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1.
Dis Model Mech ; 17(2)2024 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-38235522

RESUMEN

Motile cilia on ependymal cells that line brain ventricular walls beat in concert to generate a flow of laminar cerebrospinal fluid (CSF). Dyneins and kinesins are ATPase microtubule motor proteins that promote the rhythmic beating of cilia axonemes. Despite common consensus about the importance of axonemal dynein motor proteins, little is known about how kinesin motors contribute to cilia motility. Here, we show that Kif6 is a slow processive motor (12.2±2.0 nm/s) on microtubules in vitro and localizes to both the apical cytoplasm and the axoneme in ependymal cells, although it does not display processive movement in vivo. Using a mouse mutant that models a human Kif6 mutation in a proband displaying macrocephaly, hypotonia and seizures, we found that loss of Kif6 function causes decreased ependymal cilia motility and, subsequently, decreases fluid flow on the surface of brain ventricular walls. Disruption of Kif6 also disrupts orientation of cilia, formation of robust apical actin networks and stabilization of basal bodies at the apical surface. This suggests a role for the Kif6 motor protein in the maintenance of ciliary homeostasis within ependymal cells.


Asunto(s)
Cilios , Cinesinas , Humanos , Encéfalo/metabolismo , Cilios/metabolismo , Dineínas/metabolismo , Epéndimo , Cinesinas/metabolismo
2.
bioRxiv ; 2023 Feb 16.
Artículo en Inglés | MEDLINE | ID: mdl-36824804

RESUMEN

Ependymal cells, lining brain ventricular walls, display tufts of cilia that beat in concert promoting laminar Cerebrospinal fluid (CSF) flow within brain ventricles. The ciliary axonemes of multiciliated ependymal cells display a 9+2 microtubule array common to motile cilia. Dyneins and kinesins are ATPase microtubule motor proteins that promote the rhythmic beating of cilia axonemes. Despite common consensus about the importance of axonemal dynein motor proteins, little is known about how Kinesin motors contribute to cilia motility. Here, we define the function of Kinesin family member 6 (Kif6) using a mutation that lacks a highly conserved C-terminal tail domain ( Kif6 p.G555fs ) and which displays progressive hydrocephalus in mice. An analogous mutation was isolated in a proband displaying macrocephaly, hypotonia, and seizures implicating an evolutionarily conserved function for Kif6 in neurodevelopment. We find that loss of Kif6 function caused decreased ependymal cilia motility and subsequently decreased fluid flow on the surface of brain ventricular walls. Kif6 protein was localized at ependymal cilia and displayed processive motor movement (676 nm/s) on microtubules in vitro . Loss of the Kif6 C-terminal tail domain did not affect the initial ciliogenesis in vivo , but did result in defects in cilia orientation, the formation of robust apical actin networks, and stabilization of basal bodies at the apical surface. This suggests a novel role for the Kif6 motor in maintenance of ciliary homeostasis of ependymal cells. Summary statement: We found that Kif6 is localized to the axonemes of ependymal cells. In vitro analysis shows that Kif6 moves on microtubules and that its loss mice decrease cilia motility and cilia-driven flow, resulting in hydrocephalus.

3.
Curr Biol ; 32(7): 1485-1496.e4, 2022 04 11.
Artículo en Inglés | MEDLINE | ID: mdl-35196509

RESUMEN

Developing animals absorb nutrients either through the placenta or from ingested food; however, the mechanisms by which embryos use external nutrients for individual organ morphogenesis remain to be elucidated. In this study, we assessed nutrient-dependent thyroid follicle morphogenesis in Xenopus laevis and investigated the role of secreted gastrointestinal (GI) hormones post-feeding. We found that feeding triggers thyroid follicle formation, and the thyroid cells showed transient inactivation of cell proliferation after feeding. In addition, the thyroid cells with multi-lumina were frequently observed in the fed tadpoles. The expression of the particular GI hormone incretin, glucose-dependent insulinotropic polypeptide (GIP), responded to feeding in the intestines of Xenopus tadpoles. Inhibition of dipeptidyl peptidase 4 (Dpp4), a degradative enzyme of incretin, increased the size of the thyroid follicles by facilitating follicular lumina connection, whereas inhibition of the sodium-glucose cotransporter (SGLT) reversed the effects of Dpp4 inhibition. Furthermore, injection of GIP peptide in unfed tadpoles initiated thyroid follicle formation-without requiring feeding-and injection of an incretin receptor antagonist suppressed follicle enlargement in the fed tadpoles. Lastly, GIP receptor knockout in neonatal mice showed smaller follicles in the thyroid, suggesting that the GI hormone-dependent thyroid morphogenesis is conserved in mammals. In conclusion, our study links external nutrients to thyroid morphogenesis and provides new insights into the function of GI hormone as a regulator of organ morphology in developing animals.


Asunto(s)
Hormonas Gastrointestinales , Incretinas , Animales , Dipeptidil Peptidasa 4/metabolismo , Polipéptido Inhibidor Gástrico/metabolismo , Glucosa/metabolismo , Incretinas/metabolismo , Mamíferos , Ratones , Morfogénesis , Glándula Tiroides/metabolismo
4.
Sci Rep ; 11(1): 20224, 2021 10 12.
Artículo en Inglés | MEDLINE | ID: mdl-34642354

RESUMEN

The V-shaped arrangement of hair bundles on cochlear hair cells is critical for auditory sensing. However, regulation of hair bundle arrangements has not been fully understood. Recently, defects in hair bundle arrangement were reported in postnatal Dishevelled-associating protein (ccdc88c, alias Daple)-deficient mice. In the present study, we found that adult Daple-/- mice exhibited hearing disturbances over a broad frequency range through auditory brainstem response testing. Consistently, distorted patterns of hair bundles were detected in almost all regions, more typically in the basal region of the cochlear duct. In adult Daple-/- mice, apical microtubules were irregularly aggregated, and the number of microtubules attached to plasma membranes was decreased. Similar phenotypes were manifested upon nocodazole treatment in a wild type cochlea culture without affecting the microtubule structure of the kinocilium. These results indicate critical role of Daple in hair bundle arrangement through the orchestration of apical microtubule distribution, and thereby in hearing, especially at high frequencies.


Asunto(s)
Proteínas Portadoras/genética , Cóclea/patología , Pérdida Auditiva/patología , Microtúbulos/patología , Estereocilios/patología , Animales , Proteínas Portadoras/metabolismo , Membrana Celular/metabolismo , Cóclea/citología , Cóclea/efectos de los fármacos , Cóclea/metabolismo , Modelos Animales de Enfermedad , Potenciales Evocados Auditivos del Tronco Encefálico , Técnicas de Inactivación de Genes , Pérdida Auditiva/genética , Ratones , Microscopía Electrónica de Rastreo , Microtúbulos/metabolismo , Nocodazol/farmacología , Técnicas de Cultivo de Órganos , Estereocilios/metabolismo
5.
J Cell Biol ; 220(7)2021 07 05.
Artículo en Inglés | MEDLINE | ID: mdl-33929515

RESUMEN

Multiciliated cells (MCCs) in tracheas generate mucociliary clearance through coordinated ciliary beating. Apical microtubules (MTs) play a crucial role in this process by organizing the planar cell polarity (PCP)-dependent orientation of ciliary basal bodies (BBs), for which the underlying molecular basis remains elusive. Herein, we found that the deficiency of Daple, a dishevelled-associating protein, in tracheal MCCs impaired the planar polarized apical MTs without affecting the core PCP proteins, causing significant defects in the BB orientation at the cell level but not the tissue level. Using live-cell imaging and ultra-high voltage electron microscope tomography, we found that the apical MTs accumulated and were stabilized by side-by-side association with one side of the apical junctional complex, to which Daple was localized. In vitro binding and single-molecule imaging revealed that Daple directly bound to, bundled, and stabilized MTs through its dimerization. These features convey a PCP-related molecular basis for the polarization of apical MTs, which coordinate ciliary beating in tracheal MCCs.


Asunto(s)
Proteínas Portadoras/genética , Cilios/genética , Depuración Mucociliar/genética , Tráquea/crecimiento & desarrollo , Animales , Cuerpos Basales/metabolismo , Polaridad Celular/genética , Células Epiteliales/metabolismo , Ratones , Ratones Noqueados , Microtúbulos/genética , Tráquea/metabolismo
6.
Biochem Biophys Res Commun ; 532(3): 406-413, 2020 11 12.
Artículo en Inglés | MEDLINE | ID: mdl-32888647

RESUMEN

The canonical Wnt signaling pathway plays a crucial role in embryonic development, tissue homeostasis and cancer progression. The binding of Wnt ligands to their cognate receptors, the Frizzled (Fzd) family of proteins, recruits Dishevelled segment polarity protein (Dvl) to the plasma membrane and induces its phosphorylation via casein kinase 1 (CK1), which leads to the activation of ß-catenin. Previous studies showed that Dishevelled-associating protein with a high frequency of leucine residues (Daple) is an important component of the Wnt signaling pathway and essential for Dvl phosphorylation. However, the mechanism by which Daple promotes CK1-mediated phosphorylation of Dvl is not fully understood. In this study, we found that Daple overexpression induced CK1ε-mediated Dvl2 phosphorylation at threonine 224 (Thr224). A Daple mutant (Daple ΔGCV) that lacks a carboxyl-terminal motif to associate with Dvl, retained the ability to interact with CK1ε, but did not induce Dvl phosphorylation, suggesting the importance of the Daple/Dvl/CK1ε trimeric protein complex. We further found that Thr224 phosphorylation of Dvl was required for full activation of ß-catenin transcriptional activity. Consistent with this, wild-type Daple promoted ß-catenin transcriptional activity, following dissociation of ß-catenin and axin. Finally, Wnt3a stimulation increased the membrane localization of Daple and its association with Dvl, and Daple knockdown attenuated Wnt3a-mediated ß-catenin transcriptional activity. Collectively, these data suggested a essential role of spatial Daple localization in CK1ε-mediated activation of Dvl in the canonical Wnt signaling pathway.


Asunto(s)
Caseína Cinasa 1 épsilon/metabolismo , Proteínas Dishevelled/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de Microfilamentos/metabolismo , Vía de Señalización Wnt/fisiología , Animales , Proteínas Portadoras/metabolismo , Proteínas Dishevelled/química , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/genética , Células L , Ratones , Proteínas de Microfilamentos/antagonistas & inhibidores , Proteínas de Microfilamentos/genética , Fosforilación , Proteína Wnt3A/metabolismo , beta Catenina/metabolismo
7.
iScience ; 23(6): 101213, 2020 Jun 26.
Artículo en Inglés | MEDLINE | ID: mdl-32535020

RESUMEN

Despite common consensus about the importance of planar cell polarity (PCP) proteins in tissue orientation, little is known about the mechanisms used by PCP proteins to promote planar polarization of cytoskeletons within individual cells. One PCP protein Fzd6 asymmetrically localizes to the apical cell membrane of multi-ciliated ependymal cells lining the lateral ventricular (LV) wall on the side that contacts cerebrospinal fluid flow. Individual ependymal cells have planar polarized microtubules that connect ciliary basal bodies (BBs) with the cell cortex of the Fzd side to coordinate cilia orientation. Here, we report that cytoplasmic dynein is anchored to the cell cortex of the Fzd side via an adapter protein Daple that regulates microtubule dynamics. Asymmetric localization of cortical dynein generates a pulling force on dynamic microtubules connected to BBs, which in turn orients BBs toward the Fzd side. This is required for coordinated cilia orientation on the LV wall.

8.
Front Pharmacol ; 9: 983, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30233369

RESUMEN

Most solid tumors have their own cancer stem cells (CSCs), which are resistant to standard chemo-therapies. Recent reports have described that Wnt pathway plays a key role in self-renewal and tumorigenesis of CSCs. Regarding the Wnt/ß-catenin pathway, Dvl (mammalian Disheveled) is an attractive target of drug discovery. After analyzing the PDZ domain of human Dvl1 (Dvl1-PDZ) using NMR, we subjected it to preliminary NMR titration studies with 17 potential PDZ-binding molecules including CalBioChem-322338, a commercially available Dvl PDZ domain inhibitor. Next, we performed virtual screening (VS) using the program GOLD with nine parameter sets. Results were evaluated using the NMR-derived docking performance index (NMR-DPI). One parameter set of GOLD docking showing the best NMR-DPI was selected and used for the second VS against 5,135 compounds. The second docking trial identified more than 1,700 compounds that exhibited higher scores than CalBioChem-322338. Subsequent NMR titration experiments with five new candidate molecules (NPL-4001, 4004, 4011, 4012, and 4013), Dvl1-PDZ revealed larger chemical shift changes than those of CalBioChem-322338. Finally, these compounds showed partial proliferation inhibition activity against BT-20, a triple negative breast cancer (TNBC) cell. These compounds are promising Wnt pathway inhibitors that are potentially useful for anti-TNBC therapy.

9.
J Cell Sci ; 131(12)2018 06 28.
Artículo en Inglés | MEDLINE | ID: mdl-29777035

RESUMEN

Wounds in embryos heal rapidly through contraction of the wound edges. Despite well-recognized significance of the actomyosin purse string for wound closure, roles for other cytoskeletal components are largely unknown. Here, we report that the septin cytoskeleton cooperates with actomyosin and microtubules to coordinate circumferential contraction of the wound margin and concentric elongation of wound-proximal cells in Xenopus laevis embryos. Microtubules reoriented radially, forming bundles along lateral cell cortices in elongating wound-proximal cells. Depletion of septin 7 (Sept7) slowed wound closure by attenuating the wound edge contraction and cell elongation. ROCK/Rho-kinase inhibitor-mediated suppression of actomyosin contractility enhanced the Sept7 phenotype, whereas the Sept7 depletion did not affect the accumulation of actomyosin at the wound edge. The cortical microtubule bundles were reduced in wound-proximal cells in Sept7 knockdown (Sept7-KD) embryos, but forced bundling of microtubules mediated by the microtubule-stabilizing protein Map7 did not rescue the Sept7-KD phenotype. Nocodazole-mediated microtubule depolymerization enhanced the Sept7-KD phenotype, suggesting that Sept7 is required for microtubule reorganization during cell elongation. Our findings indicate that septins are required for the rapid wound closure by facilitating cortical microtubule reorganization and the concentric elongation of surrounding cells.


Asunto(s)
Células Epiteliales/metabolismo , Microtúbulos/metabolismo , Septinas/metabolismo , Cicatrización de Heridas/fisiología , Humanos
10.
PLoS Biol ; 16(3): e2005090, 2018 03.
Artículo en Inglés | MEDLINE | ID: mdl-29538402

RESUMEN

Amino acid signaling mediated by the activation of mechanistic target of rapamycin complex 1 (mTORC1) is fundamental to cell growth and metabolism. However, how cells negatively regulate amino acid signaling remains largely unknown. Here, we show that interaction between 4F2 heavy chain (4F2hc), a subunit of multiple amino acid transporters, and the multifunctional hub protein girders of actin filaments (Girdin) down-regulates mTORC1 activity. 4F2hc interacts with Girdin in mitogen-activated protein kinase (MAPK)- and amino acid signaling-dependent manners to translocate to the lysosome. The resultant decrease in cell surface 4F2hc leads to lowered cytoplasmic glutamine (Gln) and leucine (Leu) content, which down-regulates amino acid signaling. Consistently, Girdin depletion augments amino acid-induced mTORC1 activation and inhibits amino acid deprivation-induced autophagy. These findings uncovered the mechanism underlying negative regulation of amino acid signaling, which may play a role in tightly regulated cell growth and metabolism.


Asunto(s)
Cadena Pesada de la Proteína-1 Reguladora de Fusión/fisiología , Sistema de Señalización de MAP Quinasas , Proteínas de Microfilamentos/fisiología , Transducción de Señal , Proteínas de Transporte Vesicular/fisiología , Animales , Regulación hacia Abajo , Cadena Pesada de la Proteína-1 Reguladora de Fusión/metabolismo , Células HeLa , Humanos , Lisosomas/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/fisiología , Ratones , Proteínas de Microfilamentos/metabolismo , Fosforilación , Ubiquitinación , Proteínas de Transporte Vesicular/metabolismo
11.
Cell Rep ; 20(4): 960-972, 2017 07 25.
Artículo en Inglés | MEDLINE | ID: mdl-28746879

RESUMEN

Motile cilia in ependymal cells, which line the cerebral ventricles, exhibit a coordinated beating motion that drives directional cerebrospinal fluid (CSF) flow and guides neuroblast migration. At the apical cortex of these multi-ciliated cells, asymmetric localization of planar cell polarity (PCP) proteins is required for the planar polarization of microtubule dynamics, which coordinates cilia orientation. Daple is a disheveled-associating protein that controls the non-canonical Wnt signaling pathway and cell motility. Here, we show that Daple-deficient mice present hydrocephalus and their ependymal cilia lack coordinated orientation. Daple regulates microtubule dynamics at the anterior side of ependymal cells, which in turn orients the cilial basal bodies required for the directional cerebrospinal fluid flow. These results demonstrate an important role for Daple in planar polarity in motile cilia and provide a framework for understanding the mechanisms and functions of planar polarization in the ependymal cells.


Asunto(s)
Proteínas Portadoras/metabolismo , Epéndimo/metabolismo , Hidrocefalia/metabolismo , Microtúbulos/metabolismo , Animales , Proteínas Portadoras/genética , Movimiento Celular/genética , Movimiento Celular/fisiología , Polaridad Celular/genética , Polaridad Celular/fisiología , Cilios/metabolismo , Femenino , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL
12.
Cancer Sci ; 107(2): 133-9, 2016 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-26577606

RESUMEN

In gastric cancer, the non-canonical Wnt signaling pathway is activated by Wnt5a, which has a critical role in disease outcome. Previous studies have shown that Wnt5a mediates the expression of the extracellular matrix protein laminin γ2 through Rac and JNK activation to promote gastric cancer progression. However, the mechanism of this regulatory pathway has not been completely addressed. The scaffold protein Dvl is a major component of the Wnt signaling pathway. Here, we show that Dvl-associating protein with a high frequency of leucine residues (Daple) mediates Wnt5a-induced laminin γ2 expression. Immunohistochemical analysis showed marked expression of Daple in advanced clinical stages of gastric cancer, where it highly correlated with Wnt5a/b and laminin γ2 expression, the depth of wall invasion, and the frequency of lymph node metastasis. In cultured cancer cells, Daple depletion led to the suppression of Wnt5a-induced Rac and JNK activation, laminin γ2 expression, and cell migration and invasion. Accordingly, Daple depletion also suppressed liver metastasis in a mouse xenograft model of gastric cancer. These results suggest that the non-canonical Wnt signaling pathway contributes to gastric cancer progression at least in part via Daple, which provides a new therapeutic opportunity for the treatment of the disease.


Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de Microfilamentos/metabolismo , Neoplasias Gástricas/patología , Vía de Señalización Wnt/fisiología , Animales , Western Blotting , Movimiento Celular , Técnicas de Silenciamiento del Gen , Xenoinjertos , Humanos , Inmunohistoquímica , Ratones , Ratones Desnudos , Invasividad Neoplásica , Metástasis de la Neoplasia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
13.
EMBO J ; 33(18): 2098-112, 2014 Sep 17.
Artículo en Inglés | MEDLINE | ID: mdl-25061227

RESUMEN

In clathrin-mediated endocytosis (CME), specificity and selectivity for cargoes are thought to be tightly regulated by cargo-specific adaptors for distinct cellular functions. Here, we show that the actin-binding protein girdin is a regulator of cargo-selective CME. Girdin interacts with dynamin 2, a GTPase that excises endocytic vesicles from the plasma membrane, and functions as its GTPase-activating protein. Interestingly, girdin depletion leads to the defect in clathrin-coated pit formation in the center of cells. Also, we find that girdin differentially interacts with some cargoes, which competitively prevents girdin from interacting with dynamin 2 and confers the cargo selectivity for CME. Therefore, girdin regulates transferrin and E-cadherin endocytosis in the center of cells and their subsequent polarized intracellular localization, but has no effect on integrin and epidermal growth factor receptor endocytosis that occurs at the cell periphery. Our results reveal that girdin regulates selective CME via a mechanism involving dynamin 2, but not by operating as a cargo-specific adaptor.


Asunto(s)
Dinamina II/metabolismo , Endocitosis , Células Epiteliales/fisiología , Proteínas Activadoras de GTPasa/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animales , Línea Celular , Membrana Celular/enzimología , Membrana Celular/metabolismo , Humanos
14.
Cell Rep ; 7(4): 1156-67, 2014 May 22.
Artículo en Inglés | MEDLINE | ID: mdl-24794433

RESUMEN

For collective invasion, cancer cells form cohesive groups comprised of leading cells (LCs) at the forefront and following cells (FCs) at the rear. However, the molecular mechanisms that define LCs and FCs remain elusive. Here, we demonstrated that LCs, but not FCs, upregulated the expression of integrin ß1 after the loss of intercellular adhesion. The LC-specific expression of integrin ß1 was posttranscriptionally regulated by the TRIM27/MRTF-B complex in response to the loss of intercellular adhesion, thereby regulating the stability and translation of integrin ß1 mRNA via microRNA-124 in LCs. Accordingly, depletion of TRIM27 and MRTF-B abrogated the upregulation of integrin ß1 in LCs and blocked the invasion of cancer cell groups in vitro and in vivo. Therefore, our findings revealed that the specific function of LCs was defined by intrinsic mechanisms related to the presence of the cell's free surface, providing insights into the regulation of intratumor heterogeneity.


Asunto(s)
Carcinoma de Células Escamosas/metabolismo , Proteínas de Unión al ADN/metabolismo , Integrina beta1/biosíntesis , Proteínas Nucleares/metabolismo , Neoplasias Cutáneas/metabolismo , Factores de Transcripción/metabolismo , Animales , Carcinoma de Células Escamosas/genética , Adhesión Celular/fisiología , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Modelos Animales de Enfermedad , Femenino , Células HEK293 , Humanos , Integrina beta1/genética , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Proteínas Nucleares/genética , Transducción de Señal , Neoplasias Cutáneas/genética , Factores de Transcripción/genética , Ubiquitina-Proteína Ligasas , Regulación hacia Arriba
15.
J Cancer Res Clin Oncol ; 139(3): 379-88, 2013 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-23108890

RESUMEN

PURPOSE: Cancer-associated fibroblasts (CAFs) contribute to tumor progression through multiple pathways. However, the effect of CAFs on gene expression in lung cancer has been largely unknown. Here we systematically compared the gene expression changes in lung cancer cells induced by normal fibroblasts and CAFs. METHODS: Wound healing and cell proliferation assays were used to identify the property of CAFs used in this study. We used cDNA microarray analysis to compare gene expression in lung cancer cells cultured with either conditioned medium (CM) from lung CAFs or normal lung fibroblasts, the result of which was confirmed by RT-PCR and Western blot analysis. Immunohistochemistry on tissue sections from lung cancers was conducted to further confirm the results of cDNA microarray analysis. RESULTS: The expression of many genes was upregulated in cancer cells by CAF CM, particularly cell adhesion molecules, integrins, and anti-apoptotic protein Bcl-2. Expression of integrins appeared to be upstream from Bcl-2. We identified transforming growth factor-ß as a candidate factor that induced the expression of those genes in cancer cells. Immunohistochemical studies of clinical lung cancer tissues revealed that integrins and Bcl-2 were more highly expressed in the leading cells (LCs) than in the following cells, at the invasive front of cancer nests, which are adjacent to or in proximity to the stroma. Furthermore, the expression of integrins and Bcl-2 in LCs had a tendency to correlate with the clinical stage of cancer progression, including lymph node metastasis. CONCLUSIONS: Our results suggest that CAFs promote lung cancer progression partly through the direct regulation of gene expression in the LCs of invasive cancer nests.


Asunto(s)
Adenocarcinoma/patología , Fibroblastos/patología , Fibroblastos/fisiología , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/patología , Células Madre Neoplásicas/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Movimiento Celular/genética , Movimiento Celular/fisiología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genes bcl-2 , Humanos , Integrinas/genética , Integrinas/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Análisis por Micromatrices , Invasividad Neoplásica , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/patología , Nicho de Células Madre/genética , Factor de Crecimiento Transformador beta/farmacología , Células Tumorales Cultivadas
16.
Nat Commun ; 3: 859, 2012 May 29.
Artículo en Inglés | MEDLINE | ID: mdl-22643886

RESUMEN

Dishevelled is the common mediator of canonical and non-canonical Wnt signalling pathways, which are important for embryonic development, tissue maintenance and cancer progression. In the non-canonical Wnt signalling pathway, the Rho family of small GTPases acting downstream of Dishevelled has essential roles in cell migration. The mechanisms by which the non-canonical Wnt signalling pathway regulates Rac activation remain unknown. Here we show that Daple (Dishevelled-associating protein with a high frequency of leucine residues) regulates Wnt5a-mediated activation of Rac and formation of lamellipodia through interaction with Dishevelled. Daple increases the association of Dishevelled with an isoform of atypical protein kinase C, consequently promoting Rac activation. Accordingly, Daple deficiency impairs migration of fibroblasts and epithelial cells during wound healing in vivo. These findings indicate that Daple interacts with Dishevelled to direct the Dishevelled/protein kinase λ protein complex to activate Rac, which in turn mediates the non-canonical Wnt signalling pathway required for cell migration.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Portadoras/metabolismo , Movimiento Celular/fisiología , Fosfoproteínas/metabolismo , Proteínas Wnt/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Western Blotting , Proteínas Portadoras/genética , Línea Celular , Movimiento Celular/genética , Células Cultivadas , Proteínas Dishevelled , Fibroblastos/metabolismo , Humanos , Inmunohistoquímica , Inmunoprecipitación , Queratinocitos/metabolismo , Ratones , Ratones Noqueados , Modelos Biológicos , Fosfoproteínas/genética , Unión Proteica , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Transducción de Señal/genética , Transducción de Señal/fisiología , Proteínas Wnt/genética , Proteína Wnt-5a
17.
PLoS One ; 7(5): e36681, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22574214

RESUMEN

Cell migration is a critical cellular process that determines embryonic development and the progression of human diseases. Therefore, cell- or context-specific mechanisms by which multiple promigratory proteins differentially regulate cell migration must be analyzed in detail. Girdin (girders of actin filaments) (also termed GIV, Gα-interacting vesicle associated protein) is an actin-binding protein that regulates migration of various cells such as endothelial cells, smooth muscle cells, neuroblasts, and cancer cells. Here we show that Girdin regulates the establishment of cell polarity, the deregulation of which may result in the disruption of directional cell migration. We found that Girdin interacts with Par-3, a scaffolding protein that is a component of the Par protein complex that has an established role in determining cell polarity. RNA interference-mediated depletion of Girdin leads to impaired polarization of fibroblasts and mammary epithelial cells in a way similar to that observed in Par-3-depleted cells. Accordingly, the expression of Par-3 mutants unable to interact with Girdin abrogates cell polarization in fibroblasts. Further biochemical analysis suggests that Girdin is present in the Par protein complex that includes Par-3, Par-6, and atypical protein kinase C. Considering previous reports showing the role of Girdin in the directional migration of neuroblasts, network formation of endothelial cells, and cancer invasion, these data may provide a specific mechanism by which Girdin regulates cell movement in biological contexts that require directional cell movement.


Asunto(s)
Movimiento Celular , Polaridad Celular , Proteínas de Microfilamentos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , División Celular , Línea Celular , Citoplasma/metabolismo , Regulación hacia Abajo , Fibroblastos/citología , Técnicas de Inactivación de Genes , Humanos , Glándulas Mamarias Humanas/citología , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Ratones , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/deficiencia , Proteínas de Microfilamentos/genética , Neuronas/citología , Estructura Terciaria de Proteína , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/deficiencia , Proteínas de Transporte Vesicular/genética
18.
Dev Biol ; 349(2): 160-8, 2011 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-21070764

RESUMEN

The glial cell line-derived neurotrophic factor (GDNF)/RET tyrosine kinase signaling pathway plays crucial roles in the development of the enteric nervous system (ENS) and the kidney. Tyrosine 1062 (Y1062) in RET is an autophosphorylation residue that is responsible for the activation of the PI3K/AKT and RAS/MAPK signaling pathways. Mice lacking signaling via Ret Y1062 show renal hypoplasia and hypoganglionosis of the ENS although the phenotype is milder than the Gdnf- or Ret-deficient mice. Sprouty2 (Spry2) was found to be an antagonist for fibroblast growth factor receptor (FGFR) and acts as an inhibitory regulator of ERK activation. Spry2-deficient mice exhibit hearing loss and enteric nerve hyperplasia. In the present study, we generated Spry2-deficient and Ret Y1062F knock-in (tyrosine 1062 is replaced with phenylalanine) double mutant mice to see if abnormalities of the ENS and kidney, caused by loss of signaling via Ret Y1062, are rescued by a deficiency of Spry2. Double mutant mice showed significant recovery of ureteric bud branching and ENS development in the stomach. These results indicate that Spry2 regulates downstream signaling mediated by GDNF/RET signaling complex in vivo.


Asunto(s)
Anomalías del Sistema Digestivo/genética , Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Riñón/anomalías , Proteínas de la Membrana/deficiencia , Proteínas Proto-Oncogénicas c-ret/metabolismo , Transducción de Señal/fisiología , Proteínas Adaptadoras Transductoras de Señales , Análisis de Varianza , Animales , Western Blotting , Cartilla de ADN/genética , Anomalías del Sistema Digestivo/metabolismo , Sistema Nervioso Entérico/patología , Técnicas de Sustitución del Gen , Genotipo , Técnicas Histológicas , Hiperplasia/etiología , Hiperplasia/patología , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intracelular , Riñón/metabolismo , Riñón/patología , Ratones , Ratones Mutantes , Reacción en Cadena de la Polimerasa , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas c-ret/genética , Receptores de Factores de Crecimiento de Fibroblastos/antagonistas & inhibidores , Transducción de Señal/genética
19.
Cancer Sci ; 101(4): 836-42, 2010 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-20132219

RESUMEN

Cell migration is a fundamental aspect of a multitude of physiological and pathological processes, including embryonic development, inflammation, angiogenesis, and cancer progression. A variety of proteins are essential for cell migration, but context-specific signaling pathways and promigratory proteins must now be identified for our understanding of cancer biology to continue to advance. In this review, we focus on the emerging roles of Girdin (also designated KIAA1212, APE, GIV, and HkRP1), a novel component of the phosphatidylinositol 3-kinase (PI3-K)/Akt signaling pathway that is a core-signaling transduction pathway in cancer progression. Girdin is expressed in some types of cancer cells and immature endothelial cells, and is therefore at the crossroads of multiple intracellular processes, including reorganization of the actin cytoskeleton, endocytosis, and modulation of Akt activity, which ultimately lead to cancer invasion and angiogenesis. It also acts as a nonreceptor guanine nucleotide exchange factor (GEF) for Galphai proteins. A significant observation is that Girdin, although vital for cancer progression and postnatal vascular remodelling, is dispensable for cell migratory events during embryonic development. These findings suggest that Girdin and its interacting proteins are potential pharmaceutical targets for cancer therapies and pathological anigiogenesis, including tumor angiogenesis.


Asunto(s)
Movimiento Celular , Progresión de la Enfermedad , Neoplasias/irrigación sanguínea , Neoplasias/metabolismo , Neovascularización Patológica , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Movimiento Celular/fisiología , Células Endoteliales/metabolismo , Femenino , Humanos , Procesos Neoplásicos , Fosfatidilinositol 3-Quinasas/metabolismo , Embarazo , Transducción de Señal
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