RESUMEN
Accumulating evidence shows that protease-activated receptor-1 (PAR-1) plays an important role in the development of fibrosis, including lung fibrosis. However, whether PAR-1 also plays a role in the development of skin fibrosis remains elusive. The aim of this study was to determine the role of PAR-1 in the development of skin fibrosis. To explore possible mechanisms by which PAR-1 could play a role, human dermal fibroblasts and keratinocytes were stimulated with specific PAR-1 agonists or antagonists. To investigate the role of PAR-1 in skin fibrosis, we subjected wild-type and PAR-1-deficient mice to a model of bleomycin-induced skin fibrosis. PAR-1 activation leads to increased proliferation and extra cellular matrix (ECM) production, but not migration of human dermal fibroblasts (HDF) in vitro. Moreover, transforming growth factor (TGF)-ß production was increased in keratinocytes upon PAR-1 activation, but not in HDF. The loss of PAR-1 in vivo significantly attenuated bleomycin-induced skin fibrosis. The bleomycin-induced increase in dermal thickness and ECM production was reduced significantly in PAR-1-deficient mice compared with wild-type mice. Moreover, TGF-ß expression and the number of proliferating fibroblasts were reduced in PAR-1-deficient mice although the difference did not reach statistical significance. This study demonstrates that PAR-1 contributes to the development of skin fibrosis and we suggest that PAR-1 potentiates the fibrotic response mainly by inducing fibroblast proliferation and ECM production.
Asunto(s)
Fibroblastos/patología , Queratinocitos/patología , Receptor PAR-1/metabolismo , Enfermedades de la Piel/metabolismo , Piel/patología , Animales , Bleomicina , Línea Celular , Proliferación Celular , Células Cultivadas , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Fibrosis , Humanos , Queratinocitos/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Receptor PAR-1/agonistas , Receptor PAR-1/antagonistas & inhibidores , Receptor PAR-1/genética , Piel/efectos de los fármacos , Enfermedades de la Piel/inducido químicamente , Enfermedades de la Piel/patologíaRESUMEN
Mounting evidence suggests an important role for CCAAT-enhancer binding protein delta (C/EBPδ) in the acute-phase response after bacterial infection. However, whether C/EBPδ limits pneumonia remains elusive and is the aim of this study. Therefore, bacterial outgrowth, inflammatory responses, inflammatory cell influx, and survival were assessed in wild-type and C/EBPδ(-/-) mice infected with Klebsiella pneumoniae via the airways. We showed that C/EBPδ expression is highly induced in the lung during pulmonary infection and that Klebsiella-induced mortality was significantly increased among C/EBPδ(-/-) mice. Bacterial loads and inflammatory responses were similar in wild-type and C/EBPδ(-/-) mice early during infection, whereas bacterial loads were increased in C/EBPδ(-/-) mice later during infection. Moreover, macrophage numbers were reduced in lungs of C/EBPδ(-/-) mice. In vitro experiments showed that C/EBPδ only slightly affects macrophage function. Our data thus show that C/EBPδ contributes to host defense against Klebsiella-induced pneumonia and suggests that C/EBPδ-dependent macrophage mobilization is a key mechanism.
Asunto(s)
Proteína delta de Unión al Potenciador CCAAT/metabolismo , Movimiento Celular , Infecciones por Klebsiella/inmunología , Klebsiella pneumoniae/patogenicidad , Macrófagos/inmunología , Neumonía Bacteriana/inmunología , Animales , Carga Bacteriana , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Humanos , Infecciones por Klebsiella/patología , Klebsiella pneumoniae/inmunología , Pulmón/microbiología , Pulmón/patología , Ratones , Ratones Noqueados , Neumonía Bacteriana/patología , Análisis de SupervivenciaRESUMEN
Phoneutria nigriventer spider venom (PNV) contains Ca(2+), K(+) and Na(+) channel-acting peptides that affect neurotransmitter release and causes excitotoxicity in PNS and CNS. It has been demonstrated that PNV causes blood-brain barrier (BBB) breakdown of hippocampal microvessels time-dependently through enhanced microtubule-mediated vesicular transport. Herein, it is hypothesized that PNV can cause BBB breakdown in the hippocampus and cerebellum time-dependently through other molecular mechanisms. The BBB integrity was assessed through the analysis of expression of Poly-glycoprotein (P-gp) efflux transporter protein, laminin from basement membrane and endothelial tight junctional and adhesion junctional (TJ/AJ) proteins. Phosphatase and tensin homolog (PTEN) and protein phosphatase 2A (PP2A) expression, which are known to have a role in the phosphorylation of junctional proteins and BBB opening, were also investigated. Astrocytes P-gp activity was determined by flow cytometry. The study demonstrated temporary decreased expression of laminin, TJ and AJ proteins (ZO1//occludin//claudin-5//beta-catenin) and P-gp (more prominently in hippocampus), which was completely or partially resolved between 2 and 5 h (and more quickly for cerebellum). PNV inhibited P-gp activity in astrocytes. PP2A phosphorylation, which inhibits the enzyme activity, was increased in both regions (15-45 min); however the phosphorylation level returned to baseline after 2 h. In conclusion, PNV disrupts paracellular transport in the BBB and possesses substrates for the active P-gp efflux transporter located in the BBB complex. Further studies into cellular mechanisms of astrocyte/endothelial interactions, using PNV as tool, may identify how astrocytes regulate the BBB, a characteristic that may be useful for the temporary opening of the BBB.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Barrera Hematoencefálica/fisiología , Proteínas del Tejido Nervioso/biosíntesis , Unión Neuromuscular/metabolismo , Neurotoxinas/farmacología , Venenos de Araña/farmacología , Arañas/química , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/efectos de los fármacos , Animales , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Barrera Hematoencefálica/efectos de los fármacos , Western Blotting , Cerebelo/efectos de los fármacos , Cerebelo/metabolismo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Técnica del Anticuerpo Fluorescente , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Laminina/biosíntesis , Masculino , Proteínas del Tejido Nervioso/genética , Unión Neuromuscular/efectos de los fármacos , Síndromes de Neurotoxicidad/metabolismo , Síndromes de Neurotoxicidad/fisiopatología , Fosforilación , Ratas , Ratas WistarRESUMEN
Most aspects of leukocyte physiology are under the control of reversible tyrosine phosphorylation. It is clear that excessive phosphorylation of signal transduction elements is a pivotal element of many different pathologies including haematological malignancies and accordingly, strategies that target such phosphorylation have clinically been proven highly successful for treatment of multiple types of leukemias and lymphomas. Cellular phosphorylation status is dependent on the resultant activity of kinases and phosphatases. The cell biology of the former is now well understood; for most cellular phosphoproteins we now know the kinases responsible for their phosphorylation and we understand the principles of their aberrant activity in disease. With respect to phosphatases, however, our knowledge is much patchier. Although the sequences of whole genomes allow us to identify phosphatases using in silico methodology, whereas transcription profiling allows us to understand how phosphatase expression is regulated during disease, most functional questions as to substrate specificity, dynamic regulation of phosphatase activity and potential for therapeutic intervention are still to a large degree open. Nevertheless, recent studies have allowed us to make meaningful statements on the role of tyrosine phosphatase activity in the three major signaling pathways that are commonly affected in leukemias, i.e. the Ras-Raf-ERK1/2, the Jak-STAT and the PI3K-PKB-mTOR pathways. Lessons learned from these pathways may well be applicable elsewhere in leukocyte biology as well.
Asunto(s)
Neoplasias Hematológicas/tratamiento farmacológico , Proteínas Tirosina Fosfatasas/fisiología , Animales , Neoplasias Hematológicas/etiología , Neoplasias Hematológicas/metabolismo , Humanos , Antígenos Comunes de Leucocito/fisiología , Fosfohidrolasa PTEN/fisiología , Fosforilación , Proteína Tirosina Fosfatasa no Receptora Tipo 1/fisiología , Proteína Tirosina Fosfatasa no Receptora Tipo 11/fisiología , Proteína Tirosina Fosfatasa no Receptora Tipo 6/fisiología , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Transducción de SeñalRESUMEN
Inhibitors based on a 3-acylaminoindazole scaffold were synthesized to yield potent dual AAK1/BMP2K inhibitors. Optimization furnished a small molecule chemical probe (SGC-AAK1-1, 25) that is potent and selective for AAK1/BMP2K over other NAK family members, demonstrates narrow activity in a kinome-wide screen, and is functionally active in cells. This inhibitor represents one of the best available small molecule tools to study the functions of AAK1 and BMP2K.
RESUMEN
ß-Catenin-dependent WNT signal transduction governs development, tissue homeostasis, and a vast array of human diseases. Signal propagation through a WNT-Frizzled/LRP receptor complex requires proteins necessary for clathrin-mediated endocytosis (CME). Paradoxically, CME also negatively regulates WNT signaling through internalization and degradation of the receptor complex. Here, using a gain-of-function screen of the human kinome, we report that the AP2 associated kinase 1 (AAK1), a known CME enhancer, inhibits WNT signaling. Reciprocally, AAK1 genetic silencing or its pharmacological inhibition using a potent and selective inhibitor activates WNT signaling. Mechanistically, we show that AAK1 promotes clearance of LRP6 from the plasma membrane to suppress the WNT pathway. Time-course experiments support a transcription-uncoupled, WNT-driven negative feedback loop; prolonged WNT treatment drives AAK1-dependent phosphorylation of AP2M1, clathrin-coated pit maturation, and endocytosis of LRP6. We propose that, following WNT receptor activation, increased AAK1 function and CME limits WNT signaling longevity.
Asunto(s)
Clatrina/metabolismo , Endocitosis/fisiología , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Wnt/metabolismo , Vía de Señalización Wnt/fisiología , Animales , Retroalimentación Fisiológica , Células HEK293 , Humanos , Masculino , Ratones , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidoresRESUMEN
BACKGROUND: Low-risk patients suffering from prostate cancer (PCa) are currently placed under active surveillance rather than undergoing radical prostatectomy. However, clear parameters for selecting the right patient for each strategy are not available, and new biomarkers and treatment modalities are needed. Low-molecular-weight protein tyrosine phosphatase (LMWPTP) could present such a target. OBJECTIVE: To correlate expression levels of LMWPTP in primary PCa to clinical outcome, and determine the role of LMWPTP in prostate tumor cell biology. DESIGN, SETTING, AND PARTICIPANTS: Acid phosphatase 1, soluble (ACP1) expression was analyzed on microarray data sets, which were subsequently used in Ingenuity Pathway Analysis. Immunohistochemistry was performed on a tissue microarray containing material of 481 PCa patients whose clinicopathologic data were recorded. PCa cell line models were used to investigate the role of LMWPTP in cell proliferation, migration, adhesion, and anoikis resistance. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The association between LMWPTP expression and clinical and pathologic outcomes was calculated using chi-square correlations and multivariable Cox regression analysis. Functional consequences of LMWPTP overexpression or downregulation were determined using migration and adhesion assays, confocal microscopy, Western blotting, and proliferation assays. RESULTS AND LIMITATIONS: LMWPTP expression was significantly increased in human PCa and correlated with earlier recurrence of disease (hazard ratio [HR]:1.99; p<0.001) and reduced patient survival (HR: 1.53; p=0.04). Unbiased Ingenuity analysis comparing cancer and normal prostate suggests migratory propensities in PCa. Indeed, overexpression of LMWPTP increases PCa cell migration, anoikis resistance, and reduces activation of focal adhesion kinase/paxillin, corresponding to decreased adherence. CONCLUSIONS: Overexpression of LMWPTP in PCa confers a malignant phenotype with worse clinical outcome. Prospective follow-up should determine the clinical potential of LMWPTP overexpression. PATIENT SUMMARY: These findings implicate low-molecular-weight protein tyrosine phosphatase as a novel oncogene in prostate cancer and could offer the possibility of using this protein as biomarker or target for treatment of this disease.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Movimiento Celular , Neoplasias de la Próstata/enzimología , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Anciano , Anoicis , Biomarcadores de Tumor/genética , Adhesión Celular , Línea Celular Tumoral , Proliferación Celular , Distribución de Chi-Cuadrado , Quinasa 1 de Adhesión Focal/metabolismo , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Peso Molecular , Análisis Multivariante , Metástasis de la Neoplasia , Recurrencia Local de Neoplasia , Selección de Paciente , Paxillin/metabolismo , Valor Predictivo de las Pruebas , Pronóstico , Modelos de Riesgos Proporcionales , Prostatectomía , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/terapia , Proteínas Tirosina Fosfatasas/genética , Proteínas Proto-Oncogénicas/genética , Medición de Riesgo , Factores de Riesgo , Factores de Tiempo , Análisis de Matrices Tisulares , Transfección , Regulación hacia Arriba , Espera VigilanteRESUMEN
Phosphatases have long been regarded as tumor suppressors, however there is emerging evidence for a tumor initiating role for some phosphatases in several forms of cancer. Low Molecular Weight Protein Tyrosine Phosphatase (LMWPTP; acid phosphatase 1 [ACP1]) is an 18 kDa enzyme that influences the phosphorylation of signaling pathway mediators involved in cancer and is thus postulated to be a tumor-promoting enzyme, but neither unequivocal clinical evidence nor convincing mechanistic actions for a role of LMWPTP have been identified. In the present study, we show that LMWPTP expression is not only significantly increased in colorectal cancer (CRC), but also follows a step-wise increase in different levels of dysplasia. Chemical inhibition of LMWPTP significantly reduces CRC growth. Furthermore, downregulation of LMWPTP in CRC leads to a reduced migration ability in both 2D- and 3D-migration assays, and sensitizes tumor cells to the chemotherapeutic agent 5-FU. In conclusion, this study shows that LMWPTP is not only overexpressed in colorectal cancer, but it is correlated with the malignant potential of this cancer, suggesting that this phosphatase may act as a predictive biomaker of CRC stage and represents a rational novel target in the treatment of this disease.
Asunto(s)
Neoplasias Colorrectales/enzimología , Proteínas Tirosina Fosfatasas/metabolismo , Supervivencia Celular , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Humanos , Peso Molecular , Metástasis de la Neoplasia , Fosforilación , Proteínas Tirosina Fosfatasas/genética , Transducción de Señal , Transcriptoma , Regulación hacia ArribaRESUMEN
Severe accidents caused by the "armed" spider Phoneutria nigriventer cause neurotoxic manifestations in victims. In experiments with rats, P. nigriventer venom (PNV) temporarily disrupts the properties of the BBB by affecting both the transcellular and the paracellular route. However, it is unclear how cells and/or proteins participate in the transient opening of the BBB. The present study demonstrates that PNV is a substrate for the multidrug resistance protein-1 (MRP1) in cultured astrocyte and endothelial cells (HUVEC) and increases mrp1 and cx43 and down-regulates glut1 mRNA transcripts in cultured astrocytes. The inhibition of nNOS by 7-nitroindazole suggests that NO derived from nNOS mediates some of these effects by either accentuating or opposing the effects of PNV. In vivo, MRP1, GLUT1 and Cx43 protein expression is increased differentially in the hippocampus and cerebellum, indicating region-related modulation of effects. PNV contains a plethora of Ca(2+), K(+) and Na(+) channel-acting neurotoxins that interfere with glutamate handling. It is suggested that the findings of the present study are the result of a complex interaction of signaling pathways, one of which is the NO, which regulates BBB-associated proteins in response to PNV interference on ions physiology. The present study provides additional insight into PNV-induced BBB dysfunction and shows that a protective mechanism is activated against the venom. The data shows that PNV has qualities for potential use in drug permeability studies across the BBB.
Asunto(s)
Venenos de Araña/toxicidad , Animales , Astrocitos/metabolismo , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/metabolismo , Permeabilidad Capilar/efectos de los fármacos , Células Cultivadas , Cerebelo/efectos de los fármacos , Cerebelo/metabolismo , Conexina 43/metabolismo , Transportador de Glucosa de Tipo 1/metabolismo , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Masculino , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Ratas Wistar , ArañasRESUMEN
It is now generally recognised that different modes of programmed cell death (PCD) are intimately linked to the cancerous process. However, the mechanism of PCD involved in cancer chemoprevention is much less clear and may be different between types of chemopreventive agents and tumour cell types involved. Therefore, from a pharmacological view, it is crucial during the earlier steps of drug development to define the cellular specificity of the candidate as well as its capacity to bypass dysfunctional tumoral signalling pathways providing insensitivity to death stimuli. Studying the cytotoxic effects of violacein, an antibiotic dihydro-indolone synthesised by an Amazon river Chromobacterium, we observed that death induced in CD34(+)/c-Kit(+)/P-glycoprotein(+)/MRP1(+) TF1 leukaemia progenitor cells is not mediated by apoptosis and/or autophagy, since biomarkers of both types of cell death were not significantly affected by this compound. To clarify the working mechanism of violacein, we performed kinome profiling using peptide arrays to yield comprehensive descriptions of cellular kinase activities. Pro-death activity of violacein is actually carried out by inhibition of calpain and DAPK1 and activation of PKA, AKT and PDK, followed by structural changes caused by endoplasmic reticulum stress and Golgi apparatus collapse, leading to cellular demise. Our results demonstrate that violacein induces kinome reprogramming, overcoming death signaling dysfunctions of intrinsically resistant human leukaemia cells.
Asunto(s)
Muerte Celular/efectos de los fármacos , Aparato de Golgi/efectos de los fármacos , Indoles/uso terapéutico , Leucemia/tratamiento farmacológico , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/antagonistas & inhibidores , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Calpaína/antagonistas & inhibidores , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas Quinasas Asociadas a Muerte Celular , Estrés del Retículo Endoplásmico , Humanos , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
The development of multidrug resistance (MDR) limits the efficacy of continuous chemotherapeutic treatment in chronic myelogenous leukemia (CML). Low molecular weight protein tyrosine phosphatase (LMW-PTP) is up-regulated in several cancers and has been associated to poor prognosis. This prompted us to investigate the involvement of LMW-PTP in MDR. In this study, we investigated the role of LMW-PTP in a chemoresistant CML cell line, Lucena-1. Our results showed that LMW-PTP is highly expressed and 7-fold more active in Lucena-1 cells compared to K562 cells, the non-resistant cell line. Knocking down LMW-PTP in Lucena-1 cells reverted chemoresistance to vincristine and imatinib mesylate, followed by a decrease of Src and Bcr-Abl phosphorylation at the activating sites, inactivating both kinases. On the other hand, overexpression of LMW-PTP in K562 cells led to chemoresistance to vincristine. Our findings describe, for the first time, that LMW-PTP cooperates with MDR phenotype, at least in part, through maintaining Src and Bcr-Abl kinases in more active statuses. These findings suggest that inhibition of LMW-PTP may be a useful strategy for the development of therapies for multidrug resistant CML.