RESUMEN
G proteincoupled receptors (GPCRs) are the largest family of membrane receptors and activate several downstream signaling pathways involved in numerous physiological cellular processes. GPCRs are usually internalized and desensitized by intracellular signals. Numerous studies have shown that several GPCRs interact with sorting nexin 27 (SNX27), a cargo selector of the retromer complex, and are recycled from endosomes to the plasma membrane. Recycled GPCRs usually contain specific Cterminal postsynaptic density protein 95/Discs large protein/Zonula occludens 1 (PDZ) binding motifs, which are specifically recognized by SNX27, and return to the cell surface as functionally naïve receptors. Aberrant endosometomembrane recycling of GPCRs mediated by SNX27 may serve a critical role in cancer growth and development. Therefore, SNX27 may be a novel target for cancer therapies.
Asunto(s)
Antineoplásicos/farmacología , Carcinogénesis/patología , Neoplasias/tratamiento farmacológico , Receptores Acoplados a Proteínas G/metabolismo , Nexinas de Clasificación/metabolismo , Animales , Antineoplásicos/uso terapéutico , Carcinogénesis/efectos de los fármacos , Línea Celular Tumoral , Membrana Celular/metabolismo , Progresión de la Enfermedad , Endosomas/metabolismo , Humanos , Ratones , Terapia Molecular Dirigida/métodos , Neoplasias/patología , Nexinas de Clasificación/antagonistas & inhibidores , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The glucagon-like peptide 1 (GLP-1) receptor (GLP-1R) is a key target for type 2 diabetes (T2D) treatment. Because endocytic trafficking of agonist-bound receptors is one of the most important routes for regulation of receptor signaling, a better understanding of this process may facilitate the development of new T2D therapeutic strategies. Here, we screened 29 proteins with known functions in G protein-coupled receptor trafficking for their role in GLP-1R potentiation of insulin secretion in pancreatic ß-cells. We identify five (clathrin, dynamin1, AP2, sorting nexins [SNX] SNX27, and SNX1) that increase and four (huntingtin-interacting protein 1 [HIP1], HIP14, GASP-1, and Nedd4) that decrease insulin secretion from murine insulinoma MIN6B1 cells in response to the GLP-1 analog exendin-4. The roles of HIP1 and the endosomal SNX1 and SNX27 were further characterized in mouse and human ß-cell lines and human islets. While HIP1 was required for the coupling of cell surface GLP-1R activation with clathrin-dependent endocytosis, the SNXs were found to control the balance between GLP-1R plasma membrane recycling and lysosomal degradation and, in doing so, determine the overall ß-cell incretin responses. We thus identify key modulators of GLP-1R trafficking and signaling that might provide novel targets to enhance insulin secretion in T2D.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Endocitosis , Receptor del Péptido 1 Similar al Glucagón/agonistas , Células Secretoras de Insulina/metabolismo , Sistemas de Mensajero Secundario , Nexinas de Clasificación/metabolismo , Animales , Señalización del Calcio/efectos de los fármacos , Línea Celular , AMP Cíclico/metabolismo , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Endocitosis/efectos de los fármacos , Exenatida , Receptor del Péptido 1 Similar al Glucagón/genética , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Humanos , Incretinas/farmacología , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/ultraestructura , Lisosomas/efectos de los fármacos , Lisosomas/enzimología , Lisosomas/metabolismo , Lisosomas/ultraestructura , Ratones , Microscopía Electrónica de Transmisión , Péptidos/farmacología , Interferencia de ARN , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Sistemas de Mensajero Secundario/efectos de los fármacos , Nexinas de Clasificación/antagonistas & inhibidores , Nexinas de Clasificación/genética , Técnicas de Cultivo de Tejidos , Ponzoñas/farmacologíaRESUMEN
The tumor suppressor PTEN executes cellular functions predominantly through its phosphatase activity. Here we identified a phosphatase-independent role for PTEN during vesicular trafficking of the glucose transporter GLUT1. PTEN physically interacts with SNX27, a component of the retromer complex that recycles transmembrane receptors such as GLUT1 from endosomes to the plasma membrane. PTEN binding with SNX27 prevents GLUT1 accumulation at the plasma membrane because of defective recycling and thus reduces cellular glucose uptake. Mechanistically, PTEN blocks the association of SNX27 with VPS26 and thereby hinders assembly of a functional retromer complex during the receptor recycling process. Importantly, we found a PTEN somatic mutation (T401I) that is defective in disrupting the association between SNX27 and VPS26, suggesting a critical role for PTEN in controlling optimal GLUT1 levels at the membrane to prevent tumor progression. Together, our results reveal a fundamental role of PTEN in the regulation of the SNX27 retromer pathway, which governs glucose transport and might contribute to PTEN tumor suppressor function.
Asunto(s)
Transportador de Glucosa de Tipo 1/metabolismo , Fosfohidrolasa PTEN/metabolismo , Nexinas de Clasificación/metabolismo , Membrana Celular/metabolismo , Dicroismo Circular , Endosomas/metabolismo , Glucosa/metabolismo , Células HEK293 , Células HeLa , Humanos , Inmunoprecipitación , Proteínas de Membrana de los Lisosomas/metabolismo , Mutagénesis , Dominios PDZ , Fosfohidrolasa PTEN/antagonistas & inhibidores , Fosfohidrolasa PTEN/genética , Unión Proteica , Estructura Secundaria de Proteína , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Nexinas de Clasificación/antagonistas & inhibidores , Nexinas de Clasificación/genética , Proteínas de Transporte Vesicular/metabolismoRESUMEN
After endocytosis, transmembrane cargo reaches endosomes, where it encounters complexes dedicated to opposing functions: recycling and degradation. Microdomains containing endosomal sorting complexes required for transport (ESCRT)-0 component Hrs [hepatocyte growth factor-regulated tyrosine kinase substrate (HGRS-1) in Caenorhabditis elegans] mediate cargo degradation, concentrating ubiquitinated cargo and organizing the activities of ESCRT. At the same time, retromer associated sorting nexin one (SNX-1) and its binding partner, J-domain protein RME-8, sort cargo away from degradation, promoting cargo recycling to the Golgi. Thus, we hypothesized that there could be important regulatory interactions between retromer and ESCRT that balance degradative and recycling functions. Taking advantage of the naturally large endosomes of the C. elegans coelomocyte, we visualized complementary ESCRT-0 and RME-8/SNX-1 microdomains in vivo and assayed the ability of retromer and ESCRT microdomains to regulate one another. We found in snx-1(0) and rme-8(ts) mutants increased endosomal coverage and intensity of HGRS-1-labeled microdomains, as well as increased total levels of HGRS-1 bound to membranes. These effects are specific to SNX-1 and RME-8, as loss of other retromer components SNX-3 and vacuolar protein sorting-associated protein 35 (VPS-35) did not affect HGRS-1 microdomains. Additionally, knockdown of hgrs-1 had little to no effect on SNX-1 and RME-8 microdomains, suggesting directionality to the interaction. Separation of the functionally distinct ESCRT-0 and SNX-1/RME-8 microdomains was also compromised in the absence of RME-8 and SNX-1, a phenomenon we observed to be conserved, as depletion of Snx1 and Snx2 in HeLa cells also led to greater overlap of Rme-8 and Hrs on endosomes.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Endosomas/metabolismo , Fosfoproteínas/metabolismo , Nexinas de Clasificación/metabolismo , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/antagonistas & inhibidores , Proteínas de Caenorhabditis elegans/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/antagonistas & inhibidores , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Chaperonas Moleculares , Fosfoproteínas/antagonistas & inhibidores , Fosfoproteínas/genética , Dominios y Motivos de Interacción de Proteínas , Transporte de Proteínas , Proteolisis , ARN Interferente Pequeño/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Nexinas de Clasificación/antagonistas & inhibidores , Nexinas de Clasificación/genéticaRESUMEN
ß1-Adrenergic receptor (ß1-AR) agonists and antagonists are widely used in the treatment of major cardiovascular diseases such as heart failure and hypertension. The ß1-AR like other G protein-coupled receptors (GPCRs) are endocytosed in response to intense agonist activation. Recycling of the agonist-internalized ß1-AR is dependent on its carboxy-terminal type-1 PSD-95/DLG/ZO1 (PDZ) and on phospho-serine312 in the third intracellular loop of the ß1-AR. Progressive elongation of the ß1-AR at its C-tail inactivated the PDZ-biding domain and inhibited the recycling of the ß1-AR. However, fusing a twenty amino acid peptide derived from the multiple cloning region of the mammalian expression vector pCDNA3 to the C-tail of the ß1-AR (ß1-AR[+20]) produced a chimeric ß1-AR that recycled rapidly and efficiently. The ß1-AR[+20] recycled in a type-1 PDZ and phospho-Ser312-independent manner, indicating that this peptide provided a general GPCR recycling signal. Fusing the enhanced yellow fluorescent protein (EYFP) down-stream of ß1-AR[+20] generated a ß1-AR-EYFP chimera that was expressed on the membrane and recycled efficiently after agonist-induced internalization. This construct trafficked in a PDZ-SNX27/retromer-independent manner. We also fused EYFP to the N-terminus of the ß1-AR to created EYFP-WT ß1-AR. This construct recycled in PDZ and SNX27/retromer dependent manner. These ß1-AR-EYFP constructs would be useful for high throughput screening (HTS) programs to identify new entities that would interfere with the recycling of agonist internalized GPCR that traffic in PDZ-dependent vs. PDZ-independent roadmaps.
Asunto(s)
Agonistas de Receptores Adrenérgicos beta 1/farmacología , Descubrimiento de Drogas/métodos , Endocitosis/efectos de los fármacos , Modelos Moleculares , Receptores Adrenérgicos beta 1/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Endosomas/efectos de los fármacos , Endosomas/metabolismo , Células HEK293 , Ensayos Analíticos de Alto Rendimiento , Humanos , Inmunoprecipitación , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía Confocal , Microscopía Fluorescente , Oligopéptidos/genética , Oligopéptidos/metabolismo , Dominios PDZ , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fosfoserina/metabolismo , Plásmidos/química , Plásmidos/genética , Plásmidos/metabolismo , Interferencia de ARN , Receptores Adrenérgicos beta 1/química , Receptores Adrenérgicos beta 1/genética , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Nexinas de Clasificación/antagonistas & inhibidores , Nexinas de Clasificación/química , Nexinas de Clasificación/genética , Nexinas de Clasificación/metabolismoRESUMEN
UNLABELLED: Previous studies have demonstrated an interaction between sorting nexin 17 and the L2 capsid proteins from a variety of papillomavirus types. This interaction is required for late endosomal trafficking of the L2 protein and entry of the L2/DNA complex into the nucleus during infection. Here we show an interaction between papillomavirus L2 proteins and the related PX-FERM family member sorting nexin 27 (SNX27), which is mediated in part by a novel interaction between the PDZ domain of SNX27 and sequences in a central portion of L2. The interaction is direct and, unlike that with SNX17, is variable in strength depending on the papillomavirus type. We show that small interfering RNA (siRNA)-mediated knockdown of SNX27 alone leads to a marginal reduction in the efficiency of viral infection but that double knockdown of both sorting nexins results in a striking reduction in infection, greater than that observed for the knockdown of either sorting nexin alone. These results suggest that the HPV L2 proteins can interact through distinct mechanisms with multiple components of the cellular cargo-sorting machinery. IMPORTANCE: The trafficking of papillomaviruses to the host cell nucleus during their natural infectious life cycle is an incompletely understood process. Studies have suggested that the virus minor capsid protein L2 can interact with the endosomal recycling pathway, in part by association with sorting nexin 17, to ensure that virus DNA bound to L2 is recycled through the trans-Golgi network rather than back to the plasma membrane. In this study, we characterize the interaction between L2 and a second sorting nexin, SNX27, which is also part of the retromer complex. The study furthers our understanding of papillomavirus infection dynamics and provides potential tools for the further dissection of endosomal structure and function.
Asunto(s)
Proteínas de la Cápside/metabolismo , Papillomavirus Humano 16/genética , Proteínas Oncogénicas Virales/metabolismo , Dominios PDZ/genética , Nexinas de Clasificación/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Transporte Biológico , Cápside/química , Cápside/metabolismo , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Línea Celular , Membrana Celular/metabolismo , Membrana Celular/virología , Núcleo Celular/metabolismo , Núcleo Celular/virología , ADN/genética , ADN/metabolismo , Endosomas/metabolismo , Endosomas/virología , Expresión Génica , Células HEK293 , Interacciones Huésped-Patógeno , Papillomavirus Humano 16/metabolismo , Humanos , Queratinocitos , Datos de Secuencia Molecular , Proteínas Oncogénicas Virales/química , Proteínas Oncogénicas Virales/genética , Unión Proteica , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Nexinas de Clasificación/antagonistas & inhibidores , Nexinas de Clasificación/genética , Replicación Viral/genéticaRESUMEN
This study demonstrated the feasibility and benefit of an antibody-based experimental approach to identify microRNA functional targets from hundreds of predicted genes using miR-206 as an example. Using a receptor tyrosine kinase (RTK) antibody array, we identified 7 phosphorylated RTKs that were significantly differentially regulated after miR-206-mimic transfection. We then focused on MET, the most varied RTK, and bioinformatically constructed a MET-centred network using computationally predicted miR-206 targets. Within this network, we analyzed two validated targets, PAX3 and SNX2, and one candidate target, EIF4E, may account for the inhibitory effect of miR-206 on MET phosphorylation. Luciferase and Western-blot assays indicated that EIF4E was a direct target of miR-206. This concept may also be applicable for other microRNAs and other antibody array platforms.
Asunto(s)
Anticuerpos Fosfo-Específicos/metabolismo , Factor 4E Eucariótico de Iniciación/antagonistas & inhibidores , MicroARNs/metabolismo , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Interferencia de ARN , Especificidad de Anticuerpos , Sitios de Unión , Línea Celular Tumoral , Biología Computacional , Factor 4E Eucariótico de Iniciación/genética , Factor 4E Eucariótico de Iniciación/metabolismo , Genes Reporteros , Humanos , Proteínas Mutantes/antagonistas & inhibidores , Proteínas Mutantes/metabolismo , Factor de Transcripción PAX3 , Factores de Transcripción Paired Box/antagonistas & inhibidores , Factores de Transcripción Paired Box/genética , Factores de Transcripción Paired Box/metabolismo , Fosforilación , Análisis por Matrices de Proteínas , Procesamiento Proteico-Postraduccional , Proteómica/métodos , Proteínas Proto-Oncogénicas c-met/genética , Proteínas Proto-Oncogénicas c-met/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Nexinas de Clasificación/antagonistas & inhibidores , Nexinas de Clasificación/genética , Nexinas de Clasificación/metabolismoRESUMEN
Gefitinib is known to suppress the activation of EGFR signaling, which is required for cell survival and proliferation in non-small cell lung cancer (NSCLC) cell lines. We previously demonstrated that the gefitinib-sensitive NSCLC cell line PC9 shows efficient ligand-induced endocytosis of phosphorylated EGFR (pEGFR). In contrast, the gefitinib-resistant NSCLC cell lines QG56 and A549 showed internalized pEGFR accumulation in the aggregated early endosomes, and this was associated with SNX1, a protein that interacts with and enhances the degradation of EGFR upon EGF stimulation. In the present study, to investigate the role of SNX1 on EGF-stimulated EGFR/pEGFR endocytosis via the endocytic pathway, we examined the effect of depletion of SNX1 expression by siRNA in human NSCLC cell lines. Using immunofluorescence, we demonstrated that transfection of SNX1 siRNA into gefitinib-resistant NSCLC cells resulted in the disappearance of a large amounts of SNX1 staining. In addition, upon 15 min of EGF stimulation, we observed an efficient EGFR phosphorylation and a rapid endocytic delivery of pEGFR from early endosomes to late endosomes. Further, western blot analysis revealed that silencing of SNX1 expression by siRNA in the gefitinib-resistant cells leads to an accelerated degradation of EGFR along with a dramatic increase in the amounts of pEGFR after EGF stimulation. Based on these findings, we suggest that SNX1 is involved in the negative regulation of ligand-induced EGFR phosphorylation and mediates EGFR/pEGFR trafficking out of early endosomes for targeting to late endosomes/lysosomes via the early/late endocytic pathway in human lung cancer cells.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Receptores ErbB/genética , Quinazolinas/administración & dosificación , Nexinas de Clasificación/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Endocitosis/efectos de los fármacos , Endosomas/efectos de los fármacos , Endosomas/metabolismo , Receptores ErbB/biosíntesis , Gefitinib , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Fosforilación/efectos de los fármacos , ARN Interferente Pequeño/genética , Transducción de Señal/efectos de los fármacos , Nexinas de Clasificación/antagonistas & inhibidores , Nexinas de Clasificación/biosíntesisRESUMEN
Sorting nexins (SNXs) are phosphoinositide-binding proteins implicated in the sorting of various membrane proteins in vitro, but the in vivo functions of them remain largely unknown. We reported previously that SNX10 is a unique member of the SNX family genes in that it has vacuolation activity in cells. We investigate the biological function of SNX10 by loss-of-function assay in this study and demonstrate that SNX10 is required for the formation of primary cilia in cultured cells. In zebrafish, SNX10 is involved in ciliogenesis in the Kupffer's vesicle and essential for left-right patterning of visceral organs. Mechanistically, SNX10 interacts with V-ATPase complex and targets it to the centrosome where ciliogenesis is initiated. Like SNX10, V-ATPase regulates ciliogenesis in vitro and in vivo and does so synergistically with SNX10. We further discover that SNX10 and V-ATPase regulate the ciliary trafficking of Rab8a, which is a critical regulator of ciliary membrane extension. These results identify an SNX10/V-ATPase-regulated vesicular trafficking pathway that is crucial for ciliogenesis, and reveal that SNX10/V-ATPase, through the regulation of cilia formation in various organs, play an essential role during early embryonic development.
Asunto(s)
Nexinas de Clasificación/metabolismo , ATPasas de Translocación de Protón Vacuolares/metabolismo , Proteínas de Pez Cebra/metabolismo , Animales , Tipificación del Cuerpo , Línea Celular Tumoral , Centrosoma/fisiología , Cilios/fisiología , Desarrollo Embrionario , Humanos , Morfogénesis , Unión Proteica , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Nexinas de Clasificación/antagonistas & inhibidores , Nexinas de Clasificación/genética , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , ATPasas de Translocación de Protón Vacuolares/genética , Vacuolas/metabolismo , Vacuolas/fisiología , Pez Cebra , Proteínas de Pez Cebra/antagonistas & inhibidores , Proteínas de Pez Cebra/genética , Proteínas de Unión al GTP rab/metabolismoRESUMEN
RANKL-stimulation of osteoclast precursors results in up-regulation of genes involved in the process of differentiation and activation. In this report we describe the expression and functional characterization of Sorting Nexin 10 (snx10). Snx10 belongs to the sorting nexin (SNX) family, a diverse group of proteins with a common feature: the PX domain, which is involved in membrane trafficking and cargo sorting in endosomes. Snx10 is strongly up-regulated during RANKL-induced osteoclast differentiation in vitro and expressed in osteoclasts in vivo. qPCR analysis confirmed a significant increase in the expression of snx10 in in vitro-derived osteoclasts, as well as in femur and calvaria. Immunohistochemical analysis of mouse embryo sections showed expression in long bone, calvariae, and developing teeth. The expression was limited to cells that also expressed TRAP, demonstrating osteoclastic localization. Confocal immunofluorescence and subcellular fractionation analysis revealed Snx10 localization in the nucleus and in the endoplasmic reticulum (ER). To study a possible role for snx10 in osteoclast differentiation and function we silenced snx10 expression and found that snx10 silencing inhibited RANKL-induced osteoclast formation and osteoclast resorption on hydroxyapatite. Silencing also inhibited TRAP secretion. Taken together, these results confirm that snx10 is expressed in osteoclasts and is required for osteoclast differentiation and activity in vitro. Since inhibition of vesicular trafficking is essential for osteoclast formation and activity and SNX10 is involved in intracellular vesicular trafficking, these studies may identify a new candidate gene involved in the development of human bone diseases including osteoporosis.
Asunto(s)
Resorción Ósea/patología , Resorción Ósea/fisiopatología , Osteoclastos/citología , Osteoclastos/fisiología , Nexinas de Clasificación/fisiología , Secuencia de Aminoácidos , Animales , Diferenciación Celular/fisiología , Línea Celular , Células Cultivadas , Cartilla de ADN/genética , Regulación del Desarrollo de la Expresión Génica , Silenciador del Gen , Humanos , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Ligando RANK/fisiología , ARN Interferente Pequeño/genética , Homología de Secuencia de Aminoácido , Nexinas de Clasificación/antagonistas & inhibidores , Nexinas de Clasificación/genéticaRESUMEN
Since being introduced globally as aspirin in 1899, acetylsalicylic acid has been widely used as an analgesic, anti-inflammation, anti-pyretic, and anti-thrombotic drug for years. Aspirin had been reported to down-regulate surface expression of CD40, CD80, CD86, and MHCII in myeloid dendritic cells (DC), which played essential roles in regulating the immune system. We hypothesized that the down-regulation of these surface membrane proteins is partly due to the ability of aspirin in regulating trafficking/sorting of endocytosed surface membrane proteins. By using an established epidermoid carcinoma cell line (A-431), which overexpresses the epidermal growth factor receptor (EGFR) and transferrin receptor (TfnR), we show that aspirin (1) reduces cell surface expression of EGFR and (2) accumulates endocytosed-EGFR and -TfnR in the early/sorting endosome (ESE). Further elucidation of the mechanism suggests that aspirin enhances recruitment of SNX3 and SNX5 to membranes and consistently, both SNX3 and SNX5 play essential roles in the aspirin-mediated accumulation of endocytosed-TfnR at the ESE. This study sheds light on how aspirin may down-regulate surface expression of EGFR by inhibiting/delaying the exit of endocytosed-EGFR from the ESE and recycling of endocytosed-EGFR back to the cell surface.
Asunto(s)
Aspirina/farmacología , Carcinoma de Células Escamosas/metabolismo , Receptores ErbB/metabolismo , Nexinas de Clasificación/metabolismo , Secuencia de Bases , Transporte Biológico Activo/efectos de los fármacos , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/ultraestructura , Línea Celular Tumoral , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Cartilla de ADN/genética , Endocitosis/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Microscopía Inmunoelectrónica , Modelos Biológicos , Receptores de Transferrina/metabolismo , Transducción de Señal/efectos de los fármacos , Nexinas de Clasificación/antagonistas & inhibidores , Nexinas de Clasificación/genéticaRESUMEN
Wnt proteins are lipid-modified glycoproteins that play a central role in development, adult tissue homeostasis and disease. Secretion of Wnt proteins is mediated by the Wnt-binding protein Wntless (Wls), which transports Wnt from the Golgi network to the cell surface for release. It has recently been shown that recycling of Wls through a retromer-dependent endosome-to-Golgi trafficking pathway is required for efficient Wnt secretion, but the mechanism of this retrograde transport pathway is poorly understood. Here, we report that Wls recycling is mediated through a retromer pathway that is independent of the retromer sorting nexins SNX1-SNX2 and SNX5-SNX6. We have found that the unrelated sorting nexin, SNX3, has an evolutionarily conserved function in Wls recycling and Wnt secretion and show that SNX3 interacts directly with the cargo-selective subcomplex of the retromer to sort Wls into a morphologically distinct retrieval pathway. These results demonstrate that SNX3 is part of an alternative retromer pathway that functionally separates the retrograde transport of Wls from other retromer cargo.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Nexinas de Clasificación/metabolismo , Proteínas Wnt/metabolismo , Animales , Animales Modificados Genéticamente , Transporte Biológico Activo , Caenorhabditis elegans/genética , Caenorhabditis elegans/crecimiento & desarrollo , Caenorhabditis elegans/metabolismo , Drosophila/genética , Drosophila/crecimiento & desarrollo , Drosophila/metabolismo , Endosomas/metabolismo , Células HeLa , Humanos , Modelos Biológicos , Interferencia de ARN , Transducción de Señal , Nexinas de Clasificación/antagonistas & inhibidores , Nexinas de Clasificación/genética , Red trans-Golgi/metabolismoRESUMEN
MicroRNAs (miRNAs) are strongly implicated in cancer but their specific roles and functions in the major cancers have yet to be fully elucidated. In this study, we defined the oncogenic significance and function of miR-95, which we found to be elevated in colorectal cancer (CRC) tissues by microarray analysis. Evaluation of an expanded CRC cohort revealed that miR-95 expression was up-regulated in nearly half of the tumors examined (42/87) compared with the corresponding noncancerous tissues. Ectopic overexpression of miR-95 in human CRC cell lines promoted cell growth in vitro and tumorigenicity in vivo, whereas RNAi-mediated silencing of miR-95 decreased cell growth ratio. Mechanistic studies revealed that miR-95 repressed the expression of reporter gene coupled to the 3'-untranslated region of sorting nexin 1 (SNX1), whereas miR-95 silencing up-regulated SNX1 expression. Moreover, miR-95 expression levels correlated inversely with SNX1 protein levels in human CRC tissues. RNAi-mediated knockdown of SNX1 phenocopied the proliferation-promoting effect of miR-95, whereas overexpression of SNX1 blocked miR-95-induced proliferation of CRC cells. Taken together, these results demonstrated that miR-95 increases proliferation by directly targeting SNX1, defining miR-95 as a new oncogenic miRNA in CRC.