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1.
EMBO Rep ; 22(12): e51503, 2021 12 06.
Artículo en Inglés | MEDLINE | ID: mdl-34585824

RESUMEN

ß-Catenin is a multifunctional protein and participates in numerous processes required for embryonic development, cell proliferation, and homeostasis through various molecular interactions and signaling pathways. To date, however, there is no direct evidence that ß-catenin contributes to cytokinesis. Here, we identify a novel p-S60 epitope on ß-catenin generated by Plk1 kinase activity, which can be found at the actomyosin contractile ring of early telophase cells and at the midbody of late telophase cells. Depletion of ß-catenin leads to cytokinesis-defective phenotypes, which eventually result in apoptotic cell death. In addition, phosphorylation of ß-catenin Ser60 by Plk1 is essential for the recruitment of Ect2 to the midbody, activation of RhoA, and interaction between ß-catenin, Plk1, and Ect2. Time-lapse image analysis confirmed the importance of ß-catenin phospho-Ser60 in furrow ingression and the completion of cytokinesis. Taken together, we propose that phosphorylation of ß-catenin Ser60 by Plk1 in cooperation with Ect2 is essential for the completion of cytokinesis. These findings may provide fundamental knowledge for the research of cytokinesis failure-derived human diseases.


Asunto(s)
Actomiosina , Citocinesis , Actomiosina/metabolismo , Proteínas de Ciclo Celular/metabolismo , Células HeLa , Humanos , Fosforilación , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas/metabolismo , Huso Acromático/metabolismo , beta Catenina/metabolismo , Quinasa Tipo Polo 1
2.
Cell Mol Life Sci ; 78(7): 3725-3741, 2021 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-33687501

RESUMEN

Protein arginylation is a critical regulator of a variety of biological processes. The ability to uncover the global arginylation pattern and its associated signaling pathways would enable us to identify novel disease targets. Here, we report the development of a tool able to capture the N-terminal arginylome. This tool, termed R-catcher, is based on the ZZ domain of p62, which was previously shown to bind N-terminally arginylated proteins. Mutating the ZZ domain enhanced its binding specificity and affinity for Nt-Arg. R-catcher pulldown coupled to LC-MS/MS led to the identification of 59 known and putative arginylated proteins. Among these were a subgroup of novel ATE1-dependent arginylated ER proteins that are linked to diverse biological pathways, including cellular senescence and vesicle-mediated transport as well as diseases, such as Amyotrophic Lateral Sclerosis and Alzheimer's disease. This study presents the first molecular tool that allows the unbiased identification of arginylated proteins, thereby unlocking the arginylome and provide a new path to disease biomarker discovery.


Asunto(s)
Aminoaciltransferasas/metabolismo , Arginina/metabolismo , Retículo Endoplásmico/metabolismo , Vectores Genéticos/genética , Proteínas de la Membrana/metabolismo , Procesamiento Proteico-Postraduccional , Aminoaciltransferasas/química , Aminoaciltransferasas/genética , Arginina/química , Arginina/genética , Células HeLa , Humanos , Proteínas de la Membrana/genética , Especificidad por Sustrato
3.
Proc Natl Acad Sci U S A ; 115(12): E2716-E2724, 2018 03 20.
Artículo en Inglés | MEDLINE | ID: mdl-29507222

RESUMEN

The conjugation of amino acids to the protein N termini is universally observed in eukaryotes and prokaryotes, yet its functions remain poorly understood. In eukaryotes, the amino acid l-arginine (l-Arg) is conjugated to N-terminal Asp (Nt-Asp), Glu, Gln, Asn, and Cys, directly or associated with posttranslational modifications. Following Nt-arginylation, the Nt-Arg is recognized by UBR boxes of N-recognins such as UBR1, UBR2, UBR4/p600, and UBR5/EDD, leading to substrate ubiquitination and proteasomal degradation via the N-end rule pathway. It has been a mystery, however, why studies for the past five decades identified only a handful of Nt-arginylated substrates in mammals, although five of 20 principal amino acids are eligible for arginylation. Here, we show that the Nt-Arg functions as a bimodal degron that directs substrates to either the ubiquitin (Ub)-proteasome system (UPS) or macroautophagy depending on physiological states. In normal conditions, the arginylated forms of proteolytic cleavage products, D101-CDC6 and D1156-BRCA1, are targeted to UBR box-containing N-recognins and degraded by the proteasome. However, when proteostasis by the UPS is perturbed, their Nt-Arg redirects these otherwise cellular wastes to macroautophagy through its binding to the ZZ domain of the autophagic adaptor p62/STQSM/Sequestosome-1. Upon binding to the Nt-Arg, p62 acts as an autophagic N-recognin that undergoes self-polymerization, facilitating cargo collection and lysosomal degradation of p62-cargo complexes. A chemical mimic of Nt-Arg redirects Ub-conjugated substrates from the UPS to macroautophagy and promotes their lysosomal degradation. Our results suggest that the Nt-Arg proteome of arginylated proteins contributes to reprogramming global proteolytic flux under stresses.


Asunto(s)
Arginina/metabolismo , Autofagia/fisiología , Proteínas de Ciclo Celular/metabolismo , Proteínas Nucleares/metabolismo , Proteolisis , Proteínas de Unión al ARN/metabolismo , Aminoaciltransferasas/genética , Aminoaciltransferasas/metabolismo , Animales , Autofagia/efectos de los fármacos , Proteína BRCA1/metabolismo , Femenino , Células HEK293 , Células HeLa , Humanos , Hidroxicloroquina/farmacología , Ratones , Ratones Endogámicos C57BL , Complejo de la Endopetidasa Proteasomal/metabolismo , Dominios Proteicos , Ubiquitina/metabolismo
4.
Phytother Res ; 35(11): 6377-6388, 2021 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-34545650

RESUMEN

Harmaline is a naturally occurring ß-carboline alkaloid that is isolated from Peganum harmala. It has shown efficacy in treating Parkinson's disease and has been reported to exhibit antimicrobial and anticancer properties. However, the molecular mechanism of harmaline in the context of esophageal squamous cell carcinoma (ESCC) has not been characterized. Here, we report that harmaline attenuates ESCC growth by directly targeting the mammalian target of rapamycin (mTOR). Harmaline strongly reduced cell proliferation and anchorage-independent cell growth. Additionally, harmaline treatment induced G2/M phase cell-cycle arrest through upregulation of p27. The results of in vitro and cell-based assays showed that harmaline directly inhibited the activity of mTOR kinase and the phosphorylation of its downstream pathway components. Depletion of mTOR using an shRNA-mediated strategy in ESCC cell lines indicated that reduced mTOR protein expression levels are correlated with decreased cell proliferation. Additionally, we observed that the inhibitory effect of harmaline was dependent upon mTOR expression. Notably, oral administration of harmaline suppressed ESCC patient-derived tumor growth in vivo. Taken together, harmaline is a potential mTOR inhibitor that might be used for therapeutically treating ESCC.


Asunto(s)
Neoplasias Esofágicas , Carcinoma de Células Escamosas de Esófago , Neoplasias de Cabeza y Cuello , Peganum , Línea Celular Tumoral , Proliferación Celular , Neoplasias Esofágicas/tratamiento farmacológico , Carcinoma de Células Escamosas de Esófago/tratamiento farmacológico , Harmalina/farmacología , Humanos , Sirolimus , Serina-Treonina Quinasas TOR
5.
J Cell Sci ; 131(17)2018 09 10.
Artículo en Inglés | MEDLINE | ID: mdl-30111582

RESUMEN

The N-end rule pathway is a proteolytic system in which single N-terminal residues of proteins act as N-degrons. These degrons are recognized by N-recognins, facilitating substrate degradation via the ubiquitin (Ub) proteasome system (UPS) or autophagy. We have previously identified a set of N-recognins [UBR1, UBR2, UBR4 (also known as p600) and UBR5 (also known as EDD)] that bind N-degrons through their UBR boxes to promote proteolysis by the proteasome. Here, we show that the 570 kDa N-recognin UBR4 is associated with maturing endosomes through an interaction with Ca2+-bound calmodulin. The endosomal recruitment of UBR4 is essential for the biogenesis of early endosomes (EEs) and endosome-related processes, such as the trafficking of endocytosed protein cargos and degradation of extracellular cargos by endosomal hydrolases. In mouse embryos, UBR4 marks and plays a role in the endosome-lysosome pathway that mediates the heterophagic proteolysis of endocytosed maternal proteins into amino acids. By screening 9591 drugs through the DrugBank database, we identify picolinic acid as a putative ligand for UBR4 that inhibits the biogenesis of EEs. Our results suggest that UBR4 is an essential modulator in the endosome-lysosome system.This article has an associated First Person interview with the first author of the paper.


Asunto(s)
Proteínas de Unión a Calmodulina/metabolismo , Proteínas del Citoesqueleto/metabolismo , Endosomas/metabolismo , Calcio/metabolismo , Calmodulina/genética , Calmodulina/metabolismo , Proteínas de Unión a Calmodulina/genética , Proteínas del Citoesqueleto/genética , Endosomas/genética , Humanos , Lisosomas/genética , Lisosomas/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Biogénesis de Organelos , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas
6.
Int J Mol Sci ; 21(13)2020 Jul 07.
Artículo en Inglés | MEDLINE | ID: mdl-32645923

RESUMEN

In the past, several microtubule targeting agents (MTAs) have been developed into successful anticancer drugs. However, the usage of these drugs has been limited by the acquisition of drug resistance in many cancers. Therefore, there is a constant demand for the development of new therapeutic drugs. Here we report the discovery of 5-5 (3-cchlorophenyl)-N-(3-pyridinyl)-2-furamide (CPPF), a novel microtubule targeting anticancer agent. Using both 2D and 3D culture systems, we showed that CPPF was able to suppress the proliferation of diverse cancer cell lines. In addition, CPPF was able to inhibit the growth of multidrug-resistant cell lines that are resistant to other MTAs, such as paclitaxel and colchicine. Our results showed that CPPF inhibited growth by depolymerizing microtubules leading to mitotic arrest and apoptosis. We also confirmed CPPF anticancer effects in vivo using both a mouse xenograft and a two-step skin cancer mouse model. Using established zebrafish models, we showed that CPPF has low toxicity in vivo. Overall, our study proves that CPPF has the potential to become a successful anticancer chemotherapeutic drug.


Asunto(s)
Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Microtúbulos/metabolismo , Neoplasias/tratamiento farmacológico , Células A549 , Animales , Apoptosis/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Colchicina/farmacología , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales/métodos , Células HeLa , Células Hep G2 , Humanos , Células Jurkat , Células K562 , Células MCF-7 , Masculino , Ratones , Mitosis/efectos de los fármacos , Neoplasias/metabolismo , Células PC-3 , Paclitaxel/farmacología , Células U937 , Ensayos Antitumor por Modelo de Xenoinjerto/métodos , Pez Cebra
7.
EMBO Rep ; 18(1): 150-168, 2017 01.
Artículo en Inglés | MEDLINE | ID: mdl-27993939

RESUMEN

Although proteasome inhibitors (PIs) are used as anticancer drugs to treat various cancers, their relative therapeutic efficacy on stem cells vs. bulk cancers remains unknown. Here, we show that stem cells derived from gliomas, GSCs, are up to 1,000-fold more sensitive to PIs (IC50, 27-70 nM) compared with their differentiated controls (IC50, 47 to ¼100 µM). The stemness of GSCs correlates to increased ubiquitination, whose misregulation readily triggers apoptosis. PI-induced apoptosis of GSCs is independent of NF-κB but involves the phosphorylation of c-Jun N-terminal kinase as well as the transcriptional activation of endoplasmic reticulum (ER) stress-associated proapoptotic mediators. In contrast to the general notion that ER stress-associated apoptosis is signaled by prolonged unfolded protein response (UPR), GSC-selective apoptosis is instead counteracted by the UPR ATF3 is a key mediator in GSC-selective apoptosis. Pharmaceutical uncoupling of the UPR from its downstream apoptosis sensitizes GSCs to PIs in vitro and during tumorigenesis in mice. Thus, a combinational treatment of a PI with an inhibitor of UPR-coupled apoptosis may enhance targeting of stem cells in gliomas.


Asunto(s)
Glioma/metabolismo , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/farmacología , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Biomarcadores , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glioma/tratamiento farmacológico , Glioma/genética , Glioma/patología , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Ratones , Modelos Biológicos , FN-kappa B/metabolismo , Transducción de Señal/efectos de los fármacos , Células Tumorales Cultivadas , Ubiquitinación/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto
8.
Exp Dermatol ; 27(3): 285-288, 2018 03.
Artículo en Inglés | MEDLINE | ID: mdl-29392819

RESUMEN

Skin cancer is the most common type of cancer. The incidence rate of skin cancer has continuously increased over the past decades. In an effort to discover novel anticancer agents, we identified a novel tubulin inhibitor STK899704, which is structurally distinct from other microtubule-binding agents such as colchicine, vinca alkaloids and taxanes. STK899704 inhibited microtubule polymerization leading to mitotic arrest and suppressed the proliferation of various cancer cell lines as well as multidrug resistance cancer cell lines. In this study, our investigation is further extended into animal model to evaluate the effect of STK899704 on skin carcinogenesis in vivo. Surprisingly, almost 80% of the tumors treated with STK899704 were regressed with a one-fifth reduction in tumor volume. Furthermore, the efficacy of STK899704 was nearly 2 times higher than that of 5-fluorouracil, a widely used skin cancer therapeutic. Overall, our results suggest that STK899704 is a promising anticancer chemotherapeutic that may replace existing therapies, particularly for skin cancer.


Asunto(s)
Benzofuranos/uso terapéutico , Neoplasias Cutáneas/tratamiento farmacológico , Moduladores de Tubulina/uso terapéutico , 9,10-Dimetil-1,2-benzantraceno , Animales , Antimetabolitos Antineoplásicos/uso terapéutico , Carcinogénesis , Colchicina/uso terapéutico , Modelos Animales de Enfermedad , Fluorouracilo/uso terapéutico , Masculino , Ratones , Neoplasias Cutáneas/inducido químicamente , Neoplasias Cutáneas/patología , Acetato de Tetradecanoilforbol , Tubulina (Proteína)/metabolismo
9.
Exp Dermatol ; 27(11): 1304-1308, 2018 11.
Artículo en Inglés | MEDLINE | ID: mdl-30092122

RESUMEN

Atopic dermatitis (AD) is a chronic inflammatory skin disease whose prevalence is increasing worldwide. Filaggrin (FLG) is essential for the development of the skin barrier, and its genetic mutations are major predisposing factors for AD. In this study, we developed a convenient and practical method to detect FLG mutations in AD patients using peptide nucleic acid (PNA) probes labelled with fluorescent markers for rapid analysis. Fluorescence melting curve analysis (FMCA) precisely identified FLG mutations based on the distinct difference in the melting temperatures of the wild-type and mutant allele. Moreover, PNA probe-based FMCA easily and accurately verified patient samples with both heterozygote and homozygote FLG mutations, providing a high-throughput method to reliable screen AD patients. Our method provides a convenient, rapid and accurate diagnostic tool to identify potential AD patients allowing for early preventive treatment, leading to lower incidence rates of AD, and reducing total healthcare expenses.


Asunto(s)
Análisis Mutacional de ADN/métodos , Sondas de ADN , Dermatitis Atópica/diagnóstico , Dermatitis Atópica/genética , Proteínas de Filamentos Intermediarios/genética , Alelos , Estudios de Casos y Controles , Proteínas Filagrina , Fluorescencia , Heterocigoto , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Homocigoto , Humanos , Mutación , Ácidos Nucleicos de Péptidos/genética , Temperatura de Transición
11.
Proc Natl Acad Sci U S A ; 110(10): 3800-5, 2013 Mar 05.
Artículo en Inglés | MEDLINE | ID: mdl-23431188

RESUMEN

The N-end rule pathway is a proteolytic system in which destabilizing N-terminal residues of short-lived proteins act as degradation determinants (N-degrons). Substrates carrying N-degrons are recognized by N-recognins that mediate ubiquitylation-dependent selective proteolysis through the proteasome. Our previous studies identified the mammalian N-recognin family consisting of UBR1/E3α, UBR2, UBR4/p600, and UBR5, which recognize destabilizing N-terminal residues through the UBR box. In the current study, we addressed the physiological function of a poorly characterized N-recognin, 570-kDa UBR4, in mammalian development. UBR4-deficient mice die during embryogenesis and exhibit pleiotropic abnormalities, including impaired vascular development in the yolk sac (YS). Vascular development in UBR4-deficient YS normally advances through vasculogenesis but is arrested during angiogenic remodeling of primary capillary plexus associated with accumulation of autophagic vacuoles. In the YS, UBR4 marks endoderm-derived, autophagy-enriched cells that coordinate differentiation of mesoderm-derived vascular cells and supply autophagy-generated amino acids during early embryogenesis. UBR4 of the YS endoderm is associated with a tissue-specific autophagic pathway that mediates bulk lysosomal proteolysis of endocytosed maternal proteins into amino acids. In cultured cells, UBR4 subpopulation is degraded by autophagy through its starvation-induced association with cellular cargoes destined to autophagic double membrane structures. UBR4 loss results in multiple misregulations in autophagic induction and flux, including synthesis and lipidation/activation of the ubiquitin-like protein LC3 and formation of autophagic double membrane structures. Our results suggest that UBR4 plays an important role in mammalian development, such as angiogenesis in the YS, in part through regulation of bulk degradation by lysosomal hydrolases.


Asunto(s)
Proteínas Asociadas a Microtúbulos/fisiología , Ubiquitina-Proteína Ligasas/fisiología , Saco Vitelino/irrigación sanguínea , Saco Vitelino/enzimología , Animales , Autofagia/genética , Autofagia/fisiología , Proteínas de Unión a Calmodulina/antagonistas & inhibidores , Proteínas de Unión a Calmodulina/genética , Proteínas de Unión a Calmodulina/fisiología , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Proteínas del Citoesqueleto/antagonistas & inhibidores , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/fisiología , Desarrollo Embrionario/genética , Desarrollo Embrionario/fisiología , Endodermo/irrigación sanguínea , Endodermo/citología , Endodermo/enzimología , Femenino , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Mesodermo/irrigación sanguínea , Mesodermo/citología , Mesodermo/enzimología , Redes y Vías Metabólicas , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/deficiencia , Proteínas Asociadas a Microtúbulos/genética , Neovascularización Fisiológica/genética , Embarazo , Ubiquitina-Proteína Ligasas/deficiencia , Ubiquitina-Proteína Ligasas/genética , Saco Vitelino/citología , Saco Vitelino/embriología
12.
Mol Carcinog ; 54(9): 751-60, 2015 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-24700667

RESUMEN

Phosphatase and tensin homolog (PTEN) loss or mutation consistently activates the phosphatidylinositol 3-kinase (PI3-K)/Akt signaling pathway, which contributes to the progression and invasiveness of prostate cancer. Furthermore, the PTEN/PI3-K/Akt and Ras/MAPK pathways cooperate to promote the epithelial-mesenchymal transition (EMT) and metastasis initiated from prostate stem/progenitor cells. For these reasons, the PTEN/PI3-K/Akt pathway is considered as an attractive target for both chemoprevention and chemotherapy. Herein we report that eupafolin, a natural compound found in common sage, inhibited proliferation of prostate cancer cells. Protein content analysis indicated that phosphorylation of Akt and its downstream kinases was inhibited by eupafolin treatment. Pull-down assay and in vitro kinase assay results indicated that eupafolin could bind with PI3-K and attenuate its kinase activity. Eupafolin also exhibited tumor suppressive effects in vivo in an athymic nude mouse model. Overall, these results suggested that eupafolin exerts antitumor effects by targeting PI3-K.


Asunto(s)
Antineoplásicos Fitogénicos/uso terapéutico , Flavonas/uso terapéutico , Fosfatidilinositol 3-Quinasas/metabolismo , Próstata/efectos de los fármacos , Neoplasias de la Próstata/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Proliferación Celular/efectos de los fármacos , Humanos , Masculino , Ratones Endogámicos BALB C , Ratones Desnudos , Modelos Moleculares , Próstata/metabolismo , Próstata/patología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología
14.
J Biol Chem ; 288(36): 25924-25937, 2013 Sep 06.
Artículo en Inglés | MEDLINE | ID: mdl-23888052

RESUMEN

Chrysin (5,7-dihydroxyflavone), a natural flavonoid widely distributed in plants, reportedly has chemopreventive properties against various cancers. However, the anticancer activity of chrysin observed in in vivo studies has been disappointing. Here, we report that a chrysin derivative, referred to as compound 69407, more strongly inhibited EGF-induced neoplastic transformation of JB6 P(+) cells compared with chrysin. It attenuated cell cycle progression of EGF-stimulated cells at the G1 phase and inhibited the G1/S transition. It caused loss of retinoblastoma phosphorylation at both Ser-795 and Ser-807/811, the preferred sites phosphorylated by Cdk4/6 and Cdk2, respectively. It also suppressed anchorage-dependent and -independent growth of A431 human epidermoid carcinoma cells. Compound 69407 reduced tumor growth in the A431 mouse xenograft model and retinoblastoma phosphorylation at Ser-795 and Ser-807/811. Immunoprecipitation kinase assay results showed that compound 69407 attenuated endogenous Cdk4 and Cdk2 kinase activities in EGF-stimulated JB6 P(+) cells. Pulldown and in vitro kinase assay results indicated that compound 69407 directly binds with Cdk2 and Cdk4 in an ATP-independent manner and inhibited their kinase activities. A binding model between compound 69407 and a crystal structure of Cdk2 predicted that compound 69407 was located inside the Cdk2 allosteric binding site. The binding was further verified by a point mutation binding assay. Overall results indicated that compound 69407 is an ATP-noncompetitive cyclin-dependent kinase inhibitor with anti-tumor effects, which acts by binding inside the Cdk2 allosteric pocket. This study provides new insights for creating a general pharmacophore model to design and develop novel ATP-noncompetitive agents with chemopreventive or chemotherapeutic potency.


Asunto(s)
Carcinoma de Células Escamosas/tratamiento farmacológico , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Flavonoides/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Neoplasias Cutáneas/tratamiento farmacológico , Regulación Alostérica/efectos de los fármacos , Animales , Sitios de Unión , Carcinoma de Células Escamosas/enzimología , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Cristalografía por Rayos X , Quinasas Ciclina-Dependientes/genética , Quinasas Ciclina-Dependientes/metabolismo , Factor de Crecimiento Epidérmico/genética , Factor de Crecimiento Epidérmico/metabolismo , Flavonoides/química , Fase G1/efectos de los fármacos , Fase G1/genética , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Modelos Moleculares , Trasplante de Neoplasias , Inhibidores de Proteínas Quinasas/química , Proteína de Retinoblastoma/genética , Proteína de Retinoblastoma/metabolismo , Fase S/efectos de los fármacos , Fase S/genética , Neoplasias Cutáneas/enzimología , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología
16.
Proc Natl Acad Sci U S A ; 108(28): 11566-71, 2011 Jul 12.
Artículo en Inglés | MEDLINE | ID: mdl-21709238

RESUMEN

In an effort to understand the role of Distal-less 3 (Dlx3) in cutaneous biology and pathophysiology, we generated and characterized a mouse model with epidermal ablation of Dlx3. K14cre;Dlx3(Kin/f) mice exhibited epidermal hyperproliferation and abnormal differentiation of keratinocytes. Results from subsequent analyses revealed cutaneous inflammation that featured accumulation of IL-17-producing CD4(+) T, CD8(+) T, and γδ T cells in the skin and lymph nodes of K14cre;Dlx3(Kin/f) mice. The gene expression signature of K14cre;Dlx3(Kin/f) skin shared features with lesional psoriatic skin, and Dlx3 expression was markedly and selectively decreased in psoriatic skin. Interestingly, cultured Dlx3 null keratinocytes triggered cytokine production that is potentially linked to inflammatory responses in K14cre;Dlx3(Kin/f) mice. Thus, Dlx3 ablation in epidermis is linked to altered epidermal differentiation, barrier development, and IL-17-associated skin inflammation. This model provides a platform that will allow the systematic exploration of the contributions of keratinocytes to cutaneous inflammation.


Asunto(s)
Dermatitis/etiología , Proteínas de Homeodominio/inmunología , Interleucina-17/metabolismo , Factores de Transcripción/deficiencia , Factores de Transcripción/inmunología , Animales , Diferenciación Celular , Citocinas/biosíntesis , Dermatitis/genética , Dermatitis/inmunología , Dermatitis/patología , Modelos Animales de Enfermedad , Epidermis/inmunología , Epidermis/patología , Femenino , Proteínas de Homeodominio/genética , Humanos , Hiperplasia , Mediadores de Inflamación/metabolismo , Queratinocitos/inmunología , Queratinocitos/patología , Leucocitos/inmunología , Leucocitos/patología , Ratones , Ratones Noqueados , Embarazo , Células Th17/inmunología , Células Th17/patología , Factores de Transcripción/genética
17.
J Biol Chem ; 287(15): 12230-40, 2012 Apr 06.
Artículo en Inglés | MEDLINE | ID: mdl-22351765

RESUMEN

During development, Dlx3 is expressed in ectodermal appendages such as hair and teeth. Thus far, the evidence that Dlx3 plays a crucial role in tooth development comes from reports showing that autosomal dominant mutations in DLX3 result in severe enamel and dentin defects leading to abscesses and infections. However, the normal function of DLX3 in odontogenesis remains unknown. Here, we use a mouse model to demonstrate that the absence of Dlx3 in the neural crest results in major impairment of odontoblast differentiation and dentin production. Mutant mice develop brittle teeth with hypoplastic dentin and molars with an enlarged pulp chamber and underdeveloped roots. Using this mouse model, we found that dentin sialophosphoprotein (Dspp), a major component of the dentin matrix, is strongly down-regulated in odontoblasts lacking Dlx3. Using ChIP-seq, we further demonstrate the direct binding of Dlx3 to the Dspp promoter in vivo. Luciferase reporter assays determined that Dlx3 positively regulates Dspp expression. This establishes a regulatory pathway where the transcription factor Dlx3 is essential in dentin formation by directly regulating a crucial matrix protein.


Asunto(s)
Dentina/patología , Proteínas de la Matriz Extracelular/genética , Eliminación de Gen , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Cresta Neural/metabolismo , Fosfoproteínas/genética , Sialoglicoproteínas/genética , Factores de Transcripción/genética , Ameloblastos/metabolismo , Ameloblastos/fisiología , Animales , Secuencia de Bases , Diferenciación Celular , Línea Celular , Esmalte Dental/crecimiento & desarrollo , Esmalte Dental/metabolismo , Dentina/crecimiento & desarrollo , Dentina/metabolismo , Displasia de la Dentina/genética , Displasia de la Dentina/patología , Regulación hacia Abajo , Proteínas de la Matriz Extracelular/metabolismo , Genes Reporteros , Proteínas de Homeodominio/metabolismo , Luciferasas de Renilla/biosíntesis , Luciferasas de Renilla/genética , Mandíbula/metabolismo , Mesodermo/metabolismo , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Odontoblastos/metabolismo , Odontoblastos/fisiología , Fosfoproteínas/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Sialoglicoproteínas/metabolismo , Diente/crecimiento & desarrollo , Diente/metabolismo , Diente/patología , Factores de Transcripción/metabolismo
18.
J Cell Physiol ; 228(3): 654-64, 2013 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-22886599

RESUMEN

Mutations in DLX3 in humans lead to defects in craniofacial and appendicular bones, yet the in vivo activities related to Dlx3 function during normal skeletal development have not been fully elucidated. Here we used a conditional knockout approach to analyze the effects of neural crest deletion of Dlx3 on craniofacial bones development. At birth, mutant mice exhibit a normal overall positioning of the skull bones, but a change in the shape of the calvaria was observed. Molecular analysis of the genes affected in the frontal bones and mandibles from these mice identified several bone markers known to affect bone development, with a strong prediction for increased bone formation and mineralization in vivo. Interestingly, while a subset of these genes were similarly affected in frontal bones and mandibles (Sost, Mepe, Bglap, Alp, Ibsp, Agt), several genes, including Lect1 and Calca, were specifically affected in frontal bones. Consistent with these molecular alterations, cells isolated from the frontal bone of mutant mice exhibited increased differentiation and mineralization capacities ex vivo, supporting cell autonomous defects in neural crest cells. However, adult mutant animals exhibited decreased bone mineral density in both mandibles and calvaria, as well as a significant increase in bone porosity. Together, these observations suggest that mature osteoblasts in the adult respond to signals that regulate adult bone mass and remodeling. This study provides new downstream targets for Dlx3 in craniofacial bone, and gives additional evidence of the complex regulation of bone formation and homeostasis in the adult skeleton.


Asunto(s)
Huesos Faciales/anomalías , Proteínas de Homeodominio/genética , Cresta Neural/anomalías , Cráneo/anomalías , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Animales , Secuencia de Bases , Densidad Ósea/genética , Densidad Ósea/fisiología , Femenino , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/fisiología , Humanos , Masculino , Mandíbula/anomalías , Ratones , Ratones Noqueados , Osteogénesis/genética , Osteogénesis/fisiología , Embarazo , Factores de Transcripción/fisiología
19.
Cancers (Basel) ; 13(4)2021 Feb 18.
Artículo en Inglés | MEDLINE | ID: mdl-33670717

RESUMEN

Cancer-associated fibroblasts (CAFs) are important in tumor progression. The autophagy adaptor protein, p62/SQSTM1/Sequestosome-1, is up-regulated in tumors, but down-regulated in CAFs in the early stages of lung adenocarcinoma. We investigated whether p62-induced autophagy might control CAF activation. Under CAF-inducing conditions, like hypoxia or cancer cell co-cultures, p62 ablation or autophagy inhibition with hydroxychloroquine (HCQ) impaired CAF activation and reduced transforming growth factor beta (TGFß) production, which impeded tumor growth. During CAF activation, p62-induced autophagy up-regulated the expression of the anti-oxidant signaling protein, nuclear factor erythroid 2-related factor 2 (Nrf2), and the ER-stress response regulator, activating transcription factor 6 (ATF6). Genetically or pharmacologically inhibiting the Nrf2-ATF6 pathway totally blocked CAF activation and tumor progression. These results demonstrate that p62 is a key modulator of primary lung adenocarcinoma progression. Thus, targeting the p62-Nrf2 autophagy signaling pathway might be a novel, stroma-focused, cancer prevention and/or treatment strategy.

20.
Cell Rep ; 30(5): 1447-1462.e5, 2020 02 04.
Artículo en Inglés | MEDLINE | ID: mdl-32023461

RESUMEN

Primary cilium is an antenna-like microtubule-based cellular sensing structure. Abnormal regulation of the dynamic assembly and disassembly cycle of primary cilia is closely related to ciliopathy and cancer. The Wnt signaling pathway plays a major role in embryonic development and tissue homeostasis, and defects in Wnt signaling are associated with a variety of human diseases, including cancer. In this study, we provide direct evidence of Wnt3a-induced primary ciliogenesis, which includes a continuous pathway showing that the stimulation of Wnt3a, a canonical Wnt ligand, promotes the generation of ß-catenin p-S47 epitope by CK1δ, and these events lead to the reorganization of centriolar satellites resulting in primary ciliogenesis. We have also confirmed the application of our findings in MCF-7/ADR cells, a multidrug-resistant tumor cell model. Thus, our data provide a Wnt3a-induced primary ciliogenesis pathway and may provide a clue on how to overcome multidrug resistance in cancer treatment.


Asunto(s)
Centriolos/metabolismo , Cilios/metabolismo , Organogénesis , Proteína Wnt3A/metabolismo , beta Catenina/metabolismo , Secuencia de Aminoácidos , Animales , Caseína Quinasas/metabolismo , Centrosoma/metabolismo , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Epítopos/metabolismo , Células HEK293 , Células HeLa , Humanos , Ligandos , Células MCF-7 , Ratones , Fosforilación , Fosfoserina/metabolismo , Proteína Wnt3A/química
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