Cytoskeleton-membrane interactions.

Associations between the cytoskeleton and cellular membranes, both within the cell and at points of cell contact, play a central role in determining cell shape and tissue integrity. During the past few years, it has become clear that many of these cytoskeleton-membrane interactions go far beyond simple mechanical linkages. For example, proteins that act as linker molecules at the adherens junctions and desmosomes in the plasma membrane have newly recognized functions in signal transduction pathways. These functions have profound effects on cell behaviour during development. In addition, within the nucleus, the lamin branch of the intermediate filament protein family appears to have a key role in defining the protein composition of the inner nuclear membrane by means of extensive interactions with integral membrane proteins. The identities of these integral membrane proteins are only now coming to light.

Expression of Wnt-1 in PC12 cells results in modulation of plakoglobin and E-cadherin and increased cellular adhesion.

The Wnt-1 gene plays an essential role in fetal brain development and encodes a secreted protein whose signaling mechanism is presently unknown. In this report we have investigated intracellular mechanisms by which the Wnt-1 gene induces morphological changes in PC12 pheochromocytoma cells. PC12 cells expressing Wnt-1 show increased steady-state levels of the adhesive junction protein plakoglobin, and an altered distribution of this protein within the cell. This effect appears similar to a modulation of the plakoglobin homolog, Armadillo, that occurs in Drosophila embryos in response to the Wnt-1 homolog, wingless (Riggleman, B., P. Schedl, and E. Wieschaus. 1990. Cell. 63:549-560). In addition, PC12/Wnt-1 cells show elevated expression of E-cadherin and increased calcium-dependent cell-cell adhesion. These results imply evolutionary conservation of cellular responses to Wnt-1/wingless and indicate that in certain cell types Wnt-1 may act to modulate cell adhesion mechanisms.

The complement of desmosomal plaque proteins in different cell types.

Desmosomal plaque proteins have been identified in immunoblotting and immunolocalization experiments on a wide range of cell types from several species, using a panel of monoclonal murine antibodies to desmoplakins I and II and a guinea pig antiserum to desmosomal band 5 protein. Specifically, we have taken advantage of the fact that certain antibodies react with both desmoplakins I and II, whereas others react only with desmoplakin I, indicating that desmoplakin I contains unique regions not present on the closely related desmoplakin II. While some of these antibodies recognize epitopes conserved between chick and man, others display a narrow species specificity. The results show that proteins whose size, charge, and biochemical behavior are very similar to those of desmoplakin I and band 5 protein of cow snout epidermis are present in all desmosomes examined. These include examples of simple and pseudostratified epithelia and myocardial tissue, in addition to those of stratified epithelia. In contrast, in immunoblotting experiments, we have detected desmoplakin II only among cells of stratified and pseudostratified epithelial tissues. This suggests that the desmosomal plaque structure varies in its complement of polypeptides in a cell-type specific manner. We conclude that the obligatory desmosomal plaque proteins, desmoplakin I and band 5 protein, are expressed in a coordinate fashion but independently from other differentiation programs of expression such as those specific for either epithelial or cardiac cells.

Delta N89 beta-catenin induces precocious development, differentiation, and neoplasia in mammary gland.

To investigate the role of beta-catenin in mammary gland development and neoplasia, we expressed a stabilized, transcriptionally active form of beta-catenin lacking the NH(2)-terminal 89 amino acids (Delta N 89 beta-catenin) under the control of the mouse mammary tumor virus long terminal repeat. Our results show that Delta N 89 beta-catenin induces precocious lobuloalveolar development and differentiation in the mammary glands of both male and female mice. Virgin Delta N 89 beta-catenin mammary glands resemble those found in wild-type (wt) pregnant mice and inappropriately express cyclin D1 mRNA. In contrast to wt mammary glands, which resume a virgin appearance after cessation of lactation, transgenic mammary glands involute to a midpregnant status. All transgenic females develop multiple aggressive adenocarcinomas early in life. Surprisingly, the Delta N89 beta-catenin phenotype differs from those elicited by overexpression of Wnt genes in this gland. In particular, Delta N 89 beta-catenin has no effect on ductal side branching. This suggests that Wnt induction of ductal branching involves additional downstream effectors or modulators.

Plakoglobin suppresses epithelial proliferation and hair growth in vivo.

Plakoglobin regulates cell adhesion by providing a modulatable connection between both classical and desmosomal cadherins and their respective cytoskeletal linker proteins. Both plakoglobin and the related protein beta-catenin are posttranscriptionally upregulated in response to Wnt-1 in cultured cells. Upregulation of beta-catenin has been implicated in potentiating hyperproliferation and tumor formation. To investigate the role of plakoglobin in these functions we expressed a full-length (PG) and an NH(2)-terminally truncated form of plakoglobin (DeltaN80PG) in mouse epidermis and hair follicles, tissues which undergo continuous and easily observed postnatal renewal and remodeling. Expression of these constructs results in stunted hair growth, a phenotype that has also been observed in transgenic mice expressing Wnt3 and Dvl2 (Millar et al. 1999). Hair follicles from PG and DeltaN80PG mice show premature termination of the growth phase (anagen) of the hair cycle, an event that is regulated in part by FGF5 (Hebert et al. 1994). The proliferative rate of the epidermal cells was reduced and apoptotic changes, which are associated with entry into the regressive phase of the hair follicle cycle (catagen), occurred earlier than usual.

Protein zero, a nervous system adhesion molecule, triggers epithelial reversion in host carcinoma cells.

Protein zero (P(o)) is the immunoglobulin gene superfamily glycoprotein that mediates the self-adhesion of the Schwann cell plasma membrane that yields compact myelin. HeLa is a poorly differentiated carcinoma cell line that has lost characteristic morphological features of the cervical epithelium from which it originated. Normally, HeLa cells are not self-adherent. However, when P(o) is artificially expressed in this line, cells rapidly aggregate, and P(o) concentrates specifically at cell-cell contact sites. Rows of desmosomes are generated at these interfaces, the plasma membrane localization of cingulin and ZO-1, proteins that have been shown to be associated with tight junctions, is substantially increased, and cytokeratins coalesce into a cohesive intracellular network. Immunofluorescence patterns for the adherens junction proteins N-cadherin, alpha-catenin, and vinculin, and the desmosomal polypeptides desmoplakin, desmocollin, and desmoglein, are also markedly enhanced at the cell surface. Our data demonstrate that obligatory cell-cell adhesion, which in this case is initially brought about by the homophilic association of P(o) molecules across the intercellular cleft, triggers pronounced augmentation of the normally sluggish or sub-basal cell adhesion program in HeLa cells, culminating in suppression of the transformed state and reversion of the monolayer to an epithelioid phenotype. Furthermore, this response is apparently accompanied by an increase in mRNA and protein levels for desmoplakin and N-cadherin which are normally associated with epithelial junctions. Our conclusions are supported by analyses of ten proteins we examined immunochemically (P(o), cingulin, ZO-1, desmoplakin, desmoglein, desmocollin, N-cadherin, alpha-catenin, vinculin, and cytokeratin-18), and by quantitative polymerase chain reactions to measure relative amounts of desmoplakin and N-cadherin mRNAs. P(o) has no known signaling properties; the dramatic phenotypic changes we observed are highly likely to have developed in direct response to P(o)-induced cell adhesion. More generally, the ability of this "foreign" membrane adhesion protein to stimulate desmosome and adherens junction formation by augmenting well-studied cadherin-based adhesion mechanisms raises the possibility that perhaps any bona fide cell adhesion molecule, when functionally expressed, can engage common intracellular pathways and trigger reversion of a carcinoma to an epithelial-like phenotype.

17beta-estradiol induces apoptosis in the developing rodent prostate independently of ERalpha or ERbeta.

Estrogens induce both proliferative and antiproliferative responses in the prostate gland. To date, antiproliferative effects of estrogens are generally considered to be due to systemic antiandrogenic actions. However, estrogen action mediated through estrogen receptor (ER) beta was recently suggested as another mechanism of induction of apoptosis in the prostate. This study aimed to explore the hypothesis that the antiproliferative effects of estrogen are directly mediated through ERbeta using a prostate organ culture system. We previously reported effects of 17beta-estradiol (E2) using rat ventral prostate (VP) tissues, and adapted the system for culturing mouse tissues. In both rat and mouse models, estrogen-induced apoptosis was detected that was spatially and regionally localized to the epithelium of the distal tips. Using organ cultures of alphaER knockout (alphaERKO) and betaERKO prostates, we failed to demonstrate that apoptosis induced by E2 was mediated through either receptor subtype. Activation of ER-selective ligands (ERalpha, propyl pyrazole triol, ERbeta, diaryl-proprionitrile, and 5alpha-androstane-3beta,17beta-diol) in organ culture experiments failed to induce apoptosis, as did the membrane impermeable conjugate E2:BSA, discounting the possibility of nongenomic effects. Consequently, E2 regulation of androgen receptor (AR) expression was examined and, in the presence of nanomolar testosterone levels, E2 caused a specific reduction in AR protein expression in wild-type, alphaERKO, and betaERKO mice, particularly in the distal region where apoptosis was detected. This down-regulation of AR protein provides a possible mechanism for the proapoptotic action of E2 that is independent of ERs or nongenomic effects.

Alterations in beta-catenin phosphorylation and plakoglobin expression in human breast cancer cells.

Because the cell adhesion molecule epithelial cadherin (E-cadherin) is absent in many invasive carcinomas, we transfected the E-cadherin gene into E-cadherin-negative, invasive breast cancer cell lines BT549 and HS578t to investigate the role of E-cadherin in invasive behavior. Although the transfected E-cadherin could mediate calcium-dependent aggregation to E-cadherin-transfected L-cells, morphology and invasiveness of the breast cancer cells were not altered. We investigated the strength of the linkage of the transfected E-cadherin to the actin cytoskeleton by examining the Triton X-100 solubility of the transfected E-cadherin. In BT549 and HS578t cells, a large proportion of the transfected E-cadherin was Triton soluble, whereas in E-cadherin-positive MCF-7 cells, Triton-insoluble E-cadherin was apparent at cell-cell borders. Interaction of E-cadherin with the actin cytoskeleton is thought to be mediated by the E-cadherin-binding proteins alpha-catenin, beta-catenin, and plakoglobin. We found normal levels of alpha-catenin and beta-catenin in BT549 and HS578t cells; however, low levels of plakoglobin were expressed in these cells compared to those found in weakly invasive MCF-7 cells. Furthermore, levels of tyrosine phosphorylation of beta-catenin were elevated in E-cadherin-transfected BT549 and HS578t cells compared to MCF-7 cells. We conclude that other factors such as the expression and appropriate posttranslational modification of cadherin-associated proteins must be in place for E-cadherin to be fully functional, i.e., to alter invasiveness. During cancer progression, loss of E-cadherin expression itself or multiple other mechanisms that lead to loss of cell-cell adhesion (mutation, loss of catenin expression, alterations in phosphorylation) may contribute to a more metastatic phenotype.

Plakoglobin is a component of the filamentous subplasmalemmal coat of lens cells.

The plasma membranes of the cells of the superficial layer of the eye lens and the lens fibres are in close intercellular contact, leaving an intermembrane space of approximately 20 nm or less throughout their entire length. This plasma membrane is underlaid by a filamentous, cytoplasmic web containing actin, proteins of the spectrin and band 4.1 families, alpha-actinin and vinculin. Using immunofluorescence microscopy and immunoblotting of gel electrophoretically separated proteins, we show that plakoglobin, the plaque protein common to desmosomal and nondesmosomal adhering junctions, is present in lens cells and is also a component of the subplasmalemmal coat of these cells. Plakoglobin also exists in the extended regions of intercellular contacts between cultured lenticular cells where it often colocalizes with vinculin but does not occur in other vinculin-rich plasma membrane regions such as the focal adhesions at the ventral cell surface. Plakoglobin associated with plasma membrane regions can also be identified in various other adhesive cultured cells, but it is not detected in cells and tissues that do not establish firm intercellular junctions such as erythrocytes, platelets, cultured myeloma cells and smooth muscle tissue. We conclude that plakoglobin occurs, at least in lens cells, throughout the entire subplasmalemmal coat, coexisting in this situation not only with vinculin but also with spectrin and 4.1 protein(s). This colocalization infers the presence of a distinct, complex type of membrane-skeleton assembly involving the actin filament-associated junctional plaque elements plakoglobin and vinculin together with actin-associated proteins of the spectrin and band 4.1 protein families.

A constitutive transmembrane glycoprotein of Mr 165,000 (desmoglein) in epidermal and non-epidermal desmosomes. II. Immunolocalization and microinjection studies.

Using two monoclonal antibodies described in the preceding paper we determined by immunofluorescence microscopy the distribution of an integral membrane protein of the desmosomal domain, the major glycopolypeptide of Mr 165,000 (bovine muzzle epidermal desmosome band 3; desmoglein) in various normal tissues, tumors and cultured cell lines from several mammalian species. This protein was detected in dotted or streak-like arrays along cell boundary structures which were known to contain non-membrane-integrated desmosomal plaque proteins such as desmoplakins. This is true for epithelial, i.e. cytokeratin-expressing cell types, for the desmin-producing myocardiac and Purkinje fiber cells of the heart, and for certain vimentin-containing cells such as arachnoidal and meningiomal cells and dendritic follicular cells of lymph nodes. However, on the basis of both immunoblot and immunocytochemical reactions, the protein is absent from non-desmosomal adhering junctions, including those devoid of desmoplakin but containing another plaque protein, plakoglobin ("band 5 protein"). We have used these antibodies to localize their epitopes with respect to the cell membrane. By immunoelectron microscopy we found that both epitopes are located in the desmosomal plaques, and this was confirmed by microinjection of purified antibodies into living cultured cells which resulted in labelling of the plaques. From these findings, taken together with previous analyses and localizations of the carbohydrate moieties of this glycoprotein, we conclude that desmoglein is a transmembrane glycoprotein which projects into--and contributes to--the desmosomal plaque structure. This glycoprotein represents a general component of true desmosomes and it is coexpressed with obligatory desmosome-specific plaque proteins such as desmoplakin I. The potential value of this glycoprotein as a desmosomal and cell type marker in histology and tumor diagnosis is discussed.

A constitutive transmembrane glycoprotein of Mr 165,000 (desmoglein) in epidermal and non-epidermal desmosomes. I. Biochemical identification of the polypeptide.

Two murine monoclonal antibodies (DG 3.4 and DG 3.10) raised against a major glycoprotein ("band 3 component") from desmosomes of bovine muzzle epidermis were used in immunoblot experiments following SDS-polyacrylamide gel electrophoresis or two-dimensional gel electrophoresis to identify this or immunologically related proteins in other bovine tissues and cultured cell lines. In all desmosome-bearing cells, i.e. cells also expressing desmoplakins, including representative of stratified, transitional and simple epithelia as well as myocardium, only a single distinct polypeptide of identical Mr value (165,000) and electrical charge was detected. These findings, together with the immunolocalization results reported in the companion paper indicate that this glycoprotein (desmoglein) is a general constituent protein of desmosomes, providing a case of an integral membrane protein co-expressed with non-membranous desmosomal proteins such as the plaque component, desmoplakin I. Our results further suggest that, contrary to previous suggestions, desmoglein is very similar, if not identical in different cells of the same species and does not display significant cell type diversity.

Biochemical characterization of desmosomal proteins isolated from bovine muzzle epidermis: amino acid and carbohydrate composition.

The seven major desmosomal polypeptides from isolated bovine muzzle desmosomes ranging from Mr 75 000 to 250 000 were separated by gel electrophoresis, isolated and characterized with respect to their amino acid composition and sugar content. The two largest polypeptides (bands 1 and 2), i.e. desmoplakins I and II, are similar in their amino acid composition, confirming our previous immunological and biochemical data, and display a relatively high glycine content. In contrast, the other two cytoplasmic components also believed to be associated with the desmosomal plaque, i.e. polypeptides of bands 5 (Mr 83 000) and 6 (Mr 75 000), differ significantly in their amino acid composition from the desmoplakins and from each other. All four candidate polypeptides for plaque association, i.e. bands 1, 2, 5, and 6, show no significant glycosylation. The glycoproteins 4a and 4b (Mr 115 000 and 130 000) are similar in their amino acid composition, peptide analysis and immunological reactivity. Both are relatively rich in mannose and galactose but also contain sialic acid. Our determinations also indicate that the two polypeptides differ significantly in their N-acetylglucosamine and mannose content. Most, if not all, of the sugar residues are associated with a water-soluble fragment of Mr 15 500 obtained after limited digestion with V8 protease. The glycopolypeptides obtained in band 3 (Mr 164 000-175 000) are distinct from the glycopolypeptides 4a and 4b in amino acid composition, sugar content, isoelectric pH values, certain antigenic determinants and in their pattern of cleavage products obtained by treatment with proteases or cyanogen bromide. The results identify polypeptides of bands 3, 4a and 4b as glycosylated with characteristic sugar compositions. It is suggested that the major glycoproteins (bands 3, 4a, 4b) of the desmosome are integral membrane components arranged in a special way conferring resistance to detergent treatment. The possible roles of these glycoproteins in cell recognition and in adhesive functions of the desmosome are discussed.

Appearance of Langerhans cells in the epidermis of Tgfb1(-/-) SCID mice: paracrine and autocrine effects of transforming growth factor-beta 1 and -beta 2(1).

A striking immunologic abnormality of normal and SCID Tgfb1(-/-) mice is the total absence of Langerhans cells in their epidermis. Here we show that transfer of Tgfb1(+/-) SCID bone marrow causes, within a few weeks, the appearance of Langerhans cells in the epidermis of gamma-irradiated and unirradiated Tgfb1(-/-) SCID recipients. In addition, local injection of 2 x 10(5) latent transforming growth factor-beta1 cDNA-transduced cloned CD4+ T lymphocytes causes the appearance of Langerhans cells in the ear epidermis of Tgfb1(-/-) SCID mice. This effect is enhanced by antigen-specific activation of these T cells. Injection of recombinant active transforming growth factor-beta 2 into the ear of Tgfb1(-/-) SCID mice also results in the migration of Langerhans cells into the epidermis locally, but no epidermal Langerhans cells are seen after systemic injections of transforming growth factor-beta 2. Our results suggest that transforming growth factor-beta can act in paracrine as well as autocrine fashion to induce the differentiation of precursors into Langerhans cells. Furthermore, these results indicate that the relative roles of different transforming growth factor-beta isoforms in vivo may be influenced by their local availability and/or the regulation of their conversion from latent into active form.

Measurement of antibodies to tubulin by radioimmunoassay.

A solid-phase double antibody radioimmunoassay capable of measuring antibody to tubulin, the principal component of microtubules, is described. This assay is simple, combining sensitivity with specificity and also allowing determination of antibody subclasses.

Dilutions of corticosteroid creams and ointments - a stability study.

A study was carried out on the stability of dilutions of creams and ointments of two corticosteroids, betamethasone valerate and beclomethasone dipropionate. A reversed-phase high-performance liquid chromatographic method was developed that is simple, efficient and stability-indicating in respect of the main decomposition products and has the advantage of being carried out at ambient temperature. The effect of water content of the sample solutions and the influence of large injection volumes (200-250 mul) on the resolution of the substances on the chromatogram was investigated. Use of the diluents, cetomacrogol cream (formula A) BP and white soft paraffin BP, resulted in satisfactory products in terms of chemical stability and efficacy of antimicrobial preservation.

Structure of DSG1, the bovine desmosomal cadherin gene encoding the pemphigus foliaceus antigen. Evidence of polymorphism.

The cadherin superfamily of calcium-dependent cell-cell adhesion and recognition proteins can be categorized into a number of subsets on the basis of the distinct cytoplasmic sequences of their members. Currently these families include classical cadherins, desmogleins, desmocollins, protocadherins, and the products of the Drosophila genes FAT and Dachsous. Dsg1, the prototype of the desmoglein family, is a major component of epidermal desmosomes and the antigenic target of antibodies found in the sera of patients with the blistering disease, pemphigus foliaceus. In this study, we determined the organization of the bovine DSG1 gene. This gene consists of 15 exons distributed over > 37.5 kilobases of genomic DNA. A comparison of DSG1 with genes encoding classical cadherins revealed a striking conservation of exon boundaries in regions encoding the ectodomain and to a more limited extent among those encoding the cytoplasmic domain. Polymorphism was found in a sequence of DSG1 encoding protein proximal to the external face of the plasma membrane. This region is topologically equivalent to a domain of classical cadherins that harbors epitopes recognized by adhesion-disrupting antibodies. We discuss these results with regard to the evolution of the cadherin superfamily and their implications for the definition of pemphigus epitopes.

Interactions of the cytoplasmic domain of the desmosomal cadherin Dsg1 with plakoglobin.

Dsg1 is a 165-kDa glycoprotein component of suprabasal epidermal desmosomes and the prototype of a subset of the cadherin superfamily of cell-cell adhesion proteins known as desmogleins. The adhesive function of classical cadherins is known to be dependent upon their association with cytoplasmic components called catenins. In the case of desmogleins, a single interaction has been described with a protein called plakoglobin that is found in desmosomal plaques, adherens junctions, and the cytosol. Several proteins with homology to plakoglobin have been described that regulate junction assembly and implement morphoregulatory signals. To address the functional significance of plakoglobin-desmoglein interaction, we have mapped the sequences of Dsg1 that are crucial for this association by using blot overlay techniques. By examining the binding of plakoglobin to a deletion series of the Dsg1 cytoplasmic domain expressed as fusion proteins, we have defined a 19-amino acid sequence that is important for association. This region of Dsg1 sequence shows significant similarity to the catenin-binding domain of classical cadherins, suggesting a common mechanism for the association of plakoglobin with desmosomes and adherens junctions.

Antibodies to epithelial desmosomes show wide tissue and species cross-reactivity.

Many workers regard cell adhesion as a highly specific phenomenon, believing that different molecular mechanisms are involved in the adhesion of cells of different tissues and different species. We believe that the evidence from cell behaviour is against this view and that cells share common adhesion mechanisms (for reviews see refs 1, 2); however, molecular evidence is lacking. As an approach to providing such evidence we have begun to study desmosomes, the cell-surface organelles responsible for strong intercellular adhesion in epithelia. We have raised antisera against each of five high-molecular weight (MW) desmosomal components. Having determined the specificity of our antisera by immunoblotting, we show here that each gives a staining pattern corresponding to the distribution of desmosomes in a range of tissues from different vertebrate species, demonstrating that desmosomal components are widely shared and highly conserved.