RESUMEN
Epidermal growth factor receptor (EGFR) and human EGFR 2 (HER2) phosphorylation drives HER2-positive breast cancer cell proliferation. Enforced activation of phosphatases for those receptors could be a therapeutic option for HER2-positive breast cancers. Here, we report that degradation of an endosomal small GTPase, RhoB, by the ubiquitin ligase complex cullin-3 (CUL3)/KCTD10 is essential for both EGFR and HER2 phosphorylation in HER2-positive breast cancer cells. Using human protein arrays produced in a wheat cell-free protein synthesis system, RhoB-GTP, and protein tyrosine phosphatase receptor type H (PTPRH) were identified as interacting proteins of connector enhancer of kinase suppressor of Ras1 (CNKSR1). Mechanistically, constitutive degradation of RhoB, which is mediated by the CUL3/KCTD10 E3 complex, enabled CNKSR1 to interact with PTPRH at the plasma membrane resulting in inactivation of EGFR phosphatase activity. Depletion of CUL3 or KCTD10 led to the accumulation of RhoB-GTP at the plasma membrane followed by its interaction with CNKSR1, which released activated PTPRH from CNKSR1. This study suggests a mechanism of PTPRH activation through the exclusive binding of RhoB-GTP to CNKSR1.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteína de Unión al GTP rhoB/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias de la Mama/etiología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proteínas Portadoras , Línea Celular Tumoral , Proteínas Cullin/metabolismo , Receptores ErbB/agonistas , Receptores ErbB/genética , Receptores ErbB/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Persona de Mediana Edad , Modelos Biológicos , Fosforilación , Canales de Potasio con Entrada de Voltaje/metabolismo , Pronóstico , Análisis por Matrices de Proteínas , Unión Proteica , Proteolisis , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/metabolismoRESUMEN
Here, we report the discovery of the first plant-derived and noncanonical epidermal growth factor receptor (EGFR) agonist, the 36-residue bleogen pB1 from Pereskia bleo of the Cactaceae family. We show that bleogen pB1 is a low-affinity EGFR agonist using a suite of chemical, biochemical, cellular, and animal experiments which include incisor eruption and wound-healing mouse models. A focused positional scanning pB1 library of Ala- and d-amino acid scans yielded a high-affinity pB1 analog, [K29k]pB1, with a 60-fold-improved EGFR affinity and mitogenicity. We show that the potency of [K29k]pB1 and the epidermal growth factor (EGF) is comparable in a diabetic mouse wound-healing model. We also show that both bleogen pB1 and [K29k]pB1 are hyperstable, being >100-fold more stable than EGF against proteolytic degradation. Overall, our discovery of a noncanonical proteolytic-resistant EGFR agonist scaffold could open new avenues for developing wound healing and skin regeneration therapeutics and biomaterials.
Asunto(s)
Cactaceae/química , Receptores ErbB/agonistas , Péptidos/química , Secuencias de Aminoácidos , Animales , Sitios de Unión , Cactaceae/metabolismo , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/patología , Evaluación Preclínica de Medicamentos , Receptores ErbB/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Simulación del Acoplamiento Molecular , Péptidos/metabolismo , Péptidos/farmacología , Hojas de la Planta/química , Hojas de la Planta/metabolismo , Proteínas de Plantas/química , Estabilidad Proteica , Transducción de Señal/efectos de los fármacos , Relación Estructura-Actividad , Cicatrización de Heridas/efectos de los fármacosRESUMEN
Pepsin refluxate is considered a risk factor for laryngopharyngeal carcinogenesis. Non-acidic pepsin was previously linked to an inflammatory and tumorigenic effect on laryngopharyngeal cells in vitro. Yet there is no clear evidence of the pepsin-effect on a specific oncogenic pathway and the importance of pH in this process. We hypothesized that less acidic pepsin triggers the activation of a specific oncogenic factor and related-signalling pathway. To explore the pepsin-effect in vitro, we performed intermittent exposure of 15 min, once per day, for a 5-day period, of human hypopharyngeal primary cells (HCs) to pepsin (1 mg/mL), at a weakly acidic pH of 5.0, a slightly acidic pH of 6.0, and a neutral pH of 7.0. We have documented that the extracellular environment at pH 6.0, and particularly pH 7.0, vs. pH 5.0, promotes the pepsin-effect on HCs, causing increased internalized pepsin and cell viability, a pronounced activation of EGFR accompanied by NF-κB and STAT3 activation, and a significant upregulation of EGFR, AKT1, mTOR, IL1ß, TNF-α, RELA(p65), BCL-2, IL6 and STAT3. We herein provide new evidence of the pepsin-effect on oncogenic EGFR activation and its related-signaling pathway at neutral and slightly acidic pH in HCs, opening a window to further explore the prevention and therapeutic approach of laryngopharyngeal reflux disease.
Asunto(s)
Transformación Celular Neoplásica/metabolismo , Receptores ErbB/metabolismo , Concentración de Iones de Hidrógeno , Pepsina A/metabolismo , Transducción de Señal , Supervivencia Celular , Transformación Celular Neoplásica/genética , Células Cultivadas , Receptores ErbB/agonistas , Receptores ErbB/genética , Humanos , Hipofaringe/citología , Hipofaringe/metabolismo , FN-kappa B/metabolismo , Pepsina A/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Factor de Transcripción STAT3/metabolismoRESUMEN
Transactivation of the epidermal growth factor receptor (EGFR) by the angiotensin II (AngII) type 1 (AT1) receptor is involved in AT1 receptor-dependent growth effects and cardiovascular pathologies, however the mechanisms underpinning this transactivation are yet to be fully elucidated. Recently, a potential intermediate of this process was identified following the discovery that a kinase called TRIO was involved in AngII/AT1 receptor-mediated transactivation of EGFR. To investigate the mechanisms by which TRIO acts as an intermediate in AngII/AT1 receptor-mediated EGFR transactivation we used bioluminescence resonance energy transfer (BRET) assays to investigate proximity between the AT1 receptor, EGFR, TRIO and other proteins of interest. We found that AngII/AT1 receptor activation caused a Gαq-dependent increase in proximity of TRIO with Gγ2 and the AT1-EGFR heteromer, as well as trafficking of TRIO towards the Kras plasma membrane marker and into early, late and recycling endosomes. In contrast, we found that AngII/AT1 receptor activation caused a Gαq-independent increase in proximity of TRIO with Grb2, GRK2 and PKCζ, as well as trafficking of TRIO up to the plasma membrane from the Golgi. Furthermore, we confirmed the proximity between the AT1 receptor and the EGFR using the Receptor-Heteromer Investigation Technology, which showed AngII-induced recruitment of Grb2, GRK2, PKCζ, Gγ2 and TRIO to the EGFR upon AT1 coexpression. In summary, our results provide further evidence for the existence of the AT1-EGFR heteromer and reveal potential mechanisms by which TRIO contributes to the transactivation process.
Asunto(s)
Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Receptor de Angiotensina Tipo 2/metabolismo , Transducción de Señal/fisiología , Angiotensina II/farmacología , Relación Dosis-Respuesta a Droga , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/agonistas , Receptores ErbB/metabolismo , Células HEK293 , Humanos , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/fisiología , Receptor de Angiotensina Tipo 2/agonistas , Transducción de Señal/efectos de los fármacos , Activación Transcripcional/efectos de los fármacos , Activación Transcripcional/fisiologíaRESUMEN
The endosomal system provides rich signal processing capabilities for responses elicited by growth factor receptors and their ligands. At the single cell level, endosomal trafficking becomes a critical component of signal processing, as exemplified by the epidermal growth factor (EGF) receptors. Activated EGFRs are trafficked to the phosphatase-enriched peri-nuclear region (PNR), where they are dephosphorylated and degraded. The details of the mechanisms that govern the movements of stimulated EGFRs towards the PNR, are not completely known. Here, exploiting the advantages of lattice light-sheet microscopy, we show that EGFR activation by EGF triggers a transient calcium increase causing a whole-cell level redistribution of Adaptor Protein, Phosphotyrosine Interacting with PH Domain And Leucine Zipper 1 (APPL1) from pre-existing endosomes within one minute, the rebinding of liberated APPL1 directly to EGFR, and the dynein-dependent translocation of APPL1-EGF-bearing endosomes to the PNR within ten minutes. The cell spanning, fast acting network that we reveal integrates a cascade of events dedicated to the cohort movement of activated EGF receptors. Our findings support the intriguing proposal that certain endosomal pathways have shed some of the stochastic strategies of traditional trafficking and have evolved processes that provide the temporal predictability that typify canonical signaling.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Calcio/metabolismo , Dineínas/metabolismo , Endosomas/metabolismo , Microscopía Confocal , Microscopía Fluorescente , Análisis de la Célula Individual , Proteínas Adaptadoras Transductoras de Señales/genética , Endosomas/efectos de los fármacos , Endosomas/genética , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/agonistas , Receptores ErbB/genética , Receptores ErbB/metabolismo , Células HeLa , Humanos , Fosforilación , Unión Proteica , Transporte de Proteínas , Factores de TiempoRESUMEN
Chlamydia psittaci (C. psittaci) is an obligate intracellular, gram-negative bacterium, and mainly causes systemic disease in psittacine birds, domestic poultry, and wild fowl. The pathogen is threating to human beings due to closely contacted to employees in poultry industry. The polymorphic membrane proteins (Pmps) enriched in C. psittaci includes six subtypes (A, B/C, D, E/F, G/I and H). Compared to that of the 1 pmpG gene in Chlamydia trachomatis (C. trachomatis), the diverse pmpG gene-coding proteins of C. psittaci remain elusive. In the present study, polymorphic membrane protein 17G (Pmp17G) of C. psittaci mediated adhesion to different host cells. More importantly, expression of Pmp17G in C. trachomatis upregulated infections to host cells. Afterwards, crosstalk between Pmp17G and EGFR was screened and identified by MALDI-MS and Co-IP. Subsequently, EGFR overexpression in CHO-K1 cells and EGFR knockout in HeLa 229 cells were assessed to determine whether Pmp17G directly correlated with EGFR during Chlamydial adhesion. Finally, the EGFR phosphorylation was recognized by Grb2, triggering chlamydial invasion. Based on above evidence, Pmp17G possesses adhesive property that serves as an adhesin and activate intracellular bacterial internalization by recognizing EGFR during C. psittaci infection.
Asunto(s)
Chlamydophila psittaci/fisiología , Receptores ErbB/metabolismo , Interacciones Huésped-Patógeno , Proteínas de la Membrana/metabolismo , Psitacosis/metabolismo , Psitacosis/microbiología , Adhesión Celular , Línea Celular , Receptores ErbB/agonistas , Humanos , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Fosforilación , Unión ProteicaRESUMEN
The canonical model of agonist-stimulated phosphatidylinositol-3-OH kinase (PI3K)-Akt signalling proposes that PI3K is activated at the plasma membrane, where receptors are activated and phosphatidylinositol-4,5-bisphosphate is concentrated. Here we show that phosphatidylinositol-3,4,5-trisphosphate generation and activated Akt are instead largely confined to intracellular membranes upon receptor tyrosine kinase activation. Microtubule-associated protein 4 (MAP4) interacts with and controls localization of membrane vesicle-associated PI3Kα to microtubules. The microtubule-binding domain of MAP4 binds directly to the C2 domain of the p110α catalytic subunit. MAP4 controls the interaction of PI3Kα with activated receptors at endosomal compartments along microtubules. Loss of MAP4 results in the loss of PI3Kα targeting and loss of PI3K-Akt signalling downstream of multiple agonists. The MAP4-PI3Kα assembly defines a mechanism for spatial control of agonist-stimulated PI3K-Akt signalling at internal membrane compartments linked to the microtubule network.
Asunto(s)
Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Endosomas/enzimología , Proteínas Asociadas a Microtúbulos/metabolismo , Transducción de Señal , Animales , Células COS , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Chlorocebus aethiops , Fosfatidilinositol 3-Quinasa Clase I/genética , Endosomas/efectos de los fármacos , Activación Enzimática , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/agonistas , Receptores ErbB/metabolismo , Células HEK293 , Humanos , Insulina/farmacología , Proteínas Asociadas a Microtúbulos/genética , Fosfatos de Fosfatidilinositol/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Epidermal growth factor receptor (EGFR) activation plays a pivotal role in EGFR-driven non-small cell lung cancer (NSCLC) and is considered as a key target of molecular targeted therapy. EGFR tyrosine kinase inhibitors (TKIs) have been canonically used in NSCLC treatment. However, prevalent innate and acquired resistances and EGFR kinase-independent pro-survival properties limit the clinical efficacy of EGFR TKIs. Therefore, the discovery of novel EGFR degraders is a promising approach towards improving therapeutic efficacy and overcoming drug resistance. Here, we identified a 23-hydroxybetulinic acid derivative, namely DPBA, as a novel EGFR small-molecule ligand. It exerted potent in vitro and in vivo anticancer activity in both EGFR wild type and mutant NSCLC by degrading EGFR. Mechanistic studies disclosed that DPBA binds to the EGFR extracellular domain at sites differing from those of EGF and EGFR. DPBA did not induce EGFR dimerization, phosphorylation, and ubiquitination, but it significantly promoted EGFR degradation and repressed downstream survival pathways. Further analyses showed that DPBA induced clathrin-independent EGFR endocytosis mediated by flotillin-dependent lipid rafts and unaffected by EGFR TKIs. Activation of the early and late endosome markers rab5 and rab7 but not the recycling endosome marker rab11 was involved in DPBA-induced EGFR lysosomal degradation. The present study offers a new EGFR ligand for EGFR pharmacological degradation and proposes it as a potential treatment for EGFR-positive NSCLC, particularly NSCLC with innate or acquired EGFR TKI resistance. DPBA can also serve as a chemical probe in the studies on EGFR trafficking and degradation.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Proteínas de Neoplasias , Proteolisis/efectos de los fármacos , Triterpenos , Células A549 , Animales , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/enzimología , Carcinoma de Pulmón de Células no Pequeñas/genética , Descubrimiento de Drogas , Receptores ErbB/agonistas , Receptores ErbB/genética , Receptores ErbB/metabolismo , Células HCT116 , Células HEK293 , Células HT29 , Células Hep G2 , Humanos , Ligandos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/genética , Células MCF-7 , Masculino , Ratones , Ratones Endogámicos NOD , Ratones Desnudos , Ratones SCID , Proteínas de Neoplasias/agonistas , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Triterpenos/química , Triterpenos/farmacologíaRESUMEN
In this study, we aimed to develop a multifunctional drug/gene delivery system for the treatment of glioblastoma multiforme by combining the ligand-mediated active targeting and the pH-triggered drug release features of graphene oxide (GO). Toward this end, we load irinotecan (CPT-11) to cetuximab (CET)-conjugated GO (GO-CET/CPT11) for pH-responsive drug release after endocytosis by epidermal growth factor receptor (EGFR) over-expressed U87 human glioblastoma cells. The ultimate injectable drug/gene delivery system was designed by co-entrapping stomatin-like protein 2 (SLP2) short hairpin RNA (shRNA) and GO-CET/CPT11 in thermosensitive chitosan-g-poly(N-isopropylacrylamide) (CPN) polymer solution, which offers a hydrogel depot for localized, sustained delivery of the therapeutics after the in situ formation of CPN@GO-CET/CPT11@shRNA hydrogel. An optimal drug formulation was achieved by considering both the loading efficiency and loading content of CPT-11 on GO-CET. A sustained and controlled release behavior was found for CPT-11 and shRNA from CPN hydrogel. Confocal microscopy analysis confirmed the intracellular trafficking for the targeted delivery of CPT-11 through interactions of CET with EGFR on the U87 cell surface. The efficient transfection of U87 using SLP2 shRNA was achieved using CPN as a delivery milieu, possibly by the formation of shRNA/CPN polyplex after hydrogel degradation. In vitro cell culture experiments confirmed cell apoptosis induced by CPT-11 released from acid organelles in the cytoplasm by flow cytometry, as well as reduced SLP2 protein expression and inhibited cell migration due to gene silencing. Finally, in vivo therapeutic efficacy was demonstrated using the xenograft of U87 tumor-bearing nude mice through non-invasive intratumoral delivery of CPN@GO-CET/CPT11@shRNA by injection. Overall, we have demonstrated the novelty of this thermosensitive hydrogel to be an excellent depot for the co-delivery of anticancer drugs and siRNA. The in situ forming hydrogel will not only provide extended drug release but also combine the advantages offered by the chitosan-based copolymer structure for siRNA delivery to broaden treatment modalities in cancer therapy.
Asunto(s)
Proteínas Sanguíneas , Quitosano , Sistemas de Liberación de Medicamentos , Técnicas de Transferencia de Gen , Glioblastoma , Grafito , Irinotecán , Proteínas de la Membrana , Proteínas de Neoplasias , ARN Interferente Pequeño , Proteínas Sanguíneas/antagonistas & inhibidores , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/metabolismo , Línea Celular Tumoral , Quitosano/química , Quitosano/farmacología , Receptores ErbB/agonistas , Receptores ErbB/genética , Receptores ErbB/metabolismo , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patología , Glioblastoma/terapia , Grafito/química , Grafito/farmacología , Humanos , Hidrogeles/química , Hidrogeles/farmacología , Irinotecán/química , Irinotecán/farmacología , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , ARN Interferente Pequeño/química , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacologíaRESUMEN
CD148 is a transmembrane protein tyrosine phosphatase (PTP) that is expressed in the renal vasculature, including the glomerulus. Previous studies have shown that CD148 plays a role in the negative regulation of growth factor signals (including epidermal growth factor and vascular endothelial growth factor), suppressing cell proliferation and transformation. However, the role of CD148 in kidney disease remains unknown. Here, we generated an agonistic anti-CD148 antibody and evaluated its effects in murine diabetic nephropathy (DN). Monoclonal antibodies (mAbs) against the mouse CD148 ectodomain sequence were generated by immunizing CD148 knockout (CD148KO) mice. The mAbs that increased CD148 activity were selected by biological (proliferation) and biochemical (PTP activity) assays. The mAb (18E1) that showed strong agonistic activity was injected (10 mg/kg ip) in streptozotocin-induced wild-type and CD148KO diabetic mice for 6 wk, and the renal phenotype was then assessed. The effects of 18E1 mAb in podocyte growth factor signals were also assessed in culture. Compared with control IgG, 18E1 mAb significantly decreased albuminuria and mesangial expansion without altering hyperglycemia and blood pressure in wild-type diabetic mice. Immunohistochemical evaluation showed that 18E1 mAb significantly prevented the reduction of podocyte number and nephrin expression and decreased glomerular fibronectin expression and renal macrophage infiltration. The 18E1 mAb showed no effects in CD148KO diabetic mice. Furthermore, we demonstrated that 18E1 mAb reduces podocyte epidermal growth factor receptor signals in culture and in diabetic mice. These findings suggest that agonistic anti-CD148 mAb attenuates DN in mice, in part by reducing epidermal growth factor receptor signals in podocytes. This antibody may be used for the treatment of early DN.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Nefropatías Diabéticas/terapia , Albuminuria , Animales , Línea Celular , Diabetes Mellitus Experimental/complicaciones , Receptores ErbB/agonistas , Receptores ErbB/genética , Receptores ErbB/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Inmunoglobulina G/uso terapéutico , Ratones , Ratones Noqueados , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/genética , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/inmunología , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/metabolismo , Transducción de SeñalRESUMEN
Spinal cord injury (SCI) is a devastating disease. Strategies that enhance the intrinsic regenerative ability are very important for the recovery of SCI to radically prevent the occurrence of sensory disorders. Epidermal growth factor (EGF) showed a limited effect on the growth of primary sensory neuron neurites due to the degradation of phosphorylated-epidermal growth factor receptor (p-EGFR) in a manner dependent on Casitas B-lineage lymphoma (CBL) (an E3 ubiquitin-protein ligase). MiR-22-3p predicted from four databases could target CBL to inhibit the expression of CBL, increase p-EGFR levels and neurites length via STAT3/GAP43 pathway rather than Erk1/2 axis. EGF, EGFR, and miR-22-3p were downregulated sharply after injury. In vivo miR-22-3p Agomir application could regulate CBL/p-EGFR/p-STAT3/GAP43/p-GAP43 axis, and restore spinal cord sensory conductive function. This study clarified the mechanism of the limited promotion effect of EGF on adult primary sensory neuron neurite and targeting miR-22-3p could be a novel strategy to treat sensory dysfunction after SCI.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Receptores ErbB/metabolismo , Proteína GAP-43/metabolismo , MicroARNs/metabolismo , Regeneración Nerviosa , Proteínas Proto-Oncogénicas c-cbl/metabolismo , Factor de Transcripción STAT3/metabolismo , Células Receptoras Sensoriales/enzimología , Traumatismos de la Médula Espinal/enzimología , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/agonistas , Potenciales Evocados Somatosensoriales , Femenino , MicroARNs/genética , Regeneración Nerviosa/efectos de los fármacos , Proyección Neuronal , Oligonucleótidos/farmacología , Fosforilación , Cultivo Primario de Células , Proteínas Proto-Oncogénicas c-cbl/genética , Ratas Wistar , Recuperación de la Función , Células Receptoras Sensoriales/efectos de los fármacos , Células Receptoras Sensoriales/patología , Transducción de Señal , Traumatismos de la Médula Espinal/tratamiento farmacológico , Traumatismos de la Médula Espinal/patología , Traumatismos de la Médula Espinal/fisiopatologíaRESUMEN
BACKGROUND: Sustained activation of EGF receptor (EGFR) in proximal tubule cells is a hallmark of progressive kidney fibrosis after AKI and in CKD. However, the molecular mechanisms and particular EGFR ligands involved are unknown. METHODS: We studied EGFR activation in proximal tubule cells and primary tubular cells isolated from injured kidneys in vitro. To determine in vivo the role of amphiregulin, a low-affinity EGFR ligand that is highly upregulated with injury, we used ischemia-reperfusion injury or unilateral ureteral obstruction in mice with proximal tubule cell-specific knockout of amphiregulin. We also injected soluble amphiregulin into knockout mice with proximal tubule cell-specific deletion of amphiregulin's releasing enzyme, the transmembrane cell-surface metalloprotease, a disintegrin and metalloprotease-17 (ADAM17), and into ADAM17 hypomorphic mice. RESULTS: Yes-associated protein 1 (YAP1)-dependent upregulation of amphiregulin transcript and protein amplifies amphiregulin signaling in a positive feedback loop. YAP1 also integrates signals of other moderately injury-upregulated, low-affinity EGFR ligands (epiregulin, epigen, TGFα), which also require soluble amphiregulin and YAP1 to induce sustained EGFR activation in proximal tubule cells in vitro. In vivo, soluble amphiregulin injection sufficed to reverse protection from fibrosis after ischemia-reperfusion injury in ADAM17 hypomorphic mice; injected soluble amphiregulin also reversed the corresponding protective proximal tubule cell phenotype in injured proximal tubule cell-specific ADAM17 knockout mice. Moreover, the finding that proximal tubule cell-specific amphiregulin knockout mice were protected from fibrosis after ischemia-reperfusion injury or unilateral ureteral obstruction demonstrates that amphiregulin was necessary for the development of fibrosis. CONCLUSIONS: Our results identify amphiregulin as a key player in injury-induced kidney fibrosis and suggest therapeutic or diagnostic applications of soluble amphiregulin in kidney disease.
Asunto(s)
Lesión Renal Aguda/metabolismo , Anfirregulina/fisiología , Receptores ErbB/agonistas , Túbulos Renales Proximales/metabolismo , Insuficiencia Renal Crónica/patología , Proteína ADAM17/deficiencia , Proteína ADAM17/genética , Lesión Renal Aguda/complicaciones , Lesión Renal Aguda/patología , Proteínas Adaptadoras Transductoras de Señales/fisiología , Anfirregulina/deficiencia , Animales , Proteínas de Ciclo Celular/fisiología , Células Cultivadas , Familia de Proteínas EGF/metabolismo , Células Epiteliales/metabolismo , Fibrosis , Riñón/irrigación sanguínea , Masculino , Ratones , Ratones Noqueados , Procesamiento Proteico-Postraduccional , Insuficiencia Renal Crónica/etiología , Insuficiencia Renal Crónica/metabolismo , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Regulación hacia Arriba , Obstrucción Ureteral/metabolismo , Obstrucción Ureteral/patología , Proteínas Señalizadoras YAPRESUMEN
Cetuximab is a monoclonal antibody developed to inhibit the binding of growth factors and the subsequent activation of epidermal growth factor receptor (EGFR). Triplenegative breast cancer (TNBC) is resistant to cetuximab treatment. The aim of the present study was to examine the partial agonistic properties of cetuximab, which not only blocks ligand binding, but also partially triggers EGFR activation, which may lead to cetuximab resistance in TNBC. The phosphorylation of growth factor receptors and their signalling pathways were evaluated by determining the phosphorylation of EGFR, insulinlike growth factor receptor (IGF1R), vascular endothelial growth factor receptor (VEGFR)2, Src kinase, phosphoinositide3kinase (PI3K), extracellular signalregulated kinase (ERK1/2) and serine/threoninespecific protein kinase (Akt) and the degradation of EGFR, and by assessing the morphology and proliferation of MDAMB231 and MDAMB468 cells. Cetuximab treatment led to the phosphorylation of EGFR, VEGFR2, IGF1R and downstream signalling molecules, Src kinase and PI3K in these cells, as well as Akt in the MDAMB231 cells. The cetuximabmediated phosphorylation of IGF1R, VEGFR2 and Akt was inhibited by the EGFR kinase inhibitor, AG1478, and the Src kinase inhibitor, PP2. Cetuximab treatment led to the degradation of EGFR. The cetuximabinduced phosphorylation and EGFR degradation were less prominent compared with those induced by EGF. Cetuximab partially inhibited EGFmediated responses. Cetuximab, similar with EGF, altered cellular morphology in a serumfree medium. In both cell lines, the Src kinase inhibitor enhanced the cetuximabinduced antiproliferative response. These results indicate that cetuximab exerts a partial agonistic effect on EGFR, which activates Src kinase and subsequently transactivates IGF1R and VEGFR2. This partial agonistic property is likely one of the mechanisms underlying the resistance of TNBC to cetuximab.
Asunto(s)
Cetuximab/farmacología , Resistencia a Antineoplásicos , Neoplasias de la Mama Triple Negativas/metabolismo , Familia-src Quinasas/metabolismo , Línea Celular Tumoral , Proliferación Celular , Receptores ErbB/agonistas , Receptores ErbB/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Fosforilación/efectos de los fármacos , Receptor IGF Tipo 1 , Receptores de Somatomedina/metabolismo , Transducción de Señal/efectos de los fármacos , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
To explore thermofluorimetric analysis (TFA) in detail, we compared two related aptamers. The first, LINN2, is a DNA aptamer previously selected against EGFR recombinant protein. In this work we selected a second aptamer, KM4, against EGFR-overexpressing A549 cells. The two aptamers were derived from the same pool and bind the same target but behave differently in TFA. Our results suggest four overall conclusions about TFA of aptamers: 1. Some aptamers show reduced fluorescence upon target binding suggesting that target-bound aptamer is not always fluorescent. 2. Many aptamers do not obey the intuitive assumptions that aptamer-target interactions stabilize a folded conformation. 3. TFA may be most appropriate for aptamers with significant double-stranded structure. 4. Kinetic effects may be significant and the order of operations in preparing samples should be carefully optimized.
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Aptámeros de Nucleótidos/análisis , Fluorometría/métodos , Células A549 , Receptores ErbB/agonistas , Citometría de Flujo , HumanosRESUMEN
Melatonin is a natural hormone involved in the regulation of circadian rhythm, immunity, and cardiovascular function. In the present study, we focused on the mechanism of melatonin in the regulation of vascular permeability. We found that melatonin could inhibit both VEGF- and EGF-induced monolayer permeability of human umbilical vein endothelial cells (HUVECs) and change the tyrosine phosphorylation of vascular-endothelial (VE-)cadherin, which was related to endothelial barrier function. In addition, phospho-AKT (Ser473) and phospho-ERK(1/2) played significant roles in the regulation of VE-cadherin phosphorylation. Both the phosphatidylinositol 3-kinase/AKT inhibitor LY49002 and MEK/ERK inhibitor U0126 could inhibit the permeability of HUVECs, but with different effects on tyrosine phosphorylation of VE-cadherin. Melatonin can influence the two growth factor-induced phosphorylation of AKT (Ser473) but not ERK(1/2). Our results show that melatonin can inhibit growth factor-induced monolayer permeability of HUVECs by influencing the phosphorylation of AKT and VE-cadherin. Melatonin can be a potential treatment for diseases associated with abnormal vascular permeability. NEW & NOTEWORTHY We found that melatonin could inhibit both EGF- and VEGF-induced monolayer permeability of human umbilical vein endothelial cells, which is related to phosphorylation of vascular-endothelial cadherin. Blockade of phosphatidylinositol 3-kinase/AKT and MEK/ERK pathways could inhibit the permeability of human umbilical vein endothelial cells, and phosphorylation of AKT (Ser473) might be a critical event in the changing of monolayer permeability and likely has cross-talk with the MEK/ERK pathway.
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Permeabilidad Capilar/efectos de los fármacos , Factor de Crecimiento Epidérmico/farmacología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Melatonina/farmacología , Factor A de Crecimiento Endotelial Vascular/farmacología , Antígenos CD/metabolismo , Cadherinas/metabolismo , Células Cultivadas , Receptores ErbB/agonistas , Receptores ErbB/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Tirosina , Receptor 2 de Factores de Crecimiento Endotelial Vascular/agonistas , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
The transcriptional regulators YAP and TAZ play important roles in development, physiology, and tumorigenesis and are negatively controlled by the Hippo pathway. It is yet unknown why the YAP/ TAZ proteins are frequently activated in human malignancies in which the Hippo pathway is still active. Here, by a gain-of-function cancer metastasis screen, we discovered OTUB2 as a cancer stemness and metastasis-promoting factor that deubiquitinates and activates YAP/TAZ. We found OTUB2 to be poly-SUMOylated on lysine 233, and this SUMOylation enables it to bind YAP/TAZ. We also identified a yet-unknown SUMO-interacting motif (SIM) in YAP and TAZ required for their association with SUMOylated OTUB2. Importantly, EGF and oncogenic KRAS induce OTUB2 poly-SUMOylation and thereby activate YAP/TAZ. Our results establish OTUB2 as an essential modulator of YAP/TAZ and also reveal a novel mechanism via which YAP/TAZ activity is induced by oncogenic KRAS.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Neoplasias de la Mama/enzimología , Movimiento Celular , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Células Madre Neoplásicas/enzimología , Fosfoproteínas/metabolismo , Tioléster Hidrolasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Diferenciación Celular , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/agonistas , Receptores ErbB/metabolismo , Femenino , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Lisina , Ratones Endogámicos BALB C , Ratones Desnudos , Mutación , Metástasis de la Neoplasia , Células Madre Neoplásicas/patología , Fenotipo , Fosfoproteínas/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteolisis , Proteínas Proto-Oncogénicas p21(ras)/genética , Transducción de Señal , Sumoilación , Tioléster Hidrolasas/genética , Factores de Tiempo , Transactivadores , Factores de Transcripción , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ , Proteínas Señalizadoras YAPRESUMEN
This study is aimed to reveal morphological and functional changes in multipotent mesenchymal stromal cells (MSCs) isolated from the rat bone marrow after: (i) activation of Toll-like receptors (TLRs) with teichoic acid (TA), (ii) impact on epidermal growth factor (EGF) receptors with activator EGF or inhibitor Herceptin, and (iii) treatment with DNA intercalator Cisplatin. According to our results, TA and EGF cause an increase in the synthesis of glycosaminoglycans, c-Myc content, and protein in the MSC cytoplasm. It was observed that the cell population in G0 phase decreased and the cell population in G1 phase increased, when compared with control. At the same time, the cell population with a higher nuclear-cytoplasmic ratio (NCR) in S and G2 phases also increased. This indicates the manifestation of the MSC mesenchymal phenotype, exhibiting indirect metabolic signs of the regenerative potential increase. In other experiments, Herceptin was shown to suppress only the stemness signs of MSCs, while Cisplatin seriously affected cell viability in general, reducing synthetic and proliferative activities and causing cell morphology disturbances. © 2018 International Society for Advancement of Cytometry.
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Cisplatino/farmacología , Factor de Crecimiento Epidérmico/farmacología , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Animales , Células de la Médula Ósea/química , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Receptores ErbB/agonistas , Receptores ErbB/antagonistas & inhibidores , Citometría de Flujo , Glucógeno/metabolismo , Glicosaminoglicanos/biosíntesis , Glicosaminoglicanos/metabolismo , Humanos , Sustancias Intercalantes/farmacología , Masculino , Células Madre Mesenquimatosas/química , Proteínas Proto-Oncogénicas c-myc/metabolismo , Ratas , Ácidos Teicoicos/farmacología , Receptores Toll-Like/metabolismo , Trastuzumab/farmacologíaRESUMEN
The costimulation of immune cells using first-generation anti-4-1BB monoclonal antibodies (mAbs) has demonstrated anti-tumor activity in human trials. Further clinical development, however, is restricted by significant off-tumor toxicities associated with FcγR interactions. Here, we have designed an Fc-free tumor-targeted 4-1BB-agonistic trimerbody, 1D8N/CEGa1, consisting of three anti-4-1BB single-chain variable fragments and three anti-EGFR single-domain antibodies positioned in an extended hexagonal conformation around the collagen XVIII homotrimerization domain. The1D8N/CEGa1 trimerbody demonstrated high-avidity binding to 4-1BB and EGFR and a potent in vitro costimulatory capacity in the presence of EGFR. The trimerbody rapidly accumulates in EGFR-positive tumors and exhibits anti-tumor activity similar to IgG-based 4-1BB-agonistic mAbs. Importantly, treatment with 1D8N/CEGa1 does not induce systemic inflammatory cytokine production or hepatotoxicity associated with IgG-based 4-1BB agonists. These results implicate FcγR interactions in the 4-1BB-agonist-associated immune abnormalities, and promote the use of the non-canonical antibody presented in this work for safe and effective costimulatory strategies in cancer immunotherapy.
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Linfocitos T CD8-positivos/efectos de los fármacos , Citotoxicidad Inmunológica/efectos de los fármacos , Receptores ErbB/inmunología , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Anticuerpos de Cadena Única/farmacología , Neoplasias Cutáneas/terapia , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunología , Inmunidad Adaptativa , Animales , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Línea Celular Tumoral , Mapeo Epitopo , Epítopos/química , Epítopos/inmunología , Epítopos de Linfocito B/química , Epítopos de Linfocito B/inmunología , Receptores ErbB/agonistas , Receptores ErbB/genética , Femenino , Humanos , Inmunoglobulina G/administración & dosificación , Inmunoglobulina G/biosíntesis , Linfocitos Infiltrantes de Tumor/citología , Linfocitos Infiltrantes de Tumor/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Anticuerpos de Cadena Única/genética , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/patología , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/agonistas , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Oestrogen receptor beta (ERß) and epidermal growth factor receptor (EGFR) pathway can synergistically promote the proliferation, invasion, and metastasis of non-small-cell lung cancer (NSCLC) cells. ERß has five subtypes, and the selective splicing of exon 8 in ERß5 transcription translational phase makes its biological function different from other subtypes. The following study investigates whether ERß5 interacts with EGFR pathway in lung cancer. Briefly, we found that the overexpression of ERß5 and EGFR is associated with poor prognosis and decreased overall survival in NSCLC patients. Furthermore, the effects of ERß5 and EGFR on cell biological behaviour were investigated in vitro. These results indicated that the combination of ERß5 and EGF induces cell proliferation and invasion, while the combination of ERß5 and Gefitinib (EGFR inhibitors, Gef) induces cell apoptosis and promotes cell mitosis in A549 cell line. In addition, the combination of ERß5 and EGF increases the expression of ERß5, EGFR, and p-ERK1/2 in lung cancer cells. To sum up, the obtained results suggest that ERß5 and EGFR synergistically promote the progression of lung cancer by activating MEK/ERK signalling pathway, which provides a theoretical basis for more accurate combined targeted therapy.
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Carcinoma de Pulmón de Células no Pequeñas/inmunología , Receptor beta de Estrógeno/inmunología , Neoplasias Pulmonares/inmunología , Proteínas de Neoplasias/inmunología , Células A549 , Empalme Alternativo/efectos de los fármacos , Empalme Alternativo/inmunología , Apoptosis/efectos de los fármacos , Apoptosis/inmunología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Supervivencia sin Enfermedad , Receptores ErbB/agonistas , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/inmunología , Receptores ErbB/metabolismo , Receptor beta de Estrógeno/agonistas , Receptor beta de Estrógeno/metabolismo , Femenino , Gefitinib/farmacología , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/inmunología , Masculino , Persona de Mediana Edad , Mitosis/efectos de los fármacos , Mitosis/inmunología , Terapia Molecular Dirigida , Invasividad Neoplásica , Metástasis de la Neoplasia , Proteínas de Neoplasias/metabolismo , Isoformas de Proteínas/inmunología , Isoformas de Proteínas/metabolismo , Tasa de SupervivenciaRESUMEN
Current treatments for inflammatory bowel disease (IBD) target the overactive immune response of the intestinal mucosa. However, epidermal growth factor (EGF), an activating ligand of the EGF receptor (EGFR), has been shown to induce disease remission through direct targeting of intestinal mucosal healing. Despite promising preclinical and clinical results, this EGFR-activating therapy has not progressed, in part due to the potential for carcinogenesis associated with long-term use and the increased risk of colitis-associated cancer (CAC) in IBD. Here we tested whether pharmacological modulation of EGFR altered outcomes of CAC in the murine azoxymethane/dextran sulfate sodium model. We found that administering EGF during the period of maximum colitis severity ("early"), coincident with the initiation and early promotion of tumors, improved outcomes of colitis and reduced tumor size. In contrast, daily EGF administration beginning ~2 months after tumor initiation ("late") increased tumor size. Administration of the EGFR kinase inhibitor gefitinib increased the tumor size when the drug was given early and decreased the tumor size when the drug was administered late. EGF administration not only reduced colonic cytokine and chemokine expression during injury, but also baseline chemokine expression in homeostasis. These results suggest that EGFR activation during acute bouts of colitis may reduce the long-term burden of CAC.