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1.
Hum Genet ; 139(4): 447-459, 2020 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-32076828

RESUMEN

Low-density lipoprotein receptor-related protein 6 (LRP6) is a member of the low-density lipoprotein receptors (LDLRs) family and accumulating evidence points to the critical role of LRP6 in cardiovascular health and homeostasis. In addition to presenting the well-appreciated roles in canonical signaling regulating blood pressure, blood glucose, lipid metabolism, atherosclerosis, cardiac valve disease, cardiac development, Alzheimer's disease and tumorigenesis, LRP6 also inhibits non-canonical Wnt signals that promote arterial smooth muscle cell proliferation and vascular calcification. Noticeably, the role of LRP6 is displayed in cardiometabolic disease, an increasingly important clinical burden with aging and obesity. The prospect for cardiovascular diseases treatment via targeting LRP6-mediated signaling pathways may improve central blood pressure and lipid metabolism, and reduce neointima formation and myocardial ischemia-reperfusion injury. Thus, a deep and comprehensive understanding of LRP6 structure, function and signaling pathways will contribute to clinical diagnosis, therapy and new drug development for LRP6-related cardiovascular diseases.


Asunto(s)
Enfermedades Cardiovasculares , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad , Transducción de Señal , Calcificación Vascular , Vía de Señalización Wnt , Envejecimiento/metabolismo , Envejecimiento/patología , Animales , Enfermedades Cardiovasculares/diagnóstico , Enfermedades Cardiovasculares/metabolismo , Enfermedades Cardiovasculares/patología , Enfermedades Cardiovasculares/terapia , Humanos , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/química , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Obesidad/metabolismo , Obesidad/patología , Relación Estructura-Actividad , Calcificación Vascular/diagnóstico , Calcificación Vascular/metabolismo , Calcificación Vascular/patología , Calcificación Vascular/terapia
2.
Nat Struct Mol Biol ; 26(5): 372-379, 2019 05.
Artículo en Inglés | MEDLINE | ID: mdl-31036956

RESUMEN

Wnt signaling plays fundamental roles in organogenesis, tissue regeneration and cancer, but high-resolution structural information of mammalian Wnt proteins is lacking. We solved a 2.8-Å resolution crystal structure of human Wnt3 in complex with mouse Frizzled 8 Cys-rich domain (CRD). Wnt3 grabs the receptor in a manner very similar to that found in Xenopus Wnt8 complexed with the same receptor. Unlike Xenopus Wnt8-bound CRD, however, Wnt3-bound CRD formed a symmetrical dimer in the crystal by exchanging the tip of the unsaturated acyl chain attached to each Wnt3, confirming the ability of Wnt and Frizzled CRD to form a 2:2 complex. The hypervariable 'linker' region of Wnt3 formed a ß-hairpin protrusion opposite from the Frizzled binding interface, consistent with its proposed role in the coreceptor recognition. Direct binding between this segment and the Wnt coreceptor LRP6 was confirmed, enabling us to build a structural model of the Wnt-Frizzled-LRP6 ternary complex.


Asunto(s)
Receptores Acoplados a Proteínas G/química , Proteína Wnt3/química , Animales , Sitios de Unión , Cristalografía por Rayos X , Dimerización , Humanos , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Ratones , Conformación Proteica , Receptores Acoplados a Proteínas G/metabolismo , Proteína Wnt3/metabolismo , Xenopus
3.
Mol Carcinog ; 58(7): 1168-1180, 2019 07.
Artículo en Inglés | MEDLINE | ID: mdl-30834575

RESUMEN

Ras-association domain family (RASSF) proteins exert distinct cellular functions. The expression of RASSF10 in non-small cell lung cancer and its underlying mechanism have not been reported. Herein, we explored the roles of RASSF10 in lung cancer cells and potential molecular mechanisms. We found low RASSF10 expression in lung cancer specimens, which was associated with low differentiation, advanced pTNM stage, positive lymph node metastasis, and poor prognosis in patients. Furthermore, RASSF10 overexpression inhibited the proliferation and invasion of lung cancer cells, which was the result of Wnt signaling suppression. However, we found that RASSF10 had no influence on Hippo signaling, while RASSF10 bound to LRP6 via the coiled-coil domains and reduced p-LRP6 level, eventually prohibiting ß-catenin nuclear translocation. However, deleting the coiled-coil domains ablated this function. These findings expound the interaction between RASSF10 and LRP6 and uncover a potential link between N-terminal RASSFs and the Wnt pathway.


Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/patología , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Neoplasias Pulmonares/patología , Proteínas Supresoras de Tumor/genética , Vía de Señalización Wnt/genética , Células A549 , Transporte Activo de Núcleo Celular/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/mortalidad , Línea Celular Tumoral , Proliferación Celular/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidad , Metástasis Linfática/patología , Masculino , Persona de Mediana Edad , Invasividad Neoplásica/genética , Fosforilación/genética , Pronóstico , Unión Proteica/genética , Proteínas Serina-Treonina Quinasas/metabolismo , beta Catenina/metabolismo
4.
BMC Gastroenterol ; 19(1): 38, 2019 Mar 06.
Artículo en Inglés | MEDLINE | ID: mdl-30841855

RESUMEN

BACKGROUND: An altered Wnt-signaling activation has been reported during Barrett's esophagus progression, but with rarely detected mutations in APC and ß-catenin (CTNNB1) genes. METHODS: In this study, a robust in-depth expression pattern analysis of frizzled receptors, co-receptors, the Wnt-ligands Wnt3a and Wnt5a, the Wnt-signaling downstream targets Axin2, and CyclinD1, as well as the activation of the intracellular signaling kinases Akt and GSK3ß was performed in an in vitro cell culture model of Barrett's esophagus. Representing the Barrett's sequence, we used normal esophageal squamous epithelium (EPC-1, EPC-2), metaplasia (CP-A) and dysplasia (CP-B) to esophageal adenocarcinoma (EAC) cell lines (OE33, OE19) and primary specimens of squamous epithelium, metaplasia and EAC. RESULTS: A loss of Wnt3a expression was observed beginning from the metaplastic cell line CP-A towards dysplasia (CP-B) and EAC (OE33 and OE19), confirmed by a lower staining index of WNT3A in Barrett's metaplasia and EAC, than in squamous epithelium specimens. Frizzled 1-10 expression analysis revealed a distinct expression pattern, showing the highest expression for Fzd2, Fzd3, Fzd4, Fzd5, Fzd7, and the co-receptor LRP5/6 in EAC cells, while Fzd3 and Fzd7 were rarely expressed in primary specimens from squamous epithelium. CONCLUSION: Despite the absence of an in-depth characterization of Wnt-signaling-associated receptors in Barrett's esophagus, by showing variations of the Fzd- and co-receptor profiles, we provide evidence to have a significant role during Barrett's progression and the underlying pathological mechanisms.


Asunto(s)
Esófago de Barrett/genética , Esófago de Barrett/metabolismo , Vía de Señalización Wnt/genética , beta Catenina/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína Axina/genética , Proteína Axina/metabolismo , Esófago de Barrett/patología , Línea Celular , Ciclina D1/genética , Ciclina D1/metabolismo , Progresión de la Enfermedad , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patología , Receptores Frizzled/genética , Receptores Frizzled/metabolismo , Expresión Génica , Humanos , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Proteína Wnt-5a/genética , Proteína Wnt-5a/metabolismo , Proteína Wnt3A/genética , Proteína Wnt3A/metabolismo , beta Catenina/metabolismo
5.
Metabolism ; 94: 1-8, 2019 05.
Artículo en Inglés | MEDLINE | ID: mdl-30711569

RESUMEN

BACKGROUND: Hepatic lipogenesis dysregulation is essential for the development of non-alcoholic fatty liver disease (NAFLD). Emerging evidence indicates the importance of the involvement of long non-coding RNAs (LncRNAs) in lipogenesis. However, the specific mechanism underlying this process is not clear. OBJECTIVE: This study aimed to investigate the functional implication of LncRNA MEG3 (MEG3) in fatty degeneration of hepatocytes and in the pathogenesis of NAFLD. METHODS: The expression of MEG3 was analysed in in vitro and in vivo models of NAFLD, which were established by free fatty acid (FFA)-challenged HepG2 cells and high-fat diet-fed mice, respectively. Endogenous MEG3 was over-expressed by a specific pcDNA3.1-MEG3 to evaluate the regulatory function of MEG3 on triglyceride (TG)- and lipogenesis-related genes. Bioinformatic analysis was used to predict the target genes and binding sites, and the targeted regulatory relationship was verified with a dual luciferase assay. Finally, the possible pathway that regulates MEG3 was also evaluated. RESULTS: We found that the downregulation of MEG3 in vitro and in vivo models of NAFLD was negatively correlated with lipogenesis-related genes and that overexpression of MEG3 reversed FFA-induced lipid accumulation in HepG2 cells. miR-21 was upregulated in the FFA-challenged HepG2 cells and was physically associated with MEG3 in the process of lipogenesis. Our mechanistic studies demonstrated that MEG3 competitively binds to miR-21 with LRP6, followed by the inhibition of the mTOR pathway, which induces intracellular lipid accumulation. CONCLUSION: Our data are the first to document the working model of MEG3 functions as a potential hepatocyte lipid degeneration suppressor. MEG3 helps to alleviate lipid over-deposition, probably by binding to miR-21 to regulate the expression of LRP6. Our results suggest the potency of MEG3 as a biomarker for NAFLD and as a therapeutic target for treatment.


Asunto(s)
Lipogénesis , Hígado/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , MicroARNs/metabolismo , ARN Largo no Codificante/fisiología , Animales , Unión Competitiva , Células Hep G2 , Humanos , Lipogénesis/efectos de los fármacos , Lipogénesis/genética , Ratones , Enfermedad del Hígado Graso no Alcohólico/diagnóstico , Enfermedad del Hígado Graso no Alcohólico/etiología , Enfermedad del Hígado Graso no Alcohólico/metabolismo , ARN Largo no Codificante/farmacología
6.
Mol Cell Probes ; 44: 21-28, 2019 04.
Artículo en Inglés | MEDLINE | ID: mdl-30684559

RESUMEN

Preeclampsia (PE), a special type of hypertensive disorder complicating pregnancy (HDCP), is highly associated with the migratory and invasive capacity of extravillous trophoblasts (EVTs). Here, we aimed to study the functions of low-density lipoprotein receptor-related protein 6 (LRP6) in PE pathogenesis. A comparative analysis of cellular gene expressions between placenta tissues collected from PE patients and normal pregnant women showed that the expressions of LRP6, ß-catenin and matrix metallopeptidases/TIMP metallopeptidase inhibitors (MMPs/TIMPs) ratio in placentas of PE patients were much lower than the normal. Then, we constructed and transfected LRP6 siRNA (siLRP6) and LRP6 overexpression vectors into HTR6/SVneo cells. On the contrary to siLRP6, LRP6 overexpression could significant enhance cell viability, and strengthen the abilities of cell migration and invasion. Importantly, the overexpression of LRP6 could induce the upregulation of MMP-2 and MMP-9 levels, and downregulation of TIMPs. The mRNA and protein levels of ß-catenin, an intracellular signal transducer of Wnt signaling pathway, were significantly up-regulated under the effects of LRP6 overexpression. XAV939, a Wnt/ß-catenin pathway inhibitor, was introduced to confirm the involvement of Wnt/ß-catenin pathway in functions of LRP6. The results of cell viability detection showed that XAV939 could significantly inhibit the positive effects of LRP6 overexpression on cell viability. Taken together, low-expressed LRP6 may be responsible of lower migration and invasion of EVTs and subsequent PE, and the mechanisms show a highly association with Wnt/ß-catenin pathway.


Asunto(s)
Regulación hacia Abajo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/genética , Trofoblastos/metabolismo , Vía de Señalización Wnt , Línea Celular , Movimiento Celular , Supervivencia Celular , Femenino , Silenciador del Gen , Humanos , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Preeclampsia/genética , Embarazo , Trofoblastos/patología
7.
Nat Commun ; 10(1): 130, 2019 01 10.
Artículo en Inglés | MEDLINE | ID: mdl-30631061

RESUMEN

Aggressive behaviours of solid tumours are highly influenced by the tumour microenvironment. Multiple signalling pathways can affect the normal function of stromal fibroblasts in tumours, but how these events are coordinated to generate tumour-promoting cancer-associated fibroblasts (CAFs) is not well understood. Here we show that stromal expression of Dickkopf-3 (DKK3) is associated with aggressive breast, colorectal and ovarian cancers. We demonstrate that DKK3 is a HSF1 effector that modulates the pro-tumorigenic behaviour of CAFs in vitro and in vivo. DKK3 orchestrates a concomitant activation of ß-catenin and YAP/TAZ. Whereas ß-catenin is dispensable for CAF-mediated ECM remodelling, cancer cell growth and invasion, DKK3-driven YAP/TAZ activation is required to induce tumour-promoting phenotypes. Mechanistically, DKK3 in CAFs acts via canonical Wnt signalling by interfering with the negative regulator Kremen and increasing cell-surface levels of LRP6. This work reveals an unpredicted link between HSF1, Wnt signalling and YAP/TAZ relevant for the generation of tumour-promoting CAFs.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Fibroblastos Asociados al Cáncer/metabolismo , Factores de Transcripción del Choque Térmico/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Fosfoproteínas/metabolismo , Factores de Transcripción/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Línea Celular , Células Cultivadas , Quimiocinas , Perfilación de la Expresión Génica , Factores de Transcripción del Choque Térmico/genética , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intracelular , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones Desnudos , Ratones Transgénicos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Fosfoproteínas/genética , Factores de Transcripción/genética , Vía de Señalización Wnt/genética , beta Catenina/genética , beta Catenina/metabolismo
8.
Nat Commun ; 10(1): 365, 2019 01 21.
Artículo en Inglés | MEDLINE | ID: mdl-30664649

RESUMEN

Wnt-induced ß-catenin-mediated transcription is a driving force for stem cell self-renewal during adult tissue homeostasis. Enhanced Wnt receptor expression due to mutational inactivation of the ubiquitin ligases RNF43/ZNRF3 recently emerged as a leading cause for cancer development. Consequently, targeting canonical Wnt receptors such as LRP5/6 holds great promise for treatment of such cancer subsets. Here, we employ CIS display technology to identify single-domain antibody fragments (VHH) that bind the LRP6 P3E3P4E4 region with nanomolar affinity and strongly inhibit Wnt3/3a-induced ß-catenin-mediated transcription in cells, while leaving Wnt1 responses unaffected. Structural analysis reveal that individual VHHs variably employ divergent antigen-binding regions to bind a similar surface in the third ß-propeller of LRP5/6, sterically interfering with Wnt3/3a binding. Importantly, anti-LRP5/6 VHHs block the growth of Wnt-hypersensitive Rnf43/Znrf3-mutant intestinal organoids through stem cell exhaustion and collective terminal differentiation. Thus, VHH-mediated targeting of LRP5/6 provides a promising differentiation-inducing strategy for treatment of Wnt-hypersensitive tumors.


Asunto(s)
Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad/química , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/química , Organoides/efectos de los fármacos , Anticuerpos de Dominio Único/química , Células Madre/efectos de los fármacos , Proteína Wnt3A/genética , Animales , Sitios de Unión , Diferenciación Celular , Línea Celular Tumoral , Proliferación Celular , Cristalografía por Rayos X , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Células HEK293 , Humanos , Intestino Delgado/citología , Intestino Delgado/efectos de los fármacos , Intestino Delgado/metabolismo , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad/antagonistas & inhibidores , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/antagonistas & inhibidores , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Ratones , Modelos Moleculares , Organoides/citología , Organoides/metabolismo , Unión Proteica , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Anticuerpos de Dominio Único/genética , Anticuerpos de Dominio Único/metabolismo , Células Madre/citología , Células Madre/metabolismo , Transcripción Genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Proteína Wnt3A/metabolismo , beta Catenina/genética , beta Catenina/metabolismo
9.
Cell Tissue Res ; 376(2): 165-177, 2019 May.
Artículo en Inglés | MEDLINE | ID: mdl-30610453

RESUMEN

Fenofibrate has been shown to have therapeutic effects on diabetic retinopathy (DR). Our previous studies demonstrated that the oxidative stress-activated Wnt/ß-catenin pathway plays a pathogenic role in diabetic complications. In the present study, we evaluate the effect and mechanism of fenofibrate on regulating the oxidative stress-activated Wnt/ß-catenin pathway by using the genetic type 1 diabetes model of C57BL/6J-Ins2Akita mice and high glucose (HG)-treated ARPE-19. Our results demonstrated that retinal phosphorylation of LRP6 and nuclear ß-catenin were increased in C57BL/6J-Ins2Akita mice suggesting activation of Wnt/ß-catenin signaling. Meanwhile, C57BL/6J-Ins2Akita showed upregulation of oxidant enzyme Nox4 and Nox2 and downregulation of antioxidant enzyme SOD1 and SOD2. All these alterations were reversed in C57BL/6J-Ins2Akita mice with fenofibrate treatment. Moreover, fenofibrate significantly ameliorated diabetes-induced retinal vascular leakage in C57BL/6J-Ins2Akita mice. In cultured ARPE-19, fenofibrate decreased HG-induced Nox2 and Nox4 upregulation, attenuated SOD1 and SOD2 downregulation and inhibited LRP6 phosphorylation. Moreover, activation of Wnt/ß-catenin by Wnt3a conditional medium (WCM) reduced SOD1 and SOD2 and did not affect Nox2 and Nox4. Fenofibrate suppressed WCM-induced LRP6 phosphorylation and reversed SOD downregulation. Importantly, Nox4 overexpression directly phosphorylated LPR6 in ARPE19; conversely, Nox4 knockdown suppressed HG-induced LPR6 phosphorylation. Taken together, Nox-mediated oxidative stress contributes to Wnt/ß-catenin activation in DR. Fenofibrate ameliorated DR through coordinate attenuation of oxidative stress and blockade of Wnt/ß-catenin signaling.


Asunto(s)
Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Tipo 1/complicaciones , Retinopatía Diabética/tratamiento farmacológico , Fenofibrato/farmacología , Estrés Oxidativo/efectos de los fármacos , Vía de Señalización Wnt/efectos de los fármacos , Animales , Células Cultivadas , Diabetes Mellitus Experimental/genética , Retinopatía Diabética/etiología , Fenofibrato/uso terapéutico , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Ratones , Ratones Endogámicos C57BL , NADPH Oxidasas/metabolismo , Superóxido Dismutasa/metabolismo
10.
Artículo en Inglés | MEDLINE | ID: mdl-30476623

RESUMEN

INTRODUCTION: Dickkopf-related protein 1 (Dkk1) is a secreted protein ligand of low-density lipoprotein receptor-related protein 6 (LRP6), which antagonises canonical Wnt signalling. Elevated Dkk1 levels have been linked to Alzheimer's disease (AD), with protein blockade protective in pre-clinical AD models, suggesting inhibitors of Dkk1-LRP6 binding may have therapeutic utility against AD. Cell-based Dkk1-LRP6 assays reported in the literature use either modified Dkk1 protein and/or do not possess suitable throughput for drug screening. Here we report a novel immunocytochemical-based assay utilising high-content imaging (HCI) and automated data analysis suitable for the screening of protein and small-molecule inhibitors of Dkk1-LRP6 binding. METHODS: We developed an immunocytochemical (ICC) protocol to detect specific binding of exogenous human Dkk1 protein to human LRP6 transiently expressed in HEK293 cells. Images were generated using the PerkinElmer Operetta HCI System, after which quantitative data was generated using the PerkinElmer Columbus™ System. RESULTS: Our ICC technique and analysis pipeline allowed measurement of cell membrane-localised, LRP6-specific Dkk1 binding, normalised at individual cellular events. Saturation binding demonstrated concentration-dependent Dkk1 binding to LRP6, with a KD in keeping with reported values. Association kinetic experiments demonstrated the utility of the technique to investigate Dkk1 binding kinetics. Human Dkk members Dkk2 and Dkk4 fully displaced Dkk1 binding in a competition assay, while Dkk3 and Soggy-1/DkkL1 exhibited non-complete displacement of Dkk1. Finally gallocyanine, a previously reported inhibitor of Dkk1-LRP6 binding, fully displaced Dkk1 near the expected IC50. DISCUSSION: In conclusion, we provide a validated cell-based assay, suitable for the screening of inhibitors of Dkk1-LRP6 binding, and provide the basis for additional assay development, investigating Dkk1-LRP6 pharmacology.


Asunto(s)
Péptidos y Proteínas de Señalización Intercelular/metabolismo , Microscopía Intravital/métodos , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Oxazinas/farmacología , Sitios de Unión , Membrana Celular , Evaluación Preclínica de Medicamentos/instrumentación , Evaluación Preclínica de Medicamentos/métodos , Células HEK293 , Humanos , Procesamiento de Imagen Asistida por Computador , Inmunohistoquímica/instrumentación , Inmunohistoquímica/métodos , Concentración 50 Inhibidora , Microscopía Intravital/instrumentación , Ligandos , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/antagonistas & inhibidores , Microscopía Fluorescente/instrumentación , Microscopía Fluorescente/métodos , Transducción de Señal/efectos de los fármacos , Programas Informáticos
11.
Cancer Lett ; 444: 60-69, 2019 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-30583072

RESUMEN

Hepatitis B virus X protein mutants, particularly truncated at C-terminal (HBxΔC), generated during random viral integration, are frequently detected in hepatocellular carcinoma (HCC) and exert a more potent oncogenic effect than full-length form (FL). Here, we showed that caveolin-1 (Cav1), a robust metastasis promoter, is transcriptionally upregulated by HBxΔC but not by FL HBx. Promoting effect of HBxΔC in HCC cell aggressiveness is abolished when Cav1 is suppressed. Expression profiling identified FERM domain containing 5 (FRMD5) protein as a downstream target of Cav1. In accordance with the regulation of Cav1, HBxΔC upregulates FRMD5. Knockdown of FRMD5 in HBxΔC cells recapitulated the functional effect of Cav1 knockdown in HBxΔC cells. The regulation of FRMD5 by HBxΔC-induced Cav1 is mediated by the protein stablilization of LRP6 leading to the activation of ß-catenin. Expression of a constitutively active ß-catenin in Cav1 knockdown cells rescued FRMD5 expression and HCC tumorigenesis and metastasis. Clinical relevance of HBxΔC/Cav1/LRP6/FRMD5 pathway is demonstrated by the significant correlation of Cav1, LRP6 and FRMD5 expressions in HCC. The findings of this study uncover a novel HBxΔC-regulated molecular pathway which has profound implications in HCC therapeutics.


Asunto(s)
Carcinoma Hepatocelular/patología , Caveolina 1/metabolismo , Neoplasias Hepáticas/patología , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Transactivadores/metabolismo , Proteínas Supresoras de Tumor/metabolismo , beta Catenina/metabolismo , Animales , Apoptosis , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Carcinogénesis , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Caveolina 1/genética , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/genética , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Pronóstico , Transactivadores/genética , Células Tumorales Cultivadas , Proteínas Supresoras de Tumor/genética , Ensayos Antitumor por Modelo de Xenoinjerto , beta Catenina/genética
12.
Acta Physiol (Oxf) ; 225(2): e13177, 2019 02.
Artículo en Inglés | MEDLINE | ID: mdl-30136377

RESUMEN

AIMS: Clinical trials have shown the beneficial effects of exercise training against pulmonary fibrosis. This study aimed to investigate whether prophylactic intervention with exercise training attenuates lung fibrosis via modulating endogenous hydrogen sulphde (H2 S) generation. METHODS: First, ICR mice were allocated to Control, Bleomycin, Exercise, and Bleomycin + Exercise groups. Treadmill exercise began on day 1 and continued for 4 weeks. A single intratracheal dose of bleomycin (3 mg/kg) was administered on day 15. Second, ICR mice were allocated to Control, Bleomycin, H2 S, and Bleomycin + H2 S groups. H2 S donor NaHS (28 µmol/kg) was intraperitoneally injected once daily for 2 weeks. RESULTS: Bleomycin-treated mice exhibited increased levels of collagen deposition, hydroxyproline, collagen I, transforming growth factor (TGF)-ß1, Smad2/Smad3/low-density lipoprotein receptor-related proteins (LRP-6)/glycogen synthase kinase-3ß (GSK-3ß) phosphorylation, and Smad4/ß-catenin expression in lung tissues (P < 0.01), which was alleviated by exercise training (P < 0.01 except for Smad4 and phosphorylated GSK-3ß: P < 0.05). Bleomycin-induced lung fibrosis was associated with increased α smooth muscle actin (α-SMA) and decreased E-cadherin expression (P < 0.01). Double immunofluorescence staining showed the co-localization of E-cadherin/α-SMA, indicating epithelial-mesenchymal transition (EMT) formation, which was ameliorated by exercise training. Moreover, exercise training restored bleomycin-induced downregulation of cystathionine-ß-synthase (CBS) and cystathionine-γ-lyase (CSE) expression, as well as H2 S generation in lung tissue (P < 0.01). NaHS treatment attenuated bleomycin-induced TGF-ß1 production, activation of LRP-6/ß-catenin signalling, EMT and lung fibrosis (P < 0.01 except for ß-catenin: P < 0.05). CONCLUSION: Exercise training restores bleomycin-induced downregulation of pulmonary CBS/CSE expression, thus contributing to the increased H2 S generation and suppression of TGF-ß1/Smad and LRP-6/ß-catenin signalling pathways, EMT and lung fibrosis.


Asunto(s)
Transición Epitelial-Mesenquimal , Condicionamiento Físico Animal , Fibrosis Pulmonar/prevención & control , Sulfitos/metabolismo , Animales , Bleomicina , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Masculino , Ratones , Ratones Endogámicos ICR , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/patología , Transducción de Señal , Factor de Crecimiento Transformador beta1/metabolismo , beta Catenina/metabolismo
13.
Sci Rep ; 8(1): 17506, 2018 11 30.
Artículo en Inglés | MEDLINE | ID: mdl-30504774

RESUMEN

Regulation of the Wnt pathway in stem cells and primary tissues is still poorly understood. Here we report that Usp16, a negative regulator of Bmi1/PRC1 function, modulates the Wnt pathway in mammary epithelia, primary human fibroblasts and MEFs, affecting their expansion and self-renewal potential. In mammary glands, reduced levels of Usp16 increase tissue responsiveness to Wnt, resulting in upregulation of the downstream Wnt target Axin2, expansion of the basal compartment and increased in vitro and in vivo epithelial regeneration. Usp16 regulation of the Wnt pathway in mouse and human tissues is at least in part mediated by activation of Cdkn2a, a regulator of senescence. At the molecular level, Usp16 affects Rspo-mediated phosphorylation of LRP6. In Down's Syndrome (DS), triplication of Usp16 dampens the activation of the Wnt pathway. Usp16 copy number normalization restores normal Wnt activation in Ts65Dn mice models. Genetic upregulation of the Wnt pathway in Ts65Dn mice rescues the proliferation defect observed in mammary epithelial cells. All together, these findings link important stem cell regulators like Bmi1/Usp16 and Cdkn2a to Wnt signaling, and have implications for designing therapies for conditions, like DS, aging or degenerative diseases, where the Wnt pathway is hampered.


Asunto(s)
Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Regulación de la Expresión Génica , Ubiquitina Tiolesterasa/metabolismo , Vía de Señalización Wnt , Animales , Línea Celular , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Fibroblastos/metabolismo , Perfilación de la Expresión Génica , Humanos , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Ratones , Ratones Noqueados , Modelos Biológicos , Ubiquitina Tiolesterasa/genética , Proteína Wnt3A/metabolismo
14.
Theranostics ; 8(20): 5575-5592, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30555565

RESUMEN

Lineage differentiation of bone marrow mesenchymal stem cells (BMMSCs) is the key to bone-fat reciprocity in bone marrow. To date, the regulators of BMMSC lineage switching have all been identified to be transcription factors, and researchers have not determined whether other genes control this process. This study aims to reveal a previously unknown role of tissue-nonspecific alkaline phosphatase (TNSALP) in controlling BMMSC lineage selection. Methods: We compared the characteristics of cultured BMMSCs from patients with hypophosphatasia (HPP), which is caused by mutations in the liver/bone/kidney alkaline phosphatase (ALPL) gene, and an ALPL knockout (ko) mouse model. We performed ALPL downregulation and overexpression experiments to investigate the regulatory role of ALPL in BMMSC lineage switching. Using the PathScan array, coimmunoprecipitation experiments and pathway-guided small molecule treatments, we explored the possible mechanism underlying the regulatory effects of ALPL on cell differentiation and evaluated its therapeutic effect on ALPL ko mice. Results: BMMSCs from both patients with HPP and ALPL ko mice exhibited defective lineage differentiation, including a decrease in osteogenic differentiation and a parallel increase in adipogenic differentiation. Mechanistically, TNSALP directly interacted with LRP6 and regulated the phosphorylation of GSK3ß, subsequently resulting in lineage switching of BMMSCs. Re-phosphorylation of GSK3ß induced by LiCl treatment restored differentiation of BMMSCs and attenuated skeletal deformities in Alpl +/- mice. Conclusion: Based on our findings, TNSALP acts as a signal regulator to control lineage switching of BMMSCs by regulating the LRP6/GSK3ß cascade.


Asunto(s)
Fosfatasa Alcalina/metabolismo , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Hipofosfatasia/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Adolescente , Fosfatasa Alcalina/genética , Animales , Niño , Modelos Animales de Enfermedad , Glucógeno Sintasa Quinasa 3 beta/genética , Humanos , Hipofosfatasia/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/genética , Masculino , Ratones Noqueados
15.
Phytomedicine ; 51: 48-57, 2018 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-30466627

RESUMEN

BACKGROUND: Drug therapy plays an important role in the treatment of cervical cancer, which is one of the most common solid tumors in women. Therefore, it is important to seek more effective and less toxic therapies. PURPOSE: The aim of this study is to investigate the therapeutic potential of HMQ-T-F5 (1-(4-(2-aminoquinazolin-7-yl)phenyl)-3-(2­bromo­5-(trifluoromethoxy) phenyl)thiourea) (F5) for cervical cancer and explore the related mechanism. METHODS: By performing MTT assay, colony formation assay, flow cytometry, wound-healing assay, transwell assay, immunofluorescent staining and siRNA assay, we study the effect of F5 on human cervical HeLa cells. The mechanism of F5 was also investigated. RESULTS: We found that F5 significantly inhibited HeLa cell proliferation, led to accumulation of cells in the S phase, and induced apoptosis and inhibited migration. Mechanistically, F5 inhibited HeLa cell growth and migration through repressing the expression and nuclear translocation of ß-catenin, enhancing Axin expression, inhibiting the phosphorylation of LRP5/6 and GSK3ß, as well as downregulating the Wnt downstream targeted proteins. Knockdown of a checkpoint ß-catenin by siRNA significantly attenuated HeLa cell proliferation. Furthermore, XAV939, an inhibitor of ß-catenin, was used to treat HeLa cells and the results demonstrated that F5 inhibited proliferation and migration via the inhibition of the Wnt/ß-catenin pathway. CONCLUSION: Our findings demonstrated that F5 can target ß-catenin potentially and is useful in the treatment of cervical cancer.


Asunto(s)
Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Tiourea/farmacología , Vía de Señalización Wnt/efectos de los fármacos , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Regulación hacia Abajo , Femenino , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Células HeLa , Humanos , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , ARN Interferente Pequeño/farmacología , Tiourea/análogos & derivados , beta Catenina/metabolismo
16.
Mol Genet Genomic Med ; 6(6): 1124-1133, 2018 11.
Artículo en Inglés | MEDLINE | ID: mdl-30411539

RESUMEN

BACKGROUND: The remodeling of maternal spiral artery following embryo implantation, which relies on well-regulated trophoblast functions, is a pivotal process to ensure a successful pregnancy. Low-density lipoprotein receptor-related protein 6 (LRP6) and microRNAs (miRNAs, miRs) are suggested to be involved in angiogenesis and several vascular diseases; however, their functions in the control of trophoblast remain elusive. We therefore aimed to examine the roles of LRP6 and miR-590-3p in the regulation of trophoblast during the remodeling of maternal spiral artery. METHODS: HTR-8/SVneo cell, a trophoblast cell line, was utilized to study the effects of LRP6 and miR-590-3p on apoptosis, cell proliferation, migration, invasion, as well as tube formation. Expression of angiogenic factors placental growth factor (PlGF), matrix metalloproteinases (MMPs), vascular endothelial growth factor (VEGF), and activities of canonical Wnt/ß-catenin signaling pathway, which were implicated in the process of artery remodeling, were also examined. RESULTS: MiR-590-3p directly targeted 3' untranslated region (3'-UTR) of LRP6 mRNA and repressed LRP6 expression, which in turn inhibited proliferation, migration, invasion, as well as tube formation, and resulted in apoptosis in HTR-8/SVneo cells. Further, inhibition of LRP6 through miR-590-3p significantly suppressed the expression of PlGF, MMPs, and VEGF and reduced the activation of Wnt/ß-catenin signaling pathway. CONCLUSION: MicroRNAs-590-3p may inhibit trophoblast-dependent maternal spiral artery remodeling, via both trophoblast invasion and endovascular formation, by repressing LRP6.


Asunto(s)
Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , MicroARNs/genética , Neovascularización Fisiológica , Trofoblastos/metabolismo , Proteínas Angiogénicas/genética , Proteínas Angiogénicas/metabolismo , Apoptosis , Línea Celular , Proliferación Celular , Humanos , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/genética , MicroARNs/metabolismo , Trofoblastos/fisiología , Vía de Señalización Wnt
17.
Clin Exp Rheumatol ; 36 Suppl 113(4): 45-49, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30277866

RESUMEN

OBJECTIVES: The activity of the Wnt pathway, a critical mediator of fibrosis, is regulated by Dickkopf-1 (Dkk-1). Dkk-1 is absent from scleroderma skin in contrast to skin from healthy subjects where it is clearly expressed. There are no data on circulating levels and function of Dkk-1 in patients with systemic sclerosis (SSc). Our objectives are to assess: i) circulating and functional levels of Dkk-1 in patients with SSc and ii) whether the striking lack of Dkk-1 skin expression is also evident in a) clinically uninvolved skin from patients with SSc and b) very early disease prior to skin thickening. METHODS: Circulating Dkk-1 levels were measured in 50 patients with SSc and 50 controls. Skin biopsies were obtained from SSc patients from a) clinically involved skin b) clinically uninvolved skin, c) oedematous skin prior to skin thickening. RESULTS: Circulating and functional Dkk-1 levels were similar in patients with SSc and controls. Healthy skin displayed a high Dkk-1 immuno-expression in the epidermis and dermal fibroblasts in contrast to clinically involved scleroderma skin where Dkk-1 was totally absent. In all biopsies of clinically uninvolved skin Dkk-1 was only moderately expressed whereas skin from very early disease displayed only a weak Dkk-1 immunoreactivity. CONCLUSIONS: The downregulation of Dkk-1 at the oedematous phase of the disease indicates that the Wnt pathway is involved early in the disease process and may play a role in driving fibrosis. The decrease in Dkk-1 expression in clinically uninvolved scleroderma skin indicates that skin in SSc is universally affected.


Asunto(s)
Fibroblastos/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Esclerodermia Sistémica/metabolismo , Piel/metabolismo , Biomarcadores/metabolismo , Estudios de Casos y Controles , Progresión de la Enfermedad , Regulación hacia Abajo , Femenino , Fibroblastos/patología , Fibrosis , Humanos , Péptidos y Proteínas de Señalización Intercelular/sangre , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Masculino , Persona de Mediana Edad , Esclerodermia Sistémica/sangre , Esclerodermia Sistémica/patología , Piel/patología , Vía de Señalización Wnt
18.
Proc Natl Acad Sci U S A ; 115(39): E9135-E9144, 2018 09 25.
Artículo en Inglés | MEDLINE | ID: mdl-30209221

RESUMEN

The Xenopus laevis embryo has been subjected to almost saturating screens for molecules specifically expressed in dorsal Spemann organizer tissue. In this study, we performed high-throughput RNA sequencing of ectodermal explants, called animal caps, which normally give rise to epidermis. We analyzed dissociated animal cap cells that, through sustained activation of MAPK, differentiate into neural tissue. We also microinjected mRNAs for Cerberus, Chordin, FGF8, BMP4, Wnt8, and Xnr2, which induce neural or other germ layer differentiations. The searchable database provided here represents a valuable resource for the early vertebrate cell differentiation. These analyses resulted in the identification of a gene present in frog and fish, which we call Bighead. Surprisingly, at gastrula, it was expressed in the Spemann organizer and endoderm, rather than in ectoderm as we expected. Despite the plethora of genes already mined from Spemann organizer tissue, Bighead encodes a secreted protein that proved to be a potent inhibitor of Wnt signaling in a number of embryological and cultured cell signaling assays. Overexpression of Bighead resulted in large head structures very similar to those of the well-known Wnt antagonists Dkk1 and Frzb-1. Knockdown of Bighead with specific antisense morpholinos resulted in embryos with reduced head structures, due to increased Wnt signaling. Bighead protein bound specifically to the Wnt coreceptor lipoprotein receptor-related protein 6 (Lrp6), leading to its removal from the cell surface. Bighead joins two other Wnt antagonists, Dkk1 and Angptl4, which function as Lrp6 endocytosis regulators. These results suggest that endocytosis plays a crucial role in Wnt signaling.


Asunto(s)
Endocitosis/fisiología , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Proteínas Wnt/metabolismo , Vía de Señalización Wnt/fisiología , Angiopoyetinas/genética , Angiopoyetinas/metabolismo , Animales , Endodermo/citología , Endodermo/metabolismo , Gástrula/citología , Gástrula/metabolismo , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/genética , Unión Proteica , Proteínas Wnt/genética , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis
19.
Life Sci ; 211: 102-117, 2018 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-30201296

RESUMEN

AIMS: Although decades of research have revealed numerous molecular abnormalities in the hippocampus associated with depression, the different mechanisms involved in the susceptibility and resilience of mice to chronic social defeat stress (CSDS)-induced depression remain poorly understand. Through the social defeat model, we can study the differences in molecular changes between the susceptible and resilient mice. MAIN METHODS: We used a proteomic-based platform to compare hippocampal proteins in CSDS mice with those in control mice. Differentially expressed proteins were identified through isobaric tags for relative and absolute quantitation (iTRAQ) combined with LC-MS/MS. We then analyzed the results by ingenuity pathway analysis (IPA) and verified five proteins by western blotting. KEY FINDINGS: Mice were exposed to 10 days of CSDS, which successfully induced stress-susceptible and -resilient phenotypes. 161 and 134 proteins were significantly differentially expressed in the susceptible and resilient groups, respectively, compared with the levels in the control group. The Rac signaling and the GABA receptors signaling pathways were the common top-ranking pathways. We found that low-density lipoprotein receptor-related protein 6 (LRP6) was upregulated in resilient mice and neuropeptide Y (NPY) was downregulated in susceptible mice compared with the levels in control mice. Moreover, neuropeptide Y receptor type 2 (NPY2R) protein expression in susceptible mice was downregulated compared with that in the resilient group. SIGNIFICANCE: Our findings in the three groups potentially reveal the differences in molecular mechanisms underlying depression between susceptible and resilient mice. The results provide insight into molecular abnormalities of the hippocampus in CSDS mice and some potential drug targets for treating depression.


Asunto(s)
Trastorno Depresivo/patología , Susceptibilidad a Enfermedades , Hipocampo/patología , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Neuropéptido Y/metabolismo , Proteómica/métodos , Receptores de Neuropéptido Y/metabolismo , Estrés Psicológico , Animales , Trastorno Depresivo/metabolismo , Modelos Animales de Enfermedad , Hipocampo/metabolismo , Marcaje Isotópico/métodos , Masculino , Ratones , Ratones Endogámicos C57BL , Conducta Social
20.
EMBO J ; 37(20)2018 10 15.
Artículo en Inglés | MEDLINE | ID: mdl-30217955

RESUMEN

Uncontrolled cell division is a hallmark of cancer. Deregulation of Wnt components has been linked to aberrant cell division by multiple mechanisms, including Wnt-mediated stabilisation of proteins signalling, which was notably observed in mitosis. Analysis of Wnt components revealed an unexpected role of B-cell CLL/lymphoma 9 (BCL9) in maintaining mitotic Wnt signalling to promote precise cell division and growth of cancer cell. Mitotic interactome analysis revealed a mechanistic role of BCL9 in inhibiting clathrin-mediated degradation of LRP6 signalosome components by interacting with clathrin and the components in Wnt destruction complex; this function was further controlled by CDK1-driven phosphorylation of BCL9 N-terminal, especially T172. Interestingly, T172 phosphorylation was correlated with cancer patient prognosis and enriched in tumours. Thus, our results revealed a novel role of BCL9 in controlling mitotic Wnt signalling to promote cell division and growth.


Asunto(s)
Proteína Quinasa CDC2/metabolismo , Clatrina/metabolismo , Mitosis , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Vía de Señalización Wnt , Células HCT116 , Células HeLa , Humanos , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Neoplasias/patología , Fosforilación , Dominios Proteicos , Factores de Transcripción
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