RESUMEN
Gestational diabetes mellitus (GDM) is a condition of diabetes with onset or first recognition in pregnancy. Its incidence is increasing, and GDM deleteriously affects both mother and the fetus during and even after pregnancy. Previous studies in mice have shown that during pregnancy, ß-cell proliferation increases in the middle and late stages of pregnancy and returns to normal levels after delivery. Hormones, such as prolactin, estradiol, and progesterone as well as protein kinases, play important roles in regulating gestation-mediated ß-cell proliferation; however, the regulatory relationship between them is uncertain. We previously found that protein kinase Pbk was crucial for basal proliferation of mouse islet cells. Herein we show that Pbk is upregulated during pregnancy in mice and Pbk kinase activity is required for enhanced ß- cell proliferation during pregnancy. Notably, knock-in (KI) of a kinase-inactivating Pbk mutation leads to impaired glucose tolerance and reduction of ß-cell proliferation and islet mass in mice during pregnancy. Prolactin upregulates the expression of Pbk, but the upregulation is diminished by knockdown of the prolactin receptor and by the inhibitors of JAK and STAT5, which mediate prolactin receptor signaling, in ß-cells. Treatment of ß-cells with prolactin increases STAT5 binding to the Pbk locus, as well as the recruitment of RNA polymerase II, resulting in increased Pbk transcription. These results demonstrate that Pbk is upregulated during pregnancy, at least partly by prolactin-induced and STAT5-mediated enhancement of gene transcription, and Pbk is essential for pregnancy-induced ß-cell proliferation, increase in islet mass, and maintenance of normal blood glucose during pregnancy in preclinical models. These findings provide new insights into the interplay between hormones and protein kinases that ultimately prevent the development of GDM.
Asunto(s)
Células Secretoras de Insulina/fisiología , Quinasas de Proteína Quinasa Activadas por Mitógenos/fisiología , Embarazo/fisiología , Animales , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Células Cultivadas , Diabetes Gestacional/genética , Diabetes Gestacional/metabolismo , Femenino , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Intolerancia a la Glucosa/genética , Intolerancia a la Glucosa/metabolismo , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Prolactina/metabolismo , Prolactina/farmacología , RatasRESUMEN
PURPOSE: While chemotherapy remains the standard treatment for triple-negative breast cancer (TNBC), identifying and managing chemoresistant tumors has proven elusive. We sought to discover hallmarks and therapeutically actionable features of refractory TNBC through molecular analysis of primary chemoresistant TNBC specimens. EXPERIMENTAL DESIGN: We performed transcriptional profiling of tumors from a phase II clinical trial of platinum chemotherapy for advanced TNBC (TBCRC-009), revealing a gene expression signature that identified de novo chemorefractory tumors. We then employed pharmacogenomic data mining, proteomic and other molecular studies to define the therapeutic vulnerabilities of these tumors. RESULTS: We reveal the RAS-GTPase-activating protein (RAS-GAP) RASAL2 as an upregulated factor that mediates chemotherapy resistance but also an exquisite collateral sensitivity to combination MAP kinase kinase (MEK1/2) and EGFR inhibitors in TNBC. Mechanistically, RASAL2 GAP activity is required to confer kinase inhibitor sensitivity, as RASAL2-high TNBCs sustain basal RAS activity through suppression of negative feedback regulators SPRY1/2, together with EGFR upregulation. Consequently, RASAL2 expression results in failed feedback compensation upon co-inhibition of MEK1/2 and EGFR that induces synergistic apoptosis in vitro and in vivo. In patients with TNBC, high RASAL2 levels predict clinical chemotherapy response and long-term outcomes, and are associated via direct transcriptional regulation with activated oncogenic Yes-Associated Protein (YAP). Accordingly, chemorefractory patient-derived TNBC models exhibit YAP activation, high RASAL2 expression, and tumor regression in response to MEK/EGFR inhibitor combinations despite well-tolerated intermittent dosing. CONCLUSIONS: These findings identify RASAL2 as a mediator of TNBC chemoresistance that rewires MAPK feedback and cross-talk to confer profound collateral sensitivity to combination MEK1/2 and EGFR inhibitors.
Asunto(s)
Resistencia a Antineoplásicos , Proteínas Activadoras de GTPasa/fisiología , Quinasas de Proteína Quinasa Activadas por Mitógenos/fisiología , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/genética , Línea Celular Tumoral , Receptores ErbB/fisiología , Femenino , HumanosRESUMEN
In diabetic pathology, insufficiency in ß-cell mass, unable to meet peripheral insulin demand, and functional defects of individual ß-cells in production of insulin are often concurrently observed, collectively causing hyperglycemia. Here we show that the phosphorylation of ERK1/2 is significantly decreased in the islets of db/db mice as well as in those of a cohort of subjects with type 2 diabetes. In mice with abrogation of ERK signaling in pancreatic ß-cells through deletion of Mek1 and Mek2, glucose intolerance aggravates under high-fat diet-feeding conditions due to insufficient insulin production with lower ß-cell proliferation and reduced ß-cell mass, while in individual ß-cells dampening of the number of insulin exocytosis events is observed, with the molecules involved in insulin exocytosis being less phosphorylated. These data reveal bifunctional roles for MEK/ERK signaling in ß-cells for glucose homeostasis, i.e., in regulating ß-cell mass as well as in controlling insulin exocytosis in individual ß-cells, thus providing not only a novel perspective for the understanding of diabetes pathophysiology but also a potential clue for new drug development for diabetes treatment.
Asunto(s)
Glucemia/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/fisiología , Homeostasis , Secreción de Insulina , Células Secretoras de Insulina/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/fisiología , Animales , Línea Celular , Dieta Alta en Grasa , Exocitosis , Humanos , Células Secretoras de Insulina/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Fosforilación , Transducción de SeñalRESUMEN
BACKGROUND/AIM: To investigate the impact of PDZ-binding kinase (PBK) on the clinical outcome of patients with oral squamous cell carcinoma (OSCC) who received radiotherapy. PATIENTS AND METHODS: PBK immunoreactivity of cancer specimens obtained from 179 patients with primary OSCC was analyzed by immunohistochemistry. RESULTS: High PBK expression in tumor cells tended to be associated with advanced N-stage. The 5-year survival rate was greater for patients with high total PBK expression than in those with low PBK expression. After adjustment, high PBK remained associated with a favorable outcome. In subgroups according to tumor stage, the prognostic role was significant in patients with stage III/IV rather than those with stage I/II disease. CONCLUSION: We suggest that PBK expression should be used as an independent prognostic marker for patients with OSCC treated with radiotherapy, especially for those with advanced-stage disease.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/radioterapia , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Neoplasias de la Boca/diagnóstico , Neoplasias de la Boca/radioterapia , Anciano , Carcinoma de Células Escamosas/mortalidad , Carcinoma de Células Escamosas/patología , Femenino , Humanos , Inmunohistoquímica , Estilo de Vida , Masculino , Persona de Mediana Edad , Quinasas de Proteína Quinasa Activadas por Mitógenos/fisiología , Neoplasias de la Boca/mortalidad , Neoplasias de la Boca/patología , Metástasis de la Neoplasia , Estadificación de Neoplasias , Pronóstico , Estudios Retrospectivos , Factores de Riesgo , Carcinoma de Células Escamosas de Cabeza y Cuello/diagnóstico , Carcinoma de Células Escamosas de Cabeza y Cuello/mortalidad , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Carcinoma de Células Escamosas de Cabeza y Cuello/radioterapia , Análisis de Supervivencia , Taiwán/epidemiologíaRESUMEN
Stachydrine is a main active component of Leonurus japonicus (Chinese motherwort), which has traditionally been used to promote postpartum recovery and alleviate myocardial and cerebral ischemic injuries due to its pro-angiogenic effect. Our prior study demonstrated that stachydrine increased angiogenesis in zebrafish embryos, but its pro-angiogenic effect and underlying mechanisms on human umbilical vein endothelial cells (HUVECs) remain largely unknown. In the present study, we further investigated the role of stachydrine in sunitinib-injured HUVECs and its potential molecular mechanisms. The results showed that stachydrine exhibited a protective effect on sunitinib-injured HUVECs and significantly promoted their proliferation, migration, and tube formation, all central events of angiogenesis. In addition, stachydrine inhibited apoptosis and ROS production in sunitinib-injured HUVECs. Furthermore, our findings illustrated for the first time that stachydrine's molecular mechanisms for promoting angiogenesis might correlate with activation of the VEGFR2/MEK/ERK and inhibition of the mitochondrial-mediated apoptosis signaling pathway.
Asunto(s)
Apoptosis/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/fisiología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Mitocondrias/fisiología , Quinasas de Proteína Quinasa Activadas por Mitógenos/fisiología , Neovascularización Fisiológica/efectos de los fármacos , Prolina/análogos & derivados , Receptor 2 de Factores de Crecimiento Endotelial Vascular/fisiología , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Células Endoteliales de la Vena Umbilical Humana/fisiología , Humanos , Prolina/farmacología , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Dugesia japonica, belonging to Platyhelminthes, plays an important role in the animal evolution and is well known for its extraordinary regenerative ability. Mitogen activated protein kinase (MAPK) pathway is an important cell signaling pathway that converts extracellular stimuli into a wide range of cellular responses. The MAP-extracellular signal-regulated kinase (MEK) is a main component of MAPK/ERK signaling, but there are few studies on mek gene in planarians. In this study, we observe the expression patterns of Djmek1 and Djmek2 in planarians, and find that both of the two genes are required for the planarian regeneration. At the same time, we also find that both Djmek1 and Djmek2 are involved in the planarian regeneration by regulation of cell proliferation and apoptosis. Together, our findings show that the functions of the two genes are similar and complementary, and they play an important role in the regeneration of planarians.
Asunto(s)
Proteínas del Helminto/genética , Proteínas del Helminto/fisiología , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/fisiología , Planarias/genética , Planarias/fisiología , Regeneración/genética , Regeneración/fisiología , Animales , Apoptosis/genética , Apoptosis/fisiología , Proliferación Celular/genética , Proliferación Celular/fisiología , Regulación de la Expresión Génica , Proteínas del Helminto/antagonistas & inhibidores , Sistema de Señalización de MAP Quinasas , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Interferencia de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Autophagy is an important mechanism for cellular homeostasis and survival during pathologic stress conditions in the kidney, such as ischemia-reperfusion (IR) injury. In this study, renal IR was induced in female C57BL/6 mice after melatonin administration. Renal function, histological damage, inflammatory infiltration, cytokine production, oxidative stress, antioxidant capacity, autophagy changing, apoptosis levels, and autophagy-associated intracellular signaling pathway were assessed to evaluate the impact of antecedent melatonin treatment on IR-induced renal injury. The administration of melatonin resulted in significantly preserved renal function, and the protective effect was associated with ameliorated oxidative stress, limited pro-inflammatory cytokine production, and neutrophil and macrophage infiltration. Moreover, autophagic flux was increased after melatonin administration while the apoptosis levels were decreased in the melatonin-pretreated mice. Using TAK-242 and CRX-527, we confirmed that MyD88-dependent TLR4 and MEK/ERK/mTORC1 signaling participated in melatonin-induced autophagy in IR mice. Collectively, our results provide novel evidence that antecedent melatonin treatment provides protection for the kidney against IR injury by enhancing autophagy, as regulated by the TLR4/MyD88/MEK/ERK/mTORC1 signaling pathway. Therefore, melatonin preconditioning offers a potential therapeutic approach to prevent renal IR injury related to various clinical conditions.
Asunto(s)
Autofagia/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/fisiología , Riñón/irrigación sanguínea , Diana Mecanicista del Complejo 1 de la Rapamicina/fisiología , Quinasas de Proteína Quinasa Activadas por Mitógenos/fisiología , Factor 88 de Diferenciación Mieloide/fisiología , Daño por Reperfusión/prevención & control , Receptor Toll-Like 4/fisiología , Animales , Autofagia/fisiología , Femenino , Inflamación/prevención & control , Melatonina/farmacología , Ratones , Ratones Endogámicos C57BL , Infiltración Neutrófila/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
Adventitious roots form from non-root tissues as part of normal development or in response to stress or wounding. The root primordia form in the source tissue, and during emergence the adventitious roots penetrate the inner cell layers and the epidermis; however, the mechanisms underlying this emergence remain largely unexplored. Here, we report that a regulatory module composed of the AP2/ERF transcription factor ABSCISIC ACID INSENSITIVE 4 (ABI4), the MAP kinases MPK3 and MPK6, and the phosphatase PP2C12 plays an important role in the emergence of junction adventitious roots (J-ARs) from the root-hypocotyl junctions in Arabidopsis thaliana. ABI4 negatively regulates J-AR emergence, preventing the accumulation of reactive oxygen species and death of epidermal cells, which would otherwise facilitate J-AR emergence. Phosphorylation by MPK3/MPK6 activates ABI4 and dephosphorylation by PP2C12 inactivates ABI4. MPK3/MPK6 also directly phosphorylate and inactivate PP2C12 during J-AR emergence. We propose that this "double-check" mechanism increases the robustness of MAP kinase signaling and finely regulates the local programmed cell death required for J-AR emergence.
Asunto(s)
Proteínas de Arabidopsis/fisiología , Arabidopsis/crecimiento & desarrollo , Hipocótilo/crecimiento & desarrollo , Quinasas de Proteína Quinasa Activadas por Mitógenos/fisiología , Proteínas Quinasas Activadas por Mitógenos/fisiología , Raíces de Plantas/crecimiento & desarrollo , Factores de Transcripción/fisiología , Factores de Transcripción/metabolismoRESUMEN
The PDZ-binding kinase (PBK) protein is localised exclusively in spermatogenic cells, such as spermatogonia, spermatocytes and round spermatids, of the adult testis. However, its role in male fertility remains unknown. Analysis of adult Pbk-knockout (KO) male mice showed no significant difference in the weight of the testes, epididymis and seminal vesicle compared with adult wild-type (WT) mice. There were no significant differences in testis morphology, tubule diameter and the number of offspring born to females mated with KO or WT male mice. Sperm number, motility and morphology did not differ significantly between KO and WT mice. The oocyte fertilisation rate and embryo development following IVF were comparable between groups fertilised using spermatozoa from KO versus WT mice (P>0.05). Further analysis revealed that the phosphorylation of the mitogen-activated protein kinases (MAPKs) p38 kinase, c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinases was dysregulated in the testis of KO mice. In conclusion, Pbk-KO male mice are fertile and their spermatozoa and testis do not show any morphological and functional abnormalities despite the dysregulated phosphorylation of MAPKs. It is likely that functional redundancy of PBK and overlapping substrate specificities of the MAPK superfamily compensated for the loss of PBK from the testis.
Asunto(s)
Fertilidad/fisiología , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/fisiología , Animales , Femenino , Fertilización , Masculino , Ratones , Ratones Noqueados , Quinasas de Proteína Quinasa Activadas por Mitógenos/deficiencia , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Oocitos/fisiología , Tamaño de los Órganos , Fosforilación , Espermatozoides/enzimología , Espermatozoides/fisiología , Testículo/anatomía & histología , Testículo/enzimologíaRESUMEN
BACKGROUND: A phase Ib study of binimetinib and capecitabine for gemcitabine-pretreated biliary tract cancer (BTC) patients was conducted. METHODS: Binimetinib and capecitabine were dosed twice daily on days 1-14, in 3-week cycles. In the dose-escalation (DE) part, three dose levels (DL) were tested (DL1: binimetinib/capecitabine, 15 mg/1000 mg/m2; DL2: 30 mg/1000 mg/m2; DL3: 30 mg/1250 mg/m2). RESULTS: In the DE part, nine patients were recruited and no dose-limiting toxicity was noted. Therefore, the recommended phase 2 dose was determined as DL3. In the expansion part, 25 patients were enrolled. In total, 34 patients, 25 (73.5%) and 9 patients (26.5%) were second-line and third-line settings, respectively. The 3-month progression-free survival (PFS) rate was 64.0%, and the median PFS and overall survival (OS) were 4.1 and 7.8 months. The objective response rate and disease control rate were 20.6% and 76.5%. In total, 68.4% of stable diseases were durable (> 12 weeks). Furthermore, patients with RAS/RAF/MEK/ERK pathway mutations (38.5%) showed significantly better tumour response (p = 0.028), PFS (5.4 vs. 3.5 months, p = 0.010) and OS (10.8 vs. 5.9 months, p = 0.160) than wild type. Most of the adverse events were grade 1/2 and manageable. CONCLUSIONS: A combination of binimetinib and capecitabine shows acceptable tolerability and promising antitumor efficacy for gemcitabine-pretreated BTC, especially in patients with RAS/RAF/MEK/ERK pathway mutations. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov (Identifier: NCT02773459).
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias del Sistema Biliar/tratamiento farmacológico , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Mutación , Quinasas raf/genética , Proteínas ras/genética , Anciano , Bencimidazoles/administración & dosificación , Bencimidazoles/efectos adversos , Neoplasias del Sistema Biliar/genética , Neoplasias del Sistema Biliar/mortalidad , Neoplasias del Sistema Biliar/psicología , Capecitabina/administración & dosificación , Capecitabina/efectos adversos , Línea Celular Tumoral , Quinasas MAP Reguladas por Señal Extracelular/fisiología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Quinasas de Proteína Quinasa Activadas por Mitógenos/fisiología , Calidad de Vida , Transducción de Señal , Quinasas raf/fisiología , Proteínas ras/fisiologíaRESUMEN
Lateral root (LR) emergence is a highly coordinated process involving precise cell-cell communication. Here, we show that MITOGEN-ACTIVATED PROTEIN KINASE3 (MPK3) and MPK6, and their upstream MAP-kinase kinases (MAPKKs), MKK4 and MKK5, function downstream of HAESA (HAE)/HAESA-LIKE2 (HSL2) and their ligand INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) during LR emergence. Loss of function of MKK4/MKK5 or MPK3/MPK6 results in restricted passage of the growing lateral root primordia (LRP) through the overlaying endodermal, cortical and epidermal cell layers, leading to reduced LR density. The MKK4/MKK5-MPK3/MPK6 module regulates the expression of cell wall remodelling genes in cells overlaying LRP and therefore controls pectin degradation in the middle lamella. Expression of constitutively active MKK4 or MKK5 driven by the HAE or HSL2 promoter fully rescues the LR emergence defect in the ida and hae hsl2 mutants. In addition, the MKK4/MKK5-MPK3/MPK6 module is indispensable in auxin-facilitated LR emergence. Our study provides insights into the auxin-governed and IDA-HAE/HLS2 ligand-receptor pair-mediated LR emergence process.
Asunto(s)
Arabidopsis/crecimiento & desarrollo , Sistema de Señalización de MAP Quinasas , Raíces de Plantas/crecimiento & desarrollo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/fisiología , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/fisiología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/fisiología , Raíces de Plantas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/fisiologíaRESUMEN
Imatinib has revolutionized the treatment of gastrointestinal stromal tumors (GIST); however, primary and secondary resistance to imatinib is still a major cause of treatment failure. Multiple mechanisms are involved in this progression. In the present study, we reported a novel mechanism for the acquired resistance to imatinib, which was induced by enhanced Ca2+ influx via stromal-interacting molecule 1 (STIM1)-mediated store-operated Ca2+ entry (SOCE). We found that the STIM1 expression level was related to the acquired resistance to imatinib in our studied cohort. The function of STIM1 in imatinib-resistant GIST cells was also confirmed both in vivo and in vitro. The results showed that STIM1 overexpression contributed to SOCE and drug response in imatinib-sensitive GIST cells. Blockage of SOCE by STIM1 knockdown suppressed the proliferation of imatinib-resistant GIST cell lines and xenografts. In addition, STIM1-mediated SOCE exerted an antiapoptotic effect via the MEK/ERK pathway. The results from this study provide a basis for further research into potential novel therapeutic strategies in acquired imatinib-resistant GIST.
Asunto(s)
Antineoplásicos/uso terapéutico , Calcio/metabolismo , Neoplasias Gastrointestinales/tratamiento farmacológico , Tumores del Estroma Gastrointestinal/tratamiento farmacológico , Mesilato de Imatinib/uso terapéutico , Proteínas de Neoplasias/fisiología , Molécula de Interacción Estromal 1/fisiología , Adulto , Anciano , Animales , Línea Celular Tumoral , Resistencia a Antineoplásicos , Femenino , Neoplasias Gastrointestinales/metabolismo , Neoplasias Gastrointestinales/patología , Tumores del Estroma Gastrointestinal/metabolismo , Tumores del Estroma Gastrointestinal/patología , Humanos , Sistema de Señalización de MAP Quinasas/fisiología , Masculino , Ratones , Persona de Mediana Edad , Quinasas de Proteína Quinasa Activadas por Mitógenos/fisiología , Proteínas de Neoplasias/antagonistas & inhibidores , Molécula de Interacción Estromal 1/antagonistas & inhibidoresRESUMEN
PfPK7 is an "orphan" kinase displaying regions of homology to multiple protein kinase families. PfPK7 functions in regulating parasite proliferation/development as evident from the phenotype analysis of knockout parasites. Despite this regulatory role, the functions of PfPK7 in signaling pathways are not known. To better understand PfPK7-regulated phosphorylation events, we performed isobaric tag-based quantitative comparative phosphoproteomics of the schizont and segmenter stages from wild-type and pfpk7 - parasite lines. This analysis identified 3,875 phosphorylation sites on 1,047 proteins. Among these phosphorylation events, 146 proteins with 239 phosphorylation sites displayed reduction in phosphorylation in the absence of PfPK7. Further analysis of the phosphopeptides revealed three motifs whose phosphorylation was down regulated in the pfpk7 - cell line in both schizonts and segmenters. Decreased phosphorylation following loss of PfPK7 indicates that these proteins may function as direct substrates of PfPK7. We demonstrated that PfPK7 is active toward three of these potential novel substrates; however, PfPK7 did not phosphorylate many of the other proteins, suggesting that decreased phosphorylation in these proteins is an indirect effect. Our phosphoproteomics analysis is the first study to identify direct substrates of PfPK7 and reveals potential downstream or compensatory signaling pathways.
Asunto(s)
Quinasas de Proteína Quinasa Activadas por Mitógenos/fisiología , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/metabolismo , Humanos , Quinasas de Proteína Quinasa Activadas por Mitógenos/deficiencia , Fosfoproteínas/metabolismo , Fosforilación , Plasmodium falciparum/química , Plasmodium falciparum/enzimología , Proteínas Quinasas , Proteínas Protozoarias/fisiología , Esquizontes/química , Esquizontes/metabolismo , Transducción de Señal , Especificidad por SustratoRESUMEN
Mitogen activated protein kinase kinase (MAPKK) is a crucial component in the MAPK signaling pathway. However, the functions of MAPKKs in foliar pathogens remain poorly understood. In the current study, a MAPKK gene designated as StPBS2 was cloned from Setosphaeria turcica and the functions of this gene were investigated by RNAi technology. Four independent StPBS2 gene silence transformants with different efficiencies were confirmed by real time PCR. Compared to the wild type strain (WT), these transformants showed decreased colony growth, shortened hyphae cell length, broadened cell width and an obvious reduction in conidium yield. Moreover, the cell wall of the transformants was thicker and they were also more sensitive to substances that interfere with cell wall biosynthesis than WT. Additionally, the transformants displayed higher sensitivity to hypertonic stress than WT and the sensitivity was associated with the level of silencing of StPBS2. They were also resistant to the fungicides iprodione, procymidone and fludioxonil, to which WT almost completely sensitive. The transformants produced more red secondary metabolites than WT and the production was enhanced with increasing silencing level and increased glucose content in PDA medium. Our results suggest that StPBS2 is involved in morphogenesis, condiogenesis, cell wall development, hypertonic stress reaction and resistance to fungicides, as well as in the biosynthesis of secondary metabolites in S. turcica.
Asunto(s)
Ascomicetos/citología , Ascomicetos/genética , Pared Celular/metabolismo , Hifa/citología , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/fisiología , Presión Osmótica/fisiología , Metabolismo Secundario/fisiología , Secuencia de Aminoácidos , Ascomicetos/crecimiento & desarrollo , Ascomicetos/metabolismo , Clonación Molecular , ADN de Hongos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/fisiología , Fungicidas Industriales/farmacología , Regulación Fúngica de la Expresión Génica , Silenciador del Gen , Genes Fúngicos/genética , Genes Fúngicos/fisiología , Glucosa/metabolismo , Hifa/crecimiento & desarrollo , Microscopía Electrónica de Transmisión , Quinasas de Proteína Quinasa Activadas por Mitógenos/clasificación , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Morfogénesis/genética , Filogenia , Enfermedades de las Plantas/microbiología , Interferencia de ARN , Reacción en Cadena en Tiempo Real de la Polimerasa , Alineación de Secuencia , Esporas Fúngicas/citología , Esporas Fúngicas/genética , Esporas Fúngicas/crecimiento & desarrollo , Zea mays/microbiologíaRESUMEN
The activity of the RAF/MEK/ERK signaling pathway is critical for the proliferation of normal and cancerous cells. Oncogenic mutations driving the development of lung adenocarcinoma often activate this signaling pathway. In contrast, pathway activity levels and their biological roles are not well established in small cell lung cancer (SCLC), a fast-growing neuroendocrine lung cancer subtype. Here we discuss the function of the RAF/MEK/ERK kinase pathway and the mechanisms leading to its activation in SCLC cells. In particular, we argue that activation of this pathway may be beneficial to the survival, proliferation, and spread of SCLC cells in response to multiple stimuli. We also consider evidence that high levels of RAF/MEK/ERK pathway activity may be detrimental to SCLC tumors, including in part by interfering with their neuroendocrine fate. On the basis of these observations, we examined when small molecules targeting kinases in the RAF/MEK/ERK pathway may be useful therapeutically in patients with SCLC, including in combination with other therapeutic agents.
Asunto(s)
Quinasas MAP Reguladas por Señal Extracelular/fisiología , Neoplasias Pulmonares/tratamiento farmacológico , Sistema de Señalización de MAP Quinasas/fisiología , Quinasas de Proteína Quinasa Activadas por Mitógenos/fisiología , Carcinoma Pulmonar de Células Pequeñas/tratamiento farmacológico , Quinasas raf/antagonistas & inhibidores , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Humanos , Neoplasias Pulmonares/patología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Metástasis de la Neoplasia , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Carcinoma Pulmonar de Células Pequeñas/patología , Quinasas raf/fisiologíaRESUMEN
BACKGROUND: The parasympathetic nervous system (PNS), via neurotransmitter acetylcholine (ACh), modulates fibrogenesis in animal models. However, the role of ACh in human hepatic fibrogenesis is unclear. AIMS: We aimed to determine the fibrogenic responses of human hepatic stellate cells (hHSC) to ACh and the relevance of the PNS in hepatic fibrosis in patients with non-alcoholic steatohepatitis (NASH). METHODS: Primary hHSC were analyzed for synthesis of endogenous ACh and acetylcholinesterase and gene expression of choline acetyltransferase and muscarinic ACh receptors (mAChR). Cell proliferation and fibrogenic markers were analyzed in hHSC exposed to ACh, atropine, mecamylamine, methoctramine, and 4-diphenylacetoxy-N-methylpiperidine methiodide. mAChR expression was analyzed in human NASH scored for fibrosis. RESULTS: We observed that hHSC synthesize ACh and acetylcholinesterase and express choline acetyltransferase and M1-M5 mAChR. We also show that M2 was increased during NASH progression, while both M2 and M3 were found upregulated in activated hHSC. Furthermore, endogenous ACh is required for hHSC basal growth. Exogenous ACh resulted in hHSC hyperproliferation via mAChR and phosphoinositide 3-kinase and Mitogen-activated protein kinase kinase (MEK) signaling pathways, as well as increased fibrogenic markers. CONCLUSION: We show that ACh regulates hHSC activation via M2 and M3 mAChR involving the phosphoinositide 3-kinase and MEK pathways in vitro. Finally, we provide evidence that the PNS may be involved in human NASH fibrosis.
Asunto(s)
Acetilcolina/efectos adversos , Acetilcolina/fisiología , Células Estrelladas Hepáticas/patología , Cirrosis Hepática/etiología , Cirrosis Hepática/patología , Enfermedad del Hígado Graso no Alcohólico/etiología , Receptores Muscarínicos/fisiología , 1-Fosfatidilinositol 4-Quinasa/fisiología , Acetilcolina/biosíntesis , Acetilcolinesterasa/biosíntesis , Células Cultivadas , Colina O-Acetiltransferasa/genética , Colina O-Acetiltransferasa/metabolismo , Progresión de la Enfermedad , Fibrosis , Expresión Génica , Células Estrelladas Hepáticas/metabolismo , Humanos , Quinasas de Proteína Quinasa Activadas por Mitógenos/fisiología , Enfermedad del Hígado Graso no Alcohólico/patología , Sistema Nervioso Parasimpático/fisiología , Receptores Muscarínicos/genética , Receptores Muscarínicos/metabolismo , Transducción de Señal/fisiología , Regulación hacia ArribaRESUMEN
Gain-of-function mutations of FLT3 (FLT3-ITD), comprises up to 30% of normal karyotype acute myeloid leukemia (AML) and is associated with an adverse prognosis. Current FLT3 kinase inhibitors have been tested extensively, but have not yet resulted in a survival benefit and novel therapies are awaited. Here we show that T-LAK cell-originated protein kinase (TOPK), a mitotic kinase highly expressed in and correlated with more aggressive phenotype in several types of cancer, is expressed in AML but not in normal CD34+ cells and that TOPK knockdown decreased cell viability and induced apoptosis. Treatment of AML cells with TOPK inhibitor (OTS514) resulted in a dose-dependent decrease in cell viability with lower IC50 in FLT3-mutated cells, including blasts obtained from patients relapsed after FLT3-inhibitor treatment. Using a MV4-11-engrafted mouse model, we found that mice treated with 7.5 mg/kg IV daily for 3 weeks survived significantly longer than vehicle treated mice (median survival 46 vs 29 days, P < 0.001). Importantly, we identified TOPK as a FLT3-ITD and CEBPA regulated kinase, and that modulating TOPK expression or activity resulted in significant decrease of FLT3 expression and CEBPA phosphorylation. Thus, targeting TOPK in FLT3-ITD AML represents a novel therapeutic approach for this adverse risk subset of AML.
Asunto(s)
Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Terapia Molecular Dirigida , Mutación , Inhibidores de Proteínas Quinasas/uso terapéutico , Tirosina Quinasa 3 Similar a fms/genética , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Humanos , Leucemia Mieloide Aguda/patología , Ratones , Quinasas de Proteína Quinasa Activadas por Mitógenos/fisiología , Inhibidores de Proteínas Quinasas/farmacología , ARN Interferente Pequeño/farmacología , ARN Interferente Pequeño/uso terapéutico , Células Tumorales Cultivadas , Células U937RESUMEN
BACKGROUND: The nerve growth factor (NGF) receptor tyrosine-kinase TrkA is a well-known determinant of the melanocytic lineage, through modulation of the MAPK and AKT cascades. While TrkA gene is frequently rearranged in cancers, its involvement in malignant melanoma (MM) development is still unclear. METHODS: We analyzed a dataset of primary cutaneous MM (n = 31) by array comparative genomic hybridization (aCGH), to identify genomic amplifications associated with tumor progression. The analysis was validated by genomic quantitative PCR (qPCR) on an extended set of cases (n = 64) and the results were correlated with the clinical outcome. To investigate TrkA molecular pathways and cellular function, we generated inducible activation of the NGF-TrkA signaling in human MM cell lines. RESULTS: We identified amplification of 1q23.1, where the TrkA locus resides, as a candidate hotspot implicated in the progression of MM. Across 40 amplicons detected, segmental amplification of 1q23.1 showed the strongest association with tumor thickness. By validation of the analysis, TrkA gene amplification emerged as a frequent event in primary melanomas (50 % of patients), and correlated with worse clinical outcome. However, experiments in cell lines revealed that induction of the NGF-TrkA signaling produced a phenotype of dramatic suppression of cell proliferation through inhibition of cell division and pronounced intracellular vacuolization, in a way straightly dependent on NGF activation of TrkA. These events were triggered via MAPK activity but not via AKT, and involved p21(cip1) protein increase, compatibly with a mechanism of oncogene-induced growth arrest. CONCLUSIONS: Taken together, our findings point to TrkA as a candidate oncogene in MM and support a model in which the NGF-TrkA-MAPK pathway may mediate a trade-off between neoplastic transformation and adaptive anti-proliferative response.
Asunto(s)
Melanoma/genética , Receptor trkA/genética , Neoplasias Cutáneas/genética , Análisis de Varianza , Ciclo Celular/fisiología , Línea Celular Tumoral , Proliferación Celular/fisiología , Hibridación Genómica Comparativa/métodos , Progresión de la Enfermedad , Amplificación de Genes , Perfilación de la Expresión Génica , Humanos , Estimación de Kaplan-Meier , Melanoma/patología , Quinasas de Proteína Quinasa Activadas por Mitógenos/fisiología , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptor trkA/metabolismo , Transducción de Señal/fisiología , Neoplasias Cutáneas/patología , Melanoma Cutáneo MalignoRESUMEN
The Drosophila bone morphogenetic protein encoded by decapentaplegic (dpp) controls ventral head morphogenesis by expression in the head primordia, eye-antennal imaginal discs. These are epithelial sacs made of two layers: columnar disc proper cells and squamous cells of the peripodial epithelium. dpp expression related to head formation occurs in the peripodial epithelium; cis-regulatory mutations disrupting this expression display defects in sensory vibrissae, rostral membrane, gena, and maxillary palps. Here we document that disruption of this dpp expression causes apoptosis in peripodial cells and underlying disc proper cells. We further show that peripodial Dpp acts directly on the disc proper, indicating that Dpp must cross the disc lumen to act. We demonstrate that palp defects are mechanistically separable from the other mutant phenotypes; both are affected by the c-Jun N-terminal kinase pathway but in opposite ways. Slight reduction of both Jun N-terminal kinase and Dpp activity in peripodial cells causes stronger vibrissae, rostral membrane, and gena defects than Dpp alone; additionally, strong reduction of Jun N-terminal kinase activity alone causes identical defects. A more severe reduction of dpp results in similar vibrissae, rostral membrane, and gena defects, but also causes mutant maxillary palps. This latter defect is correlated with increased peripodial Jun N-terminal kinase activity and can be caused solely by ectopic activation of Jun N-terminal kinase. We conclude that formation of sensory vibrissae, rostral membrane, and gena tissue in head morphogenesis requires the action of Jun N-terminal kinase in peripodial cells, while excessive Jun N-terminal kinase signaling in these same cells inhibits the formation of maxillary palps.