Quantitative changes in integrin and focal adhesion signaling regulate myoblast cell cycle withdrawal.
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We previously demonstrated contrasting roles for integrin alpha subunits and their cytoplasmic domains in controlling cell cycle withdrawal and the onset of terminal differentiation (Sastry, S., M. Lakonishok, D. Thomas, J. Muschler, and A.F. Horwitz. 1996. J. Cell Biol. 133:169-184). Ectopic expression of the integrin alpha5 or alpha6A subunit in primary quail myoblasts either decreases or enhances the probability of cell cycle withdrawal, respectively. In this study, we addressed the mechanisms by which changes in integrin alpha subunit ratios regulate this decision. Ectopic expression of truncated alpha5 or alpha6A indicate that the alpha5 cytoplasmic domain is permissive for the proliferative pathway whereas the COOH-terminal 11 amino acids of alpha6A cytoplasmic domain inhibit proliferation and promote differentiation. The alpha5 and alpha6A cytoplasmic domains do not appear to initiate these signals directly, but instead regulate beta1 signaling. Ectopically expressed IL2R-alpha5 or IL2R-alpha6A have no detectable effect on the myoblast phenotype. However, ectopic expression of the beta1A integrin subunit or IL2R-beta1A, autonomously inhibits differentiation and maintains a proliferative state. Perturbing alpha5 or alpha6A ratios also significantly affects activation of beta1 integrin signaling pathways. Ectopic alpha5 expression enhances expression and activation of paxillin as well as mitogen-activated protein (MAP) kinase with little effect on focal adhesion kinase (FAK). In contrast, ectopic alpha6A expression suppresses FAK and MAP kinase activation with a lesser effect on paxillin. Ectopic expression of wild-type and mutant forms of FAK, paxillin, and MAP/erk kinase (MEK) confirm these correlations. These data demonstrate that (a) proliferative signaling (i.e., inhibition of cell cycle withdrawal and the onset of terminal differentiation) occurs through the beta1A subunit and is modulated by the alpha subunit cytoplasmic domains; (b) perturbing alpha subunit ratios alters paxillin expression and phosphorylation and FAK and MAP kinase activation; (c) quantitative changes in the level of adhesive signaling through integrins and focal adhesion components regulate the decision of myoblasts to withdraw from the cell cycle, in part via MAP kinase. (+info)
Extracellular matrix regulates apoptosis in mammary epithelium through a control on insulin signaling.
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Adherent epithelial cells require interactions with the extracellular matrix for their survival, though the mechanism is ill-defined. In long term cultures of primary mammary epithelial cells, a laminin-rich basement membrane (BM) but not collagen I suppresses apoptosis, indicating that adhesion survival signals are specific in their response (. J. Cell Sci. 109:631-642). We now demonstrate that the signal from BM is mediated by integrins and requires both the alpha6 and beta1 subunits. In addition, a hormonal signal from insulin or insulin-like growth factors, but not hydrocortisone or prolactin, is necessary to suppress mammary cell apoptosis, indicating that BM and soluble factors cooperate in survival signaling. Insulin induced autophosphorylation of its receptor whether mammary cells were cultured on collagen I or BM substrata. However, both the tyrosine phosphorylation of insulin receptor substrate-1 and its association with phosphatidylinositol 3-kinase were enhanced in cells cultured on BM, as was the phosphorylation of the phosphatidylinositol 3-kinase effector, protein kinase B. These results suggest a novel extracellular matrix-dependent restriction point in insulin signaling in mammary epithelial cells. The proximal signal transduction event of insulin receptor phosphorylation is not dependent on extracellular matrix, but the activation of downstream effectors requires adhesion to BM. Since phosphatidylinositol 3-kinase was required for mammary epithelial cell survival, we propose that a possible mechanism for BM-mediated suppression of apoptosis is through its facilitative effects on insulin signaling. (+info)
beta1- and alpha6-integrin are surface markers on mouse spermatogonial stem cells.
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Although spermatogenesis is essential for reproduction, little is known about spermatogonial stem cells. These cells provide the basis for spermatogenesis throughout adult life by undergoing self-renewal and by providing progeny that differentiate into spermatozoa. A major impediment to our understanding of the biology of these stem cells is the inability to distinguish them from spermatogonia that are committed to differentiation. We made use of the known association of stem cells with basement membranes and our spermatogonial transplantation assay system to identify specific molecular markers on the stem cell surface. Selection of mouse testis cells with anti-beta1- or anti-alpha6-integrin antibody, but not anti-c-kit antibody, produced cell populations with a significantly enhanced ability to colonize recipient testes and generate donor cell-derived spermatogenesis. We demonstrate spermatogonial stem cell-associated antigens by using an assay system based on biological function. Furthermore, the presence of surface integrins on spermatogonial stem cells suggests that these cells share elements of a common molecular machinery with stem cells in other tissues. (+info)
alpha6 Integrin is regulated with lens cell differentiation by linkage to the cytoskeleton and isoform switching.
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The developing chicken embryo lens provides a unique model for examining the relationship between alpha6 integrin expression and cell differentiation, since multiple stages of differentiation are expressed concurrently at one stage of development. We demonstrate that alpha6 integrin is likely to mediate the inductive effects of laminin on lens differentiation as well as to function in a matrix-independent manner along the cell-cell interfaces of the differentiating cortical lens fiber cells. Both alpha6 isoform expression and its linkage to the cytoskeleton were regulated in a differentiation-specific manner. The association of alpha6 integrin with the Triton-insoluble cytoskeleton increased as the lens cells differentiated, reaching its highest levels in the cortical fiber region where the lens fiber cells are formed. In this region of the lens alpha6 integrin was uniquely localized along the cell-cell borders of the differentiating fiber cells, similar to beta1. alpha6beta4, the primary transmembrane protein of hemidesmosomes, is also expressed in the lens, but in the absence of hemidesmosomes. Differential expression of alpha6A and alpha6B isoforms with lens cell differentiation was seen at both the mRNA and the protein levels. RT-PCR studies demonstrated that alpha6B was the predominant isoform expressed both early in development, embryonic day 4, and in the epithelial regions of the day 10 embryonic lens. Isoform switching, with alpha6A now the predominant isoform, occurred in the fiber cell zones. Immunoprecipitation studies showed that alpha6B, which is characteristic of undifferentiated cells, was expressed by the lens epithelial cells but was dramatically reduced in the lens fiber zones. Expression of alpha6B began to drop as the cells initiated their differentiation and then dropped precipitously in the cortical fiber zone. In contrast, expression of the alpha6A isoform remained high until the cells became terminally differentiated. alpha6A was the predominant isoform expressed in the cortical fiber region. The down-regulation of alpha6B relative to alpha6A provides a developmental switch in the process of lens fiber cell differentiation. (+info)
Synergistic activities of alpha3 and alpha6 integrins are required during apical ectodermal ridge formation and organogenesis in the mouse.
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Integrins alpha6beta1 and alpha6beta4 are cell surface receptors for laminins. Integrin alpha6-null mice die at birth with severe skin blistering and defects in the cerebral cortex and in the retina. Integrin alpha3beta1 can associate with laminins and other ligands. Integrin alpha3-null mice also die at birth, with kidney and lung defects at late stages of development, and moderate skin blistering. To investigate possible overlapping functions between alpha3 and alpha6 integrins, we analyzed the phenotype of compound alpha3-/-/alpha6-/- mutant embryos. Double homozygous mutant embryos were growth-retarded and displayed several developmental defects not observed in the single mutant animals. First, limb abnormalities characterized by an absence of digit separation and the fusion of preskeletal elements were observed. Further analyses indicated a defect in the apical ectodermal ridge, an essential limb organizing center. In the double mutant, the ridge appeared flattened, and ridge cells did not show a columnar morphology. A strong reduction in ridge cell proliferation and alterations of the basal lamina underlying the ectoderm were observed. These results suggest that alpha3 and alpha6 integrins are required for the organization or compaction of presumptive apical ectodermal ridge cells into a distinct differentiated structure. Additional defects were present: an absence of neural tube closure, bilateral lung hypoplasia, and several abnormalities in the urogenital tract. Finally, an aggravation of brain and eye lamination defects was observed. The presence of novel phenotypes in double mutant embryos demonstrates the synergism between alpha3 and alpha6 integrins and their essential roles in multiple processes during embryogenesis. (+info)
Expression of the alpha6 integrin confers papillomavirus binding upon receptor-negative B-cells.
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Papillomaviruses (PV) bind to a wide range of cell lines in a specific and saturable manner. We have recently identified a candidate receptor for papillomavirus as the alpha6 integrin (Evander et al., J. Virol. 71, 2449-2456, 1997). We have further investigated the role the alpha6 integrin plays in PV binding. Here we show that the cells expressing the alpha6 integrin, partnered with either the beta4 integrin or the beta1 integrin, are equally able to bind PV HPV6b L1 virus-like particles, indicating that the beta partner does not play a major role in virus binding. In order to provide definitive evidence that the alpha6 integrin is required for PV binding we undertook to genetically complement the receptor-negative B-cell line DG75 by expressing the human alpha6A gene. The transduction of the alpha6 integrin gene into DG75 cells results in the cell surface expression of the alpha6 protein and this expression confers upon DG75 cells the ability to bind laminin, a normal ligand for alpha6 integrin. Furthermore, the alpha6 protein is partnered with the beta1 integrin in DG75 cells. Finally, we show that the DG75-alpha6 cells were able to bind papillomavirus VLPs and this binding was inhibited by a functionally blocking anti-alpha6 antibody. Together these data indicate that the alpha6 integrin is a primary cell receptor for papillomaviruses and is both necessary and sufficient for PV binding. (+info)
Interleukin-10 receptors are expressed by basement membrane anchored, alpha(6) integrin(+) cytotrophoblast cells in early human placenta.
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Cytotrophoblast cells produce interleukin (IL)-10 and express IL-10 receptor mRNA in culture. Furthermore, IL-10 dramatically reduces the synthesis of matrix metalloproteinase (MMP)-9 and the invasivity of cytotrophoblast cells in vitro, suggesting that an autocrine regulatory role in vivo is also possible. To test this hypothesis we investigated the expression of IL-10 receptor protein by first trimester cytotrophoblasts both in vitro and in situ, using flow cytometry and immunohistochemistry. Flow cytometric analyses demonstrated that 75-80% of cytotrophoblasts are able to bind labelled IL-10, suggesting that these cells possess IL-10 receptors in vitro. Serial sections of early human placentae stained for either alpha(5) and alpha(6) integrin subunits, or for IL-10 receptors respectively, revealed that placental cytotrophoblasts possess cell surface IL-10 receptors not only in vitro, but also in vivo. IL-10 receptors were present mainly on alpha(6) integrin expressing villous cytotrophoblast cells and on alpha(6)-positive cells of invasive cell columns located nearest the villous stroma. Differentiated trophoblasts (i.e. alpha(5)-positive cells and villous syncytiotrophoblasts) showed no reactivity. This differential expression of IL-10 receptors suggests that IL-10 might suppress the invasivity of undifferentiated cytotrophoblast cells, in vivo, preserving their non-invasive state in an autocrine manner. The possible involvement in cytotrophoblast proliferation and/or differentiation is also discussed. (+info)
Partial rescue of epithelial phenotype in integrin beta4 null mice by a keratin-5 promoter driven human integrin beta4 transgene.
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Integrin beta4 null mice exhibit extensive epidermal detachment, reminiscent of the human skin blistering disease junctional epidermolysis bullosa associated with pyloric atresia. Hemidesmosomes, the stable adhesion structures of squamous epithelia, are not formed in the absence of alpha6beta4. Null mutant mice die shortly after birth, but apart from their striking epithelial phenotype, no obvious developmental defects have been observed. To elucidate the cause of death in these mice, we generated transgenic mice with a heterologous construct consisting of the squamous epithelial-specific keratin-5 promoter and a human integrin beta4 subunit cDNA. The transgene was not expressed in the presence of endogenous beta4, probably as a result of competition for a limited pool of alpha6 subunits. In a beta4 null background, however, the transgene was expressed, and its expression pattern followed that of squamous epithelial-specific keratins. These rescued pups appeared healthy and ultrastructural analysis revealed that the interspecies heterodimer alpha6(mouse)/beta4(human) was sufficient to trigger the assembly of hemidesmosomes. After a variable period of up to 48 hours after birth these animals began to exhibit haemorrhages at the plantar and palmar areas. We observed the formation of small blisters and found that the transgene was not detectably expressed in this region, which is devoid of hair follicles. The rescued neonates became increasingly cyanotic and died soon after the onset of this phenomenon. We performed a developmental study of the expression of beta4 in the complete respiratory tract, but we found no correlation between the spatiotemporal distribution of beta4 and the onset of the respiratory insufficiency. It became clear, however, that there was a gradual detachment of squamous epithelia in the oral and nasal cavities which led to obstruction of the respiratory tract, suggesting that in beta4 null and rescued mice, neonatal death was a direct consequence of decreased adhesion properties of hairless squamous epithelia, rather than a developmental defect of the lungs. (+info)