Division of labor among the alpha6beta4 integrin, beta1 integrins, and an E3 laminin receptor to signal morphogenesis and beta-casein expression in mammary epithelial cells.

Contact of cultured mammary epithelial cells with the basement membrane protein laminin induces multiple responses, including cell shape changes, growth arrest, and, in the presence of prolactin, transcription of the milk protein beta-casein. We sought to identify the specific laminin receptor(s) mediating the multiple cell responses to laminin. Using assays with clonal mammary epithelial cells, we reveal distinct functions for the alpha6beta4 integrin, beta1 integrins, and an E3 laminin receptor. Signals from laminin for beta-casein expression were inhibited in the presence of function-blocking antibodies against both the alpha6 and beta1 integrin subunits and by the laminin E3 fragment. The alpha6-blocking antibody perturbed signals mediated by the alpha6beta4 integrin, and the beta1-blocking antibody perturbed signals mediated by another integrin, the alpha subunit(s) of which remains to be determined. Neither alpha6- nor beta1-blocking antibodies perturbed the cell shape changes resulting from cell exposure to laminin. However, the E3 laminin fragment and heparin both inhibited cell shape changes induced by laminin, thereby implicating an E3 laminin receptor in this function. These results elucidate the multiplicity of cell-extracellular matrix interactions required to integrate cell structure and signaling and ultimately permit normal cell function.

[LagoZ and LagZ, 2 genes depleted of CpG dinucleotides, derived from the LacZ gene for the study of epigenetic control]. / LagoZ et LagZ, deux gènes appauvris en dinucléotides CpG dérivés du gène LacZ pour l'étude des contrôles épigénétiques.

The methylation of 5'CpG 3' dinucleotides within genes creates potential targets for protein complexes that bind to methylated DNA sequences and to histone deacetylases (MBD-HDAC). This can lead to transcriptional repression by modification of chromatic. To test the importance of this repression in vivo and to determine when during development these epigenetic controls are placed on genes, two novel genes have been engineered by directed mutagenesis of the CpG-rich LacZ gene that are depleted of (LagZ) or completely lacking (LagoZ) CpG sequences. We report that the expression (transcriptional and translational) of the three genes is indistinguishable in transient assays in cleaving mouse embryos. Therefore, the complete absence of CpG sequences within three kilobases of coding sequence is compatible with its maintenance in the nucleus and with its expression. These molecules can now be used to study the ontogenesis of the CpG-dependent repressive system in intact organisms.

Immunopurification of a sarcomeric junctional protein complex containing GAPDH.

We have isolated a monoclonal antibody, P4B2, which localizes to multiple anchorage junctions, namely, a subset of focal adhesions, the Z-disk of muscle, and neuromuscular junctions. Immunopurification of the antigen to this antibody from chicken brain tissue yielded a complex of three prominent proteins with mobilities of 36, 30, and 18 kDa. Amino acid sequencing of the purified proteins identified the 36-kDa protein as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The other two protein bands were heterogeneous, containing proteins found in the synaptic vesicle fusion core complex. Immunolocalization of P4B2 antigen in developing cultured muscle cells showed that the antigen is incorporated into Z-lines soon after the sarcomeric architecture was positive for alpha-actinin. Together, the data indicate the P4B2 antigen is part of a unique GAPDH-containing protein complex that may be involved in reinforcement of established cytoskeletal structures.

Matrix metalloproteinase stromelysin-1 triggers a cascade of molecular alterations that leads to stable epithelial-to-mesenchymal conversion and a premalignant phenotype in mammary epithelial cells.

Matrix metalloproteinases (MMPs) regulate ductal morphogenesis, apoptosis, and neoplastic progression in mammary epithelial cells. To elucidate the direct effects of MMPs on mammary epithelium, we generated functionally normal cells expressing an inducible autoactivating stromelysin-1 (SL-1) transgene. Induction of SL-1 expression resulted in cleavage of E-cadherin, and triggered progressive phenotypic conversion characterized by disappearance of E-cadherin and catenins from cell-cell contacts, downregulation of cytokeratins, upregulation of vimentin, induction of keratinocyte growth factor expression and activation, and upregulation of endogenous MMPs. Cells expressing SL-1 were unable to undergo lactogenic differentiation and became invasive. Once initiated, this phenotypic conversion was essentially stable, and progressed even in the absence of continued SL-1 expression. These observations demonstrate that inappropriate expression of SL-1 initiates a cascade of events that may represent a coordinated program leading to loss of the differentiated epithelial phenotype and gain of some characteristics of tumor cells. Our data provide novel insights into how MMPs function in development and neoplastic conversion.

Integrin alpha subunit ratios, cytoplasmic domains, and growth factor synergy regulate muscle proliferation and differentiation.

The role of integrins in muscle differentiation was addressed by ectopic expression of integrin alpha subunits in primary quail skeletal muscle, a culture system particularly amenable to efficient transfection and expression of exogenous genes. Ectopic expression of either the human alpha5 subunit or the chicken alpha6 subunit produced contrasting phenotypes. The alpha5-transfected myoblasts remain in the proliferative phase and are differentiation inhibited even in confluent cultures. In contrast, myoblasts that overexpress the alpha6 subunit exhibit inhibited proliferation and substantial differentiation. Antisense suppression of endogenous quail alpha6 expression inhibits myoblast differentiation resulting in sustained proliferation. These effects of ectopic alpha subunit expression are mediated, to a large extent, by the cytoplasmic domains. Ectopic expression of chimeric alpha subunits, alpha5ex/6cyto and alpha6ex/5cyto, produced phenotypes opposite to those observed with ectopic alpha5 or alpha6 expression. Myoblasts that express alpha5ex/6cyto show decreased proliferation while differentiation is partially restored. In contrast, the alpha6ex/5cyto transfectants remain in the proliferative phase unless allowed to become confluent for at least 24 h. Furthermore, expression of human alpha5 subunit cytoplasmic domain truncations, before and after the conserved GFFKR motif, shows that this sequence is important in alpha5 regulation of differentiation. Ectopic alpha5 and alpha6 expression also results in contrasting responses to the mitogenic effects of serum growth factors. Myoblasts expressing the human alpha5 subunit differentiate only in the absence of serum while differentiation of untransfected and alpha6-transfected myoblasts is insensitive to serum concentration. Addition of individual, exogenous growth factors to alpha5-transfected myoblasts results in unique responses that differ from their effects on untransfected cells. Both bFGF or TGFbeta inhibit the serum-free differentiation of alpha5-transfected myoblasts, but differ in that bFGF stimulates proliferation whereas TGF-beta inhibits it. Insulin or TGF-alpha promote proliferation and differentiation of alpha5-transfected myoblasts; however, insulin alters myotube morphology. TGF-alpha or PDGF-BB enhance muscle alpha-actinin organization into myofibrils, which is impaired in differentiated alpha5 cultures. With the exception of TGF-alpha, these growth factor effects are not apparent in untransfected myoblasts. Finally, myoblast survival under serum-free conditions is enhanced by ectopic alpha5 expression only in the presence of bFGF and insulin while TGF-alpha and TGF-beta promote survival of untransfected myoblasts. Our observations demonstrate (1) a specificity for integrin alpha subunits in regulating myoblast proliferation and differentiation; (2) that the ratio of integrin expression can affect the decision to proliferate or differentiate; (3) a role for the alpha subunit cytoplasmic domain in mediating proliferative and differentiative signals; and (4) the regulation of proliferation, differentiation, cytoskeletal assembly, and cell survival depend critically on the expression levels of different integrins and the growth factor environment in which the cells reside.

The alpha 5 beta 1 integrin associates with a dystrophin-containing lattice during muscle development.

The organization of the alpha 5 beta 1 integrin on skeletal muscle was studied in culture and in sections from adult and embryonic tissue using monoclonal antibodies specific for the alpha 5 subunit. The alpha 5 beta 1 integrin showed changes in organization and in the molecules with which it colocalizes. On early myoblasts, possessing a fibroblast-like morphology, the alpha 5 integrin organization was indistinguishable from that on fibroblasts; it was expressed prominently and localized in numerous focal contacts around the cell periphery. In bipolar myoblasts and early myotubes, the alpha 5 integrin was expressed only weakly and localized in a small number of focal contact-like structures. As myogenesis proceeded there was an apparent increase in integrin expression and a change in organization. In addition to the focal contact-like structures that persist throughout myogenesis in vitro, a dense lattice-like structure of integrin appeared. Fibrillar fibronectin, talin, and non-muscle alpha-actinin did not colocalize with the alpha 5 beta 1 integrin in the lattice structure as they did in the focal contact-like structures. However, dystrophin, which displayed a diffuse distribution earlier, now colocalized with the alpha 5 beta 1 integrin in the punctate lattice. Coincident with the registration of myofibrils into visible sarcomeres, the prominent dense, lattice structure disappeared leaving the focal contact-like structures as the only regions of organized alpha 5 beta 1 integrin. Despite the presence of the beta 1 integrin in neuromuscular or myotendinous junctions in vivo and on myotubes in vitro, the alpha 5 beta 1 integrin was not present in either junction. These observations suggest that the alpha 5 beta 1 integrin is involved in the adhesion of muscle to the extracellular matrix, the organization of the dystrophin-containing lattice, and the organization of nascent myofibrils which emanate from the focal contact- and stress fiber-like structures in muscle. Other integrins appear to anchor myofibrils at the myotendinous and neuromuscular junctions.

Developmentally regulated expression of alpha 6 integrin in avian embryos.

The distribution pattern of the avian alpha 6 integrin subunit was examined during early stages of development. The results show that this subunit is prevalent in cells of the developing nervous system and muscle. alpha 6 is first observed on neuroepithelial cells of the cranial neural plate and trunk neural tube. With time, immunoreactivity becomes prominent near the lumen and ventrolateral portions of the neural tube, co-distributing with neurons and axons, particularly notable on commissural neurons. The alpha 6 expression pattern is dynamic in the neural tube, with immunoreactivity peaking by embryonic day 6 (stage 30) and decreasing thereafter. The ventral roots and retina exhibit high levels of immunoreactivity throughout development. In the peripheral nervous system, alpha 6 immunoreactivity first appears on a subpopulation of sympathoadrenal cells around the dorsal aorta and later in the dorsal root ganglia shortly after gangliogenesis. Immunoreactivity appears on prospective myotomal cells as the somites delaminate into the dermomyotome and sclerotome, remaining prominent on myoblasts and differentiated muscle at all stages. The mesonephros also has intense immunoreactivity. In the periphery, alpha 6 immunoreactive regions often in proximity to laminin, which is thought to be the ligand of alpha 6 beta 1 integrin.

LBL, a novel, developmentally regulated, laminin-binding lectin.

A 190/220-kDa complex found in integrin preparations was purified, and monoclonal antibodies were raised against it. The immunoaffinity-purified complex appears to be a trimer of very similar or identical 70-kDa subunits. It is a novel extracellular matrix molecule as determined by its subunit composition, N-terminal amino acid sequence, and in vivo localization. It is distributed widely in basement membranes including those from muscle, nerve, and kidney. It is also present in connective tissue regions such as perineurium and perimysium. It has the unusual property that it is initially expressed very late in avian development near the time of hatching. This protein is found to copurified with integrin because it binds to the carbohydrate support in Sepharose. Hemagglutination assays with mono- and disaccharides show that it functions as a lectin with galactoside-binding specificity. This protein is also found to bind strongly and specifically to laminin at a site distinct from its lectin activity, but does not bind to fibronectin or type IV collagen. The protein appears to be conserved and is a common contaminant of many laminin preparations. We call this novel protein "LBL" for laminin-binding lectin.

Down-regulation of the chicken alpha 5 beta 1 integrin fibronectin receptor during development.

We have characterized the diversity of the chicken beta 1 integrin family and studied the expression of individual receptors during development. The diversity of the beta 1 integrin family was investigated by affinity purifying the beta 1 integrins from a variety of adult and embryonic tissues. These purifications reveal the relative levels of expression and also the differential expression of the alpha subunits in those tissues. Monoclonal antibodies were generated against the prominent 'band 1' of the embryonic chicken integrins and used to characterize the expression of this alpha subunit in embryonic and adult tissues. This alpha subunit is shown to be the chicken homologue of human alpha 5 fibronectin receptor. The chicken alpha 5 beta 1 integrin is the most prominent beta 1 integrin in the embryo and is expressed on the majority of cell types through the day 17 stage. The distribution of this receptor in the embryo closely parallels the distribution of its ligand, fibronectin. In adult tissues, expression of this receptor is greatly diminished relative to the expression of other alpha subunits. The cell type distribution is highly restricted: limited primarily to the vasculature and to connective tissue regions. These studies reveal a prominent role for the alpha 5 beta 1 integrin in embryonic cell types and a down-regulation of this receptor on many cell types during development.

Integrin on developing and adult skeletal muscle.

Avian integrin is a complex of integral membrane glycoproteins that appears to function as a dual receptors for both intracellular cytoskeletal and extracellular matrix components. Antibodies were raised against this complex and used to (1) immunolocalize integrin on cryosections of developing and adult muscle tissue and on developing myotube cultures in vitro and (2) immunoaffinity purify integrin from various fiber-type specific muscles. Integrin localization was compared with that of its putative cytoskeletal-associated and extracellular matrix ligands, talin and vinculin and fibronectin and laminin, respectively. The goal was to identify putative sites of interaction between the muscle sarcolemma and the cytoskeleton and the extracellular matrix and to reveal any differences in the molecular composition at these sites. Integrin's distribution on the sarcolemma of early (Day 12) embryonic limb muscle was random and punctate. On late embryonic (Days 17-19) limb muscle tissue its distribution was generally uniform but with occasional increased densities at specific sites along the sarcolemma. Posthatch (greater than 3 weeks) fast twitch muscle showed a highly regionalized distribution. These regions of integrin concentration coincided with densities of acetylcholine receptors, revealed by TRITC alpha-bungarotoxin labeling, and regions of muscle-tendon interaction, identified by morphological criteria. Tissue culture studies also demonstrated integrin densities at analogous sites in vitro, e.g., acetylcholine receptor clusters and sites at which myofibrils terminate at the sarcolemma. These integrin-rich sites were also shown to be Triton X-100 insoluble and therefore presumably are linked to the cytoskeleton or extracellular matrix. The localization of integrin on developing and adult muscle tissue was compared with that of fibronectin, laminin, vinculin, and talin using double, immunofluorescently labeled cryosections. In general, integrin did not colocalize exclusively with any one of its putative ligands. In the embryo, discrete densities of both talin and vinculin were observed at the myotendinous junction, whereas integrin immunoreactivity was widely distributed on muscle, vasculature, nerve, and connective tissue with no discernible sites of increased density. Laminin was primarily associated with muscle and nerve whereas fibronectin was prominent on connective tissue. On posthatch tissue, the distributions of talin, vinculin, laminin, and fibronectin were similar to those in the embryo, whereas the distribution of integrin was restricted to specific sites. The distribution of integrin was also examined for fiber-type specific differences on adu