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1.
J Biol Chem ; 299(10): 105248, 2023 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-37703992

RESUMEN

Rho in filopodia (Rif), a member of the Rho family of small GTPases, induces filopodia formation primarily on the dorsal surface of cells; however, its function remains largely unclear. Here, we show that Rif interacts with Ror1, a receptor for Wnt5a that can also induce dorsal filopodia. Our immunohistochemical analysis revealed a high frequency of coexpression of Ror1 and Rif in lung adenocarcinoma. Lung adenocarcinoma cells cultured on Matrigel established front-rear polarity with massive filopodia on their front surfaces, where Ror1 and Rif were accumulated. Suppression of Ror1 or Rif expression inhibited cell proliferation, survival, and invasion, accompanied by the loss of filopodia and cell polarity in vitro, and prevented tumor growth in vivo. Furthermore, we found that Rif was required to activate Wnt5a-Ror1 signaling at the cell surface leading to phosphorylation of the Wnt signaling pathway hub protein Dvl2, which was further promoted by culturing the cells on Matrigel. Our findings reveal a novel function of Rif in mediating Wnt5a-Ror1-Dvl2 signaling, which is associated with the formation of polarized filopodia on 3D matrices in lung adenocarcinoma cells.

2.
Biochem Biophys Res Commun ; 709: 149829, 2024 05 21.
Artículo en Inglés | MEDLINE | ID: mdl-38552553

RESUMEN

The microRNA-200 (miR-200) family is a potent suppressor of epithelial-to-mesenchymal transition (EMT). While its role as a tumor suppressor has been well documented, recent studies suggested that it can promote cancer progression in several stages. In this study, we investigated whether the miR-200 family members play a role in the acquisition of a hybrid epithelial/mesenchymal (E/M) state, which is reported to be associated with cancer malignancy, in mesenchymal MDA-MB-231 cells. Our results demonstrated that the induction of miR-200c-141, a cluster of the miR-200 family member, can induce the expression of epithelial gene and cell-cell junction while mesenchymal markers are retained. Moreover, induction of miR-200c-141 promoted collective migration accompanied by the formation of F-actin cables anchored by adherens junction. These results suggest that the miR-200 family can induce a hybrid E/M state and endows with the ability of collective cell migration in mesenchymal cancer cells.


Asunto(s)
Células MDA-MB-231 , MicroARNs , Línea Celular Tumoral , MicroARNs/genética , MicroARNs/metabolismo , Genes Supresores de Tumor , Movimiento Celular/genética , Transición Epitelial-Mesenquimal/genética , Regulación Neoplásica de la Expresión Génica
3.
J Biol Chem ; 298(7): 102090, 2022 07.
Artículo en Inglés | MEDLINE | ID: mdl-35654143

RESUMEN

Invadopodia on cancer cells play crucial roles in tumor invasion and metastasis by degrading and remodeling the surrounding extracellular matrices and driving cell migration in complex 3D environments. Previous studies have indicated that microtubules (MTs) play a crucial role in elongation of invadopodia, but not their formation, probably by regulating delivery of membrane and secretory proteins within invadopodia. However, the identity of the responsible MT-based molecular motors and their regulation has been elusive. Here, we show that KIF1C, a member of kinesin-3 family, is localized to the tips of invadopodia and is required for their elongation and the invasion of cancer cells. We also found that c-Src phosphorylates tyrosine residues within the stalk domain of KIF1C, thereby enhancing its association with tyrosine phosphatase PTPD1, that in turn activates MT-binding ability of KIF1C, probably by relieving the autoinhibitory interaction between its motor and stalk domains. These findings shed new insights into how c-Src signaling is coupled to the MT-dependent dynamic nature of invadopodia and also advance our understanding of the mechanism of KIF1C activation through release of its autoinhibition.


Asunto(s)
Genes src , Cinesinas , Invasividad Neoplásica , Podosomas , Línea Celular Tumoral , Humanos , Cinesinas/genética , Microtúbulos/metabolismo , Fosforilación , Podosomas/metabolismo , Proteínas Tirosina Fosfatasas no Receptoras , Tirosina/metabolismo
4.
Genes Cells ; 27(5): 368-375, 2022 May.
Artículo en Inglés | MEDLINE | ID: mdl-35261108

RESUMEN

Accumulating evidence demonstrates that bone marrow (BM)-derived mesenchymal stem cells (MSCs) play critical roles in regulating progression of various types of cancer. We have previously shown that Wnt5a-Ror2 signaling in MSCs induces expression of CXCL16, and that CXCL16 secreted from MSCs then binds to its cognate receptor CXCR6 on the surface of an undifferentiated gastric cancer cell line MKN45 cells, eventually leading to proliferation and migration of MKN45 cells. However, it remains unclear about a possible involvement of another (other) cytokine(s) in regulating progression of gastric cancer. Here, we show that CXCL16-CXCR6 signaling is also activated in MSCs through cell-autonomous machinery, leading to upregulated expression of CCL5. We further show that CCR1 and CCR3, receptors of CCL5, are expressed on the surface of MKN45 cells, and that CCL5 secreted from MSCs promotes migration of MKN45 cells presumably via its binding to CCR1/CCR3. These data indicate that cell-autonomous CXCL16-CXCR6 signaling activated in MSCs upregulates expression of CCL5, and that subsequent activation of CCL5-CCR1/3 signaling in MKN45 cells through intercellular machinery can promote migration of MKN45 cells. Collectively, these findings postulate the presence of orchestrated chemokine signaling emanated from MSCs to regulate progression of undifferentiated gastric cancer cells.


Asunto(s)
Células Madre Mesenquimatosas , Neoplasias Gástricas , Línea Celular Tumoral , Quimiocina CXCL16/metabolismo , Humanos , Células Madre Mesenquimatosas/metabolismo , Transducción de Señal , Neoplasias Gástricas/metabolismo
5.
Cancer Sci ; 111(4): 1254-1265, 2020 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-32012403

RESUMEN

Bone marrow-derived mesenchymal stem or stromal cells (MSC) have been shown to be recruited to various types of tumor tissues, where they interact with tumor cells to promote their proliferation, survival, invasion and metastasis, depending on the type of the tumor. We have previously shown that Ror2 receptor tyrosine kinase and its ligand, Wnt5a, are expressed in MSC, and Wnt5a-Ror2 signaling in MSC induces expression of CXCL16, which, in turn, promotes proliferation of co-cultured MKN45 gastric cancer cells via the CXCL16-CXCR6 axis. However, it remains unclear how CXCL16 regulates proliferation of MKN45 cells. Here, we show that knockdown of CXCL16 in MSC by siRNA suppresses not only proliferation but also migration of co-cultured MKN45 cells. We also show that MSC-derived CXCL16 or recombinant CXCL16 upregulates expression of Ror1 through activation of STAT3 in MKN45 cells, leading to promotion of proliferation and migration of MKN45 cells in vitro. Furthermore, co-injection of MSC with MKN45 cells in nude mice promoted tumor formation in a manner dependent on expression of Ror1 in MKN45 cells, and anti-CXCL16 neutralizing antibody suppressed tumor formation of MKN45 cells co-injected with MSC. These results suggest that CXCL16 produced through Ror2-mediated signaling in MSC within the tumor microenvironment acts on MKN45 cells in a paracrine manner to activate the CXCR6-STAT3 pathway, which, in turn, induces expression of Ror1 in MKN45 cells, thereby promoting tumor progression.


Asunto(s)
Quimiocina CXCL16/genética , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/genética , Factor de Transcripción STAT3/genética , Neoplasias Gástricas/genética , Animales , Anticuerpos Neutralizantes/farmacología , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Quimiocina CXCL16/antagonistas & inhibidores , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica/genética , Xenoinjertos , Humanos , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/patología , Ratones , Unión Proteica/genética , Receptores CXCR6/genética , Transducción de Señal/genética , Neoplasias Gástricas/patología , Proteína Wnt-5a/genética
6.
Genes Cells ; 24(4): 307-317, 2019 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-30801848

RESUMEN

Mutations in the human receptor tyrosine kinase ROR2 are associated with Robinow syndrome (RRS) and brachydactyly type B1. Amongst others, the shortened limb phenotype associated with RRS is recapitulated in Ror2-/- mutant mice. In contrast, Ror1-/- mutant mice are viable and show no limb phenotype. Ror1-/- ;Ror2-/- double mutants are embryonic lethal, whereas double mutants containing a hypomorphic Ror1 allele (Ror1hyp ) survive up to birth and display a more severe shortened limb phenotype. Both orphan receptors have been shown to act as possible Wnt coreceptors and to mediate the Wnt5a signal. Here, we analyzed genetic interactions between the Wnt ligand, Wnt9a, and Ror2 or Ror1, as Wnt9a has also been implicated in skeletal development. Wnt9a-/- single mutants display a mild shortening of the long bones, whereas these are severely shortened in Ror2-/- mutants. Ror2-/- ;Wnt9a-/- double mutants displayed even more severely shortened long bones, and intermediate phenotypes were observed in compound Ror2;Wnt9a mutants. Long bones were also shorter in Ror1hyp/hyp ;Wnt9a-/- double mutants. In addition, Ror1hyp/hyp ;Wnt9a-/- double mutants displayed a secondary palate cleft phenotype, which was not present in the respective single mutants. Interestingly, 50% of compound mutant pups heterozygous for Ror2 and homozygous mutant for Ror1 also developed a secondary palate cleft phenotype.


Asunto(s)
Fisura del Paladar/genética , Epistasis Genética , Deformidades Congénitas de las Extremidades/genética , Mutación , Osteogénesis/genética , Proteínas Wnt/genética , Animales , Ratones , Ratones Endogámicos C57BL , Fenotipo , Receptores Huérfanos Similares al Receptor Tirosina Quinasa
7.
Cancer Sci ; 110(4): 1306-1316, 2019 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-30742741

RESUMEN

Collective invasion is an important strategy of cancers of epithelial origin, including colorectal cancer (CRC), to infiltrate efficiently into local tissues as collective cell groups. Within the groups, cells at the invasive front, called leader cells, are highly polarized and motile, thereby providing the migratory traction that guides the follower cells. However, its underlying mechanisms remain unclear. We have previously shown that signaling emanating from the receptor tyrosine kinase Ror2 can promote invasion of human osteosarcoma cells and that intraflagellar transport 20 (IFT20) mediates its signaling to regulate Golgi structure and transport. Herein, we investigated the role of Ror2 and IFT20 in collective invasion of CRC cells, where Ror2 expression is either silenced or nonsilenced. We show by cell biological analyses that IFT20 promotes collective invasion of CRC cells, irrespective of expression and function of Ror2. Intraflagellar transport 20 is required for organization of Golgi-associated, stabilized microtubules, oriented toward the direction of invasion in leader cells. Our results also indicate that IFT20 promotes reorientation of the Golgi apparatus toward the front side of leader cells. Live cell imaging of the microtubule plus-end binding protein EB1 revealed that IFT20 is required for continuous polarized microtubule growth in leader cells. These results indicate that IFT20 plays an important role in collective invasion of CRC cells by regulating organization of Golgi-associated, stabilized microtubules and Golgi polarity in leader cells.


Asunto(s)
Proteínas Portadoras/metabolismo , Aparato de Golgi/metabolismo , Microtúbulos/metabolismo , Neoplasias/metabolismo , Línea Celular Tumoral , Movimiento Celular , Expresión Génica , Humanos , Neoplasias/genética , Neoplasias/patología , ARN Interferente Pequeño/genética , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/genética , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/metabolismo
8.
Cancer Sci ; 110(10): 3340-3349, 2019 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-31342590

RESUMEN

Aberrant activation of the MET/hepatocyte growth factor (HGF) receptor participates in the malignant behavior of cancer cells, such as invasion-metastasis and resistance to molecular targeted drugs. Many mutations in the MET extracellular region have been reported, but their significance is largely unknown. Here, we report the dysregulation of mutant MET originally found in a lung cancer patient with Val370 to Asp370 (V370D) replacement located in the extracellular SEMA domain. MET-knockout cells were prepared and reconstituted with WT-MET or V370D-MET. HGF stimulation induced MET dimerization and biological responses in cells reconstituted with WT-MET, but HGF did not induce MET dimerization and failed to induce biological responses in V370D-MET cells. The V370D mutation abrogated HGF-dependent drug resistance of lung cancer cells to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKI). Compared with WT-MET cells, V370D-MET cells showed different activation patterns in receptor tyrosine kinases upon exposure to survival/growth-stressed conditions. Surface plasmon resonance analysis indicated that affinity between the extracellular region of V370D-MET and HGF was reduced compared with that for WT-MET. Further analysis of the association between V370D-MET and the separate domains of HGF indicated that the SP domain of HGF was unchanged, but its association with the NK4 domain of HGF was mostly lost in V370D-MET. These results indicate that the V370D mutation in the MET receptor impairs the functional association with HGF and is therefore a loss-of-function mutation. This mutation may change the dependence of cancer cell growth/survival on signaling molecules, which may promote cancer cell characteristics under certain conditions.


Asunto(s)
Factor de Crecimiento de Hepatocito/metabolismo , Neoplasias Pulmonares/genética , Mutación Missense , Proteínas Proto-Oncogénicas c-met/química , Proteínas Proto-Oncogénicas c-met/genética , Animales , Células CHO , Línea Celular Tumoral , Cricetulus , Resistencia a Antineoplásicos , Técnicas de Inactivación de Genes , Humanos , Mutación con Pérdida de Función , Dominios Proteicos , Inhibidores de Proteínas Quinasas/farmacología , Multimerización de Proteína , Proteínas Proto-Oncogénicas c-met/metabolismo , Activación Transcripcional
9.
Genes Cells ; 23(7): 606-613, 2018 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-29845703

RESUMEN

Malignant pleural mesothelioma (MPM) is a highly aggressive tumor with poor prognosis and closely related to exposure to asbestos. MPM is a heterogeneous tumor with three main histological subtypes, epithelioid, sarcomatoid, and biphasic types, among which sarcomatoid type shows the poorest prognosis. The Ror-family of receptor tyrosine kinases, Ror1 and Ror2, is expressed in various types of tumor cells at higher levels and affects their aggressiveness. However, it is currently unknown whether they are expressed in and involved in aggressiveness of MPM. Here, we show that Ror1 and Ror2 are expressed in clinical specimens and cell lines of MPM with different histological features. Studies using MPM cell lines indicate that expression of Ror2 is associated tightly with high invasiveness of MPM cells, whereas Ror1 can contribute to their invasion in the absence of Ror2. However, both Ror1 and Ror2 promote proliferation of MPM cells. We also show that promoted invasion and proliferation of MPM cells by Ror signaling can be mediated by the Rho-family of small GTPases, Rac1, and Cdc42. These findings elucidate the critical role of Ror signaling in promoting invasion and proliferation of MPM cells.


Asunto(s)
Neoplasias Pulmonares/metabolismo , Mesotelioma/metabolismo , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/metabolismo , Línea Celular Tumoral , Proliferación Celular , Humanos , Neoplasias Pulmonares/genética , Mesotelioma/genética , Mesotelioma Maligno , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/genética , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/fisiología , Transducción de Señal
10.
Proc Natl Acad Sci U S A ; 113(21): 5993-8, 2016 May 24.
Artículo en Inglés | MEDLINE | ID: mdl-27162350

RESUMEN

Hair cells of the inner ear, the mechanosensory receptors, convert sound waves into neural signals that are passed to the brain via the auditory nerve. Little is known about the molecular mechanisms that govern the development of hair cell-neuronal connections. We ascertained a family with autosomal recessive deafness associated with a common cavity inner ear malformation and auditory neuropathy. Via whole-exome sequencing, we identified a variant (c.2207G>C, p.R736T) in ROR1 (receptor tyrosine kinase-like orphan receptor 1), cosegregating with deafness in the family and absent in ethnicity-matched controls. ROR1 is a tyrosine kinase-like receptor localized at the plasma membrane. At the cellular level, the mutation prevents the protein from reaching the cellular membrane. In the presence of WNT5A, a known ROR1 ligand, the mutated ROR1 fails to activate NF-κB. Ror1 is expressed in the inner ear during development at embryonic and postnatal stages. We demonstrate that Ror1 mutant mice are severely deaf, with preserved otoacoustic emissions. Anatomically, mutant mice display malformed cochleae. Axons of spiral ganglion neurons show fasciculation defects. Type I neurons show impaired synapses with inner hair cells, and type II neurons display aberrant projections through the cochlear sensory epithelium. We conclude that Ror1 is crucial for spiral ganglion neurons to innervate auditory hair cells. Impairment of ROR1 function largely affects development of the inner ear and hearing in humans and mice.


Asunto(s)
Células Ciliadas Auditivas/metabolismo , Pérdida Auditiva Sensorineural/metabolismo , Mutación , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/metabolismo , Ganglio Espiral de la Cóclea/metabolismo , Animales , Axones/metabolismo , Axones/patología , Línea Celular , Células Ciliadas Auditivas/patología , Pérdida Auditiva Sensorineural/genética , Pérdida Auditiva Sensorineural/patología , Humanos , Ratones , Ratones Mutantes , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/genética , Ganglio Espiral de la Cóclea/patología , Proteína Wnt-5a/genética , Proteína Wnt-5a/metabolismo
11.
Clin Calcium ; 29(3): 291-297, 2019.
Artículo en Japonés | MEDLINE | ID: mdl-30814373

RESUMEN

Non-canonical Wnt signaling, including planar cell polarity and Ca2+ pathways, plays crucial roles in developmental processes, including morphogenesis and tissue-/organo-genesis, in animals. Ror2 receptor tyrosine kinase mediates non-canonical Wnt signaling by acting as a receptor for Wnt5a, which also inhibits canonical Wnt signaling. Dysregulation of Wnt5a-Ror2 signaling causes a wide range of developmental defects and cancer progression. Recently, Ror2-mediated non-canonical Wnt signaling has also been shown to induce formation of filopodia that facilitates transport of Wnt to neighboring cells, thereby activating canonical Wnt signaling there.


Asunto(s)
Calcio/metabolismo , Polaridad Celular , Proteínas Wnt , Vía de Señalización Wnt , Proteína Wnt-5a/metabolismo , Animales , Humanos
12.
Cell Struct Funct ; 42(2): 159-167, 2017 Dec 20.
Artículo en Inglés | MEDLINE | ID: mdl-29070775

RESUMEN

The submandibular gland (SMG) is one of the major salivary glands that play important roles for variety of physiological functions, such as digestion of foods, prevention of infection, and lubrication of the mouth. Dysfunction of the SMG, often associated with a salivary inflammation, adversely influences a person's quality of life. However, the mechanism underlying inflammation-driven dysfunction of the SMG is largely unknown. Here, we used a mouse model in which the main excretory duct of the SMG is ligated unilaterally to induce inflammation of the gland and examined the expression of Wnt5a, Ror1 and Ror2 genes, encoding Wnt5a ligand and its cognate receptors, which have been implicated in tissue damage or inflammatory responses in variety of tissues. We show that expression levels of Ror1, Ror2, and Wnt5a are increased in the ligated SMG undergoing interstitial fibrosis, which is accompanied by robust expression of fibrosis-associated genes, such as TGF-ß1, TNF-α, IL-1ß, and MMP-2. Increased immunostaining signal of Ror2 was detected in the fibrotic tissues with abundant accumulation of fibroblasts and collagen fibers in the ligated SMG, suggesting that Ror2-mediated signaling might be activated in response to tissue damage and associated with progression of fibrosis in the SMG.Key words: submandibular gland, Ror2, Wnt5a, fibrosis, inflammation.


Asunto(s)
Fibrosis/genética , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/genética , Glándula Submandibular/metabolismo , Animales , Modelos Animales de Enfermedad , Fibrosis/metabolismo , Fibrosis/patología , Perfilación de la Expresión Génica , Masculino , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/análisis , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/metabolismo , Glándula Submandibular/patología
13.
Genes Cells ; 21(4): 325-34, 2016 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-26840931

RESUMEN

Spatiotemporally regulated interaction between the metanephric mesenchyme (MM) and Wolffian duct (WD) is essential for the induction of a single ureteric bud (UB). The MM then interacts with the tip of the UB to induce outgrowth and branching of the UB, which in turn promotes growth of the adjacent MM. The Ror family receptor tyrosine kinases, Ror1 and Ror2, have been shown to act as receptors for Wnt5a to mediate noncanonical Wnt signaling. Previous studies have shown that Ror2-mutant mice exhibit ectopic formation of the UB, due to abnormal juxtaposition of the MM to the WD. We show here that both Ror1 and Ror2 are expressed in the mesenchyme between the MM and WD during UB formation. Although Ror1-mutant mice show no apparent defects in UB formation, Ror1;Ror2-double-mutant mice exhibit either defects in UB outgrowth and branching morphogenesis, associated with the loss of the MM from the UB domain, or ectopic formation of the UB. We also show genetic interactions between Ror1 and Wnt5a during UB formation. These findings suggest that Wnt5a-Ror1/Ror2 signaling regulates cooperatively the formation of the MM at the proper position to ensure normal development of the UB.


Asunto(s)
Riñón/embriología , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/metabolismo , Transducción de Señal , Uréter/embriología , Animales , Embrión de Mamíferos/metabolismo , Riñón/metabolismo , Mesodermo/metabolismo , Ratones , Mutación , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/genética , Uréter/metabolismo
14.
Cancer Sci ; 107(3): 290-7, 2016 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-26708384

RESUMEN

Wnt5a-Ror2 signaling has been shown to play important roles in promoting aggressiveness of various cancer cells in a cell-autonomous manner. However, little is known about its function in cancer-associated stromal cells, including mesenchymal stem cells (MSCs). Thus, we examined the role of Wnt5a-Ror2 signaling in bone marrow-derived MSCs in regulating proliferation of undifferentiated gastric cancer cells. Coculture of a gastric cancer cell line, MKN45, with MSCs either directly or indirectly promotes proliferation of MKN45 cells, and suppressed expression of Ror2 in MSCs prior to coculture inhibits enhanced proliferation of MKN45 cells. In addition, conditioned media from MSCs, treated with control siRNA, but not siRNAs against Ror2, can enhance proliferation of MKN45 cells. Interestingly, it was found that expression of CXCL16 in MSCs is augmented by Wnt5a-Ror2 signaling, and that recombinant chemokine (C-X-C motif) ligand (CXCL)16 protein can enhance proliferation of MKN45 cells in the absence of MSCs. In fact, suppressed expression of CXCL16 in MSCs or an addition of a neutralizing antibody against CXCL16 fails to promote proliferation of MKN45 cells in either direct or indirect coculture with MSCs. Importantly, we show that MKN45 cells express chemokine (C-X-C motif) receptor (CXCR)6, a receptor for CXCL16, and that suppressed expression of CXCR6 in MKN45 cells results in a failure of its enhanced proliferation in either direct or indirect coculture with MSCs. These findings indicate that Wnt5a-Ror2 signaling enhances expression of CXCL16 in MSCs and, as a result, enhanced secretion of CXCL16 from MSCs might act on CXCR6 expressed on MKN45, leading to the promotion of its proliferation.


Asunto(s)
Quimiocinas CXC/fisiología , Células Madre Mesenquimatosas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/metabolismo , Receptores de Quimiocina/metabolismo , Receptores Depuradores/fisiología , Receptores Virales/metabolismo , Neoplasias Gástricas/metabolismo , Proteínas Wnt/metabolismo , Línea Celular Tumoral , Proliferación Celular , Quimiocina CXCL16 , Técnicas de Cocultivo , Regulación Neoplásica de la Expresión Génica , Humanos , Receptores CXCR6 , Transducción de Señal , Neoplasias Gástricas/patología , Activación Transcripcional , Proteína Wnt-5a
15.
J Biol Chem ; 289(38): 26302-26313, 2014 Sep 19.
Artículo en Inglés | MEDLINE | ID: mdl-25100728

RESUMEN

Cofilin plays an essential role in cell migration and morphogenesis by enhancing actin filament dynamics via its actin filament-severing activity. Slingshot-1 (SSH1) is a protein phosphatase that plays a crucial role in regulating actin dynamics by dephosphorylating and reactivating cofilin. In this study, we identified insulin receptor substrate (IRS)-4 as a novel SSH1-binding protein. Co-precipitation assays revealed the direct endogenous binding of IRS4 to SSH1. IRS4, but not IRS1 or IRS2, was bound to SSH1. IRS4 was bound to SSH1 mainly through the unique region (amino acids 335-400) adjacent to the C terminus of the phosphotyrosine-binding domain of IRS4. The N-terminal A, B, and phosphatase domains of SSH1 were bound to IRS4 independently. Whereas in vitro phosphatase assays revealed that IRS4 does not directly affect the cofilin phosphatase activity of SSH1, knockdown of IRS4 increased cofilin phosphorylation in cultured cells. Knockdown of IRS4 decreased phosphatidylinositol 3-kinase (PI3K) activity, and treatment with an inhibitor of PI3K increased cofilin phosphorylation. Akt preferentially phosphorylated SSH1 at Thr-826, but expression of a non-phosphorylatable T826A mutant of SSH1 did not affect insulin-induced cofilin dephosphorylation, and an inhibitor of Akt did not increase cofilin phosphorylation. These results suggest that IRS4 promotes cofilin dephosphorylation through sequential activation of PI3K and SSH1 but not through Akt. In addition, IRS4 co-localized with SSH1 in F-actin-rich membrane protrusions in insulin-stimulated cells, which suggests that the association of IRS4 with SSH1 contributes to localized activation of cofilin in membrane protrusions.


Asunto(s)
Cofilina 1/metabolismo , Proteínas Sustrato del Receptor de Insulina/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Procesamiento Proteico-Postraduccional , Extensiones de la Superficie Celular/metabolismo , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Insulina/fisiología , Proteínas Sustrato del Receptor de Insulina/química , Proteínas Sustrato del Receptor de Insulina/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfoproteínas Fosfatasas/química , Fosforilación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Transporte de Proteínas , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal
16.
Genes Cells ; 19(4): 287-96, 2014 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-24475942

RESUMEN

Activation of Wnt/ß-catenin signal in muscle satellite cells (mSCs) of aged mice during myogenic differentiation has been appreciated as an important age-related feature of the skeletal muscles, resulting in impairment of their regenerative ability following muscle injury. However, it remains elusive about molecules involved in this age-related alteration of Wnt/ß-catenin signal in myogenic cells. To clarify this issue, we carried out expression analyses of Wnt receptor genes using real-time RT-PCR in mSCs isolated from the skeletal muscles of young and aged mice. Here, we show that expression of Frizzled1 (Fzd1) was detected at high levels in mSCs of aged mice. Higher expression levels of Fzd1 were also detected in mSC-derived myogenic cells from aged mice and associated with activation of Wnt/ß-catenin signal during their myogenic differentiation in vitro. We also provide evidence that suppressed expression of Fzd1 in myogenic cells from aged mice results in a significant increase in myogenic differentiation, and its forced expression in those from young mice results in its drastic inhibition. These findings indicate the critical role of Fzd1 in altered myogenic differentiation associated with aging.


Asunto(s)
Diferenciación Celular/fisiología , Receptores Frizzled/metabolismo , Células Satélite del Músculo Esquelético/citología , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Factores de Edad , Envejecimiento , Animales , Células Cultivadas , Masculino , Ratones Endogámicos ICR , Células Satélite del Músculo Esquelético/metabolismo , Transducción de Señal
17.
J Cell Sci ; 125(Pt 8): 2017-29, 2012 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-22328498

RESUMEN

The Ror family receptor tyrosine kinases (RTKs), Ror1 and Ror2, have been shown to play crucial roles in developmental morphogenesis by acting as receptors or co-receptors to mediate Wnt5a-induced signaling. Although Ror1, Ror2 and Wnt5a are expressed in the developing brain, little is known about their roles in the neural development. Here we show that Ror1, Ror2 and their ligand Wnt5a are highly expressed in neocortical neural progenitor cells (NPCs). Small interfering RNA (siRNA)-mediated suppression of Ror1, Ror2 or Wnt5a in cultured NPCs isolated from embryonic neocortex results in the reduction of ßIII-tubulin-positive neurons that are produced from NPCs possibly through the generation of T-box brain 2 (Tbr2)-positive intermediate progenitors. BrdU-labeling experiments further reveal that the proportion of proliferative and neurogenic NPCs, which are positive for neural progenitor cell marker (Pax6) but negative for glial cell marker (glial fibrillary acidic protein; GFAP), is reduced within a few days in culture following knockdown of these molecules, suggesting that Ror1, Ror2 and Wnt5a regulate neurogenesis through the maintenance of NPCs. Moreover, we show that Dishevelled 2 (Dvl2) is involved in Wnt5a-Ror1 and Wnt5a-Ror2 signaling in NPCs, and that suppressed expression of Dvl2 indeed reduces the proportion of proliferative and neurogenic NPCs. Interestingly, suppressed expression of either Ror1 or Ror2 in NPCs in the developing neocortex results in the precocious differentiation of NPCs into neurons, and their forced expression results in delayed differentiation. Collectively, these results indicate that Wnt5a-Ror1 and Wnt5a-Ror2 signaling pathways play roles in maintaining proliferative and neurogenic NPCs during neurogenesis of the developing neocortex.


Asunto(s)
Neocórtex/embriología , Neocórtex/enzimología , Células-Madre Neurales/enzimología , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/metabolismo , Animales , Proliferación Celular , Células Cultivadas , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Ratones , Ratones Endogámicos ICR , Neocórtex/citología , Células-Madre Neurales/citología , Neurogénesis , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box/genética , Factores de Transcripción Paired Box/metabolismo , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/genética , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Transducción de Señal , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Proteína Wnt-5a
18.
Genes Cells ; 18(7): 608-19, 2013 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-23755735

RESUMEN

Activation of Wnt5a-Ror2 signaling has been shown to be associated with epithelial-to-mesenchymal transition (EMT) of epidermoid carcinoma cells via induction of matrix metalloproteinase-2 (MMP-2). Because EMT has also been implicated in the progression of tissue fibrosis, we examined the possible association of Wnt5a-Ror2 signaling with renal fibrosis. Here, we show that expression of Wnt5a and Ror2 is induced in a damaged mouse kidney after unilateral ureteral obstruction (UUO) treatment. Immunofluorescent analysis showed that Ror2 expression is clearly induced in tubular epithelial cells during renal fibrosis, and these Ror2-expressing cells also express Snail and vimentin, markers of mesenchymal cells, suggesting that Ror2 might be induced in epithelial cells undergoing EMT. We also found that MMP-2 expression is induced at Ror2-positive epithelium adjacent to significantly disrupted tubular basement membrane (TBM). Interestingly, reduced expression of MMP-2 is detected at epithelium in damaged kidneys from Ror2(+/-) mice compared with those from wild-type Ror2(+/+) mice. Importantly, extents of TBM disruption are apparently reduced in damaged kidneys from Ror2(+/-) mice compared with those from wild-type mice. Collectively, these findings indicate that activation of Wnt5a-Ror2 signaling in epithelial cells undergoing EMT may play an important role in disrupting TBM via MMP-2 induction during renal fibrosis.


Asunto(s)
Células Epiteliales/metabolismo , Transición Epitelial-Mesenquimal , Fibrosis/metabolismo , Enfermedades Renales/metabolismo , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/metabolismo , Proteínas Wnt/metabolismo , Animales , Modelos Animales de Enfermedad , Células Epiteliales/patología , Fibrosis/patología , Enfermedades Renales/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/genética , Transducción de Señal , Proteínas Wnt/genética , Proteína Wnt-5a
19.
In Vitro Cell Dev Biol Anim ; 60(5): 489-501, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38587578

RESUMEN

Ror-family receptors, Ror1 and Ror2, are type I transmembrane proteins that possess an extracellular cysteine-rich domain, which is conserved throughout the Frizzled-family receptors and is a binding site for Wnt ligands. Both Ror1 and Ror2 function primarily as receptors or co-receptors for Wnt5a to activate the ß-catenin-independent, non-canonical Wnt signaling, thereby regulating cell polarity, migration, proliferation, and differentiation depending on the context. Ror1 and Ror2 are expressed highly in many tissues during embryogenesis but minimally or scarcely in adult tissues, with some exceptions. In contrast, Ror1 and Ror2 are expressed in many types of cancers, and their high expression often contributes to the progression of the disease. Therefore, Ror1 and Ror2 have been proposed as potential targets for the treatment of the malignancies. In this review, we provide an overview of the regulatory mechanisms of Ror1/Ror2 expression and discuss how Wnt5a-Ror1/Ror2 signaling is mediated and regulated by their interacting proteins.


Asunto(s)
Receptores Huérfanos Similares al Receptor Tirosina Quinasa , Proteína Wnt-5a , Humanos , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/metabolismo , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/genética , Proteína Wnt-5a/metabolismo , Proteína Wnt-5a/genética , Animales , Vía de Señalización Wnt , Transducción de Señal , Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/patología
20.
J Biol Chem ; 287(2): 1588-99, 2012 Jan 06.
Artículo en Inglés | MEDLINE | ID: mdl-22128168

RESUMEN

It has been shown that constitutively active Wnt5a-Ror2 signaling in osteosarcoma cell lines plays crucial roles in induced expression of matrix metalloproteinase-13 (MMP-13), required for their invasiveness; however, it remains largely unclear about the molecular basis of MMP-13 gene induction by Wnt5a-Ror2 signaling. Here we show by reporter assay that the activator protein 1 (AP1) (binding site in the promoter region of MMP-13 gene is primarily responsible for its transcriptional activation by Wnt5a-Ror2 signaling in osteosarcoma cell lines SaOS-2 and U2OS. Chromatin immunoprecipitation assays revealed that c-Jun and ATF2 are crucial transcription factors recruited to the AP1-binding site in the MMP-13 gene promoter during Wnt5a-Ror2 signaling in SaOS-2 cells. Using siRNA-mediated suppression or specific inhibitors, we also show that Dishevelled2 (Dvl2) and c-Jun N-terminal kinase are required for MMP-13 gene induction presumably via phosphorylation of c-Jun and ATF2 during Wnt5a-Ror2 signaling in SaOS-2 cells. Interestingly, Dvl2 and Rac1, but not Dvl3, are required for MMP-13 expression in SaOS-2 cells, whereas Dvl3, but not Dvl2 and Rac1, is required for its expression in U2OS cells, indicating the presence of distinct intracellular signaling machineries leading to expression of the same gene, in this case MMP-13 gene in different osteosarcoma cell lines. Moreover, we provide evidence suggesting that Wnt5a-Ror2 signaling might also be required for expression of MMP-13 gene during the development of the cartilaginous tissue.


Asunto(s)
Regulación Enzimológica de la Expresión Génica/fisiología , Metaloproteinasa 13 de la Matriz/biosíntesis , Proteínas Proto-Oncogénicas/metabolismo , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/metabolismo , Proteínas Wnt/metabolismo , Vía de Señalización Wnt/fisiología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Línea Celular Tumoral , Proteínas Dishevelled , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/genética , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Masculino , Metaloproteinasa 13 de la Matriz/genética , Ratones , Ratones Noqueados , Neuropéptidos/genética , Neuropéptidos/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilación/fisiología , Regiones Promotoras Genéticas/fisiología , Proteínas Proto-Oncogénicas/genética , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/genética , Factor de Transcripción AP-1/genética , Factor de Transcripción AP-1/metabolismo , Proteínas Wnt/genética , Proteína Wnt-5a , Proteínas de Unión al GTP rac/genética , Proteínas de Unión al GTP rac/metabolismo , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismo
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