The homeobox gene Otx of the jellyfish Podocoryne carnea: role of a head gene in striated muscle and evolution

In many bilaterian animals members of the Otx gene family are expressed in head or brain structures. Cnidarians, however, have no clearly homologous head and no distinct brain; but an Otx homolog from the jellyfish Podocoryne carnea is highly conserved in sequence and domain structure. Sequence similarities extend well beyond the homeodomain and Podocoryne Otx can be aligned over its entire length to human OTX1, OTX2, and CRX. The overall structure of Otx is better conserved from Podocoryne to deuterostomes while protostomes appear to be more derived. In contrast, functions seem to be conserved from protostomes to vertebrates but not in Podocoryne or echinoderms. Podocoryne Otx is expressed only during medusa bud formation and becomes restricted to the striated muscle of medusae. Cnidaria are the most basal animals with striated muscle. Podocoryne polyps have no striated muscle and no Otx expression; both appear only during the asexual medusa budding process. The common ancestor of all animals that gave rise to cnidarians, protostomes, and deuterostomes already had an Otx gene more similar to today's Podocoryne and human homologs than to Drosophila otd, while the head-specific function appears to have evolved only later.     

The germ line and somatic stem cell gene Cniwi in the jellyfish Podocoryne carnea

In most animal phyla from insects to mammals, there is a clear division of somatic and germ line cells. This is however not the case in plants and some animal phyla including tunicates, flatworms and the basal phylum Cnidaria, where germ stem cells arise de novo from somatic cells. Piwi-like genes represent essential stem cell genes in diverse multicellular organisms. The cnidarian Piwihomolog Cniwiwas cloned from Podocoryne carnea, a hydrozoan with a full life cycle. CniwiRNA is present in all developmental stages with highest levels in the egg and the medusa. In the adult medusa, Cniwi expression is prominent in the gonads where it likely functions as a germ stem cell gene. The gene is also expressed, albeit at low levels, in differentiated somatic cells like the striated muscle of the medusa. Isolated striated muscle cells can be induced to transdifferentiate into smooth muscle cells which proliferate and differentiate into nerve cells. Cniwi expression is upregulated transiently after induction of transdifferentiation and again when the emerging smooth muscle cells proliferate and differentiate. The continuous low-level expression of an inducible stem cell gene in differentiated somatic cells may underlie the ability to form medusa buds from polyp cells and explain the extraordinary transdifferentation and regeneration potential of Podocoryne carnea.     

Integrins in periodontal disease

Cell surface integrin receptors mediate cell adhesion, migration and cellular signaling in all nucleated cells. They are activated by binding to extracellular ligands or by intracellular proteins, such as kindlins that engage with their cytoplasmic tails. Cells in the periodontal tissues express several integrins with overlapping ligand-binding capabilities. A distinct phenotype in the periodontium has only been described for knockouts or mutations of three integrin subunits, α11, β6 and β2. Integrin α11β1 appears to have some regulatory function in the periodontal ligament of continuously erupting incisors in mice. Integrin αvβ6 is expressed in the junctional epithelium (JE) of the gingiva. Animals deficient in this receptor develop classical signs of periodontal disease, including inflammation, apical migration of the JE and bone loss, suggesting that it plays a role in the regulation of periodontal inflmmation, likely through activation of transforming growth factor-β1. Lack of integrin activation in the JE is also associated with periodontitis. Patients with kindlin-1 mutations have severe early-onset periodontal disease. Finally, patients with mutations in the leukocyte-specific β2 integrin subunit have severe periodontal problems due to lack of transiting neutrophils in the periodontal tissues.     

Reversible inactivation of cell-type-specific regulatory and structural genes in migrating isolated striated muscle cells of jellyfish.

We have investigated, by RT-PCR and in situ hybridization, expression of genes encoding regulatory and structural proteins in migrating mononucleated striated muscle cells of the medusa Podocoryne carnea. Expression of the three homeobox genes Otx, Cnox1-Pc, and Cnox3-Pc; a specific splice variant of the myosin heavy chain gene (Myo1); and a tropomyosin (Tpm2) is stable in isolated and cultured striated muscle tissue. When grafted onto cell-free extracellular matrix (ECM), muscle cells of the tissue fragments leave their native ECM and migrate as a coherent tissue onto a host ECM until a stretched cell monolayer is formed. Shortly after the first cells of the grafted isolate have made contact with the host ECM, Otx and Cnox1-Pc expression is completely turned off in all cells of the graft, including those still adhering to their native ECM. Myo1 message disappears with a delay while the expression level of Tpm2 is strongly reduced. However, expression of the homeobox gene Cnox3-Pc, a msh-like gene, and of the ubiquitously expressed elongation factor 1 alpha is not affected by the migration process. All genes are reexpressed after 12-24 h, once migration of the cells has ceased. Our results demonstrate that the first few migrating cells induce a change in gene expression which is rapidly communicated throughout the entire tissue. Furthermore, we showed that commitment of striated muscle cells remains stable despite the transient inactivation of cell-type-specific regulatory and structural genes.     

Integrins alpha5beta1, alphavbeta1, and alphavbeta6 collaborate in squamous carcinoma cell spreading and migration on fibronectin

The expression of alphavbeta6 fibronectin/tenascin receptor integrin is induced in malignant transformation of oral epithelium. In this study, we demonstrate the contribution of alphavbeta6 as well as other fibronectin receptor integrins in squamous cell carcinoma (SCC) cell adhesion and migration. Of 11 SCC cell lines isolated from the head and neck area, 8 (73%) expressed alphavbeta6 integrin on the cell surface. Three cell lines were chosen for further functional experiments: 1 with relatively high, 1 with moderate, and 1 with minimal surface expression of alphavbeta6 integrin. In addition to alphavbeta6, all 3 cell lines expressed alpha5beta1 and alphavbeta1 fibronectin receptor integrins. Function-blocking experiments with inhibitory anti-integrin antibodies showed that all these three integrins were functional in SCC cell spreading on fibronectin. Integrin alphavbeta6, however, was not used as a primary but as an alternative fibronectin receptor by SCC cells, as the inhibitory anti-beta6 integrin antibody alone had no effect on spreading. In migration, however, alphavbeta6, alpha5beta1, and alphavbeta1 integrins were all used in cooperation. The presence of alphavbeta1 integrin in SCC cells is a novel finding as is its contribution to SCC cell migration. When one or two of these three receptors were blocked, the cells demonstrated an adaptive ability to remain migratory using integrins that were not targeted by antibodies. Utilization of a combination of receptors of different affinities may be beneficial for SCC cell migration versatility.     

The effect of gamma-tocopherol on proliferation, integrin expression, adhesion, and migration of human glioma cells

The effect of vitamin E on proliferation, integrin expression, adhesion, and migration in human glioma cells has been studied. gamma-tocopherol at 50 microM concentration exerted more inhibitory effect than alpha-tocopherol at the same concentration on glioma cell proliferation. Integrin alpha5 and beta1 protein levels were increased upon both alpha- and gamma-tocopherol treatments. In parallel, an increase in the alpha5beta1 heterodimer cell surface expression was observed. The tocopherols inhibited glioma cell-binding to fibronectin where gamma-tocopherol treatment induced glioma cell migration. Taken together, the data reported here are consistent with the notion that the inhibition of glioma cell proliferation induced by tocopherols may be mediated, at least in part, by an increase in integrin alpha5 and beta1 expression. Cell adhesion is also negatively affected by tocopherols, despite a small increase in the surface appearance of the alpha5beta1 heterodimer. Cell migration is stimulated by gamma-tocopherol. It is concluded that alpha5 and beta1 integrin expression and surface appearance are not sufficient to explain all the observations and that other integrins or in general other factors may be associated with these events.     

Amygdalin Influences Bladder Cancer Cell Adhesion and Invasion In Vitro

The cyanogenic diglucoside amygdalin, derived from Rosaceae kernels, is employed by many patients as an alternative anti-cancer treatment. However, whether amygdalin indeed acts as an anti-tumor agent is not clear. Metastasis blocking properties of amygdalin on bladder cancer cell lines was, therefore, investigated. Amygdalin (10 mg/ml) was applied to UMUC-3, TCCSUP or RT112 bladder cancer cells for 24 h or for 2 weeks. Tumor cell adhesion to vascular endothelium or to immobilized collagen as well as tumor cell migration was examined. Effects of drug treatment on integrin α and β subtypes, on integrin-linked kinase (ILK) and total and activated focal adhesion kinase (FAK) were also determined. Integrin knock-down was carried out to evaluate integrin influence on migration and adhesion. A 24 h or 2 week amygdalin application distinctly reduced tumor cell adhesion and migration of UMUC-3 and RT112 cells. TCCSUP adhesion was also reduced, but migration was elevated under amygdalin. Integrin subtype expression was significantly and specifically altered by amygdalin depending on the cell line. ILK was moderately, and activated FAK strongly, lost in all tumor cell lines in the presence of amygdalin. Knock down of β1 integrin caused a significant decrease in both adhesion and migration of UMUC-3 cells, but a significant increase in TCCSUP adhesion. Knock down of β4 integrin caused a significant decrease in migration of RT112 cells. Since the different actions of amygdalin on the different cell lines was mirrored by β1 or β4 knock down, it is postulated that amygdalin influences adhesion and migratory properties of bladder cancer cells by modulating β1 or β4 integrin expression. The amygdalin induced increase in TCCSUP migratory behavior indicates that any anti-tumor benefits from amygdalin (seen with the other two cell lines) may depend upon the cancer cell type.     

Mechanisms of integrin activation and trafficking

Integrin adhesion receptors are essential for the normal function of most multicellular organisms, and defective integrin activation or integrin signaling is associated with an array of pathological conditions. Integrins are regulated by conformational changes, clustering, and trafficking, and regulatory mechanisms differ strongly between individual integrins and between cell types. Whereas integrins in circulating blood cells are activated by an inside-out-induced conformational change that favors high-affinity ligand binding, β1-integrins in adherent cells can be activated by force or clustering. In addition, endocytosis and recycling play an important role in the regulation of integrin turnover and integrin redistribution in adherent cells, especially during dynamic processes such as cell migration and invasion. Integrin trafficking is strongly regulated by their cytoplasmic tails, and the mechanisms are now being identified.     

The jellyfish and its polyp: a comparative study of gene expression monitored by the protein patterns, using two-dimensional gels with double-label autoradiography

The life cycle of Podocoryne carnea (Coelenterata, Anthomedusae) shows several distinct stages which differ considerably in terms of their ecology, morphology, cellular composition, and ultrastructure. Previously these stages had even been described as separate species. Using two-dimensional gel electrophoresis and a new method of double-label autoradiography, we show here for the first time for metagenic hydrozoans that only minor differences in gene expression exist between the various life cycle stages. Our results demonstrate the high resolution power of these techniques and show that the different life stages of P. carnea remain rather similar on the protein level. Most of the prominent spots of the two-dimensional gel protein patterns are common to all stages studied. These data show that the hydrozoan life cycle and development are regulated by only minor distinctions in gene expression which possibly explains the great morphogenetic repertoire of these animals described in many studies.     

Distribution of integrins and their ligands in the trunk of Xenopus laevis during neural crest cell migration

We have examined the distribution in Xenopus embryos of beta 1 subunits of integrin, as recognized by cross-reactive antibodies against the avian integrin beta 1 subunit. These antibodies recognize a doublet of bands of approximately 120 kD in Xenopus embryos. The distribution pattern of these integrin cell surface receptors was compared with that of two possible ligands, fibronectin and laminin, in the extracellular matrix during the time of neural crest cell migration. Integrin immunoreactivity in the early neurula was observed lightly outlining somite and epidermal cells and the notochord. The integrin immunostaining increased with developmental age and was observed on most cell types in the embryo but was particularly notable in the intersomitic clefts through which motoraxons grow. The immunoreactivity in this region was not, however, wholly on the axon surfaces, since intersomitic integrin remained detectable in embryos in which the neural tube had been ablated. Fibronectin and laminin were more extensively distributed than integrin at all stages examined. Immunoreactivity for both was observed around the neural tube, notochord, somites, epidermis, dorsal mesentery, and lateral plate mesoderm. The distribution of laminin and fibronectin around the somites was particularly interesting since it was non-uniform and similar to that of integrin. Strongest staining was observed in the intersomitic clefts, and weakest staining was observed on the medial surface of the somites, which faces the neural tube and notochord. The major differences in distribution pattern between the fibronectin and laminin immunoreactivities were that only fibronectin was detected in the mesenchyme of the dorsal fin. Our results demonstrate that a molecule homologous to avian integrin is present in Xenopus embryos during neural crest cell migration and motoraxon outgrowth. Its presence in the intersomitic clefts and on the surface of many embryonic cell types together with the abundant distribution of its ligands are consistent with a potentially important developmental function in neurite outgrowth and/or muscle development.     

整合素在细胞黏附中的生物学功能

整合素是一种跨膜蛋白,属于黏附分子家族.其主要功能是参与细胞和细胞、细胞和细胞外基质(ECM)的黏附和信号转导.整合素是含有α和β两条肽链的异源二聚体,来源不同的α、β亚基所形成的整合素具有不同的ECM结合能力.阐述了整合素的结构、生物学功能以及生理、病理学意义,并概述了其研究进展.     

Characterization of mouse integrin alpha3 subunit gene.

Integrin alpha3beta1 (VLA-3) is an adhesion receptor for extracellular matrix proteins including various isoforms of laminin. We have isolated mouse genomic clones encoding the integrin alpha3 subunit and deduced the exon/intron organization. The mouse integrin alpha3 subunit gene is encoded by 26 exons spanning 40 kb. The exon/intron structure of the integrin alpha3 subunit gene resembles that of the integrin alpha6 subunit gene, but differs somewhat from those of other members of the integrin family. We have demonstrated that the cytoplasmic domain splicing variants of the alpha3 subunits (alpha3A and alpha3B) are generated by alternative exon usage. We also cloned the 5'-flanking region and performed a preliminary analysis of its promoter activity in various tumor cell lines with different degrees of integrin alpha3 expression. Following transfection, activity in the luciferase assay was found to be roughly correlated with the expression level of integrin alpha3 as measured by flow cytometry. Furthermore, the luciferase assay was performed with normal and SV-40- or polyoma virus-transformed fibroblasts. In mouse, human, and hamster fibroblasts, higher levels of luciferase expression were observed in transformed cells than in normal cells. This result is consistent with our previous finding that integrin alpha3 expression at both the protein and mRNA levels is enhanced upon oncogenic transformation of fibroblasts by tumor viruses.     

Integrin and Cadherin Synergy Regulates Contact Inhibition of Migration and Motile Activity

Integrin receptors play a central role in cell migration through their roles as adhesive receptors for both other cells and extracellular matrix components. In this study, we demonstrate that integrin and cadherin receptors coordinately regulate contact-mediated inhibition of cell migration. In addition to promoting proliferation (Sastry, S., M. Lakonishok, D. Thomas, J. Muschler, and A. Horwitz. 1996. J. Cell Biol. 133:169–184), ectopic expression of the α5 integrin in cultures of primary quail myoblasts promotes a striking contact-mediated inhibition of cell migration. Myoblasts ectopically expressing α5 integrin (α5 myoblasts) move normally when not in contact, but upon contact, they show inhibition of migration and motile activity (i.e., extension and retraction of membrane protrusions). As a consequence, these cells tend to grow in aggregates and do not migrate to close a wound. This phenotype is also seen with ectopic expression of β1 integrin, paxillin, or activated FAK (CD2 FAK) and therefore appears to result from enhanced integrin-mediated signaling. The contact inhibition observed in the α5 myoblasts is mediated by N-cadherin, whose expression is upregulated more than fivefold. Perturbation studies using low calcium conditions, antibody inhibition, and ectopic expression of wild-type and mutant N-cadherins all implicate N-cadherin in the contact inhibition of migration. Ectopic expression of N-cadherin also produces cells that show inhibited migration upon contact; however, they do not show suppressed motile activity, suggesting that integrins and cadherins coordinately regulate motile activity. These observations have potential importance to normal and pathologic processes during embryonic development and tumor metastasis.     

Laminin alpha4 and integrin alpha6 are upregulated in regenerating dy/dy skeletal muscle: comparative expression of laminin and integrin isoforms in muscles regenerating after crush injury

The expression of laminin isoforms and laminin-binding integrin receptors known to occur in muscle was investigated during myogenic regeneration after crush injury. Comparisons were made between dystrophic 129ReJ dy/dy mice, which have reduced laminin alpha2 expression, and their normal littermates. The overall histological pattern of regeneration after crush injury was similar in dy/dy and control muscle, but proceeded faster in dy/dy mice. In vitro studies revealed a greater yield of mononuclear cells extracted from dy/dy muscle and a reduced proportion of desmin-positive cells upon in vitro cultivation, reflecting the presence of inflammatory cells and "preactivated" myoblasts due to ongoing regenerative processes within the endogenous dystrophic lesions. Laminin alpha1 was not detectable in skeletal muscle. Laminin alpha2 was present in basement membranes of mature myofibers and newly formed myotubes in control and dy/dy muscles, albeit weaker in dy/dy. Laminin alpha2-negative myogenic cells were detected in dy/dy and control muscle, suggesting the involvement of other laminin alpha chains in early myogenic differentiation, such as laminin alpha4 and alpha5 which were both transiently expressed in basement membranes of newly formed myotubes of dy/dy and control mice. Integrin beta1 was expressed on endothelial cells, muscle fibers, and peripheral nerves in uninjured muscle and broadened after crush injury to the interstitium where it occurred on myogenic and nonmyogenic cells. Integrin alpha3 was not expressed in uninjured or regenerating muscle, while integrin alpha6 was expressed mainly on endothelial cells and peripheral nerves in uninjured muscle. Upon crush injury integrin alpha6 increased in the interstitium mainly on nonmyogenic cells, including infiltrating leukocytes, endothelial cells, and fibroblasts. In dy/dy muscle, integrin alpha6 occurred on some newly formed myotubes. Integrin alpha7 was expressed on muscle fibers at the myotendinous junction and showed weak and irregular expression on muscle fibers. After crush injury, integrin alpha7 expression extended to the newly formed myotubes and some myoblasts. However, many myoblasts and newly formed myotubes were integrin alpha7 negative. No marked difference was observed in integrin alpha7 expression between dy/dy and control muscle, either uninjured or after crush injury. Only laminin alpha4 and integrin alpha6 expression patterns were notably different between dy/dy and control muscle. Expression of both molecules was more extensive in dy/dy muscle, especially in the interstitium of regenerating areas and on newly formed myotubes. In view of the faster myogenic regeneration observed in dy/dy mice, the data suggest that laminin alpha4 and integrin alpha6 support myogenic regeneration. However, whether these accelerated myogenic effects are a direct consequence of the reduced laminin alpha2 expression in dy/dy mice, or an accentuation of the ongoing regenerative events in focal lesions in the muscle, requires further investigation.