied interaction mechanisms are believed to be needed to satisfy the different structural and dynamic requirements of the particular systems.     

Changes in expression of a major sialoglycoprotein associated with ascites forms of a mammary adenocarcinoma

Glycoproteins of a cultured form (MR) of the 13762 rat mammary adenocarcinoma and its variants have been studied by analyses for peanut agglutinin receptors, [³H]glucosamine labeling, lactoperoxidase labeling and CsCl density gradient centrifugation. The 13762 MR cells, derived from 13762 MAT-B ascites cells, do not contain detectable ASGP-1, the predominant cell surface sialoglycoprotein of the ascites forms of the 13762 tumor.Transplantation and continued passage as ascites cells of MR cells or clonal lines derived from MR results in abrupt expression of ASGP-1 at about passage 16; it is absent in early passages of the ascites tumor. When these ascites cells are transferred to culture, ASGP-1 is again lost. No ASGP-1 is found in solid tumors derived from subcutaneous transplantation of the 13762 MR cells. The results suggest modulation of ASGP-1 content of the 13762 tumor cells.     

Deficient E-cadherin adhesion in C57BL/6J-Min/+ mice is associated with increased tyrosine kinase activity and RhoA-dependent actomyosin contractility

The Min/+ mouse is a model for APC-dependent colorectal cancer (CRC). We showed that tumorigenesis in this animal was associated with decreased E-cadherin adhesion and increased epidermal growth factor receptor (Egfr) activity in the non-tumor intestinal mucosa. Here, we tested whether these abnormalities correlated with changes in the actin cytoskeleton due to increased Rho-ROCK signaling. We treated Apc+/+ (WT) littermate small intestine with EGTA, an inhibitor of E-cadherin, and with LPA, an RhoA activator; both induced effects on adhesion and kinase activity that mimicked the Min/+ phenotype. GTP-bound Rho was increased in Min/+ enterocytes relative to WT. Since RhoA activity is associated with actomyosin contractility, markers of this signaling cascade were assessed including phosphorylated myosin light chain (MLC), cofilin, Pyk2, Src, and MAPK kinases. The increased actomyosin contractility characterizing Min/+ intestinal tissue was suppressed by the ROCK inhibitor, Y27632, but was inducible in the WT by EGTA or LPA. Finally, ultrastructural imaging revealed changes consistent with actomyosin contractility in Min/+ enterocytes. Thus, the positive regulation of E-cadherin adhesion provided by Apc+ in vivo allows proper negative regulation of Egfr, Src, Pyk2, and MAPK, as well as RhoA activities.