RESUMEN
Tissue self-organization into defined and well-controlled three-dimensional structures is essential during development for the generation of organs. A similar, but highly deranged process might also occur during the aberrant growth of cancers, which frequently display a loss of the orderly structures of the tissue of origin, but retain a multicellular organization in the form of spheroids, strands, and buds. The latter structures are often seen when tumors masses switch to an invasive behavior into surrounding tissues. However, the general physical principles governing the self-organized architectures of tumor cell populations remain by and large unclear. In this work, we perform in-vitro experiments to characterize the growth properties of glioblastoma budding emerging from monolayers. We further propose a theoretical model and its finite element implementation to characterize such a topological transition, that is modelled as a self-organised, non-equilibrium phenomenon driven by the trade-off of mechanical forces and physical interactions exerted at cell-cell and cell-substrate adhesions. Notably, the unstable disorder states of uncontrolled cellular proliferation macroscopically emerge as complex spatio-temporal patterns that evolve statistically correlated by a universal law.
Asunto(s)
Neoplasias , Adhesión Celular , División Celular , Humanos , Fenómenos Mecánicos , Modelos TeóricosRESUMEN
Son of Sevenless 1 (SOS1) is a dual guanine nucleotide exchange factor (GEF) that activates the small GTPases RAC and RAS. Although the molecular mechanisms of RAS GEF catalysis have been unveiled, how SOS1 acquires RAC GEF activity and what is the physio-pathological relevance of this activity is much less understood. Here we show that SOS1 is tyrosine phosphorylated on Y1196 by ABL. Phosphorylation of Y1196 controls SOS1 inter-molecular interaction, is required to promote the exchange of nucleotides on RAC in vitro and for platelet-derived growth factor (PDGF) activation of RAC- and RAC-dependent actin remodeling and cell migration. SOS1 is also phosphorylated on Y1196 by BCR-ABL in chronic myelogenous leukemic cells. Importantly, in these cells, SOS1 is required for BCR-ABL-mediated activation of RAC, cell proliferation and transformation in vitro and in a xenograft mouse model. Finally, genetic removal of Sos1 in the bone marrow-derived cells (BMDCs) from Sos1fl/fl mice and infected with BCR-ABL causes a significant delay in the onset of leukemogenesis once BMDCs are injected into recipient, lethally irradiated mice. Thus, SOS1 is required for full transformation and critically contribute to the leukemogenic potential of BCR-ABL.
Asunto(s)
Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Proteínas de Fusión bcr-abl/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteína SOS1/metabolismo , Animales , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Modelos Animales de Enfermedad , Proteínas de Fusión bcr-abl/metabolismo , Humanos , Leucemia/genética , Leucemia/metabolismo , Ratones , Fosforilación , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Tirosina/metabolismo , Proteínas de Unión al GTP rac , Proteína de Unión al GTP rac1/metabolismoRESUMEN
PURPOSE: The aim of this study is to understand whether the isokinetic strength could be a valid objective data of functional recovery evaluating a group of patients with proximal humeral fractures treated with open reduction and internal fixation with locking plate comparing the clinical and functional recovery (isokinetic strength) with the not involved side. METHODS: Seventy patients underwent surgery with locking plate placement for proximal humeral fractures. The strength of each patient's shoulders, both involved and not involved, was evaluated using isokinetic tests and Constant-Murley score. Finally, the study included 48 patients and the mean follow-up of 33 months. RESULTS: The functional outcome showed no significant differences between operated and not operated shoulder. CONCLUSIONS: This study shows that the assessment of the force can be supported by use of tools such as the evaluation with isokinetic machines with the advantage of having, in this way, an objective data on the functional recovery. LEVEL OF EVIDENCE: III.
Asunto(s)
Placas Óseas , Fijación Interna de Fracturas/instrumentación , Fracturas del Hombro/diagnóstico , Fracturas del Hombro/cirugía , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Estudios de Seguimiento , Fijación Interna de Fracturas/métodos , Curación de Fractura , Humanos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Radiografía/métodos , Rango del Movimiento Articular , Estudios Retrospectivos , Tomografía Computarizada por Rayos X , Resultado del TratamientoRESUMEN
Here we show that the fate of osteolytic bone metastasis depends on the balance among autophagy, anoikis resistance and ossification, and that the hepatocyte growth factor (HGF) signaling pathway seems to have an important role in orchestrating bone colonization. These findings are consistent with the pathophysiology of bone metastasis that is influenced by the cross-talk of supportive and neoplastic cells through molecular signaling networks. We adopted the strategy to target metastasis and stroma with the use of adenovirally expressed NK4 (AdNK4) and Dasatinib to block HGF/Met axis and Src activity. In human bone metastatic 1833 cells, HGF conferred anoikis resistance via Akt and Src activities and HIF-1α induction, leading to Bim isoforms degradation. When Src and Met activities were inhibited with Dasatinib, the Bim isoforms accumulated conferring anoikis sensitivity. The proviability effect of HGF, under low-nutrient stress condition, was related to a faster autophagy deactivation with respect to HGF plus Dasatinib. In the 1833 xenograft model, AdNK4 switched metastasis vasculature to blood lacunae, increasing HIF-1α in metastasis. The combination of AdNK4 plus Dasatinib gave the most relevant results for mice survival, and the following molecular and cellular changes were found to be responsible. In bone metastasis, we observed a hypoxic condition - marked by HIF-1α - and an autophagy failure - marked by p62 without Beclin-1. Then, osteolytic bone metastases were largely prevented, because of autophagy failure in metastasis and ossification in bone marrow, with osteocalcin deposition. The abnormal repair process was triggered by the dysfunctional autophagy/anoikis interplay. In conclusion, the concomitant blockade of HGF/Met axis and Src activity seemed to induce HIF-1α in metastasis, whereas the bone marrow hypoxic response was reduced. As a consequence, anoikis resistance might be hampered favoring, instead, autophagy failure and neoformation of woven bone trabeculae. Mice survival was, therefore, prolonged by overcoming an escape strategy adopted by metastatic cells by disruption of tumor-stroma coevolution, showing the importance of autophagy inhibition for the therapy of bone metastasis.
Asunto(s)
Anoicis , Autofagia , Neoplasias Óseas/fisiopatología , Neoplasias de la Mama/patología , Osteólisis , Animales , Neoplasias Óseas/genética , Neoplasias Óseas/metabolismo , Neoplasias Óseas/secundario , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Femenino , Factor de Crecimiento de Hepatocito/metabolismo , Humanos , Ratones , Osificación Heterotópica , Proteínas Proto-Oncogénicas c-met/genética , Proteínas Proto-Oncogénicas c-met/metabolismo , Proteínas Proto-Oncogénicas pp60(c-src)/genética , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismoRESUMEN
The dissection of the molecular circuitries at the base of cell life and the identification of their abnormal transformation during carcinogenesis rely on the characterization of biological phenotypes generated by targeted overexpression or deletion of gene products through genetic manipulation. Fluorescence microscopy provides a wide variety of tools to monitor cell life with minimal perturbations. The observation of living cells requires the selection of a correct balance between temporal, spatial and "statistical" resolution according to the process to be analyzed. In the following paper ad hoc developed optical tools for dynamical tracking from cellular to molecular resolution will be presented. Particular emphasis will be devoted to discuss how to exploit light-matter interaction to selectively target specific molecular species, understanding the relationships between their intracellular compartmentalization and function.
Asunto(s)
Microscopía Confocal/métodos , Microscopía Fluorescente/métodos , Espectrometría de Fluorescencia/métodos , Fenómenos Biomecánicos , Células , Colorantes Fluorescentes/química , Regulación de la Expresión Génica , Movimiento/efectos de la radiación , MutaciónRESUMEN
Dynamic assembly of actin filaments generates the forces supporting cell motility. Several recent biochemical and genetic studies have revealed a plethora of different actin binding proteins whose coordinated activity regulates the turnover of actin filaments, thus controlling a variety of actin-based processes, including cell migration. Additionally, emerging evidence is highlighting a scenario whereby the same basic set of actin regulatory proteins is also the convergent node of different signaling pathways emanating from extracellular stimuli, like those from receptor tyrosine kinases. Here, we will focus on the molecular mechanisms of how the machinery of actin polymerization functions and is regulated, in a signaling-dependent mode, to generate site-directed actin assembly leading to cell motility.
Asunto(s)
Citoesqueleto de Actina/fisiología , Movimiento Celular/fisiología , Transducción de Señal/fisiología , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animales , Proteínas del Citoesqueleto/fisiología , Humanos , Proteínas de Microfilamentos/fisiología , Modelos Biológicos , Seudópodos/metabolismo , Proteínas de Unión al GTP rac/fisiología , Proteínas de Unión al GTP rho/fisiologíaRESUMEN
Genetic and biochemical evidence demonstrated that Eps8 is involved in the routing of signals from Ras to Rac. This is achieved through the formation of a tricomplex consisting of Eps8-E3b1-Sos-1, which is endowed with Rac guanine nucleotide exchange activity. The catalytic subunit of this complex is represented by Sos-1, a bifunctional molecule capable of catalyzing guanine nucleotide exchange on Ras and Rac. The mechanism by which Sos-1 activity is specifically directed toward Rac remains to be established. Here, by performing a structure-function analysis we show that the Eps8 output function resides in an effector region located within its COOH terminus. This effector region, when separated from the holoprotein, activates Rac and acts as a potent inducer of actin polymerization. In addition, it binds to Sos-1 and is able to induce Rac-specific, Sos-1-dependent guanine nucleotide exchange activity. Finally, the Eps8 effector region mediates a direct interaction of Eps8 with F-actin, dictating Eps8 cellular localization. We propose a model whereby the engagement of Eps8 in a tricomplex with E3b1 and Sos-1 facilitates the interaction of Eps8 with Sos-1 and the consequent activation of an Sos-1 Rac-specific catalytic ability. In this complex, determinants of Eps8 are responsible for the proper localization of the Rac-activating machine to sites of actin remodeling.
Asunto(s)
Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/metabolismo , Proteínas/metabolismo , Proteína SOS1/metabolismo , Proteínas de Unión al GTP rac/metabolismo , Proteínas ras/metabolismo , Animales , Proteínas Portadoras/genética , Fraccionamiento Celular , Extensiones de la Superficie Celular , Células Cultivadas , Medio de Cultivo Libre de Suero , Citocalasina D/farmacología , Proteínas del Citoesqueleto , Embrión de Mamíferos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Genes Reporteros , Humanos , Immunoblotting , Péptidos y Proteínas de Señalización Intracelular , Ratones , Microscopía Fluorescente , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Estructura Terciaria de Proteína , Proteínas/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteína SOS1/genética , Transducción de Señal/fisiología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Transfección , Proteína de Unión al GTP cdc42/metabolismo , Proteínas de Unión al GTP rac/genética , Proteínas ras/genéticaRESUMEN
How epidermal growth factor receptor (EGFR) signalling is linked to EGFR trafficking is largely unknown. Signalling and trafficking involve small GTPases of the Rho and Rab families, respectively. But it remains unknown whether the signalling relying on these two classes of GTPases is integrated, and, if it is, what molecular machinery is involved. Here we report that the protein Eps8 connects these signalling pathways. Eps8 is a substrate of the EGFR, which is held in a complex with Sos1 by the adaptor protein E3bl (ref. 2), thereby mediating activation of Rac. Through its src homology-3 domain, Eps8 interacts with RN-tre. We show that RN-tre is a Rab5 GTPase-activating protein, whose activity is regulated by the EGFR. By entering in a complex with Eps8, RN-tre acts on Rab5 and inhibits internalization of the EGFR. Furthermore, RN-tre diverts Eps8 from its Rac-activating function, resulting in the attenuation of Rac signalling. Thus, depending on its state of association with E3b1 or RN-tre, Eps8 participates in both EGFR signalling through Rac, and trafficking through Rab5.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Receptores ErbB/metabolismo , Proteínas/fisiología , Transducción de Señal , Proteínas de Unión al GTP rab5/metabolismo , Proteínas de Unión al GTP rac/metabolismo , Animales , Células COS , Proteínas Portadoras/metabolismo , Catálisis , Clonación Molecular , Proteínas del Citoesqueleto , Endocitosis , Proteínas Activadoras de GTPasa , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular , Proteínas de Fusión Oncogénica/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión , Proteína SOS1/metabolismoRESUMEN
Members of a family of intracellular molecular switches, the small GTPases, sense modifications of the extracellular environment and transduce them into a variety of homeostatic signals. Among small GTPases, Ras and the Rho family of proteins hierarchically and/or coordinately regulate signaling pathways leading to phenotypes as important as proliferation, differentiation and apoptosis. Ras and Rho-GTPases are organized in a complex network of functional interactions, whose molecular mechanisms are being elucidated. Starting from the simple concept of linear cascades of events (GTPase-->activator--> GTPase), the work of several laboratories is uncovering an increasingly complex scenario in which upstream regulators of GTPases also function as downstream effectors and influence the precise biological outcome. Furthermore, small GTPases assemble into macromolecular machineries that include upstream activators, downstream effectors, regulators and perhaps even final biochemical targets. We are starting to understand how these macromolecular complexes work and how they are regulated and targeted to their proper subcellular localization. Ultimately, the acquisition of a cogent picture of the various levels of integration and regulation in small GTPase-mediated signaling should define the physiology of early signal transduction events and the pathological implication of its subversion.
Asunto(s)
GTP Fosfohidrolasas/fisiología , Transducción de Señal/fisiología , Animales , GTP Fosfohidrolasas/clasificación , Guanosina Trifosfato/fisiología , Mamíferos/metabolismo , Modelos Biológicos , Fosfatidilinositol 3-Quinasas/fisiología , Proteínas de Unión al GTP rac/fisiología , Proteínas ras/fisiología , Proteínas de Unión al GTP rho/fisiologíaRESUMEN
The main physiological role of somatostatin (SST) is the control of hormone secretion. Recently, SST has been shown to exert antiproliferative effects on some human tumors via both direct and indirect mechanisms. We have previously found that in the human neuroblastoma cell line SY5Y the SST analogue lanreotide (BIM 23014) inhibited serum-stimulated cell proliferation and MAP kinase activity. Here, we examine the effect of SST on PDGF-induced Ras activation. We found that SST suppressed PDGF-induced Ras activation in a pertussis toxin (PTx)-independent and peroxovanadate-dependent manner. Ras-specific GTPase activating protein (GAP) activities were not altered by SST treatment. On the contrary, PDGF-induced PDGF receptor phosphorylation was decreased by SST in a PTx-independent, peroxovanadate-dependent manner, likely accounting for the SST-mediated inhibition of PDGF-induced Ras activation.
Asunto(s)
Neuroblastoma/metabolismo , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Somatostatina/fisiología , Proteínas Activadoras de ras GTPasa/metabolismo , Proteínas ras/genética , Animales , Regulación de la Expresión Génica , Humanos , Ratones , Neuroblastoma/genética , Factor de Crecimiento Derivado de Plaquetas/antagonistas & inhibidores , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Células Tumorales Cultivadas , Proteínas ras/metabolismoRESUMEN
The small guanine nucleotide (GTP)-binding protein Rac regulates mitogen-induced cytoskeletal changes and c-Jun amino-terminal kinase (JNK), and its activity is required for Ras-mediated cell transformation. Epistatic analysis placed Rac as a key downstream target in Ras signalling; however, the biochemical mechanism regulating the cross-talk among these small GTP-binding proteins remains to be elucidated. Eps8 (relative molecular mass 97,000) is a substrate of receptors with tyrosine kinase activity which binds, through its SH3 domain, to a protein designated E3b1/Abi-1. Here we show that Eps8 and E3b1/Abi-1 participate in the transduction of signals from Ras to Rac, by regulating Rac-specific guanine nucleotide exchange factor (GEF) activities. We also show that Eps8, E3b1 and Sos-1 form a tri-complex in vivo that exhibits Rac-specific GEF activity in vitro. We propose a model in which Eps8 mediates the transfer of signals between Ras and Rac, by forming a complex with E3b1 and Sos-1.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas/metabolismo , Transducción de Señal , Proteínas ras/metabolismo , Células 3T3 , Animales , Células COS , Clonación Molecular , Proteínas del Citoesqueleto , GTP Fosfohidrolasas/metabolismo , Vectores Genéticos , Factores de Intercambio de Guanina Nucleótido , Guanosina Trifosfato/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Ratones , Unión Proteica , Proteínas/genética , Proteínas de Unión al GTP rac , Factores de Intercambio de Guanina Nucleótido rasRESUMEN
Retinoic acid (RA) reduced growth, fibronectin, and retinoic acid receptor (RAR alpha) in NIH 3T3 cells but not in cells transformed by the Ha-ras oncogene. RA lowered RAR alpha transcript and protein, increased RAR beta transcripts, and had no effect on RAR gamma. H-ras transformation downregulated RAR expression and abolished responsiveness to RA. Ha-ras-transformed cells were as active as normal NIH-3T3 cells in RA uptake but were unable to degrade it to medium oxidation product, so that, paradoxically, the resistant cells accumulated 20-30-fold as much RA as the sensitive cells. RA sensitivity/insensitivity correlated with RA metabolism/lack thereof in 15 cell lines in serum-free medium. These data suggest a relationship between RA inhibition of cell growth and intracellular RA metabolism.
Asunto(s)
Células 3T3/citología , Células 3T3/efectos de los fármacos , Fibronectinas/metabolismo , Receptores de Ácido Retinoico/metabolismo , Tretinoina/farmacología , Células 3T3/metabolismo , Animales , División Celular/efectos de los fármacos , Genes ras/fisiología , Ratones , Receptores de Ácido Retinoico/efectos de los fármacos , Tretinoina/metabolismoRESUMEN
We have previously reported an unexpected relationship between retinoic acid-induced inhibition of cell growth and the ability of various cell lines to metabolize the retinoid. Here, we report that stable expression of the truncated retinoic acid receptor RAR alpha403, transduced in NIH-3T3 cells by a retroviral vector, rendered the cells resistant to retinoic acid for growth inhibition and reduced their ability to metabolize the retinoid at the same time as it blunted the induction of the target gene transglutaminase II. The data suggest that retinoic acid receptors mediate the growth-inhibitory action of retinoic acid as well as its metabolism and the induction of transglutaminase II.
Asunto(s)
Receptores de Ácido Retinoico/biosíntesis , Tretinoina/metabolismo , Tretinoina/farmacología , Células 3T3 , Animales , División Celular/efectos de los fármacos , Inducción Enzimática , Vectores Genéticos , Cinética , Ratones , Receptores de Ácido Retinoico/fisiología , Proteínas Recombinantes/biosíntesis , Receptor alfa de Ácido Retinoico , Retroviridae , Eliminación de Secuencia , Transfección , Transglutaminasas/biosíntesisRESUMEN
SH3 domains are structurally well-characterized as monomeric modular units of protein structure that mediate protein-protein recognition in numerous signal transduction proteins. The X-ray crystallographic structure of the Eps8 SH3 domain reveals a novel variation of the canonical SH3 fold: the SH3 domain from Eps8 is a dimer formed by strand interchange. In addition, co-immunoprecipitation experiments show that intact Eps8 is multimeric in vivo. Hence, the SH3 domain of Eps8 may represent a dimerization motif.
Asunto(s)
Proteínas/química , Dominios Homologos src , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Animales , Células COS , Cristalografía por Rayos X , Proteínas del Citoesqueleto , Dimerización , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Pruebas de Precipitina , Conformación Proteica , Proteínas/genética , Proteínas Recombinantes de Fusión , Alineación de SecuenciaRESUMEN
Retinoic acid receptor transcripts (RARalpha and RARgamma) are decreased in benign mouse epidermal tumors relative to normal skin and are almost absent in carcinomas. In this report, the expression of RARalpha and RARgamma proteins was analyzed by immunoblotting in benign skin tumors induced by two different promotion protocols designed to yield tumors at low or high risk for malignant conversion. RARalpha was slightly reduced in papillomas promoted with 12-O-tetradecanoylphorbol-13-acetate (low risk) and markedly decreased or absent in papillomas promoted by mezerein (high risk). However, mezerein also caused substantial reduction of RARalpha in nontumorous skin. RARgamma was not detected in tumors from either protocol and was greatly reduced in skin treated by either promoter. Both RARalpha and RARgamma proteins were decreased in keratinocytes overexpressing an oncogenic v-ras(Ha) gene, and RARalpha was underexpressed in a benign keratinocyte cell line carrying a mutated c-ras(Ha) gene. Introduction of a recombinant RARalpha expression vector into benign keratinocyte tumor cells reduced the S-phase population and inhibited [3H]thymidine incorporation in response to retinoic acid. Furthermore, transactivation of B-RARE-tk-LUC by retinoic acid was markedly decreased in keratinocytes transduced with the v-ras(Ha) oncogene (v-ras(Ha)-keratinocytes). Blocking protein kinase C function in v-ras(Ha)-keratinocytes with bryostatin restored RARalpha protein to near normal levels, reflecting the involvement of protein kinase C in RARalpha regulation. Both RARalpha and RARgamma are down-regulated in cultured keratinocytes by 12-O-tetradecanoylphorbol-13-acetate, further implicating PKC in the regulation of retinoid receptors. Our data suggest that modulation of RARs could contribute to the neoplastic phenotype in mouse skin carcinogenesis and may be involved in the differential promoting activity of mezerein and 12-O-tetradecanoylphorbol-13-acetate, particularly for selecting tumors at high risk for malignant conversion.
Asunto(s)
Genes ras , Lesiones Precancerosas/etiología , Receptores de Ácido Retinoico/análisis , Neoplasias Cutáneas/etiología , Piel/química , Animales , Ciclo Celular , División Celular , Femenino , Regulación de la Expresión Génica , Queratinocitos/química , Ratones , Ratones Endogámicos BALB C , Lesiones Precancerosas/genética , Lesiones Precancerosas/metabolismo , Proteína Quinasa C/fisiología , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/metabolismo , Acetato de Tetradecanoilforbol , Activación TranscripcionalRESUMEN
All-trans-retinoic acid (RA) is a master regulator of cell differentiation and in this process it greatly influences cell adhesion and the elaboration of the extracellular matrix. Therefore, we were interested in the effect of RA on the biosynthesis of fibronectin (FN). RA reduced the level of intracellular FN in a time- and concentration-dependent fashion in NIH-3T3 cells, but not in NIH-3T3 cells transformed by an activated Ha-ras oncogene. Since the steady-state level of FN transcripts did not change after treatment of the cells with RA for various times or concentrations, RA probably acts at the translational level. In NIH-3T3 cells, RA had distinct effects on different receptors, from decreasing retinoic acid receptor (RAR)alpha to increasing RAR beta expression to no effect on RAR gamma. Transformation of NIH-3T3 cells with an activated Ha-ras oncogene downmodulated RAR expression and also abolished responsiveness to RA. A variety of approaches permitted the following conclusions: 1) RA-dependent FN downmodulation is mediated by RARs, 2) retinoid X receptors (RXRs) mediate the observed reduction of RAR alpha by RA, and 3) the blockade of RA responsiveness by Ha-ras-transfected cells cannot be overcome by overexpression of RAR alpha. These studies have identified fibronectin and RAR alpha as RA targets in fibroblast cells and have shown that oncogenic transformation renders the cells resistant to RA action.
Asunto(s)
Fibronectinas/fisiología , Genes ras/fisiología , Proteínas Proto-Oncogénicas p21(ras)/fisiología , Tretinoina/fisiología , Regulación de la Expresión Génica , Humanos , Membranas IntracelularesRESUMEN
Retinoids exert pleiotropic effects on the development of vertebrates through the action of retinoic acid receptors (RAR) and retinoid X receptors (RXR). We have investigated the effect of synthetic retinoids selective for RXR and RAR on the development of Xenopus and zebrafish embryos. In Xenopus, both ligands selective for RAR and RXR caused striking malformations along the anterior-posterior axis, whereas in zebrafish only ligands specific for RAR caused embryonic malformations. In Xenopus, RAR- and RXR-selective ligands regulated the expression of the Xlim-1, gsc, and HoxA1 genes similarly as all-trans-retinoic acid. Nevertheless, RXR-selective ligands activated only an RXR responsive reporter but not an RAR responsive reporter introduced by microinjection into the Xenopus embryo, consistent with our failure to detect conversion of an RXR-selective ligand to different derivatives in the embryo. These results suggest that Xenopus embryos possess a unique response pathway in which liganded RXR can control gene expression. Our observations further illustrate the divergence in retinoid responsiveness between different vertebrate species.
Asunto(s)
Receptores de Ácido Retinoico/fisiología , Factores de Transcripción/fisiología , Xenopus laevis/embriología , Pez Cebra/embriología , Animales , Anomalías Congénitas/embriología , Expresión Génica/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica , Ligandos , Receptores X Retinoide , Retinoides/farmacologíaRESUMEN
All-trans-retinoic acid (RA) markedly reduced the level of intracellular fibronectin (FN) in a time- and concentration-dependent fashion in NIH-3T3 cells, but not in NIH-3T3 cells transformed by an activated Ha-ras oncogene. Pulse/chase experiments indicated that RA affects FN biosynthesis rather than its turnover rate. Steady state levels of FN transcripts did not change after treatment of the cells with RA for various times or concentrations, suggesting that RA acts at the translational level. Similar effects were observed in other fibroblasts. In NIH-3T3 cells, RA had distinct effects on different receptors; it down-modulated retinoic acid receptor (RAR) a protein and transcript levels, it up-regulated RAR beta transcripts, and it had no effect on RAR gamma. Transformation of NIH-3T3 cells with an activated Ha-ras oncogene down-modulated RAR expression and abolished responsiveness to RA. We identified the retinoid signal transduction pathways responsible for the effects of RA on FN and RAR alpha proteins by the use of the retinoid X receptor-selective compound, SR11237, by stable over-expression of a truncated form of the RAR alpha gene, RAR alpha 403 with strong RAR dominant negative activity, and by overexpression of RAR alpha. We conclude that: 1) RA-dependent FN down-modulation is mediated by RARs, 2) retinoid X receptors mediate the observed reduction of RAR alpha by RA, and 3) the block of RA responsiveness in Ha-ras cells cannot be overcome by overexpression of RAR alpha. These studies have defined fibronectin and RAR alpha as targets of RA in fibroblast cells and have shown that oncogenic transformation renders the cells resistant to RA action.