RESUMEN
The protein kinase WNK1 (with-no-lysine 1) influences trafficking of ion and small-molecule transporters and other membrane proteins as well as actin polymerization state. We investigated the possibility that actions of WNK1 on both processes are related. Strikingly, we identified the E3 ligase tripartite motif-containing 27 (TRIM27) as a binding partner for WNK1. TRIM27 is involved in fine tuning the WASH (Wiskott-Aldrich syndrome protein and SCAR homologue) regulatory complex which regulates endosomal actin polymerization. Knockdown of WNK1 reduced the formation of the complex between TRIM27 and its deubiquitinating enzyme USP7 (ubiquitin-specific protease 7), resulting in significantly diminished TRIM27 protein. Loss of WNK1 disrupted WASH ubiquitination and endosomal actin polymerization, which are necessary for endosomal trafficking. Sustained receptor tyrosine kinase (RTK) expression has long been recognized as a key oncogenic signal for the development and growth of human malignancies. Depletion of either WNK1 or TRIM27 significantly increased degradation of the epidermal growth factor receptor (EGFR) following ligand stimulation in breast and lung cancer cells. Like the EGFR, the RTK AXL was also affected similarly by WNK1 depletion but not by inhibition of WNK1 kinase activity. This study uncovers a mechanistic connection between WNK1 and the TRIM27-USP7 axis and extends our fundamental knowledge about the endocytic pathway regulating cell surface receptors.
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Actinas , Endosomas , Humanos , Peptidasa Específica de Ubiquitina 7 , Factores de Transcripción , Receptores ErbB , Proteínas Tirosina Quinasas Receptoras , Proteínas de Unión al ADN , Proteínas Nucleares , Proteína Quinasa Deficiente en Lisina WNK 1RESUMEN
The with no lysine (K) 1 (WNK1) protein kinase maintains cellular ion homeostasis in many tissues through actions on ion cotransporters and channels. Increased accumulation of WNK1 protein leads to pseudohypoaldosteronism type II (PHAII), a form of familial hypertension. WNK1 can be degraded via its adaptor-dependent recruitment to the Cullin3-RBX1 E3 ligase complex by the ubiquitin-proteasome system. Disruption of this process also leads to disease. To determine if this is the primary mechanism of WNK1 turnover, we examined WNK1 protein stability and degradation by measuring its rate of decay after blockade of translation. Here, we show that WNK1 protein degradation exhibits atypical kinetics in HeLa cells. Consistent with this apparent complexity, we found that multiple degradative pathways can modulate cellular WNK1 protein amount. WNK1 protein is degraded by not only the proteasome but also the lysosome. Non-lysosomal cysteine proteases calpain and caspases also influence WNK1 degradation, as inhibitors of these proteases modestly increased WNK1 protein expression. Importantly, we discovered that the E3 ubiquitin ligase UBR5 interacts with WNK1 and its deficiency results in increased WNK1 protein. Our results further demonstrate that increased WNK1 in UBR5-depleted cells is attributable to reduced lysosomal degradation of WNK1 protein. Taken together, our findings provide insights into the multiplicity of degradative pathways involved in WNK1 turnover and uncover UBR5 as a previously unknown regulator of WNK1 protein stability that leads to lysosomal degradation of WNK1 protein.
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Proteínas Serina-Treonina Quinasas , Seudohipoaldosteronismo , Células HeLa , Humanos , Antígenos de Histocompatibilidad Menor/genética , Complejo de la Endopetidasa Proteasomal , Proteínas Serina-Treonina Quinasas/genética , Seudohipoaldosteronismo/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Proteína Quinasa Deficiente en Lisina WNK 1/genética , Proteína Quinasa Deficiente en Lisina WNK 1/metabolismoRESUMEN
Bone mass is maintained by balanced activity of osteoblasts and osteoclasts. Lrp4 (low-density lipoprotein receptor related protein 4) is a member of the LDL receptor family, whose mutations have been identified in patients with high-bone-mass disorders, such as sclerosteosis and van Buchem diseases. However, it remains unknown whether and how Lrp4 regulates bone-mass homeostasis in vivo. Here we provide evidence that Lrp4-null mutation or specific mutation in osteoblast-lineage cells increased cortical and trabecular bone mass, which was associated with elevated bone formation and impaired bone resorption. This phenotype was not observed in osteoclast-selective Lrp4 knockout mice. Mechanistic studies indicate that loss of Lrp4 function in osteoblast-lineage cells increased serum levels of sclerostin, a key factor for bone-mass homeostasis that interacts with Lrp4, but abolished the inhibition of Wnt/ß-catenin signaling and osteoblastic differentiation by sclerostin. Concomitantly, sclerostin induction of RANKL (receptor activator of nuclear kappa B ligand) was impaired, leading to a lower ratio of RANKL over OPG (osteoprotegerin) (a key factor for osteoclastogenesis). Taken together, these results support the view for Lrp4 as a receptor of sclerostin to inhibit Wnt/ß-catenin signaling and bone formation and identify Lrp4 as a critical player in bone-mass homeostasis.
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Resorción Ósea/metabolismo , Resorción Ósea/patología , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteogénesis , Receptores de LDL/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Aminoácidos/sangre , Animales , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/patología , Resorción Ósea/sangre , Diferenciación Celular , Linaje de la Célula , Fémur/diagnóstico por imagen , Fémur/patología , Glicoproteínas/sangre , Glicoproteínas/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Proteínas Relacionadas con Receptor de LDL , Ratones Noqueados , Músculos/metabolismo , Especificidad de Órganos , Osteoblastos/patología , Osteocalcina/metabolismo , Osteoclastos/patología , Osteoprotegerina/metabolismo , Ligando RANK/metabolismo , Receptores de LDL/deficiencia , Células del Estroma/metabolismo , Células del Estroma/patología , Vía de Señalización Wnt , Microtomografía por Rayos X , beta Catenina/metabolismoRESUMEN
BACKGROUND: High-grade gliomas are one of the most invasive and therapy-resistant cancers. We have recently shown that noncanonical NF-κB/RelB signaling is a potent driver of tumorigenesis and invasion in the aggressive, mesenchymal subtype of glioma. However, the relevant signals that induce activation of noncanonical NF-κB signaling in glioma and its function relative to the canonical NF-κB pathway remain elusive. METHODS: The ability of tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) to regulate NF-κB signaling and promote tumor progression was investigated in both established and primary high-grade glioma tumor lines using a three-dimensional (3-D) collagen invasion assay. The roles of specific NF-κB proteins in regulating glioma cell invasion and expression of Matrix Metalloproteinase 9 (MMP9) in response to TWEAK were evaluated using shRNA-mediated loss-of-function studies. The ability of NF-κB-inducing kinase (NIK) to promote glioma growth in vivo was investigated using an orthotopic xenograft mouse model. RESULTS: In glioma cells that display elevated noncanonical NF-κB signaling, loss of RelB attenuates invasion without affecting RelA expression or phosphorylation and RelB is sufficient to promote invasion in the absence of RelA. The cytokine TWEAK preferentially activates the noncanonical NF-κB pathway through induction of p100 processing to p52 and nuclear accumulation of both RelB and p52 without activating the canonical NF-κB pathway. Moreover, TWEAK, but not TNFα, significantly increases NIK mRNA levels. TWEAK also promotes noncanonical NFκB-dependent MMP9 expression and glioma cell invasion. Finally, expression of NIK is sufficient to increase gliomagenesis in vivo. CONCLUSIONS: Our data establish a key role for NIK and noncanonical NF-κB in mediating TWEAK-induced, MMP-dependent glioma cell invasion. The findings also demonstrate that TWEAK induces noncanonical NF-κB signaling and signal-specific regulation of NIK mRNA expression. Together, these studies reveal the important role of noncanonical NF-κB signaling in regulating glioma invasiveness and highlight the therapeutic potential of targeting activation of NIK in this deadly disease.
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Glioma/genética , Glioma/patología , FN-kappa B/genética , Invasividad Neoplásica/genética , Proteínas Serina-Treonina Quinasas/genética , Transducción de Señal/genética , Factores de Necrosis Tumoral/genética , Animales , Apoptosis/genética , Línea Celular , Línea Celular Tumoral , Citocina TWEAK , Células HEK293 , Humanos , Metaloproteinasa 9 de la Matriz/genética , Ratones , Ratones Desnudos , Invasividad Neoplásica/patología , Fosforilación/genética , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Quinasa de Factor Nuclear kappa BRESUMEN
The WNK-OSR1/SPAK protein kinase signaling pathway regulates ion homeostasis and cell volume, but its other functions are poorly understood. To uncover undefined signaling functions of the pathway we analyzed the binding specificity of the conserved C-terminal (CCT) domains of OSR1 and SPAK to find all possible interaction motifs in human proteins. These kinases bind the core consensus sequences R-F-x-V/I and R-x-F-x-V/I. Motifs were ranked based on sequence, conservation, cellular localization, and solvent accessibility. Out of nearly 3,700 motifs identified, 90% of previously published motifs were within the top 2% of those predicted. Selected candidates (TSC22D1, CAVIN1, ATG9A, NOS3, ARHGEF5) were tested. Upstream kinases WNKs 1-4 and their close relatives, the pseudokinases NRBP1/2, contain CCT-like domains as well. We identified additional distinct motif variants lacking the conserved arginine previously thought to be required, and found that the NRBP1 CCT-like domain binds TSC22D1 via the same motif as OSR1 and SPAK. Our results further highlight the rich and diverse functionality of CCT and CCT-like domains in connecting WNK signaling to cellular processes.
RESUMEN
Neogenin, a deleted in colorectal cancer (DCC) family member, has been identified as a receptor for the neuronal axon guidance cues netrins and repulsive guidance molecules repulsive guidance molecules (RGM). RGMc, also called hemojuvelin (HJV), is essential for iron homeostasis. Here we provide evidence that neogenin plays a critical role in iron homeostasis by regulation of HJV secretion and bone morphogenetic protein (BMP) signaling. Livers of neogenin mutant mice exhibit iron overload, low levels of hepcidin, and reduced BMP signaling. Mutant hepatocytes in vitro show impaired BMP2 induction of Smad1/5/8 phosphorylation and hepcidin expression. Neogenin is expressed in liver cells in a reciprocal pattern to that of hepcidin, suggesting that neogenin functions in a cell nonautonomous manner. Further studies demonstrate that neogenin may stabilize HJV, a glycosylphosphatidylinositol-anchored protein that interacts with neogenin and suppresses its secretion. Taken together, our results lead the hypothesis that neogenin regulates iron homeostasis via inhibiting secretion of HJV, an inhibitor of BMP signaling, to enhance BMP signaling and hepcidin expression. These results reveal a novel mechanism underlying neogenin regulation of HJV-BMP signaling.
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Péptidos Catiónicos Antimicrobianos/metabolismo , Proteína Morfogenética Ósea 2/farmacología , Homeostasis/efectos de los fármacos , Hierro/metabolismo , Proteínas de la Membrana/metabolismo , Animales , Péptidos Catiónicos Antimicrobianos/genética , Proteínas de Transporte de Catión/metabolismo , Espacio Extracelular/efectos de los fármacos , Espacio Extracelular/metabolismo , Proteínas Ligadas a GPI , Regulación de la Expresión Génica/efectos de los fármacos , Proteína de la Hemocromatosis , Células Hep G2 , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Hepatocitos/patología , Hepcidinas , Humanos , Sobrecarga de Hierro/genética , Sobrecarga de Hierro/patología , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Fosforilación/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Proteínas Smad/metabolismoRESUMEN
Metastasis is the major cause of mortality in cancer patients. Analyses of mouse models and patient data have implicated the protein kinase WNK1 as one of a handful of genes uniquely linked to a subset of invasive cancers. WNK1 signaling pathways are widely implicated in the regulation of ion co-transporters and in controlling cell responses to osmotic stress. In this review we will discuss its actions in tumor malignancy in human cancers and present evidence for its function in invasion, migration, angiogenesis and mesenchymal transition.
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Mesenchymal stem cells (MSCs) are attractive alternatives to conventional anti-asthmatic drugs for severe asthma. Mechanisms underlying the anti-asthmatic effects of MSCs have not yet been elucidated. This study evaluated the anti-asthmatic effects of intravenously administered MSCs, focusing on macrophages and monocytes. Seven-week-old transgenic (Tg) mice with lung-specific overexpression of IL-13 were used to simulate chronic asthma. MSCs were intravenously administered four days before sampling. We examined changes in immune cell subpopulations, gene expression, and histological phenotypes. IL-13 Tg mice exhibited diverse features of chronic asthma, including severe type 2 inflammation, airway fibrosis, and mucus metaplasia. Intravenous administration of MSCs attenuated these asthmatic features just four days after a single treatment. MSC treatment significantly reduced SiglecF-CD11c-CD11b+ monocyte-derived macrophages (MoMs) and inhibited the polarization of MoMs into M2 macrophages, especially M2a and M2c. Furthermore, MSCs downregulated the excessive accumulation of Ly6c- monocytes in the lungs. While an intravenous adoptive transfer of Ly6c- monocytes promoted the infiltration of MoM and Th2 inflammation, that of MSC-exposed Ly6c- monocytes did not. Ex vivo Ly6c- MoMs upregulated M2-related genes, which were reduced by MSC treatment. Molecules secreted by Ly6c- MoMs from IL-13 Tg mice lungs upregulated the expression of fibrosis-related genes in fibroblasts, which were also suppressed by MSC treatment. In conclusion, intravenously administered MSCs attenuate asthma phenotypes of chronic asthma by modulating macrophages. Identifying M2 macrophage subtypes revealed that exposure to MSCs transforms the phenotype and function of macrophages. We suggest that Ly6c- monocytes could be a therapeutic target for asthma management.
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Cancers, including glioblastoma multiforme (GBM), undergo coordinated reprogramming of metabolic pathways that control glycolysis and oxidative phosphorylation (OXPHOS) to promote tumor growth in diverse tumor microenvironments. Adaptation to limited nutrient availability in the microenvironment is associated with remodeling of mitochondrial morphology and bioenergetic capacity. We recently demonstrated that NF-κB-inducing kinase (NIK) regulates mitochondrial morphology to promote GBM cell invasion. Here, we show that NIK is recruited to the outer membrane of dividing mitochondria with the master fission regulator, Dynamin-related protein1 (DRP1). Moreover, glucose deprivation-mediated metabolic shift to OXPHOS increases fission and mitochondrial localization of both NIK and DRP1. NIK deficiency results in decreased mitochondrial respiration, ATP production, and spare respiratory capacity (SRC), a critical measure of mitochondrial fitness. Although IκB kinase α and ß (IKKα/ß) and NIK are required for OXPHOS in high glucose media, only NIK is required to increase SRC under glucose deprivation. Consistent with an IKK-independent role for NIK in regulating metabolism, we show that NIK phosphorylates DRP1-S616 in vitro and in vivo. Notably, a constitutively active DRP1-S616E mutant rescues oxidative metabolism, invasiveness, and tumorigenic potential in NIK-/- cells without inducing IKK. Thus, we establish that NIK is critical for bioenergetic stress responses to promote GBM cell pathogenesis independently of IKK. Our data suggest that targeting NIK may be used to exploit metabolic vulnerabilities and improve therapeutic strategies for GBM.
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Neoplasias Encefálicas/enzimología , Metabolismo Energético , Glioblastoma/enzimología , Mitocondrias/enzimología , Proteínas Serina-Treonina Quinasas/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Dinaminas/genética , Dinaminas/metabolismo , Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , Glioblastoma/patología , Humanos , Mitocondrias/genética , Mitocondrias/patología , Dinámicas Mitocondriales , Membranas Mitocondriales/enzimología , Membranas Mitocondriales/patología , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Microambiente Tumoral , Quinasa de Factor Nuclear kappa BRESUMEN
Metastasis is the major cause of mortality in patients with breast cancer. Many signaling pathways have been linked to cancer invasiveness, but blockade of few protein components has succeeded in reducing metastasis. Thus, identification of proteins contributing to invasion that are manipulable by small molecules may be valuable in inhibiting spread of the disease. The protein kinase with no lysine (K) 1 (WNK1) has been suggested to induce migration of cells representing a range of cancer types. Analyses of mouse models and patient data have implicated WNK1 as one of a handful of genes uniquely linked to invasive breast cancer. Here, we present evidence that inhibition of WNK1 slows breast cancer metastasis. We show that depletion or inhibition of WNK1 reduces migration of several breast cancer cell lines in wound healing assays and decreases invasion in collagen matrices. Furthermore, WNK1 depletion suppresses expression of AXL, a tyrosine kinase implicated in metastasis. Finally, we demonstrate that WNK inhibition in mice attenuates tumor progression and metastatic burden. These data showing reduced migration, invasion, and metastasis upon WNK1 depletion in multiple breast cancer models suggest that WNK1 contributes to the metastatic phenotype, and that WNK1 inhibition may offer a therapeutic avenue for attenuating progression of invasive breast cancers.
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Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/patología , Regulación Neoplásica de la Expresión Génica , Proteína Quinasa Deficiente en Lisina WNK 1/metabolismo , Animales , Apoptosis , Biomarcadores de Tumor/genética , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Movimiento Celular , Proliferación Celular , Femenino , Humanos , Imidazoles/farmacología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Invasividad Neoplásica , Pirrolidinas/farmacología , Células Tumorales Cultivadas , Proteína Quinasa Deficiente en Lisina WNK 1/antagonistas & inhibidores , Proteína Quinasa Deficiente en Lisina WNK 1/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Gangliosides are sialic acid-conjugated glycosphingolipids that are believed to regulate cell differentiation as well as the signals of several signal molecules, including epidermal growth factor receptors (EGFR). These compounds are localized in a glycosphingolipid-enriched microdomain on the cell surface and regulated by the glycosphingolipid composition. However, the role that gangliosides play in osteoblastogenesis is not yet clearly understood, therefore, in this study, the relationship between gangliosides and EGFR activation was investigated during osteoblast differentiation in human mesenchymal stem cells (hMSCs). The results of high-performance thin-layer chromatography (HPTLC) showed that ganglioside GM3 expression was decreased, whereas ganglioside GD1a expression was increased during the differentiation of hMSCs into osteoblasts. In addition, an increase in the activation of alkaline phosphatase (ALP) was observed in response to treatment with EGF (5 ng/ml) and GD1a (1 microM) (p<0.05). The activation of ALP was significantly elevated in response to treatment of ganglioside GD1a with EGF when compared to control cells (p<0.01). However, treatment with GM3 (1muM) resulted in decreased ALP activation (p<0.01), and treatment of hMSCs with a chemical inhibitor of EGFR, AG1478, removed the differential effect of the two gangliosides. Moreover, incubation of the differentiating cells with GD1a enhanced the phosphorylation of EGFR, whereas treatment with GM3 reduced the EGFR phosphorylation. However, AG1478 treatment inhibited the effect of ganglioside GD1a elicitation on EGFR phosphorylation. Taken together, these results indicate that GD1a promotes osteoblast differentiation through the enhancement of EGFR phosphorylation, but that GM3 inhibits osteoblast differentiation through reduced EGFR phosphorylation, suggesting that GM3 and GD1a are essential molecules for regulating osteoblast differentiation in hMSCs.
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Diferenciación Celular , Receptores ErbB/metabolismo , Gangliósido G(M3)/fisiología , Gangliósidos/fisiología , Células Madre Mesenquimatosas/citología , Osteoblastos/citología , Fosfatasa Alcalina/metabolismo , Diferenciación Celular/efectos de los fármacos , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/agonistas , Gangliósido G(M3)/farmacología , Gangliósidos/farmacología , Humanos , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Osteoblastos/metabolismo , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Quinazolinas , Tirfostinos/farmacologíaRESUMEN
Gangliosides are a family of sialic acid-containing glycosphingolipids that are abundant in neurons and have a variety of functions in developing and mature tissues. We examined the expression of ganglioside GT1b in the embryonic preimplantation stage after freezing and thawing processes to determine the regulatory roles of ganglioside GT1b in early embryonic development. ICR mouse embryos at the two-cell stage obtained by flushing the oviducts were frozen by two cryopreservation procedures, slow freezing using a programmable freezer or vitrification by direct plunging into liquid nitrogen. Slow freezing was conducted with equilibration in 1.5 M 1,2-propanediol or 5% equilibration glycerol. Vitrification was applied with a 10-15 min equilibration in 7.5% ethylene glycol (EG), 7.5% dimethylsulfoxide (DMSO), and 30 sec in a solution of 15% EG, 15% DMSO and 0.5 M sucrose. Immediately after thawing, the survival rate of the embryos was assessed by their morphology and ability to develop to blastocysts in culture. The survival rate of vitrified and thawed embryos (92%) was significantly higher than that of slow frozen and thawed embryos (76%) (P<0.05). A tendency of higher blastocyst rate was found in the vitrified and thawed embryos compared to that of the slow frozen and thawed embryos. Confocal immunofluorescence staining confirmed that surviving embryos expressed ganglioside GT1b, with the strongest expression at the compacted eight-cell or later stage embryos. Ganglioside GT1b was not observed in the TUNEL-positive, apoptotic embryos, suggesting that cryopreservation had induced DNA breaks in them. These results suggest that ganglioside GT1b may play an important role in embryo survival or development.
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Desarrollo Embrionario/fisiología , Gangliósidos/biosíntesis , Animales , Apoptosis/efectos de los fármacos , Blastocisto/metabolismo , Supervivencia Celular/efectos de los fármacos , Criopreservación , Femenino , Técnica del Anticuerpo Fluorescente , Etiquetado Corte-Fin in Situ , Masculino , Ratones , Ratones Endogámicos ICR , Mórula/metabolismo , Embarazo , Zona Pelúcida/efectos de los fármacos , Zona Pelúcida/ultraestructuraRESUMEN
Bone homeostasis depends on the functional balance of osteoblasts (OBs) and osteoclasts (OCs). Lrp4 is a transmembrane protein that is mutated in patients with high bone mass. Loss of Lrp4 in OB-lineage cells increases bone mass by elevating bone formation by OBs and reducing bone resorption by OCs. However, it is unclear how Lrp4 deficiency in OBs impairs osteoclastogenesis. Here, we provide evidence that loss of Lrp4 in the OB lineage stabilizes the prorenin receptor (PRR) and increases PRR/V-ATPase-driven ATP release, thereby enhancing the production of the ATP derivative adenosine. Both pharmacological and genetic inhibition of adenosine-2A receptor (A2AR) in culture and Lrp4 mutant mice diminishes the osteoclastogenic deficit and reduces trabecular bone mass. Furthermore, elevated adenosine-A2AR signaling reduces receptor activator of nuclear factor κB (RANK)-mediated osteoclastogenesis. Collectively, these results identify a mechanism by which osteoblastic Lrp4 controls osteoclastogenesis, reveal a cross talk between A2AR and RANK signaling in osteoclastogenesis, and uncover an unrecognized pathophysiological mechanism of high-bone-mass disorders.
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Adenosina Trifosfato/metabolismo , Proteínas Relacionadas con Receptor de LDL/metabolismo , Osteoblastos/metabolismo , Osteogénesis/fisiología , Receptor de Adenosina A2A/metabolismo , Células 3T3 , Animales , Resorción Ósea/metabolismo , Resorción Ósea/fisiopatología , Huesos/metabolismo , Huesos/fisiología , Diferenciación Celular/fisiología , Línea Celular , Linaje de la Célula , Células HEK293 , Humanos , Ratones , Osteoblastos/fisiología , Osteoclastos/metabolismo , Osteoclastos/fisiología , Osteoprotegerina/metabolismo , Ligando RANK/metabolismo , Receptor Activador del Factor Nuclear kappa-B/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Stem cells are used for the investigation of developmental processes at both cellular and organism levels and offer tremendous potentials for clinical applications as an unlimited source for transplantation. Gangliosides, sialic acid-conjugated glycosphingolipids, play important regulatory roles in cell proliferation and differentiation. However, their expression patterns in stem cells and during neuronal differentiation are not known. Here, we investigated expression of gangliosides during the growth of mouse embryonic stem cells (mESCs), mesenchymal stem cells (MSCs) and differentiated neuronal cells by using high-performance thin-layer chromatography (HPTLC). Monosialoganglioside 1 (GM1) was expressed in mESCs and MSCs, while GM3 and GD3 were expressed in embryonic bodies. In the 9-day old differentiated neuronal cells from mESCs cells and MSCs, GM1 and GT1b were expressed. Results from immunostaining were consistent with those observed by HPTLC assay. These suggest that gangliosides are specifically expressed according to differentiation of mESCs and MSCs into neuronal cells and expressional difference of gangliosides may be a useful marker to identify differentiation of mESCs and MSCs into neuronal cells.
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Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Gangliósidos/metabolismo , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Neuronas/citología , Neuronas/metabolismo , Animales , Diferenciación Celular , Células Cultivadas , RatonesRESUMEN
Although the role of NF-κB-inducing kinase (NIK) in immunity is well established, its relevance in cancer is just emerging. Here we describe novel functions for NIK in regulating mitochondrial dynamics and motility to promote cell invasion. We show that NIK is localized to mitochondria in cancer cell lines, ex vivo tumor tissue, and mouse embryonic fibroblasts (MEFs). NIK promotes mitochondrial fission, velocity, and directional migration, resulting in subcellular distribution of mitochondria to the periphery of migrating cells. Moreover, NIK is required for recruitment of Drp1 to mitochondria, forms a complex with Drp1, and regulates Drp1 phosphorylation at Ser-616 and dephosphorylation at Ser-637. Consistent with a role for NIK in regulating mitochondrial dynamics, we demonstrate that Drp1 is required for NIK-dependent, cytokine-induced invasion. Importantly, using MEFs, we demonstrate that the established downstream mediators of NIK signaling, IκB kinase α/ß (IKKα/ß) and NF-κB, are not required for NIK to regulate cell invasion, Drp1 mitochondrial localization, or mitochondrial fission. Our results establish a new paradigm for IKK-independent NIK signaling and significantly expand the current dogma that NIK is predominantly cytosolic and exclusively regulates NF-κB activity. Overall, these findings highlight the importance of NIK in tumor pathogenesis and invite new therapeutic strategies that attenuate mitochondrial dysfunction through inhibition of NIK and Drp1.
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Fibroblastos/metabolismo , Mitocondrias/metabolismo , Invasividad Neoplásica , Proteínas Serina-Treonina Quinasas/metabolismo , Transporte de Proteínas/fisiología , Animales , Línea Celular Tumoral , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/fisiología , Humanos , Ratones , Proteínas Serina-Treonina Quinasas/genética , Quinasa de Factor Nuclear kappa BRESUMEN
Reduced bone mineral density and hip fracture are frequently observed in patients with Alzheimer's disease (AD). However, mechanisms underlying their association remain poorly understood. Amyloid precursor protein (APP) is a transmembrane protein that is ubiquitously expressed in bone marrow stromal cells (BMSCs), osteoblasts (OBs), macrophages (BMMs), and osteoclasts (OCs). Mutations in the APP gene identified in early-onset AD patients are believed to cause AD. But little is known about APP's role in bone remodeling. Here, we present evidence for Swedish mutant APP (APPswe) in suppression of OB differentiation and function in culture and in mouse. APP expression in BMSCs increases during aging. Ubiquitous expression of APPswe in young adult Tg2576 transgenic mice (under the control of a prion promoter) recaptured skeletal "aging-like" deficits, including decreased OB genesis and bone formation, increased adipogenesis and bone marrow fat, and enhanced OC genesis and bone resorption. Remarkably, selective expression of APPswe in mature OB-lineage cells in TgAPPswe-Ocn mice (under the control of osteocalcin [Ocn] promoter-driven Cre) also decreased OB genesis and increased OC formation, resulting in a trabecular bone loss. These results thus suggest a cell-autonomous role for APPswe in suppressing OB formation and function, but a nonautonomous effect on OC genesis. Notably, increased adipogenesis and elevated bone marrow fat were detected in young adult Tg2576 mice, but not in TgAPPswe-Ocn mice, implying that APPswe in BMSCs and/or multicell types in bone marrow promotes bone marrow adipogenesis. Intriguingly, the skeletal aging-like deficits in young adult Tg2576 mice were prevented by treatment with N-acetyl-L-cysteine (NAC), an antioxidant, suggesting that reactive oxygen species (ROS) may underlie APPswe-induced osteoporotic deficits. Taken together, these results demonstrate a role for APPswe in suppressing OB differentiation and bone formation, implicate APPswe as a detrimental factor for AD-associated osteoporotic deficit, and reveal a potential clinical value of NAC in the treatment of osteoporotic deficits. © 2013 American Society for Bone and Mineral Research.
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Acetilcisteína/uso terapéutico , Péptidos beta-Amiloides/metabolismo , Diferenciación Celular , Mutación/genética , Osteoblastos/patología , Osteoporosis/tratamiento farmacológico , Osteoporosis/patología , Acetilcisteína/farmacología , Fosfatasa Ácida/metabolismo , Adipogénesis/efectos de los fármacos , Envejecimiento/patología , Animales , Animales Recién Nacidos , Resorción Ósea/complicaciones , Resorción Ósea/tratamiento farmacológico , Resorción Ósea/metabolismo , Resorción Ósea/patología , Huesos/efectos de los fármacos , Huesos/metabolismo , Huesos/patología , Diferenciación Celular/efectos de los fármacos , Linaje de la Célula/efectos de los fármacos , Células Cultivadas , Cricetinae , Humanos , Isoenzimas/metabolismo , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Tamaño de los Órganos/efectos de los fármacos , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Osteogénesis/efectos de los fármacos , Osteoporosis/complicaciones , Osteoporosis/metabolismo , Fosfatasa Ácida TartratorresistenteRESUMEN
Receptor activator of NF-κB (RANK) plays a critical role in osteoclastogenesis, an essential process for the initiation of bone remodeling to maintain healthy bone mass and structure. Although the signaling and function of RANK have been investigated extensively, much less is known about the negative regulatory mechanisms of its signaling. We demonstrate in this paper that RANK trafficking, signaling, and function are regulated by VPS35, a major component of the retromer essential for selective endosome to Golgi retrieval of membrane proteins. VPS35 loss of function altered RANK ligand (RANKL)-induced RANK distribution, enhanced RANKL sensitivity, sustained RANKL signaling, and increased hyperresorptive osteoclast (OC) formation. Hemizygous deletion of the Vps35 gene in mice promoted hyperresorptive osteoclastogenesis, decreased bone formation, and caused a subsequent osteoporotic deficit, including decreased trabecular bone volumes and reduced trabecular thickness and density in long bones. These results indicate that VPS35 critically deregulates RANK signaling, thus restraining increased formation of hyperresorptive OCs and preventing osteoporotic deficits.
Asunto(s)
Osteoclastos/metabolismo , Osteoporosis/metabolismo , Ligando RANK/metabolismo , Transducción de Señal , Proteínas de Transporte Vesicular/metabolismo , Animales , Huesos/metabolismo , Huesos/patología , Aparato de Golgi/genética , Aparato de Golgi/metabolismo , Ratones , Ratones Mutantes , Tamaño de los Órganos , Osteoclastos/patología , Osteoporosis/genética , Osteoporosis/patología , Transporte de Proteínas/genética , Ligando RANK/genética , Receptor Activador del Factor Nuclear kappa-B/genética , Receptor Activador del Factor Nuclear kappa-B/metabolismo , Proteínas de Transporte Vesicular/genéticaRESUMEN
Myasthenia gravis (MG) is the most common disorder affecting the neuromuscular junction (NMJ). MG is frequently caused by autoantibodies against acetylcholine receptor (AChR) and a kinase critical for NMJ formation, MuSK; however, a proportion of MG patients are double-negative for anti-AChR and anti-MuSK antibodies. Recent studies in these subjects have identified autoantibodies against low-density lipoprotein receptor-related protein 4 (LRP4), an agrin receptor also critical for NMJ formation. LRP4 autoantibodies have not previously been implicated in MG pathogenesis. Here we demonstrate that mice immunized with the extracellular domain of LRP4 generated anti-LRP4 antibodies and exhibited MG-associated symptoms, including muscle weakness, reduced compound muscle action potentials (CMAPs), and compromised neuromuscular transmission. Additionally, fragmented and distorted NMJs were evident at both the light microscopic and electron microscopic levels. We found that anti-LRP4 sera decreased cell surface LRP4 levels, inhibited agrin-induced MuSK activation and AChR clustering, and activated complements, revealing potential pathophysiological mechanisms. To further confirm the pathogenicity of LRP4 antibodies, we transferred IgGs purified from LRP4-immunized rabbits into naive mice and found that they exhibited MG-like symptoms, including reduced CMAP and impaired neuromuscular transmission. Together, these data demonstrate that LRP4 autoantibodies induce MG and that LRP4 contributes to NMJ maintenance in adulthood.
Asunto(s)
Autoanticuerpos/inmunología , Proteínas Relacionadas con Receptor de LDL/inmunología , Miastenia Gravis Autoinmune Experimental/inmunología , Miastenia Gravis/inmunología , Unión Neuromuscular/inmunología , Receptores de LDL/inmunología , Animales , Modelos Animales de Enfermedad , Células HEK293 , Humanos , Ratones , Ratones Endogámicos A , Unión Neuromuscular/ultraestructura , Estructura Terciaria de Proteína , Conejos , Ratas , Receptores de LDL/fisiologíaRESUMEN
Alzheimer's disease (AD), one of the most dreaded neurodegenerative disorders, is characterized by cortical and cerebrovascular amyloid ß peptide (Aß) deposits, neurofibrillary tangles, chronic inflammation, and neuronal loss. Increased bone fracture rates and reduced bone density are commonly observed in patients with AD, suggesting one or more common denominators between both disorders. However, very few studies are available that have addressed this issue. Here, we present evidence for a function of amyloid precursor protein (APP) and Aß in regulating osteoclast (OC) differentiation in vitro and in vivo. Tg2576 mice, which express the Swedish mutation of APP (APPswe) under the control of a prion promoter, exhibit biphasic effects on OC activation, with an increase of OCs in younger mice (< 4 months old), but a decrease in older Tg2576 mice (> 4 months old). The increase of OCs in young Tg2576 mice appears to be mediated by Aß oligomers and receptor for advanced glycation end products (RAGE) expression in bone marrow macrophages (BMMs). However, the decrease of OC formation and activity in older Tg2576 mice may be due to the increase of soluble rage (sRAGE) in aged Tg2576 mice, an inhibitor of RANKL-induced osteoclastogenesis. These results suggest an unexpected function of APPswe/Aß, reveal a mechanism underlying altered bone remodeling in AD patients, and implicate APP/Aß and RAGE as common denominators for both AD and osteoporosis.