NF2 deficiency promotes tumorigenesis and metastasis by destabilizing adherens junctions

Mutation of the Neurofibromatosis 2 (NF2) tumor suppressor gene leads to cancer development in humans and mice. Recent studies suggest that Nf2 loss also contributes to tumor metastasis. The Nf2-encoded protein, merlin, is related to the ERM (ezrin, radixin, and moesin) family of membrane:cytoskeleton-associated proteins. However, the cellular mechanism whereby merlin controls cell proliferation from this location is not known. Here we show that the major cellular consequence of Nf2 deficiency in primary cells is an inability to undergo contact-dependent growth arrest and to form stable cadherin-containing cell:cell junctions. Merlin colocalizes and interacts with adherens junction (AJ) components in confluent wild-type cells, suggesting that the lack of AJs and contact-dependent growth arrest in Nf2(-/-) cells directly results from the absence of merlin at sites of cell:cell contact. Our studies indicate that merlin functions as a tumor and metastasis suppressor by controlling cadherin-mediated cell:cell contact.     

Functional analysis of the relationship between the neurofibromatosis 2 tumor suppressor and its binding partner,hepatocyte growth factor-regulated tyrosine kinase substrate

Individuals with the neurofibromatosis 2 (NF2) inherited tumor predisposition syndrome are prone to the development of nervous system tumors, including schwannomas and meningiomas. The NF2 tumor suppressor protein, merlin or schwannomin, inhibits cell growth and motility as well as affects actin cytoskeleton-mediated processes. Merlin interacts with several proteins that might mediate merlin growth suppression, including hepatocyte growth factor-regulated tyrosine kinase substrate (HRS or HGS). Previously, we demonstrated that regulated overexpression of HRS in RT4 rat schwannoma cells had the same functional consequences as regulated overexpression of merlin. To determine the functional significance of this interaction, we generated a series of HRS truncation mutants and defined the regions of HRS required for merlin binding and HRS growth suppression. The HRS domain required for merlin binding was narrowed to a region (residues 470-497) containing the predicted coiled-coil domain whereas the major domain responsible for HRS growth suppression was distinct (residues 498-550). To determine whether merlin growth suppression required HRS, we demonstrated that merlin inhibited growth in HRS (+/+), but not HRS( -/-) mouse embryonic fibroblast cells. In contrast, HRS could suppress cell growth in the absence of Nf2 expression. These results suggest that merlin growth suppression requires HRS expression and that the binding of merlin to HRS may facilitate its ability to function as a tumor suppressor.     

The NF2 interactor, hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), associates with merlin in the "open" conformation and suppresses cell growth and motility

The neurofibromatosis 2 tumor suppressor protein, merlin or schwannomin, functions as a negative growth regulator; however, its mechanism of action is not known. In an effort to determine how merlin regulates cell growth, we analyzed a recently identified novel merlin interactor, hepatocyte growth factor-regulated tyrosine kinase substrate (HRS). We demonstrate that regulated overexpression of HRS in rat schwannoma cells results in similar effects as overexpression of merlin, including growth inhibition, decreased motility and abnormalities in cell spreading. Previously, we showed that merlin forms an intramolecular association between the N- and C-termini and exists in "open" and "closed" conformations. Merlin interacts with HRS in the unfolded, or open, conformation. This HRS binding domain maps to merlin residues 453-557. Overexpression of C-terminal merlin has no effect on HRS function, arguing that merlin binding to HRS does not negatively regulate HRS growth suppressor activity. These results suggest the possibility that merlin and HRS may regulate cell growth in schwannoma cells through interacting pathways.     

Neurofibromin (Nf1) is required for skeletal muscle development

Neurofibromatosis type 1 (NF1) is a multi-system disease caused by mutations in the NF1 gene encoding a Ras-GAP protein, neurofibromin, which negatively regulates Ras signaling. Besides neuroectodermal malformations and tumors, the skeletal system is often affected (e.g. scoliosis and long bone dysplasia) demonstrating the importance of neurofibromin for development and maintenance of the musculoskeletal system. Here, we focus on the role of neurofibromin in skeletal muscle development. Nf1 gene inactivation in the early limb bud mesenchyme using Prx1-cre (Nf1(Prx1)) resulted in muscle dystrophy characterized by fibrosis, reduced number of muscle fibers and reduced muscle force. This was caused by an early defect in myogenesis affecting the terminal differentiation of myoblasts between E12.5 and E14.5. In parallel, the muscle connective tissue cells exhibited increased proliferation at E14.5 and an increase in the amount of connective tissue as early as E16.5. These changes were accompanied by excessive mitogen-activated protein kinase pathway activation. Satellite cells isolated from Nf1(Prx1) mice showed normal self-renewal, but their differentiation was impaired as indicated by diminished myotube formation. Our results demonstrate a requirement of neurofibromin for muscle formation and maintenance. This previously unrecognized function of neurofibromin may contribute to the musculoskeletal problems in NF1 patients.     

Embryonic and fetal limb myogenic cells are derived from developmentally distinct progenitors and have different requirements for β-catenin

Vertebrate muscle arises sequentially from embryonic, fetal, and adult myoblasts. Although functionally distinct, it is unclear whether these myoblast classes develop from common or different progenitors. Pax3 and Pax7 are expressed by somitic myogenic progenitors and are critical myogenic determinants. To test the developmental origin of embryonic and fetal myogenic cells in the limb, we genetically labeled and ablated Pax3⁺ and Pax7⁺ cells. Pax3⁺Pax7⁻ cells contribute to muscle and endothelium, establish and are required for embryonic myogenesis, and give rise to Pax7⁺ cells. Subsequently, Pax7⁺ cells give rise to and are required for fetal myogenesis. Thus, Pax3⁺ and Pax7⁺ cells contribute differentially to embryonic and fetal limb myogenesis. To investigate whether embryonic and fetal limb myogenic cells have different genetic requirements we conditionally inactivated or activated β-catenin, an important regulator of myogenesis, in Pax3- or Pax7-derived cells. β-Catenin is necessary within the somite for dermomyotome and myotome formation and delamination of limb myogenic progenitors. In the limb, β-catenin is not required for embryonic myoblast specification or myofiber differentiation but is critical for determining fetal progenitor number and myofiber number and type. Together, these studies demonstrate that limb embryonic and fetal myogenic cells develop from distinct, but related progenitors and have different cell-autonomous requirements for β-catenin.     

Genetic control of muscle development: learning from Drosophila

Muscle development involves a complex sequence of time and spatially regulated cellular events leading to the formation of highly specialised syncytial muscle cells displaying a common feature, the capacity of contraction. Analyses of mechanisms controlling muscle development reveals that the main steps of muscle formation including myogenic determination, diversification of muscle precursors, myoblast fusion and terminal differentiation involve the actions of evolutionarily conserved genes. Thus dissecting the genetic control of muscle development in simple model organisms appears to be an attractive way to get insights into core genetic cascade that orchestrate myogenesis. In this respect, particularly insightful have been data generated using Drosophila as a model system. Notably, the interplay between intrinsic and extrinsic cues that determine the early myogenic decisions leading to the specification of muscle progenitors and those controlling myoblasts fusion are much better characterised in Drosophila than in vertebrate species. Also, adult Drosophila myogenesis, which leads to the formation of vertebrate-like multi-fibre muscles, emerges as a particularly well-adapted system to study normal and aberrant muscle development.     

Functional analysis of neurofibromatosis 2 (NF2) missense mutations.

Neurofibromatosis 2 (NF2) is a tumor predisposition syndrome in which affected individuals develop nervous system tumors at an increased frequency. The most common tumor in individuals with NF2 is the schwannoma, which is composed of neoplastic Schwann cells lacking NF2 gene expression. Moreover, inactivation of the NF2 gene is observed in nearly all sporadic schwannomas, suggesting that the NF2 gene is a critical growth regulator for Schwann cells. In an effort to gain insights into the function of the NF2 gene product, merlin or schwannomin, we performed a detailed functional analysis of eight naturally occurring non-conservative missense mutations in the NF2 gene. Using a regulatable expression system in rat schwannoma cells, we analyzed proliferation, actin cytoskeleton-mediated events and merlin folding. In this report, we demonstrate that mutations clustered in the predicted alpha-helical region did not impair the function of merlin whereas those in either the N- or C-terminus of the protein rendered merlin inactive as a negative growth regulator. These results suggest that the key functional domains of merlin lie within the highly conserved FERM domain and the unique C-terminus of the protein.     

不同浓度曲古抑菌素A对鸡胚翅芽发育的影响

目的 观察组蛋白去乙酰化酶(HDAC)抑制剂曲古抑菌素A(TSA)对鸡胚翅芽发育中某些基因表达水平的作用,探讨不同浓度TSA(75μmol/L、750 μmol/L、1.5mmol/L)对鸡胚翅芽发育的影响,提高我们对染色质结构重组和表观遗传对基因表达的调控作用,以及对正在开发的抗癌药物的认识. 方法以鸡胚翅芽作为实验模型,用TSA处理翅芽,对鸡胚胎实施原位杂交,检测与肌肉发育相关基因的表达,并采用硫酸耐尔蓝(Nile bluesulfate)法检测细胞凋亡状况. 结果 75 μmol/L TSA能够抑制与肌肉发生相关的基因MyoD和Myf5在鸡胚翅芽的表达,经TSA(75μmol/L)处理的胚胎没有发生明显的细胞凋亡.但是随着TSA药物浓度的提高,TSA(≥750μmol/L)不仅强烈抑制相关基因的表达,而且出现翅芽外部畸形(外形改变、体积缩小)以及细胞凋亡. 结论75μmol/LTSA调控某些重要的转录因子及有力地控制肌肉细胞的分化;高浓度TSA(≥750μmol/L)导致细胞凋亡和胚胎翅芽畸形;发育的鸡胚能够作为一个便利的,供体内研究药物(如HDAC抑制剂类等)的模型.     

Expression of slit-2 and slit-3 during chick development.

The Drosophila and vertebrate slit proteins have been characterized as secreted chemorepellents recognized by the robo receptor proteins that function principally for the guidance of neuronal axons and neurons. slit genes are also expressed in the limb. To provide a basis for the determination of slit functions in the limb we have isolated and characterized the expression of chick slit-2 and slit-3 in the developing limb and other tissues of the chick embryo. Both genes share similar expression profiles in the chick embryo when compared to that of their mammalian homologues, particularly in the neural tube. In the limb, their expression patterns suggest their involvement in many aspects of limb development. In the early limb bud, slit-2 is expressed in the peripheral mesenchyme and invading muscle precursors, while slit-3 expression is restricted to the future chondrogenic core of the limb bud. At later stages, both slit genes are expressed in interdigital mesenchyme, in inner periosteal cells, and in mesenchyme immediately radial to the periosteum and under the epidermis. slit-3 is also expressed in proliferating chondrocytes during cartilage development, while slit-2 is expressed in later muscle masses and peripherally to joints in the autopod.     

The formation of skeletal muscle: from somite to limb

During embryogenesis, skeletal muscle forms in the vertebrate limb from progenitor cells originating in the somites. These cells delaminate from the hypaxial edge of the dorsal part of the somite, the dermomyotome, and migrate into the limb bud, where they proliferate, express myogenic determination factors and subsequently differentiate into skeletal muscle. A number of regulatory factors involved in these different steps have been identified. These include Pax3 with its target c-met, Lbx1 and Mox2 as well as the myogenic determination factors Myf5 and MyoD and factors required for differentiation such as Myogenin, Mrf4 and Mef2 isoforms. Mutants for genes such as Lbx1 and Mox2, expressed uniformly in limb muscle progenitors, reveal unexpected differences between fore and hind limb muscles, also indicated by the differential expression of Tbx genes. As development proceeds, a secondary wave of myogenesis takes place, and, postnatally, satellite cells become located under the basal lamina of adult muscle fibres. Satellite cells are thought to be the progenitor cells for adult muscle regeneration, during which similar genes to those which regulate myogenesis in the embryo also play a role. In particular, Pax3 as well as its orthologue Pax7 are important. The origin of secondary/fetal myoblasts and of adult satellite cells is unclear, as is the relation of the latter to so-called SP or stem cell populations, or indeed to potential mesangioblast progenitors, present in blood vessels. The oligoclonal origin of postnatal muscles points to a small number of founder cells, whether or not these have additional origins to the progenitor cells of the somite which form the first skeletal muscles, as discussed here for the embryonic limb.     

Fibroblast growth factor signaling regulates Dach1 expression during skeletal development.

Dach1 is a mouse homologue of the Drosophila dachshund gene, which is a key regulator of cell fate determination during eye, leg, and brain development in the fly. We have investigated the expression and growth factor regulation of Dach1 during pre- and postnatal skeletal development in the mouse limb to understand better the function of Dach1. Dach1 was expressed in the distal mesenchyme of the early embryonic mouse limb bud and subsequently became restricted to the tips of digital cartilages. Dach1 protein was localized to postmitotic, prehypertrophic, and early hypertrophic chondrocytes during the initiation of ossification centers, but Dach1 was not expressed in growth plates that exhibited extensive ossification. Dach1 colocalized with Runx2/Cbfa1 in chondrocytes but not in the forming bone collar or primary spongiosa. Dach1 also colocalized with cyclin-dependent kinase inhibitors p27 (Kip1) and p57 (Kip2) in chondrocytes of the growth plate and in the epiphysis before the formation of the secondary ossification center. Because fibroblast growth factors (FGF), bone morphogenetic proteins (BMP), and hedgehog molecules (Hh) regulate skeletal patterning of the limb bud and chondrocyte maturation in developing endochondral bones, we investigated the regulation of Dach1 by these growth and differentiation factors. Expression of Dach1 in 11 days postcoitus mouse limb buds in organ culture was up-regulated by implanting beads soaked in FGF1, 2, 8, or 9 but not FGF10. BMP4-soaked beads down-regulated Dach1 expression, whereas Shh and bovine serum albumin had no effect. Furthermore, FGF4 or 8 could substitute for the apical ectodermal ridge in maintaining Dach1 expression in the limb buds. Immunolocalization of FGFR2 and FGFR3 revealed overlap with Dach1 expression during skeletal patterning and chondrocyte maturation. We conclude that Dach1 is a target gene of FGF signaling during limb skeletal development, and Dach1 may function as an intermediary in the FGF signaling pathway regulating cell proliferation or differentiation.     

Regulation of the neurofibromatosis 2 gene promoter expression during embryonic development.

Mutations in the Neurofibromatosis 2 (NF2) gene are associated with predisposition to vestibular schwannomas, spinal schwannomas, meningiomas, and ependymomas. Presently, how NF2 is expressed during embryonic development and in the tissues affected by neurofibromatosis type 2 (NF2) has not been well defined. To examine NF2 expression in vivo, we generated transgenic mice carrying a 2.4-kb NF2 promoter driving beta-galactosidase (beta-gal) with a nuclear localization signal. Whole-mount embryo staining revealed that the NF2 promoter directed beta-gal expression as early as embryonic day E5.5. Strong expression was detected at E6.5 in the embryonic ectoderm containing many mitotic cells. beta-gal staining was also found in parts of embryonic endoderm and mesoderm. The beta-gal staining pattern in the embryonic tissues was corroborated by in situ hybridization analysis of endogenous Nf2 RNA expression. Importantly, we observed strong NF2 promoter activity in the developing brain and in sites containing migrating cells including the neural tube closure, branchial arches, dorsal aorta, and paraaortic splanchnopleura. Furthermore, we noted a transient change of NF2 promoter activity during neural crest cell migration. While little beta-gal activity was detected in premigratory neural crest cells at the dorsal ridge region of the neural fold, significant activity was seen in the neural crest cells already migrating away from the dorsal neural tube. In addition, we detected considerable NF2 promoter activity in various NF2-affected tissues such as acoustic ganglion, trigeminal ganglion, spinal ganglia, optic chiasma, the ependymal cell-containing tela choroidea, and the pigmented epithelium of the retina. The NF2 promoter expression pattern during embryogenesis suggests a specific regulation of the NF2 gene during neural crest cell migration and further supports the role of merlin in cell adhesion, motility, and proliferation during development.     

Embryonic myogenesis pathways in muscle regeneration.

Embryonic myogenesis involves the staged induction of myogenic regulatory factors and positional cues that dictate cell determination, proliferation, and differentiation into adult muscle. Muscle is able to regenerate after damage, and muscle regeneration is generally thought to recapitulate myogenesis during embryogenesis. There has been considerable progress in the delineation of myogenesis pathways during embryogenesis, but it is not known whether the same signaling pathways are relevant to muscle regeneration in adults. Here, we defined the subset of embryogenesis pathways induced in muscle regeneration using a 27 time-point in vivo muscle regeneration series. The embryonic Wnt (Wnt1, 3a, 7a, 11), Shh pathway, and the BMP (BMP2, 4, 7) pathway were not induced during muscle regeneration. Moreover, antagonists of Wnt signaling, sFRP1, sFRP2, and sFRP4 (secreted frizzled-related proteins) were significantly up-regulated, suggesting active inhibition of the Wnt pathway. The pro-differentiation FGFR4 pathway was transiently expressed at day 3, commensurate with expression of MyoD, Myogenin, Myf5, and Pax7. Protein verification studies showed fibroblast growth factor receptor 4 (FGFR4) protein to be strongly expressed in differentiating myoblasts and newly formed myotubes. We present evidence that FGF6 is likely the key ligand for FGFR4 during muscle regeneration, and further suggest that FGF6 is released from necrotic myofibers where it is then sequestered by basal laminae. We also confirmed activation of Notch1 in the regenerating muscle. Finally, known MyoD coactivators (MEF2A, p/CIP, TCF12) and repressors (Twist, Id2) were strongly induced at appropriate time points. Taken together, our results suggest that embryonic positional signals (Wnt, Shh, and BMP) are not induced in postnatal muscle regeneration, whereas cell-autonomous factors (Pax7, MRFs, FGFR4) involving muscle precursor proliferation and differentiation are recapitulated by muscle regeneration.     

The NF2 tumor suppressor gene product, merlin, mediates contact inhibition of growth through interactions with CD44

The neurofibromatosis-2 (NF2) gene encodes merlin, an ezrin-radixin-moesin-(ERM)-related protein that functions as a tumor suppressor. We found that merlin mediates contact inhibition of growth through signals from the extracellular matrix. At high cell density, merlin becomes hypo-phosphorylated and inhibits cell growth in response to hyaluronate (HA), a mucopolysaccharide that surrounds cells. Merlin's growth-inhibitory activity depends on specific interaction with the cytoplasmic tail of CD44, a transmembrane HA receptor. At low cell density, merlin is phosphorylated, growth permissive, and exists in a complex with ezrin, moesin, and CD44. These data indicate that merlin and CD44 form a molecular switch that specifies cell growth arrest or proliferation.     

Striated myogenesis and peristalsis in the fetal murine esophagus occur without cell migration or interstitial cells of Cajal

Esophageal striated myogenesis progresses differently from appendicular myogenesis, but the mechanism underlying this process is incompletely understood. Early theories of transdifferentiation of smooth muscle into striated muscle are not supported by transgenic fate-mapping experiments; however, the origin of esophageal striated muscle remains unknown. To better define the process of striated myogenesis, we examined myogenesis in murine fetal cultured esophageal whole-organ explants. Embryonic day 14.5 (E14.5) esophagi maintained a functional contractile phenotype for up to 7 days in culture. Striated myogenesis, as evidenced by myogenin expression, proceeded in a craniocaudal direction along the length of the esophagus. Esophageal length did not change during this process. Complete, but not partial, mechanical disruption of the rostral esophagus inhibited myogenesis distally. Addition of fibroblast growth factor-2 (FGF-2) to the culture media failed to inhibit striated myogenesis, but attenuated smooth muscle actin expression and reduced peristaltic activity. Inhibition of c-kit failed to inhibit peristalsis. These results suggest that striated myogenic precursors are resident along the entire length of the esophagus by day 14.5 and do not migrate along the esophagus after E14.5. Induction of myogenesis craniocaudally appears to require physical continuity of the esophagus and is not inhibited by FGF-2. Finally, peristalsis in E14.5 esophagi appears not to be regulated by interstitial cells of Cajal.     

Raf kinase inhibitor protein1 is a myogenic inhibitor with conserved function in avians and mammals

Background: Raf Kinase Inhibitor Protein1 (RKIP) is a tumor suppressor that is present in several adult tissues. It functions as an inhibitor of both Raf/Mek/Erk and NF?B signaling when unphosphorylated, but following phosphorylation the ability to inhibit Raf/Mek/Erk signaling is lost and RKIP becomes an activator of G‐protein coupled receptor signaling. In neonates and adults, RKIP is known to be expressed in muscle; however, its physiological function is currently unknown. Results: In this study, we show by in situ hybridization and immunofluorescence that RKIP is also expressed in developing chick embryonic muscle, and mouse C2C12 myoblasts. Furthermore, we demonstrate that, in these systems, it functions as an inhibitor of myogenesis: increased levels of RKIP suppress myotube differentiation whereas decreasing RKIP promotes differentiation. Additionally, we show that the ability of RKIP to inhibit myogenesis is dependent upon its phosphorylation state as only the nonphosphorylated form of RKIP suppresses myogenesis. Conclusions: This study, therefore, clearly demonstrates that RKIP has conserved functions as a myogenic inhibitor in both mammalian and avian muscle. Developmental Dynamics 245:902–912, 2016. © 2016 Wiley Periodicals, Inc.