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1.
Cell migration is an integrated process that involves cell adhesion, protrusion and contraction. We recently used CAS (Crk-associated substrate, 130CAS)-deficient mouse embryo fibroblasts (MEFs) to examined contribution made to v-Crk to that process via its interaction with Rac1. v-Crk, the oncogene product of avian sarcoma virus CT10, directly affects membrane ruffle formation and is associated with Rac1 activation, even in the absence of CAS, a major substrate for Crk. In CAS-deficient MEFs, cell spreading and lamellipodium dynamics are delayed; moreover, Rac activation is significantly reduced and it is no longer targeted to the membrane. However, expression of v-Crk by CAS-deficient MEFs increased cell spreading and active lamellipodium protrusion and retraction. v-Crk expression appears to induce Rac1 activation and its targeting to the membrane, which directly affects membrane dynamics and, in turn, cell migration. It thus appears that v-Crk/Rac1 signaling contributes to the regulation of membrane dynamics and cell migration, and that v-Crk is an effector molecule for Rac1 activation that regulates cell motility.Key words: v-Crk, Rac, lamellipodia dynamics, cell migration, p130CASCell migration is a central event in a wide array of biological and pathological processes, including embryonic development, inflammatory responses, angiogenesis, tissue repair and regeneration, cancer invasion and metastasis, osteoporosis and immune responses.1,2 Although the molecular basis of cell migration has been studied extensively, the underlying mechanisms are still not fully understood. It is known that cell migration is an integrated process that involves formation of cell adhesions and/or cell polarization, membrane protrusion in the direction of migration (e.g., filopodium formation and lamellipodium extension), cell body contraction and tail detachment.1–3 Formation of cell adhesions, including focal adhesions, fibrillar adhesions and podosomes are the first step in cell migration. Cell adhesions are stabilized by attachment to the extracellular matrix (ECM) mediated by integrin transmembrane receptors, which are also linked to various cytoplasmic proteins and the actin cytoskeleton, which provide the mechanical force necessary for migration.2,4 The next steps in the process of cell migration are filopodium formation and lamellipodium extension. These are accompanied by actin polymerization and microtubule dynamics, which also contribute to the control of cell adhesion and migration.5Focal adhesions are highly dynamic structures that form at sites of membrane contact with the ECM and involve the activities of several cellular proteins, including vinculin, focal adhesion kinase (FAK), Src family kinase, paxillin, CAS (Crk-associated substrate, p130CAS) and Crk.6 A deficiency in focal adhesion protein is associated with the severe defects in cell motility and results in embryonic death. For example, FAK deficiency disrupts mesoderm development in mice and delays cell migration in vitro,7 which reflects impaired assembly and disassembly the focal adhesions.8 In addition, mouse embryonic fibroblasts (MEFs) lacking Src kinase showed a reduced rate of cell spreading that resulted in embryonic death.9 Taken together, these findings strongly support the idea that cell adhesion complexes play crucial roles in cell migration.CAS is a hyperphosphorylated protein known to be a major component of focal adhesion complexes and to be involved in the transformation of cells expressing v-Src or v-Crk.10 CAS-deficient mouse embryos die in utero and show marked systematic congestion and growth retardation,4 while MEFs lacking CAS show severely impaired formation and bundling of actin stress fibers and delayed cell motility.4,11,12 Conversely, transient expression of CAS in COS7 cells increases cell migration.11 Crk-null mice also exhibit lethal defects in embryonic development,13 which is consistent with the fact that CAS is a major substrate for v-Crk, and both CAS and v-Crk are necessary for induction of cell migration.14 v-Crk consists of a viral gag sequence fused to cellular Crk sequences, which contain Src homology 2 (SH2) and SH3 domains but no kinase domain, and both CAS and paxillin bind to SH2 domains.12,15,16 Despite the absence of a kinase domain, cell expressing v-Crk show upregulation of tyrosine phosphorylation of CAS, FAK and paxillin, which is consistent with v-Crk functioning as an adaptor protein.17 Moreover, this upregulation of tyrosine phosphorylation correlates well with the transforming activity of v-Crk.17 By contrast, tyrosine phosphorylation of FAK and CAS is diminished in Src kinase-deficient cells expressing v-Crk, and they are not targeted to the membrane, suggesting v-Crk signaling is Src kinase-dependent. After formation of the CAS/v-Crk complex, v-Crk likely transduces cellular signaling to Src kinase and FAK.12 Notably, tyrosine phosphorylation of FAK and cell migration and spreading are all enhanced when v-Crk is introduced into CAS-deficient MEFs.12 We therefore suggest that v-Crk activity, but not cellular Crk activity, during cell migration and spreading is CAS-independent.Membrane dynamics such as lamellipodium protrusion and membrane ruffling reportedly involve Rac1,18 α4β1 integrin,19 Arp2/3,6 and N-WASP,20 and are enhanced in v-Crk-expressing CAS-deficient MEFs.21 Moreover, expression in those cells of N17Rac1, a dominant defective Rac1 mutant, abolished membrane dynamics at early times and delayed cell migration.21 v-Crk-expressing, CAS-deficient MEFs transfected with N17Rac1 did not begin spreading until one hour after being plated on fibronectin, and blocking Rac activity suppressed both membrane dynamics and cell migration. We therefore suggest that v-Crk is involved in cell attachment and spreading, and that this process is mediated by Rac1 activation. In addition, v-Crk expression apparently restores lamellipodium formation and ruffle retraction in CAS-deficient MEFs. Thus v-Crk appears to participate in a variety cellular signaling pathways leading to cell spreading, Rac1 activation, membrane ruffling and cell migration, even in the absence of CAS, its major substrate protein.In fibroblasts, the Rho family of small GTP-binding proteins (e.g., Cdc42, Rac and Rho) functions to control actin cytoskeleton turnover, including filopodium extension, lamellipodium formation and generation of actin stress fibers and focal adhesions.22 These GTPases function in a cascade, such that activation of Cdc42 leads to activation of Rac1, which in turn activates Rho.22 Once activated, Rho controls cell migration. Cell adhesion to ECM leads to the translocation of Rac1 and Cdc42 from the cytosol to the plasma membrane,23 where they regulate actin polymerization at the leading edge.19,24 Dominant negative Rac and Cdc42 mutants inhibit the signaling to cell spreading initiated by the interaction of integrin with ECM.24 The fact that cellular levels of activated Rac are higher in cells adhering to ECM than in suspended cells further suggests that activation of Rac and Cdc42 is a critical step leading to membrane protrusion and ruffle formation. It is noteworthy in this regard that v-Crk is able to induce Rac activation and its translocation to plasma membrane.21Overall, the findings summarized in this article demonstrate that v-Crk participates in several steps leading to cell adhesion and spreading (Fig. 1), and the targeting of v-Crk to focal adhesion sites appears to be a prerequisite for regulation of cell migration and spreading via Rac activation. To fully understand its function, however, it will be necessary to clarify the role of v-Crk in Rac1 and Cdc42 activation initiated by integrin-ECM interactions.Open in a separate windowFigure 1Schematic diagram of v-Crk signaling in MEFs. Cell adhesion signaling initiated by the integrin-ECM interaction triggers v-Crk signaling mediated by Src kinase, after which focal adhesion proteins are tyrosine phosphorylated. These events lead to translocation of Rac from the cytosol to the membrane, where it promotes membrane protrusion and ruffle formation. Under basal conditions, Rac is bound with GDP and is inactive. Upon stimulation, Rac activation is mediated by guanine nucleotide exchange factors (GEFs) that stimulate the release of bound GDP and the binding of GTP. Activation of Rac is transient, however, as it is inactivated by GTPase activating protein (GAP). 相似文献
2.
Cell migration during wound healing is a complex process that involves the expression of a number of growth factors and cytokines. One of these factors, transforming growth factor-beta (TGFβ) controls many aspects of normal and pathological cell behavior. It induces migration of keratinocytes in wounded skin and of epithelial cells in damaged cornea. Furthermore, this TGFβ-induced cell migration is correlated with the production of components of the extracellular matrix (ECM) proteins and expression of integrins and matrix metalloproteinases (MMPs). MMP digests ECMs and integrins during cell migration, but the mechanisms regulating their expression and the consequences of their induction remain unclear. It has been suggested that MMP-14 activates cellular signaling processes involved in the expression of MMPs and other molecules associated with cell migration. Because of the manifold effects of MMP-14, it is important to understand the roles of MMP-14 not only the cleavage of ECM but also in the activation of signaling pathways.Key words: wound healing, migration, matrix metalloproteinase, transforming growth factor, skin, corneaWound healing is a well-ordered but complex process involving many cellular activities including inflammation, growth factor or cytokine secretion, cell migration and proliferation. Migration of skin keratinocytes and corneal epithelial cells requires the coordinated expression of various growth factors such as platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), transforming growth factor (TGF), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), epidermal growth factor (EGF), small GTPases, and macrophage stimulating protein (reviewed in refs. 1 and 2). The epithelial cells in turn regulate the expression of matrix metalloproteinases (MMPs), extracellular matrix (ECM) proteins and integrins during cell migration.1,3,4 TGF-β is a well-known cytokine involved in processes such as cell growth inhibition, embryogenesis, morphogenesis, tumorigenesis, differentiation, wound healing, senescence and apoptosis (reviewed in refs. 5 and 6). It is also one of the most important cytokines responsible for promoting the migration of skin keratinocytes and corneal epithelial cells.3,6,7TGFβ has two quite different effects on skin keratinocytes: it suppresses their multiplication and promotes their migration. The TGFβ-induced cell growth inhibition is usually mediated by Smad signaling, which upregulates expression of the cell cycle inhibitor p21WAF1/Cip1 or p12CDK2-AP1 in HaCaT skin keratinocyte cells and human primary foreskin keratinocytes.8,9 Keratinocyte migration in wounded skin is associated with strong expression of TGFβ and MMPs,1 and TGFβ stimulates the migration of manually scratched wounded HaCaT cells.10 TGFβ also induces cell migration and inhibits proliferation of injured corneal epithelial cells, whereas it stimulates proliferation of normal corneal epithelial cells via effects on the MAPK family and Smad signaling.2,7 Indeed, skin keratinocytes and corneal epithelial cells display the same two physiological responses to TGFβ during wound healing; cell migration and growth inhibition. However as mentioned above, TGFβ has a different effect on normal cells. For example, it induces the epithelial to mesenchymal transition (EMT) of normal mammary cells and lens epithelial cells.11,12 It also promotes the differentiation of corneal epithelial cells, and induces the fibrosis of various tissues.2,6The MMPs are a family of structurally related zinc-dependent endopeptidases that are secreted into the extracellular environment.13 Members of the MMP family have been classified into gelatinases, stromelysins, collagenases and membrane type-MMPs (MT-MMPs) depending on their substrate specificity and structural properties. Like TGFβ, MMPs influence normal physiological processes including wound healing, tissue remodeling, angiogenesis and embryonic development, as well as pathological conditions such as rheumatoid arthritis, atherosclerosis and tumor invasion.13,14The expression patterns of MMPs during skin and cornea wound healing are well studied. In rats, MMP-2, -3, -9, -11, -13 and -14 are expressed,15 and in mice, MMP-1, -2, -3, -9, -10 and -14 are expressed during skin wound healing.1 MMP-1, -3, -7 and -12 are increased in corneal epithelial cells during Wnt 7a-induced rat cornea wound healing.16 Wound repair after excimer laser keratectomy is characterized by increased expression of MMP-1, -2, -3 and -9 in the rabbit cornea, and MMP-2, -9 in the rat cornea.17,18 The expression of MMP-2 and -9 during skin keratinocyte and corneal epithelial cell migration has been the most thoroughly investigated, and it has been shown that their expression generally depends on the activity of MMP-14. MMP-14 (MT1-MMP) is constitutively anchored to the cell membrane; it activates other MMPs such as MMP-2, and also cleaves various types of ECM molecules including collagens, laminins, fibronectin as well as its ligands, the integrins.13 The latent forms of some cytokines are also cleaved and activated by MMP-14.19 Overexpression of MMP-14 protein was found to stimulate HT1080 human fibrosarcoma cell migration.20 In contrast, the attenuation of MMP-14 expression using siRNA method decreased fibroblast invasiveness,21 angiogenesis of human microvascular endothelial cells,22 and human skin keratinocyte migration.10 The latter effect was shown to result from lowering MMP-9 expression. Other studies have shown that EGF has a critical role in MMP-9 expression during keratinocyte tumorigenesis and migration.23,24 On the other hand, TGFβ modulates MMP-9 production through the Ras/MAPK pathway in transformed mouse keratinocytes and NFκB induces cell migration by binding to the MMP-9 promoter in human skin primary cultures.25,26 Enhanced levels of pro-MMP-9 and active MMP-9 have also been noted in scratched corneal epithelia of diabetic rats.27There is evidence that MMP-14 activates a number of intracellular signaling pathways including the MAPK family pathway, focal adhesion kinase (FAK), Src family, Rac and CD44, during cell migration and tumor invasion.19,20,28 In COS-7 cells, ERK activation is stimulated by overexpression of MMP-14 and is essential for cell migration.29 These observations all indicate that MMP-14 plays an important role in cell migration, not only by regulating the activity or expression of downstream MMPs but also by processing and activating migration-associated molecules such as integrins, ECMs and a variety of intracellular signaling pathays.30Cell migration during wound healing is a remarkably complex phenomenon. TGFβ is just one small component of the overall process of wound healing and yet it triggers a multitude of reactions needed for cell migration. It is important to know what kinds of molecules are expressed when cell migration is initiated, but it is equally important to investigate the roles of these molecules and how their expression is regulated. Despite the availability of some information about how MMPs and signaling molecules can influence each other, much remains to be discovered in this area. It will be especially important to clarify how MMP-14 influences other signaling pathways since its role in cell migration is not restricted to digesting ECM molecules but also includes direct or indirect activation of cellular signaling pathways. 相似文献
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4.
Fetal cells migrate into the mother during pregnancy. Fetomaternal transfer probably occurs in all pregnancies and in humans the fetal cells can persist for decades. Microchimeric fetal cells are found in various maternal tissues and organs including blood, bone marrow, skin and liver. In mice, fetal cells have also been found in the brain. The fetal cells also appear to target sites of injury. Fetomaternal microchimerism may have important implications for the immune status of women, influencing autoimmunity and tolerance to transplants. Further understanding of the ability of fetal cells to cross both the placental and blood-brain barriers, to migrate into diverse tissues, and to differentiate into multiple cell types may also advance strategies for intravenous transplantation of stem cells for cytotherapeutic repair. Here we discuss hypotheses for how fetal cells cross the placental and blood-brain barriers and the persistence and distribution of fetal cells in the mother.Key Words: fetomaternal microchimerism, stem cells, progenitor cells, placental barrier, blood-brain barrier, adhesion, migrationMicrochimerism is the presence of a small population of genetically distinct and separately derived cells within an individual. This commonly occurs following transfusion or transplantation.1–3 Microchimerism can also occur between mother and fetus. Small numbers of cells traffic across the placenta during pregnancy. This exchange occurs both from the fetus to the mother (fetomaternal)4–7 and from the mother to the fetus.8–10 Similar exchange may also occur between monochorionic twins in utero.11–13 There is increasing evidence that fetomaternal microchimerism persists lifelong in many child-bearing women.7,14 The significance of fetomaternal microchimerism remains unclear. It could be that fetomaternal microchimerism is an epiphenomenon of pregnancy. Alternatively, it could be a mechanism by which the fetus ensures maternal fitness in order to enhance its own chances of survival. In either case, the occurrence of pregnancy-acquired microchimerism in women may have implications for graft survival and autoimmunity. More detailed understanding of the biology of microchimeric fetal cells may also advance progress towards cytotherapeutic repair via intravenous transplantation of stem or progenitor cells.Trophoblasts were the first zygote-derived cell type found to cross into the mother. In 1893, Schmorl reported the appearance of trophoblasts in the maternal pulmonary vasculature.15 Later, trophoblasts were also observed in the maternal circulation.16–20 Subsequently various other fetal cell types derived from fetal blood were also found in the maternal circulation.21,22 These fetal cell types included lymphocytes,23 erythroblasts or nucleated red blood cells,24,25 haematopoietic progenitors7,26,27 and putative mesenchymal progenitors.14,28 While it has been suggested that small numbers of fetal cells traffic across the placenta in every human pregnancy,29–31 trophoblast release does not appear to occur in all pregnancies.32 Likewise, in mice, fetal cells have also been reported in maternal blood.33,34 In the mouse, fetomaternal transfer also appears to occur during all pregnancies.35 相似文献
5.
A role for SR proteins in plant stress responses 总被引:1,自引:0,他引:1
Paula Duque 《Plant signaling & behavior》2011,6(1):49-54
6.
P Vasantha Rao 《Cell Adhesion & Migration》2008,2(3):170-173
Lens development and differentiation are intricate and complex processes characterized by distinct molecular and morphological changes. The growth of a transparent lens involves proliferation of the epithelial cells and their subsequent differentiation into secondary fiber cells. Prior to differentiation, epithelial cells at the lens equator exit from the cell cycle and elongate into long, ribbon-like cells. Fiber cell elongation takes place bidirectionally as fiber tips migrate both anteriorly and posteriorly along the apical surface of the epithelium and inner surface of the capsule, respectively. The differentiating fiber cells move inward from the periphery to the center of the lens on a continuous basis as the lens grows throughout life. Finally, when fiber cells reach the center or suture line, their basal and apical tips detach from the epithelium and capsule, respectively, and interlock with cells from the opposite direction of the lens and form the suture line. Further, symmetric packing of fiber cells and degradation of most of the cellular organelle during fiber cell terminal differentiation are crucial for lens transparency. These sequential events are presumed to depend on cytoskeletal dynamics and cell adhesive interactions; however, our knowledge of regulation of lens fiber cell cytosketal reorganization, cell adhesive interactions and mechanotransduction, and their role in lens morphogenesis and function is limited at present. Recent biochemical and molecular studies have targeted cytoskeletal signaling proteins, including Rho GTPases, Abl kinase interacting proteins, cell adhesion molecules, myosin II, Src kinase and phosphoinositide 3-kinase in the developing chicken and mouse lens and characterized components of the fiber cell basal membrane complex. These studies have begun to unravel the vital role of cytoskeletal proteins and their regulatory pathways in control of lens morphogenesis, fiber cell elongation, migration, differentiation, survival and mechanical properties.Key words: lens, fiber cells, elongation, migration, adhesion, Rho GTPasesLens morphogenesis involves a complex network of regulatory genes and interplay between growth factor, mitogenic, cell adhesive and cytoskeletal signaling pathways. The lens originates from surface ectoderm near the optic vesicle and lens vesicle that is formed via invagination of lens placode differentiates into primary fibers (the posterior half ) and epithelial cells (the anterior half ). These changes in embryonic cells control the lens distinctive anterior-posterior polarity. Subsequently, the lens grows through the proliferation of epithelial cells and the differentiation of their progeny into secondary fiber cells.1,2 The continuous addition of new fiber cells at the lens periphery leads to a gradual inward movement of older cells to the center of the lens. The ectodermal basement membrane that surrounds the lens vesicle thickens to form the lens capsule and is composed of mainly proteins of extracellular matrix.2,3 Since the lens does not shed cells, they are retained throughout the lens''s life and are packed symmetrically within the lens4 (Fig. 1).Open in a separate windowFigure 1Diagram of organization of lens epithelial and differentiating fiber cells. The lens is enclosed by a thick capsule consisting of various extracellular matrix proteins. Lens epithelial cells at the equator divide and exit from the cell cycle, and as they exit from the cell cycle, they start to elongate bidirectionally by making apical (AMC) and basal (BMC) membrane complexes with epithelium and capsule, respectively. As fiber cells elongate, they are pushed down and migrate toward the center. As the fiber cells migrate toward the center, both the basal and apical membrane complexes are expected to undergo changes in a regulated manner to control fiber cell adhesive, protrusive and contractile activity. Finally, when the fiber cells reach the center or suture line, their basal and apical ends detach from the epithelium and capsule, respectively and interlock with cells from the opposite direction of the lens and form suture. During fiber cell elongation and differentiation, cell adhesive interactions are reorganized extensively, and terminally differentiated fiber cells exhibit loss of cellular organelle and extensive membrane remodeling with unique ball and socket interdigitations. Arrows indicate the direction of fiber cell movement. This schematic is a modified version of Figure 2 from Lovicu and McAvoy.1Lens fiber cell elongation and differentiation is associated with a remarkable change in cell morphology, with the length of fiber cells increasing on the order of several hundredfold. These morphological changes are associated with extensive membrane and cortical cytoskeletal remodeling, actomyosin reorganization and cell adhesion turnover.5–17 Additionally, the tips of the elongating fiber cells at both the anterior and posterior terminals slide along the lens epithelium and capsule, respectively, as these cells migrate inward, and finally detach at the suture, where they form contacts with their counterparts from the opposite side of the lens.4,12 These cell movements are fundamental for maintaining distinct lens fiber cell polarity and are temporally and spatially regulated as the lens grows continuously throughout life.1,2,12 Another unique feature of the lens is that during fiber cell terminal differentiation, all the cellular organelles, including nuclei, endoplasmic reticulum and mitochondria, are degraded in a programmed manner.18 It has been well documented that lens epithelial cell elongation and differentiation is associated with reorganization of actin cytoskeleton, increased ratio of G-actin to F-actin, integrin switching, formation of N-cadherin linked cell adhesions, and expression of actin capping protein tropomodulin.5,6,9,10,13,15,17,19–21 Importantly, disruption of actin cytoskeletal organization has been shown to impair lens epithelial differentiation and induce cataract formation, indicating the significance of actin cytoskeleton in lens differentiation and maintenance of lens optical quality.14,22 Further, during accommodation, lens shape is changed in a reversible manner. Therefore, the tensional homeostasis between actomyosin inside the fiber cell and fiber cell adhesion on the inner side of the lens capsule is considered to be crucial for accommodation.12In the developing mouse and chicken lens, the tips of the fiber cells (both apical and basal) have been reported to cluster with different cytoskeletal proteins, including actin, myosin II, actin capping protein tropomodulin, and N-cadherins.10,19,21 Similarly, adhesion regulating signaling molecules including integrins, focal adhesion kinase, Cdk5, abl kinase interacting protein (Abi-2), and Rho GTPases have been shown to localize to the fiber cell apical and basal tips.20,23–26 Moreover, isolation and characterization of the fiber cell basal membrane complexes (BMCs) had revealed a symmetric organization of N-cadherin, myosin II, actin in association with myosin light chain kinase, focal adhesion kinase, β1 integrin and caldesmon.12 The signaling activity, tensional property and dynamics of BMCs are thought to control the coordinated migration of fiber cells along the lens capsule, formation of lens suture line, and lens accommodation.12 Additionally, the BMCs have been shown to undergo a characteristic regional rearrangement (including size and shape) during lens elongation and migration along the lens capsule.27 Therefore, impaired fiber cell migration on the lens capsule is expected to induce cataractogenesis.27 Taken together, these different observations convincingly indicate the importance of cytoskeleton and cell adhesion regulatory mechanisms in lens fiber cell elongation and migration.Although important insights have emerged regarding external cues controlling lens epithelial cell proliferation, elongation and differentiation, little is known regarding the specific signaling pathways that drive the processes culminating in fiber cell formation, migration, packing and maturation.1,7,28 For example, growth factors are known to play key roles in influencing cell fates during development. Some of the major growth factor families, including FGFs and TGFβ/BMPs, have been shown to be involved in the regulation of lens developmental processes and primary fiber cell differentiation via ERK kinase activation.1,28,29 However, the identity and role of signaling pathways acting downstream to growth factors regulating lens secondary fiber cell elongation, migration, adhesion, membrane remodeling and survival are poorly understood.1,12,21,30 In particular, regulatory mechanisms involved in cytoskeletal reorganization, tensional force and cell adhesive interactions during these cellular processes have yet be identified and characterized.7,9,12,21,30–32Our laboratory has been working on a broad hypothesis that the actin cytoskeletal and cell adhesive signaling mechanisms composed of Rho GTPases (Rho, Rac and Cdc42) and their effector molecules play a critical role in controlling lens growth and differentiation, and in maintaining lens integrity.7 The Rho family of small GTPases regulates morphogenesis, polarity, migration and cell adhesion.33 These proteins bind GTP, exhibit GTPase activity, and cycle between an inactive GDP-bound form and an active GTP-bound form. This cycling is regulated by three groups of proteins: guanine-nucleotide exchange factors, which facilitate the exchange of GDP for GTP, thus rendering Rho GTPases active; GTPase-activating proteins, which regulate the inactivation of Rho by accelerating intrinsic GTPase activity and converting Rho GTPases back to their GDP-bound form; and GDP dissociation inhibitors (GDIs), which inhibit the dissociation of GDP bound to Rho GTPases.33,34 The GTP-bound form of the Rho GTPases interact with downstream effectors, which include protein kinases (e.g., ROCK and PAK), regulators of actin polymerization (e.g., N-WASP/WAVE, PI3-kinase and mDia), and other proteins with adaptor functions.33 The selective interaction of the different Rho GTPases with a variety of effectors determines the final outcome of their activation.33 For example, during cell movement, Rac and Cdc42 stimulate formation of protrusions at the leading edges of cells, and RhoA induces retraction at the tail ends of cells. This coordinated cytoskeletal reorganization permits cells to move toward a target.35 PI3-kinase and PI (3, 4, 5) P3 have also been widely implicated in controlling cell migration and polarity in a Rac GTPase-dependent manner.35 Members of the Wiskott-Aldrich syndrome protein (WASP) and WASP-family verprolin homologous protein (WAVE) families serve to link Rho GTPases signals to the ARP2/3 complex, leading to actin polymerization that is crucial for the reorganization of the actin cytoskeleton at the leading edge for processes such as cell movement and protrusions.36 Importantly, all three Rho GTPases also regulate microtubule polymerization and assembly of adherens junctions to influence polarity and cell adhesion, respectively.33,37Likewise, a tensional balance between cell adhesion on the outside and myosin II-based contractility on the inside of the cells is regulated by Rho GTPases.38To explore the role of the Rho GTPases in lens morphogenesis and differentiation, we have targeted the lens Rho GTPases by overexpressing either the C3 exoenzyme (inactivator of RhoA and RhoB) or RhoGDIα (Rho GDP dissociation inhibitor) in a lens-specific manner in transgenic mice and followed their effects developmentally. These two transgenic mouse models exhibited ocular phenotype, including lens opacity (cataract) and microphthalmic eyes. Importantly, various histological, immunofluorescence and biochemical analyses performed in these developing transgenic mice have revealed defective lens morphogenesis, abnormal fiber cell migration, elongation, disrupted cytoskeletal organization and adhesive interactions, along with changes in proteins of the fiber cell gap junctions and water channels.32,39 These lenses have also shown decreased ERM (ezrin, radixin, moesin) protein phosphorylation,40 proteins that are involved in crosslinking of the plasma membrane with actin cytoskeleton,41 and increased apoptosis.32 Defective fiber cell migration has been found to be more notable in the Rho GDI overexpressing lenses than in the C3 exoenzyme expressing lenses (Fig. 2). The Rho GDI overexpressing lenses have shown a defective membrane localization of Rho, Rac and Cdc42 confirming their inactivation. These data, together with mechanistic studies performed using the lens epithelial cells and the noted effects on cell shape, actin polymerization, myosin phosphorylation and cell adhesive interactions, reveal the importance of Rho GTPase-dependent signaling pathways in processes underlying fiber cell migration, elongation, cytoskeletal and membrane organization and survival in the developing lens.7 Lens fiber cell BMC has been found to be localized intensely with Rac GTPase involved in cell migration (our unpublished work). Additionally, the Rho GDI transgenic lenses showed an impaired apical-apical cell-cell interactions between the fiber cells and epithelial cells.32 Moreover, the ruptured posterior capsule and disrupted suture lines in these lenses are indicative of defective BMC organization and activity.32Open in a separate windowFigure 2Abnormal lens phenotype in the neonatal Rho GDIα overexpressing transgenic mouse. Hematoxylin and eosin-stained sagittal sections of P1 RhoGDIα transgenic eyes reveal abnormal migration and morphology of the posterior lens fibers as compared with the symmetric organization of lens fibers and their migration toward the lens suture in the wild type mouse (reproduced with permission from Maddala et al.)32.Further support for involvement of Rho GTPases in lens fiber cell differentiation and survival has come from studies conducted with chick lens epithelial explants and cultured epithelial cells. Inactivation of Rho kinase or Rac activation by PI3 kinase in chick lens epithelial cells has been reported to induce fiber cell differentiation and survival in association with distinct cortical actin cytoskeletal reorganization, indicating the significance of Rho GTPases in lens fiber cell differentiation and survival.9,42 Additionally, lens fiber cell elongation and differentiation has been found to be associated with increased myosin light chain (MLC) phosphorylation, and inhibition of MLC phosphorylation regulated by MLC kinase and Rho kinase has induced lens opacity and disruption of cytoskeletal integrity, supporting the importance of myosin II activity in maintaining lens architecture and transparency.10 Importantly, various growth factors that regulate lens morphogenesis, fiber cell differentiation, and survival have been found to activate Rho and Rac GTPases and to induce MLC phosphorylation, actin cytoskeletal reorganization, and focal adhesion formation in lens epithelial cells.7,30 In addition to Rho GTPases, inhibition of Src kinase has been shown to induce fiber cell differentiation in association with actin cytoskeletal reorganization and cell adhesive interactions.43 Also, the expression and activation of focal adhesion kinase has been reported to increase in differentiating and migrating lens epithelial cells.44 Both these molecules are well recognized to regulate cell migration by participating in the disassembly of cell adhesions at the front of migrating cells.35Additional evidence for the participation of actin cytoskeletal organization and Rho GTPases in lens fiber cell migration and elongation has been derived from the studies of Abi-2 deficient mouse. Abl-interactor adaptor proteins Abi-1 and Abi-2 are linked to the Rac-WAVE-Arp2/3 signaling pathway and regulate actin polymerization and cell-cell adhesive interactions.45 Homozygous deletion of Abi-2 in mice has been shown to exhibit ocular phenotype including microphthalmia and lens opacity similar to the Rho GDI overexpressing transgenic mouse eyes noted in previous studies.23,32 In the absence of Abi-2, the secondary lens fiber orientation, migration and elongation were found to be defective, supporting the importance of Rac-WAVE-Arp2/3 signaling in lens fiber cell migration and cell adhesion.23 Abi-2 has been shown to localize intensely to the both basal and apical regions of the fiber cells and adherens junctions, and suppression of Abi-2 expression in epithelial cells resulted in impaired adherens junctions and downregulation of actin nucleation promoting factors.23 The significance of cytoskeletal signaling in lens has also been implicated in Lowe syndrome, a rare X-linked disorder characterized by congenital cataracts, results from mutations in the OCRL1 gene. The OCRL1 protein product (phosphatidylinositol 4, 5 bisphosphate 5-phosphatase) has been shown to participate in Rac GTPase regulated actin cytoskeletal organization, cell migration, and cell adhesion in various cell types.46 Finally, Wnt/PCP signaling via activation of Rho GTPases has been suggested to control lens morphogenesis, fiber cell migration and differentiation.26Importantly, given how the activity of the Rho GTPases is regulated by external cues and various effector proteins, a detailed understanding of the regulation of Rho GTPase signaling is necessary for a better appreciation of their role in lens morphogenesis, fiber cell elongation and differentiation, and tensional homeostasis. Further mechanistic studies are critical to unravel the specific role(s) of Rho GTPases and other cytoskeletal regulatory mechanisms involved in regulating the formation and disassembly of fiber cell basal and apical membrane complexes, fiber cell lateral membrane remodeling, and fiber cell-cell adhesive interactions during lens differentiation. Very little is known in terms of the assembly of different cell adhesive molecules at the apical-apical interface between the lens fibers and epithelial cells. We are only beginning to glimpse the regulatory networks involved in the regulation of fiber cell elongation, polarity, migration and adhesion. Many challenging questions remain: for example, how are the pathways regulating migration, basal and apical membrane complexes, and tensional homeostasis controlled by extracellular signals, and how are they integrated during fiber cell migration, suture formation, and packing? Novel insights into the molecular mechanisms regulating these cellular processes are expected to advance our understanding of lens morphogenesis, function and cataractogenesis. 相似文献
7.
In addition to mediating cell adhesion, many cell adhesion molecules act as tumor suppressors. These proteins are capable of restricting cell growth mainly through contact inhibition. Alterations of these cell adhesion molecules are a common event in cancer. The resulting loss of cell-cell and/or cell-extracellular matrix adhesion promotes cell growth as well as tumor dissemination. Therefore, it is conventionally accepted that cell adhesion molecules that function as tumor suppressors are also involved in limiting tumor cell migration. Paradoxically, in 2005, we identified an immunoglobulin superfamily cell adhesion molecule hepaCAM that is able to suppress cancer cell growth and yet induce migration. Almost concurrently, CEACAM1 was verified to co-function as a tumor suppressor and invasion promoter. To date, the reason and mechanism responsible for this exceptional phenomenon remain unclear. Nevertheless, the emergence of these intriguing cell adhesion molecules with conflicting roles may open a new chapter to the biological significance of cell adhesion molecules.Key words: hepaCAM, cell adhesion molecules, tumor suppressor, migration, E-cadherin, CADM1, integrin α7, CEACAM1It is well known that many cell adhesion molecules function as tumor suppressors (reviewed in ref. 1). These molecules exert their tumor suppressive effect mainly through cell-adhesion-mediated contact inhibition. Cell adhesion molecules allow cells to communicate with one another or to the extracellular environment by mediating cell-cell or cell-extracellular matrix (ECM) interactions (reviewed in refs. 2 and 3). Broadly, these proteins can be classified into five families including immunoglobulin superfamily, integrins, cadherins, selectins and CD44. Apart from participating in the development and maintenance of tissue architecture, cell adhesion molecules serve as cell surface receptors critical for capturing, integrating and transmitting signals from the extracellular milieu to the cell interior (reviewed in refs. 2 and 3). These signaling events are vital for the regulation of a wide variety of cellular functions including embryogenesis, immune and inflammatory responses, tissue repair, cell migration, differentiation, proliferation and apoptosis. Alterations of these cell adhesion molecules are a common event in cancer (reviewed in refs. 1, 2, 4 and 5). The disrupted cell-cell or cell-ECM adhesion significantly contributes to uncontrolled cell proliferation and progressive distortion of normal tissue architecture. More importantly, changes in cell adhesion molecules play a causal role in tumor dissemination. Loss of cell adhesion contacts allows malignant cells to detach and to escape from the primary mass. Gaining a more motile and invasive phenotype, these cells break down the ECM and eventually invade and metastasize to distal organs.Based on the above understanding, it is conventionally accepted that cell adhesion molecules with tumor suppressor activity, when expressed in cancer cells, are able to exert inhibitory effect on cell motility. The ability of cells in migration/motility is a prerequisite for cancer invasion and metastasis (reviewed in refs. 1 and 5). Indeed, a number of cell adhesion molecule-tumor suppressors have been reported to be capable of reducing cell migration. The most classical example is E-cadherin, a calcium-dependent cell adhesion molecule. E-cadherin is expressed exclusively in epithelial cells and its expression is commonly suppressed in tumors of epithelial origins. The cytoplasmic domain of E-cadherin interacts with catenins to establish an intracellular linkage with the actin cytoskeleton (reviewed in ref. 6). The assembly of E-cadherin with the cytoskeleton via catenins at the sites of adherens junctions is important for the stabilization of cell-cell adhesions. Disruption of E-cadherin-mediated cell-cell adhesion, due to loss of expression or function of E-cadherin and/or catenins, is assocated with tumor development and progression (reviewed in ref. 7). Forced expression of E-cadherin in several cancer cell lines not only slows down cell growth8,9 but also significantly reduces the invasiveness of the cells.10,11 On the other hand, inhibition of E-cadherin by function-blocking antibodies and antisense RNA restores the invasiveness in non-invasive transformed cells.11 Furthermore, using a transgenic mouse model of pancreatic beta-cell carcinogenesis, it has been demonstrated that E-cadherin-mediated cell adhesion is important in preventing the transition from well differentiated adenoma to invasive carcinoma.12Cell adhesion molecule 1 (CADM1), another example, has also been implicated in cancer progression. CADM1 is a member of the immunoglobulin superfamily and mediates cell-cell adhesion.13 The molecule associates with the actin cytoskeleton via the differentially expressed in adenocarcinoma of the lung (DAL1) protein; and the formation of CADM1-DAL1 complex is dependent on the integrity of actin cytoskeleton.14 Inactivation of the CADM1 and/or DAL1 gene usually through methylation has been reported in diverse human cancers.15,16 A paper by Ito et al. showed that restoration of CADM1 expression in esophageal squamous cell carcinoma cells not only suppresses cell growth, but also retards cell motility and invasion.16In contrast to E-cadherin and CADM1, integrin α7 is a cell-ECM adhesion molecule which also possesses tumor suppressor activity. Ren et al. showed that integrin α7 gene is mutated in several human malignances; and the mutations are associated with an increase in cancer recurrence.17 Forced expression of integrin α7 in integrin α7-deficient leiomyosarcoma cells results in decreased colony formation and slower cell motility. Conversely, knockdown of integrin α7 in lung cancer cells expressing wild-type integrin α7 increases the colony number and cell motility rate. In addition, the researchers revealed that mice bearing xenograft tumors overexpressing integrin α7 have reduced tumor size with no obvious metastasis.In 2005, we first reported the identification of a cell adhesion molecule belonging to the immunoglobulin superfamily, designated as hepaCAM.18 To date, we have shown that the gene is frequently downregulated in a variety of human cancers.18,19 Re-expression of hepaCAM in the hepatocellular carcinoma HepG2 cells18 and breast cancer MCF7 cells19 inhibits colony formation and retards cell proliferation. In addition, expression of hepaCAM in MCF7 cells results in cell cycle arrest at the G2/M phase and cellular senescence. Concurrently, the expression of several senescence-associated proteins including p53, p21 and p27 is enhanced. Moreover, downregulation of p53 by p53-specific small interfering RNA in cells expressing hepaCAM clearly reduces p21 without changing p27 and alleviates senescence, indicating that hepaCAM induces senescence through a p53/p21-dependent pathway.19 Together, the data suggest that hepaCAM is a tumor suppressor. Interestingly, the expression of hepaCAM in both HepG2 and MCF7 cells stimulates both cell-ECM adhesion and cell migration.18,20,21 The function of hepaCAM as a tumor suppressor in cell migration is contradictory to other cell adhesion molecule-tumor suppressors. Noteworthily, hepaCAM-mediated cell motility is evidenced by its direct interaction with the actin cytoskeleton.21Evidences are currently emerging to support the contradictory roles of cell adhesion molecules that both inhibit cell growth and promote cell motility when restored in cancer cells. In addition to hepaCAM, the immunoglobulin superfamily carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is implicated to function as a tumor suppressor and a metastasis promoter. The characteristics and functions of CEACAM1 have been demonstrated in individual reports. CEACAM1 is frequently downregulated or dysregulated in multiple human tumors,22–25 and is capable of suppressing cell growth and inducing apoptosis.26–28 Ebrahimnejad et al. demonstrated that exogenous expression of CEACAM1 enhances melanoma cell invasion and migration; and this enhanced motility can be reverted by anti-CEACAM antibodies.29 The ability of CEACAM to co-stimulate tumor suppression and invasion was finally established by Liu et al. in restricting thyroid cancer growth but promoting invasiveness.30 Introduction of CEACAM1 into CEACAM1-deficient thyroid cancer cells results in G1/S phase cell cycle arrest accompanied by elevated p21 expression and diminished Rb phosphorylation. Overexpression of CEACAM1 also increases cell-ECM adhesion and promotes cell migration and tumor invasiveness. In xenografted mice, CEACAM1 expression results in reduced tumor growth but increased tumor invasiveness. Conversely, silencing of endogenous CEACAM1 accelerates tumor growth and suppresses invasiveness.30It is an exciting issue to address why a cell adhesion molecule is able to suppress tumor growth yet promote tumor progression. Could there be a molecular switch that controls the functions of the gene between a tumor suppressor and a migration promoter in cancer or are the functions executed simultaneously? The expression level, the extracellular cues as well as the interacting partners of the cell adhesion molecules may likely play a critical role in regulating its functions. The question is under what circumstances these factors come into play. To answer all these questions, and maybe more, on the intriguing findings of these proteins, more extensive and intensive experimentation is required. Nevertheless, it is obvious that the emergence of these cell adhesion molecules that function in a contradictory manner opens a new chapter to the biological significance of cell adhesion molecules. 相似文献
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The glucocorticoids (GCs), the most downstream effectors of the hypothalamic-pituitary-adrenal (HPA) axis, are the main mediators of stress response. Stress-triggered GCs as well as acute and chronic GC treatment can impair the structural plasticity and function of the brain. The exposure of perinatal animals and humans to excess stress or GCs can affect the brain development, resulting in altered behaviors in the adult offspring of animals and an increased risk of psychiatric disorders in humans. Despite the numerous studies documenting these effects, the underlying mechanism remains unclear. In this commentary we will focus on the effect of excess GCs on cortical development. We have recently showed that excess-GC-dependent retardation of the radial migration of neural progenitor cells (NPCs) is caused by the dysregulation of actin-myosin interaction via upregulation of caldesmon (CaD), an actin-linked regulatory protein. The elucidation of the molecular mechanisms that underlie the detrimental action of GCs on cortical development will expand our understanding of how stress/GCs alter the formation of neural networks and affect behaviors later in life.Key words: neuronal migration, actin-myosin interaction, cortical development, stress, psychiatric disorderIt has been well documented that the elevation of GCs in response to stress impairs the development and function of the brain.1–3 Prenatal stress is associated with an increase in abnormal behaviors of adult offspring.2 In humans, maternal stress increases the risk of psychiatric disorders, including anxiety, depression and schizophrenia, in the offspring during adolescence and adulthood.3–6 The treatment of neonatal rats with GC transiently retards their brain development,3,5,7 and giving GC to pregnant sheep retards fetal brain growth.8 Antenatal exposure to synthetic GC in humans results in a reduced cortex convolution index and surface area in infants.9 Thus, excessive stress or GC exposure during the perinatal stages affects the brain development and subsequently causes abnormal behaviors in experimental animals and an increased incidence of psychiatric disorders in humans. Despite the numerous studies documenting these detrimental effects of GCs, the underlying mechanism remains unclear.Cortical development progresses by two types of neuronal migration: radial and tangential.10,11 Postmitotic NPCs in the ventricular zone (VZ) migrate radially along radial glial fibers toward the surface of the neocortex, until they reach their final destination within the cortical plate (CP). About 80–90% of all cortical neurons arise from NPCs by radial migration. The remaining cells, which include the majority of GABAergic interneurons, migrate tangentially from the ganglionic eminence to the neocortex. Several human disorders that arise from defective neuronal migration have been identified. These disorders include periventricular nodular heterotopia and lissencephaly, which are caused by mutations in the genes involved in radial migration.12 More commonly than gene mutations, however, environmental factors such as stress-triggered GCs as described above are implicated in inducing abnormalities in brain development. Most recently, we have found that excess GCs cause a change in radial migration during cortical development via the dysregulation of actomyosin system by GC-induced upregulation of CaD.13 相似文献
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Peptide signaling regulates a variety of developmental processes and environmental responses in plants.1–6 For example, the peptide systemin induces the systemic defense response in tomato7 and defensins are small cysteine-rich proteins that are involved in the innate immune system of plants.8,9 The CLAVATA3 peptide regulates meristem size10 and the SCR peptide is the pollen self-incompatibility recognition factor in the Brassicaceae.11,12 LURE peptides produced by synergid cells attract pollen tubes to the embryo sac.9 RALFs are a recently discovered family of plant peptides that play a role in plant cell growth.Key words: peptide, growth factor, alkalinization 相似文献
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Junctional Adhesion Molecule A (JAM-A) is a member of the Ig superfamily of membrane proteins expressed in platelets, leukocytes, endothelial cells and epithelial cells. We have previously shown that in endothelial cells, JAM-A regulates basic fibroblast growth factor, (FGF-2)-induced angiogenesis via augmenting endothelial cell migration. Recently, we have revealed that in breast cancer cells, downregulation of JAM-A enhances cancer cell migration and invasion. Further, ectopic expression of JAM-A in highly metastatic MDA-MB-231 cells attenuates cell migration, and downregulation of JAM-A in low-metastatic T47D cells enhance migration. Interestingly, JAM-A expression is greatly diminished as breast cancer disease progresses. The molecular mechanism of this function of JAM-A is beyond its well-characterized barrier function at the tight junction. Our results point out that JAM-A differentially regulates migration of endothelial and cancer cells.Key words: JAM-A, integrin, αvβ3, FGF-2, breast cancer, cell migration and invasion, T47D, MDA-MB-231, siRNAEndothelial and epithelial cells exhibit cell polarity and have characteristic tight junctions (TJs) that separate apical and basal surfaces. TJs are composed of both transmembrane and cytoplasmic proteins. The three major families of transmembrane proteins include claudins, occludin and JAM family members.1–3 Additionally, interaction between the peripheral proteins such as PDS-95/Discs large/ZO family (PDZ) domain-containing proteins in TJs plays an important role in maintaining the junctional integrity.2,4,5JAMs are type I membrane proteins (Fig. 1) predominately expressed in endothelial and epithelial cell TJs, platelets and some leukocytes.6–8 The classical JAMs are JAM-A, JAM-B and JAM-C, which can all regulate leukocyte-endothelial cell interaction through their ability to undergo heterophilic binding with integrins αLβ2 or αvβ3, α4β1 and αMβ2 respectively. The cytoplasmic tail of JAMs contains a type II PDZ-domain-binding motif (Fig. 1) that can interact with the PDZ domain containing cytoplasmic molecules such as ZO-1, ASIP/PAR-3 or AF-6.9,10 Additionally, consistant with their junctional localization and their tendency to be involved in homophilic interactions, JAMs have been shown to modulate paracellular permeability and thus may play an important role in regulating the epithelial and endothelial barrier.11,12 In addition, ectopic expression of JAM-A in CHO cells promotes localization of ZO-1 and occludin at points of cell contacts, which suggests a role for JAM-A in TJ assembly.10,13,14 Recently, it has been shown that JAM-A regulates epithelial cell morphology by modulating the activity of small GTPase Rap1 suggesting a role for JAM-A in intracellular signaling.15Open in a separate windowFigure 1Schematic representation of the domain structure of JAM family proteins. V, variable Ig domain; C2, constant type 2 Ig domain; TM, transmembrane domain; T-II, Type II PDZ-domain binding motif.We have previously shown that JAM-A is a positive regulator of fibroblast growth factor-2 (FGF-2) induced angiogenesis.16 Evidence was provided to support the notion that JAM-A forms a complex with integrin αvβ3 at the cell-cell junction in quiescent human umbilical cord vein endothelial cells (HUVECs) and FGF-2 dissociates this complex.16 It was further established that inhibition of JAM-A using a function-blocking antibody also inhibits FGF-2 induced HUVECs migration in vitro and angiogenesis in vivo. Overexpression of JAM-A induced a change in HUVECs morphology similar to that observed when treated with FGF-2.17 Furthermore, overexpression of JAM-A, but not its cytoplasmic domain deletion mutant, augmented cell migration in the absence of FGF-2.17 In addition, downregulation of JAM-A in HUVECs using specific siRNA, resulted in reduced FGF-2-induced cell migration and inhibition of mitogen activated protein (MAP) kinase activation.18 These findings clearly suggested that JAM-A positively regulates FGF-2-induced endothelial cell migration. This was further confirmed in vivo by using JAM-A null mouse in which FGF-2 failed to support angiogenesis.19It is known that JAM-C, a JAM family member, is involved in the process of tumor cell metastasis.20 However, little is known about JAM-A''s role in cancer progression. We recently found that JAM-A is expressed in breast cancer tissues and cell lines.21 Based on our studies with endothelial cells it was felt that JAM-A expression in breast cancer cells may also enhance the migratory ability of these cells. Surprisingly, we found an inverse relation between the expression of JAM-A and the metastatic ability of breast cancer cells. T47D cells, which express high levels of JAM-A, are the least migratory; whereas MDA-MB-231 cells, which are highly migratory, are found to express the least amount of JAM-A.21 We also found that overexpression of JAM-A in MDA-MB-231 cells caused a change in cell morphology from spindle-like to rounded shape and formed cobblestone-like clusters.21 This is consistent with the previous report, that downregulation of JAM-A expression from epithelial cells using siRNA results in the change of epithelial cell morphology.15 This change in cell morphology by knockdown of JAM-A was attributed to the disruption of epithelial cell barrier function.15 It was further shown that knockdown of JAM-A affects epithelial cell morphology through reduction of β1integrin expression due to decreased Rap1 activity.15 Our observed effect of JAM-A downregulation in T47D cells, however, is not due to downregulation of β1integrin, since the level of this integrin was not affected in these cells. Interestingly, overexpression of JAM-A significantly affected both the cell migration and invasion of MDA-MB-231 cells. Furthermore, knockdown of JAM-A using siRNA enhanced invasiveness of MDA-MB-231 cells, as well as T47D cells.21 The ability of JAM-A to attenuate cell invasion was found to be due to the formation of functional tight junctions as observed by distinct accumulation of JAM-A and ZO-1 at the TJs and increased transepithelial resistance. These results identify, for the first time, a tight junctional cell adhesion protein as a key negative regulator of breast cancer cell migration and invasion.21JAM-A has been shown to be important in maintaining TJ integrity.15,22–25 Disruption of TJs has been implicated to play a role in cancer cell metastasis by inducing epithelial mesenchymal transition.26 Several laboratories, including ours, have shown that cytokines and growth factors redistribute JAM-A from TJs.16,27,28 Consistent with this finding, it has been shown that hepatocyte growth factor (HGF) disrupts TJs in human breast cancer cells and downregulates expression of several TJ proteins.29 It is therefore conceivable that the loss of JAM-A in highly metastatic cells is a consequence of disruption of TJs. This was further supported by the findings that overexpression of JAM-A forms functional TJs in MDA-MB-231 cells and attenuates their migratory behavior. Our result is the first report correlating an inverse relationship of JAM-A expression in breast cancer cells to their invasive ability.21Using cDNA microarray technology, it has been revealed how genes involved in cell-cell adhesion, including those of the TJ, are under or overexpressed in different carcinomas.15,30 Cell-cell adhesion molecules have been well documented to regulate cancer cell motility and invasion. Of these, the cadherin family have been studied the most.31,32 It was proposed that a cadherin switch, that is, the loss of E-cadherin and subsequent expression of N-cadherin, may be responsible for breast cancer cell invasion.33,34 Although the role of cadherins is well-documented, it remains controversial since some breast cancer cell lines that do not express these proteins still posses highly invasive characteristics.33,34 However, the observed effect of overexpression of JAM-A does not appear to be simply due to the formation of TJs, since individual cells that express increased JAM-A show reduced migration.21 This is not surprising, considering the fact that JAM-A in addition to its function of regulating TJ integrity is also shown to participate in intracellular signaling. JAM-A is capable of interacting homotypically as well as heterotypically on the cell surface.35,36 It has also been shown that it interacts with several cytoplasmic proteins through its PDZ domain-binding motif and recruits signaling proteins at the TJs.37 Recent findings using site-directed mutagenesis suggest that cis-dimerization of JAM-A is necessary for it to carry out its biological functions.38 Our own observations suggest that a JAM-A function-blocking antibody inhibits focal adhesion formation in endothelial cells (unpublished data), whereas overexpresion of JAM-A in MDA-MB-231 cells show increased and stable focal adhesions.21 It is therefore conceivable that in quiescent endothelial/epithelial cells JAM-A associates with integrin to form an inactive complex at the TJ (Fig. 2). Growth factors such as FGF-2 signaling dissociates this complex thus allowing dimerization of JAM-A and activation of integrin augmenting cell migration (Fig. 2). On the contrary, in MDA-MB-231 cancer cells, which express low levels of JAM-A and do not form tight junctions, there may not be efficient inactive complex formation between JAM-A and integrin. Overexpression of JAM-A in these cells however, may promote such inactive complex formation leading to inhibition of integrin activation and JAM-A dimerization, both necessary events for cell migration. We are currently in the process of determining the specificity of interaction of JAM-A with integrins. Further experimentation is ongoing to determine the contribution of JAM-A dependent signaling in cell migration.Open in a separate windowFigure 2Schematic representation of JAM-A regulation of cell migration. JAM-A forms an inactive complex with the integrin and sequesters it at the TJs. Growth factor signaling dissociates this complex, promoting integrin activation and JAM-A dimerization leading to cell migration via MAP kinase activation. Ectopic expression of JAM-A in cancer cells may induce its association with integrin, forming an inactive complex and hence attenuation of migration.JAM-A differentially regulates cell migration in endothelial and cancer cells due to its ability to form inactive complex with integrin, making it a metastasis suppressor. The downregulation of JAM-A in carcinoma cells may be detrimental to the survival of breast cancer patients. It is therefore very important to determine the molecular determinants that are responsible for the downregulation of JAM-A during cancer progression. Thus, JAM-A, a molecule that dictates breast cancer cell invasion, could be used as a prognostic marker for metastatic breast cancer. 相似文献
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Stephen J Demarest Mangala Hariharan Marikka Elia Jared Salbato Ping Jin Colin Bird Jay M Short Bruce E Kimmel Michael Dudley Gary Woodnutt Geneviève Hansen 《MABS-AUSTIN》2010,2(2):190-198
The pathogenicity of Clostridium difficile (C. difficile) is mediated by the release of two toxins, A and B. Both toxins contain large clusters of repeats known as cell wall binding (CWB) domains responsible for binding epithelial cell surfaces. Several murine monoclonal antibodies were generated against the CWB domain of toxin A and screened for their ability to neutralize the toxin individually and in combination. Three antibodies capable of neutralizing toxin A all recognized multiple sites on toxin A, suggesting that the extent of surface coverage may contribute to neutralization. Combination of two noncompeting antibodies, denoted 3358 and 3359, enhanced toxin A neutralization over saturating levels of single antibodies. Antibody 3358 increased the level of detectable CWB domain on the surface of cells, while 3359 inhibited CWB domain cell surface association. These results suggest that antibody combinations that cover a broader epitope space on the CWB repeat domains of toxin A (and potentially toxin B) and utilize multiple mechanisms to reduce toxin internalization may provide enhanced protection against C. difficile-associated diarrhea.Key words: Clostridium difficile, toxin neutralization, therapeutic antibody, cell wall binding domains, repeat proteins, CROPs, mAb combinationThe most common cause of nosocomial antibiotic-associated diarrhea is the gram-positive, spore-forming anaerobic bacillus Clostridium difficile (C. difficile). Infection can be asymptomatic or lead to acute diarrhea, colitis, and in severe instances, pseudomembranous colitis and toxic megacolon.1,2The pathological effects of C. difficile have long been linked to two secreted toxins, A and B.3,4 Some strains, particularly the virulent and antibiotic-resistant strain 027 with toxinotype III, also produce a binary toxin whose significance in the pathogenicity and severity of disease is still unclear.5 Early studies including in vitro cell-killing assays and ex vivo models indicated that toxin A is more toxigenic than toxin B; however, recent gene manipulation studies and the emergence of virulent C. difficile strains that do not express significant levels of toxin A (termed “A− B+”) suggest a critical role for toxin B in pathogenicity.6,7Toxins A and B are large multidomain proteins with high homology to one another. The N-terminal region of both toxins enzymatically glucosylates small GTP binding proteins including Rho, Rac and CDC42,8,9 leading to altered actin expression and the disruption of cytoskeletal integrity.9,10 The C-terminal region of both toxins is composed of 20 to 30 residue repeats known as the clostridial repetitive oligopeptides (CROPs) or cell wall binding (CWB) domains due to their homology to the repeats of Streptococcus pneumoniae LytA,11–14 and is responsible for cell surface recognition and endocytosis.12,15–17C. difficile-associated diarrhea is often, but not always, induced by antibiotic clearance of the normal intestinal flora followed by mucosal C. difficile colonization resulting from preexisting antibiotic resistant C. difficile or concomitant exposure to C. difficile spores, particularly in hospitals. Treatments for C. difficile include administration of metronidazole or vancomycin.2,18 These agents are effective; however, approximately 20% of patients relapse. Resistance of C. difficile to these antibiotics is also an emerging issue19,20 and various non-antibiotic treatments are under investigation.20–25Because hospital patients who contract C. difficile and remain asymptomatic have generally mounted strong antibody responses to the toxins,26,27 active or passive immunization approaches are considered hopeful avenues of treatment for the disease. Toxins A and B have been the primary targets for immunization approaches.20,28–33 Polyclonal antibodies against toxins A and B, particularly those that recognize the CWB domains, have been shown to effectively neutralize the toxins and inhibit morbidity in rodent infection models.31 Monoclonal antibodies (mAbs) against the CWB domains of the toxins have also demonstrated neutralizing capabilities; however, their activity in cell-based assays is significantly weaker than that observed for polyclonal antibody mixtures.33–36We investigated the possibility of creating a cocktail of two or more neutralizing mAbs that target the CWB domain of toxin A with the goal of synthetically re-creating the superior neutralization properties of polyclonal antibody mixtures. Using the entire CWB domain of toxin A, antibodies were raised in rodents and screened for their ability to neutralize toxin A in a cell-based assay. Two mAbs, 3358 and 3359, that (1) both independently demonstrated marginal neutralization behavior and (2) did not cross-block one another from binding toxin A were identified. We report here that 3358 and 3359 use differing mechanisms to modify CWB-domain association with CHO cell surfaces and combine favorably to reduce toxin A-mediated cell lysis. 相似文献
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Cell motility is a highly coordinated multistep process. Uncovering the mechanism of myosin II (MYO2) activation responsible for the contractility underlying cell protrusion and retraction provides clues on how these complementary activities are coordinated. Several protein kinases have been shown to activate MYO2 by phosphorylating the associated myosin light chain (MLC). Recent work suggests that these MLC kinases are strategically localized to various cellular regions during cell migration in a polarized manner. This localization of the kinases together with their specificity in MLC phosphorylation, their distinct enzymatic properties and the distribution of the myosin isoforms generate the specific contractile activities that separately promote the cell protrusion or retraction essential for cell motility.Key words: myosin, MLCK, ROK, MRCK, phosphorylation, cell migrationCell movement is a fundamental activity underlying many important biological events ranging from embryological development to immunological responses in the adult. A typical cell movement cycle entails polarization, membrane protrusion, formation of new adhesions, cell body translocation and finally rear retraction.1 A precise temporal and spatial coordination of these separate steps that take place in different parts of the cell is important for rapid and efficient movement.2One major event during eukaryotic cell migration is the myosin II (MYO2)-mediated contraction that underlies cell protrusion, traction and retraction.1,3 An emerging theme from collective findings is that there are distinct myosin contractile modules responsible for the different functions which are separately regulated by local myosin regulatory light chain (MLC) kinases. These kinases contribute to contractile forces that connect adhesion, protrusion and actin organization.2 Unraveling the regulation of these contractile modules is therefore pivotal to a better understanding of the coordination mechanism.At the lamellipodium, the conventional calcium/calmodulin-dependent myosin light chain kinase (MLCK) has been shown to play an essential role in a Rac-dependent lamellipodial extension.4 Inhibition of calmodulin or MLCK activity by specific photoactivatable peptides in motile eosinophils effectively blocks lamellipodia extension and net movement.5 Furthermore, there is a strong correlation between activated MLCK and phosphorylated MLC within the lamellipodia of Ptk-2 cells as revealed by fluorescence resonance energy transfer (FRET) analysis.6 More recent studies showed MLCK to regulate the formation of focal complexes during lamellipodia extension.7,8 Functionally, MLCK is thought to play a critical role in the environment-sensing mechanism that serves to guide membrane protrusion. It mediates contraction that exerts tension on integrin-extracellular matrix (ECM) interaction, which, depending on the rigidity of the substratum, will lead to either stabilization of adhesion resulting in protrusion or destabilization of attachment seen as membrane ruffling on non-permissive surfaces.8,9As a Rho effector, Rho-associated kinase (ROK/ROCK/Rho-kinase) has been shown to regulate stress fibers and focal adhesion formation by activating myosin, an effect that can be blocked by the specific ROK inhibitor Y-27632.10,11 Myosin activation by ROK is the effect of two phosphorylation events: the direct phosphorylation on MLC and the inhibition of myosin phosphatase through phosphorylation of its associated myosin-binding subunit (MBS).11 Consistent with this notion of a localization-function relationship, ROK and MBS, which can interact simultaneously with activated RhoA,11 have been shown to colocalize on stress fibers.12,13 In migrating cells, Rho and ROK activities have been mostly associated with the regulation of tail retraction, as inhibition of their activities often results in trailing tails due to the loss of contractility specifically confined to the cell rear.14,15 Tail retraction requires high contractile forces to overcome the strong integrin-mediated adhesion established at the rear end, an event which coincides with the strategic accumulation of highly stable and contractile stress fibers that assemble at the posterior region of migrating cells.MRCK was previously shown to phosphorylate MLC and promote Cdc42-mediated cell protrusion.16 More recently, it was found to colocalize extensively with and regulate the dynamics of a specific actomyosin network located in the lamella and cell center, in a Cdc42-dependent manner but independent of MLCK and ROK.17 The lamellar actomyosin network physically overlaps with, but is biochemically distinct from the lamellipodial actin meshwork.9,18 The former network consists of an array of filaments assembled in an arrangement parallel to the leading edge, undergoing continuous retrograde flow across the lamella, with their disassembly occurring at the border of the cell body zone sitting in a deeper region.17–19 Retrograde flow of the lamellar network plays a significant role in cell migration as it is responsible for generating contractile forces that support sustained membrane protrusion and cell body advancement.17–19It is therefore conceivable that these three known MLC kinases are regulated by different signaling mechanisms at different locations and on different actomyosin contractile modules. The coordination of the various modules will ensure persistent directional migration (Figure 1). Phosphorylation of MLC by PAK and ZIP kinase has also been reported, but their exact roles in this event have yet to be determined.20,21 It is also noteworthy that individual kinases can work independently of each other, as amply shown by evidence from inhibitor treatments. This is particularly true for MRCK in the lamella, whose activity on lamellar actomyosin flow is not affected by ML7 and Y-27632, the inhibitors of MLCK and ROK respectively.17 These findings further indicate that although both ROK and MRCK have been shown to upregulate phosphorylated MLC levels by inhibiting the myosins phosphatases,11,22 they are likely to act as genuine MLC kinases themselves, without the need of MLCK as previously suggested.11Open in a separate windowFigure 1Upper panel depicts a model for the specific activation of the different MLC kinases at various locations in the cell. In response to upstream signals, MLC kinases MLCK, MRCK and ROK are activated and localized to different regions. In the case of MRCK and ROK, the interaction of the GTP-bound Rho GTPase binding domain will determine the specific action of the downstream kinase, resulting in actomyosin contractility at different locations. The coordination of these signalling events is crucial for directional cell migration. Lower panel shows a typical front-rear location for Myosin 2A and 2B in a migrating U2OS cell.In conjunction with their differences in localization, the three MLC kinases show apparent individual preferences and specificity towards the MYO2 isoforms that they associate with. The two major MYO2 isoforms MYO2A and 2B are known to have distinct intracellular distributions that are linked to their individual functions (Figure 1).23,24 In motile cells, MYO2A localization that is skewed towards the protruding cell front is consistent with it being the major myosin 2 component of the lamellar filaments regulated by MRCK as well as its regulation by MLCK in lamellipodial contraction.8,17,19 In contrast, the enrichment of MYO2B at retracting cell rear conforms well with the requirement of thick and stable stress fibers capable of causing tail contraction and prevention of protrusion under the control of Rho/ROK signaling.23,25 The selection for MYO2B filaments in the cell rear stems from their more contractile and stable nature compared with MYO2A, a consequence of their higher time-averaged association with actin.26,27 Conversely, the lower tension property of MYO2A filaments suggests that they are more dynamic in nature,26,27 a characteristic which fits well with the dynamic actomyosin activities at the leading edge and lamella that regulate protrusion.It deserves special mention that the three MLC kinases display subtle differences in their specificity towards MLC. While MLCK and MRCK phosphorylate only a single Ser19 site (monophosphorylation),18,28 ROK is able to act on both Thr18 and Ser19 residues causing diphosphorylation of MLC,29 MLCK only causes diphosphorylation when present at higher concentrations.30 By further increasing its actin-activated ATPase activity, diphosphorylation of MLC has been shown to induce a higher myosin activation and filament stability.30–32 The use of specific antibodies that can differentiate between the two populations of phosphorylated MLC has been instrumental in revealing their localization and correlation with the activity of the MLC kinases. The emerging picture from these experiments is that mono and diphosphorylated MLC exhibit distinct distributions in migrating cells, with the monophosphorylated MLC localized more towards the protrusive region, while the diphosphorylated form is more enriched at the posterior end.21,33 Taking into account their biochemical properties, the polarized distributions of these differentially phosphorylated MLC coincide functionally with the segregation of the MYO2 isoforms and their corresponding regulators. These findings provide further support for the existence of segregated contractile modules in migrating cell and their distinctive regulation.The mechanisms that determine the specific segregation of the contractile modules and their regulation are unclear. However, some clues have emerged from recent studies. It has been shown that the C-terminal coiled-coil region of MYO2B is important for determining its localization in cell rear25 and which requires Rho/ROK activity as their inhibition resulted in the loss of this specific localization.23 Correspondingly, the inhibition of MRCK activity resulted in the loss of lamella-localized MYO2A.17 These findings suggest that activation of MYO2 filaments by their upstream regulators is important for their functional segregation and maintenance. It is noteworthy that both ROK and MRCK have distinct regulatory domains including the pleckstrin homology domains which have been shown to be essential for their localization, a process which may involve myosin interaction and lipid-dependent targeting as has been respectively shown for ROK and MRCK.11,13,16 Further, the specificity of MRCK for lamellar actomyosin is believed to be largely determined by the two proteins it forms a complex with: the adaptor LRAP35a, and the MYO2-related MYO18A. Activation of MYO18A by MRCK, a process bridged by LRAP35a, is a crucial step which facilitates MRCK regulation on lamellar MYO2A.17The mechanisms responsible for segregating the contractile modules and their regulators may also comprise a pathway that parallels the microtubule-modulatory Par6/aPKC/GSK3β signalling pathway which regulates cellular polarization. This notion is supported by both Cdc42 and Rho being common upstream regulators of these two pathways.34 GTPase activation may determine the localized activities of the separate contractile modules and create an actomyosin-based asymmetry across the cell body, which together with the microtubule-based activities, result in the formation of a front-back axis important for directional movement. The involvement of MRCK in MTOC reorientation and nuclear translocation events,35 and our unpublished observation that LRAP35a has a GSK3β-dependent microtubule stabilizing function are supportive of a possible cross-talk between these two pathways.In conclusion, the complex regulation of contractility in cell migration emphasizes the importance of the localization, specificity and enzymatic properties of the different MLC kinases and myosin isoforms involved. The initial excitement and confusion caused by the emergence of the different MLC kinases are fading, being now overtaken by the curiosity about how they cooperate and are coordinated while promoting cell motility. 相似文献
16.
17.
Organelle movement in plants is dependent on actin filaments with most of the organelles being transported along the actin cables by class XI myosins. Although chloroplast movement is also actin filament-dependent, a potential role of myosin motors in this process is poorly understood. Interestingly, chloroplasts can move in any direction and change the direction within short time periods, suggesting that chloroplasts use the newly formed actin filaments rather than preexisting actin cables. Furthermore, the data on myosin gene knockouts and knockdowns in Arabidopsis and tobacco do not support myosins'' XI role in chloroplast movement. Our recent studies revealed that chloroplast movement and positioning are mediated by the short actin filaments localized at chloroplast periphery (cp-actin filaments) rather than cytoplasmic actin cables. The accumulation of cp-actin filaments depends on kinesin-like proteins, KAC1 and KAC2, as well as on a chloroplast outer membrane protein CHUP1. We propose that plants evolved a myosin XI-independent mechanism of the actin-based chloroplast movement that is distinct from the mechanism used by other organelles.Key words: actin, Arabidopsis, blue light, kinesin, myosin, organelle movement, phototropinOrganelle movement and positioning are pivotal aspects of the intracellular dynamics in most eukaryotes. Although plants are sessile organisms, their organelles are quickly repositioned in response to fluctuating environmental conditions and certain endogenous signals. By and large, plant organelle movements and positioning are dependent on actin filaments, although microtubules play certain accessory roles in organelle dynamics.1,2 Actin inhibitors effectively retard the movements of mitochondria,3–6 peroxisomes,5,7–11 Golgi stacks,12,13 endoplasmic reticulum (ER),14,15 and nuclei.16–18 These organelles are co-aligned and associated with actin filaments.5,7,8,10–12,15,18 Recent progress in this field started to reveal the molecular motility system responsible for the organelle transport in plants.19Chloroplast movement is among the most fascinating models of organelle movement in plants because it is precisely controlled by ambient light conditions.20,21 Weak light induces chloroplast accumulation response so that chloroplasts can capture photosynthetic light efficiently (Fig. 1A). Strong light induces chloroplast avoidance response to escape from photodamage (Fig. 1B).22 The blue light-induced chloroplast movement is mediated by the blue light receptor phototropin (phot). In some cryptogam plants, the red light-induced chloroplast movement is regulated by a chimeric phytochrome/phototropin photoreceptor neochrome.23–25 In a model plant Arabidopsis, phot1 and phot2 function redundantly to regulate the accumulation response,26 whereas phot2 alone is essential for the avoidance response.27,28 Several additional factors regulating chloroplast movement were identified by analyses of Arabidopsis mutants deficient in chloroplast photorelocation.29–32 In particular, identification of CHUP1 (chloroplast unusual positioning 1) revealed the connection between chloroplasts and actin filaments at the molecular level.29 CHUP1 is a chloroplast outer membrane protein capable of interacting with F-actin, G-actin and profilin in vitro.29,33,34 The chup1 mutant plants are defective in both the chloroplast movement and chloroplast anchorage to the plasma membrane,22,29,33 suggesting that CHUP1 plays an important role in linking chloroplasts to the plasma membrane through the actin filaments. However, how chloroplasts move using the actin filaments and whether chloroplast movement utilizes the actin-based motility system similar to other organelle movements remained to be determined.Open in a separate windowFigure 1Schematic distribution patterns of chloroplasts in a palisade cell under different light conditions, weak (A) and strong (B) lights. Shown as a side view of mid-part of the cell and a top view with three different levels (i.e., top, middle and bottom of the cell). The cell was irradiated from the leaf surface shown as arrows. Weak light induces chloroplast accumulation response (A) and strong light induces the avoidance response (B).Here, we review the recent findings pointing to existence of a novel actin-based mechanisms for chloroplast movement and discuss the differences between the mechanism responsible for movement of chloroplasts and other organelles. 相似文献
18.
VERNALIZATION INSENSITIVE 3 (VIN3) encodes a PHD domain chromatin remodelling protein that is induced in response to cold and is required for the establishment of the vernalization response in Arabidopsis thaliana.1 Vernalization is the acquisition of the competence to flower after exposure to prolonged low temperatures, which in Arabidopsis is associated with the epigenetic repression of the floral repressor FLOWERING LOCUS C (FLC).2,3 During vernalization VIN3 binds to the chromatin of the FLC locus,1 and interacts with conserved components of Polycomb-group Repressive Complex 2 (PRC2).4,5 This complex catalyses the tri-methylation of histone H3 lysine 27 (H3K27me3),4,6,7 a repressive chromatin mark that increases at the FLC locus as a result of vernalization.4,7–10 In our recent paper11 we found that VIN3 is also induced by hypoxic conditions, and as is the case with low temperatures, induction occurs in a quantitative manner. Our experiments indicated that VIN3 is required for the survival of Arabidopsis seedlings exposed to low oxygen conditions. We suggested that the function of VIN3 during low oxygen conditions is likely to involve the mediation of chromatin modifications at certain loci that help the survival of Arabidopsis in response to prolonged hypoxia. Here we discuss the implications of our observations and hypotheses in terms of epigenetic mechanisms controlling gene regulation in response to hypoxia.Key words: arabidopsis, VIN3, FLC, hypoxia, vernalization, chromatin remodelling, survival 相似文献
19.
Konstantina Marinoglou 《The Yale journal of biology and medicine》2012,85(4):469-480
It has been estimated that a human cell is confronted with 1 million DNA lesions
every day, one fifth of which may originate from the activity of Reactive Oxygen
Species (ROS) alone [1,2]. Terminally differentiated
neurons are highly active cells with, if any, very restricted regeneration
potential [3]. In
addition, genome integrity and maintenance during neuronal development is
crucial for the organism. Therefore, highly accurate and robust mechanisms for
DNA repair are vital for neuronal cells. This requirement is emphasized by the
long list of human diseases with neurodegenerative phenotypes, which are either
caused by or associated with impaired function of proteins involved in the
cellular response to genotoxic stress [4-8]. Ataxia
Telangiectasia Mutated (ATM), one of the major kinases of the DNA Damage
Response (DDR), is a node that links DDR, neuronal development, and
neurodegeneration [2,9-12]. In humans, inactivating mutations of ATM lead to
Ataxia-Telangiectasia (A-T) disease [11,13], which is
characterized by severe cerebellar neurodegeneration, indicating an important
protective function of ATM in the nervous system [14]. Despite the large number of studies on the
molecular cause of A-T, the neuroprotective role of ATM is not well established
and is contradictory to its general proapoptotic function. This review discusses
the putative functions of ATM in neuronal cells and how they might contribute to
neuroprotection. 相似文献
20.
Olga V Dyachenko Taras V Shevchuk Leo Kretzner Yaroslav I Buryanov Steven S Smith 《Epigenetics》2010,5(7):569-572
Non-CG methylation is well characterized in plants where it appears to play a role in gene silencing and genomic imprinting. Although strong evidence for the presence of non-CG methylation in mammals has been available for some time, both its origin and function remain elusive. In this review we discuss available evidence on non-CG methylation in mammals in light of evidence suggesting that the human stem cell methylome contains significant levels of methylation outside the CG site.Key words: non-CG methylation, stem cells, Dnmt1, Dnmt3a, human methylomeIn plant cells non-CG sites are methylated de novo by Chromomethylase 3, DRM1 and DRM2. Chromomethylase 3, along with DRM1 and DRM2 combine in the maintenance of methylation at symmetric CpHpG as well as asymmetric DNA sites where they appear to prevent reactivation of transposons.1 DRM1 and DRM2 modify DNA de novo primarily at asymmetric CpH and CpHpH sequences targeted by siRNA.2Much less information is available on non-CG methylation in mammals. In fact, studies on mammalian non-CG methylation form a tiny fraction of those on CG methylation, even though data for cytosine methylation in other dinucleotides, CA, CT and CC, have been available since the late 1980s.3 Strong evidence for non-CG methylation was found by examining either exogenous DNA sequences, such as plasmid and viral integrants in mouse and human cell lines,4,5 or transposons and repetitive sequences such as the human L1 retrotransposon6 in a human embryonic fibroblast cell line. In the latter study, non-CG methylation observed in L1 was found to be consistent with the capacity of Dnmt1 to methylate slippage intermediates de novo.6Non-CG methylation has also been reported at origins of replication7,8 and a region of the human myogenic gene Myf3.9 The Myf3 gene is silenced in non-muscle cell lines but it is not methylated at CGs. Instead, it carries several methylated cytosines within the sequence CCTGG. Gene-specific non-CG methylation was also reported in a study of lymphoma and myeloma cell lines not expressing many B lineage-specific genes.10 The study focused on one specific gene, B29 and found heavy CG promoter methylation of that gene in most cell lines not expressing it. However, in two other cell lines where the gene was silenced, cytosine methylation was found almost exclusively at CCWGG sites. The authors provided evidence suggesting that CCWGG methylation was sufficient for silencing the B29 promoter and that methylated probes based on B29 sequences had unique gel shift patterns compared to non-methylated but otherwise identical sequences.10 The latter finding suggests that the presence of the non-CG methylation causes changes in the proteins able to bind the promoter, which could be mechanistically related to the silencing seen with this alternate methylation.Non-CG methylation is rarely seen in DNA isolated from cancer patients. However, the p16 promoter region was reported to contain both CG and non-CG methylation in breast tumor specimens but lacked methylation at these sites in normal breast tissue obtained at mammoplasty.11 Moreover, CWG methylation at the CCWGG sites in the calcitonin gene is not found in normal or leukemic lymphocyte DNA obtained from patients.12 Further, in DNA obtained from breast cancer patients, MspI sites that are refractory to digestion by MspI and thus candidates for CHG methylation were found to carry CpG methylation.13 Their resistance to MspI restriction was found to be caused by an unusual secondary structure in the DNA spanning the MspI site that prevents restriction.13 This latter observation suggests caution in interpreting EcoRII/BstNI or EcoRII/BstOI restriction differences as due to CWG methylation, since in contrast to the 37°C incubation temperature required for full EcoRII activity, BstNI and BstOI require incubation at 60°C for full activity where many secondary structures are unstable.The recent report by Lister et al.14 confirmed a much earlier report by Ramsahoye et al.15 suggesting that non-CG methylation is prevalent in mammalian stem cell lines. Nearest neighbor analysis was used to detect non-CG methylation in the earlier study on the mouse embryonic stem (ES) cell line,15 thus global methylation patterning was assessed. Lister et al.14 extend these findings to human stem cell lines at single-base resolution with whole-genome bisulfite sequencing. They report14 that the methylome of the human H1 stem cell line and the methylome of the induced pluripotent IMR90 (iPS) cell line are stippled with non-CG methylation while that of the human IMR90 fetal fibroblast cell line is not. While the results of the two studies are complementary, the human methylome study addresses locus specific non-CG methylation. Based on that data,14 one must conclude that non-CG methylation is not carefully maintained at a given site in the human H1 cell line. The average non-CG site is picked up as methylated in about 25% of the reads whereas the average CG methylation site is picked up in 92% of the reads. Moreover, non-CG methylation is not generally present on both strands and is concentrated in the body of actively transcribed genes.14Even so, the consistent finding that non-CG methylation appears to be confined to stem cell lines,14,15 raises the possibility that cancer stem cells16 carry non-CG methylation while their nonstem progeny in the tumor carry only CG methylation. Given the expected paucity of cancer stem cells in a tumor cell population, it is unlikely that bisulfite sequencing would detect non-CG methylation in DNA isolated from tumor cells since the stem cell population is expected to be only a very minor component of tumor DNA. Published sequences obtained by bisulfite sequencing generally report only CG methylation, and to the best of our knowledge bisulfite sequenced tumor DNA specimens have not reported non-CG methylation. On the other hand, when sequences from cell lines have been reported, bisulfite-mediated genomic sequencing8 or ligation mediated PCR17 methylcytosine signals outside the CG site have been observed. In a more recent study plasmid DNAs carrying the Bcl2-major breakpoint cluster18 or human breast cancer DNA13 treated with bisulfite under non-denaturing conditions, cytosines outside the CG side were only partially converted on only one strand18 or at a symmetrical CWG site.13 In the breast cancer DNA study the apparent CWG methylation was not detected when the DNA was fully denatured before bisulfite treatment.13In both stem cell studies, non-CG methylation was attributed to the Dnmt3a,14,15 a DNA methyltransferase with similarities to the plant DRM methyltransferase family19 and having the capacity to methylate non-CG sites when expressed in Drosophila melanogaster.15 DRM proteins however, possess a unique permuted domain structure found exclusively in plants19 and the associated RNA-directed non-CG DNA methylation has not been reproducibly observed in mammals despite considerable published20–23 and unpublished efforts in that area. Moreover, reports where methylation was studied often infer methylation changes from 5AzaC reactivation studies24 or find that CG methylation seen in plants but not non-CG methylation is detected.21,22,25,26 In this regard, it is of interest that the level of non-CG methylation reported in stem cells corresponds to background non-CG methylation observed in vitro with human DNA methyltransferase I,27 and is consistent with the recent report that cultured stem cells are epigenetically unstable.28The function of non-CG methylation remains elusive. A role in gene expression has not been ruled out, as the studies above on Myf3 and B29 suggest.9,10 However, transgene expression of the bacterial methyltransferase M.EcoRII in a human cell line (HK293), did not affect the CG methylation state at the APC and SerpinB5 genes29 even though the promoters were symmetrically de novo methylated at mCWGs within each CCWGG sequence in each promoter. This demonstrated that CG and non-CG methylation are not mutually exclusive as had been suggested by earlier reports.9,10 That observation is now extended to the human stem cell line methylome where CG and non-CG methylation co-exist.14 Gene expression at the APC locus was likewise unaffected by transgene expression of M.EcoRII. In those experiments genome wide methylation of the CCWGG site was detected by restriction analysis and bisulfite sequencing,29 however stem cell characteristics were not studied.Many alternative functions can be envisioned for non-CG methylation, but the existing data now constrains them to functions that involve low levels of methylation that are primarily asymmetric. Moreover, inheritance of such methylation patterns requires low fidelity methylation. If methylation were maintained with high fidelity at particular CHG sites one would expect that the spontaneous deamination of 5-methylcytosine would diminish the number of such sites, so as to confine the remaining sites to those positions performing an essential function, as is seen in CG methylation.30–33 However, depletion of CWG sites is not observed in the human genome.34 Since CWG sites account for only about 50% of the non-CG methylation observed in the stem cell methylome14 where methylated non-CG sites carry only about 25% methylation, the probability of deamination would be about 13% of that for CWG sites that are subject to maintenance methylation in the germ line. Since mutational depletion of methylated cytosines has to have its primary effect on the germ line, if the maintenance of non-CG methylation were more accurate and more widespread, one would have had to argue that stem cells in the human germ lines lack CWG methylation. As it is the data suggests that whatever function non-CG methylation may have in stem cells, it does not involve accurate somatic inheritance in the germ line.The extensive detail on non-CG methylation in the H1 methylome14 raises interesting questions about the nature of this form of methylation in human cell lines. A key finding in this report is the contrast between the presence of non-CG methylation in the H1 stem cell line and its absence in the IMR90 human fetal lung fibroblast cell line.14 This suggests that it may have a role in the origin and maintenance of the pluripotent lineage.14By analogy with the well known methylated DNA binding proteins specific for CG methylation,35 methylated DNA binding proteins that selectively bind sites of non-CG methylation are expected to exist in stem cells. Currently the only protein reported to have this binding specificity is human Dnmt1.36–38 While Dnmt1 has been proposed to function stoichiometrically39 and could serve a non-CG binding role in stem cells, this possibility and the possibility that other stem-cell specific non-CG binding proteins might exist remain to be been explored.Finally, the nature of the non-CG methylation patterns in human stem cell lines present potentially difficult technical problems in methylation analysis. First, based on the data in the H1 stem cell methylome,40 a standard MS-qPCR for non-CG methylation would be impractical because non-CG sites are infrequent, rarely clustered and are generally characterized by partial asymmetric methylation. This means that a PCR primer that senses the 3 adjacent methylation sites usually recommended for MS-qPCR primer design41,42 cannot be reliably found. For example in the region near Oct4 (Chr6:31,246,431), a potential MS-qPCR site exists with a suboptimal set of two adjacent CHG sites both methylated on the + strand at Chr6:31,252,225 and 31,252,237.14,40 However these sites were methylated only in 13/45 and 30/52 reads. Thus the probability that they would both be methylated on the same strand is about 17%. Moreover, reverse primer locations containing non-CG methylation sites are generally too far away for practical bisulfite mediated PCR. Considering the losses associated with bisulfite mediated PCR43 the likelihood that such an MS-qPCR system would detect non-CG methylation in the H1 cell line or stem cells present in a cancer stem cell niche44,45 is very low.The second difficulty is that methods based on the specificity of MeCP2 and similar methylated DNA binding proteins for enriching methylated DNA (e.g., MIRA,46 COMPARE-MS47) will discard sequences containing non-CG methylation since they require cooperative binding afforded by runs of adjacent methylated CG sites for DNA capture. This latter property of the methylated cytosine capture techniques makes it also unlikely that methods based on 5-methylcytosine antibodies (e.g., meDIP48) will capture non-CG methylation patterns accurately since the stem cell methylome shows that adjacent methylated non-CG sites are rare in comparison to methylated CG sites.14In summary, whether or not mammalian stem cells in general or human stem cells in particular possess functional plant-like methylation patterns is likely to continue to be an interesting and challenging question. At this point we can conclude that the non-CG patterns reported in human cells appear to differ significantly from the non-CG patterns seen in plants, suggesting that they do not have a common origin or function. 相似文献