RESUMEN
Renal cell carcinoma (RCC) mainly originates from renal proximal tubules. Intriguingly, disruption of genes frequently mutated in human RCC samples thus far has only generated RCC originated from other renal tubule parts in mouse models. This hampers our understanding of the pathogenesis of RCC. Here we show that mTOR signaling, often activated in RCC samples, initiates RCC development from renal proximal tubules. Ablation of Tsc1, encoding an mTOR suppressor, in proximal tubule cells led to multiple precancerous renal cysts. mTOR activation increased MEK1 expression and ERK activation, and Mek1 ablation or inhibition diminished cyst formation in Tsc1-deficient mice. mTOR activation also increased MKK6 expression and p38MAPK activation, and ablation of the p38α-encoding gene further enhanced cyst formation and led to RCC with clear cell RCC features. Mechanistically, Tsc1 deletion induced p53 and p16 expression in a p38MAPK-dependent manner, and deleting Tsc1 and Trp53 or Cdkn2a (encoding p16) enhanced renal cell carcinogenesis. Thus, mTOR activation in combination with inactivation of the p38MAPK-p53/p16 pathway drives RCC development from renal proximal tubules. Moreover, this study uncovers previously unidentified mechanisms by which mTOR controls cell proliferation and suggests the MEK-ERK axis to be a potential target for treatment of RCC. SIGNIFICANCE: Mouse modeling studies show that mTOR activation in combination with inactivation of the p38MAPK-p53/p16 axis initiates renal cell carcinoma that mimics human disease, identifying potential therapeutic targets for RCC treatment.
Asunto(s)
Carcinoma de Células Renales/patología , Inhibidor p16 de la Quinasa Dependiente de Ciclina/fisiología , MAP Quinasa Quinasa 1/fisiología , Proteína Quinasa 14 Activada por Mitógenos/fisiología , Serina-Treonina Quinasas TOR/metabolismo , Proteína 1 del Complejo de la Esclerosis Tuberosa/fisiología , Proteína p53 Supresora de Tumor/fisiología , Animales , Apoptosis , Carcinoma de Células Renales/etiología , Carcinoma de Células Renales/metabolismo , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Neoplasias Renales/etiología , Neoplasias Renales/metabolismo , Neoplasias Renales/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosforilación , Serina-Treonina Quinasas TOR/genética , Células Tumorales CultivadasRESUMEN
Hematopoietic stem cells (HSCs) and their progeny sustain lifetime hematopoiesis. Aging alters HSC function, number, and composition and increases risk of hematological malignancies, but how these changes occur in HSCs remains unclear. Signaling via p38 mitogen-activated kinase (p38MAPK) has been proposed as a candidate mechanism underlying induction of HSC aging. Here, using genetic models of both chronological and premature aging, we describe a multimodal role for p38α, the major p38MAPK isozyme in hematopoiesis, in HSC aging. We report that p38α regulates differentiation bias and sustains transplantation capacity of HSCs in the early phase of chronological aging. However, p38α decreased HSC transplantation capacity in the late progression phase of chronological aging. Furthermore, codeletion of p38α in mice deficient in ataxia-telangiectasia mutated, a model of premature aging, exacerbated aging-related HSC phenotypes seen in ataxia-telangiectasia mutated single-mutant mice. Overall, these studies provide new insight into multiple functions of p38MAPK, which both promotes and suppresses HSC aging context dependently.
Asunto(s)
Envejecimiento/patología , Diferenciación Celular , Senescencia Celular , Células Madre Hematopoyéticas/metabolismo , Proteína Quinasa 14 Activada por Mitógenos/fisiología , Envejecimiento/metabolismo , Animales , Proteínas de la Ataxia Telangiectasia Mutada/fisiología , Proliferación Celular , Femenino , Hematopoyesis , Células Madre Hematopoyéticas/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
BACKGROUND: The cyclin-dependent kinase 7 (CDK7) subunit of TFIIH regulates RNA polymerase-II-based transcription and promotes tumor progression. However, the mechanisms involved in CDK7-mediated immune evasion are unclear in non-small cell lung cancer (NSCLC). METHODS: RNA silencing and pharmacologic inhibitors were used to evaluate the functions of CDK7/p38α/MYC/PD-L1 axis in cancer cell proliferation and antiPD-1 therapy resistance. Flow cytometry was performed to detect the status of the immune microenvironment after CDK7 inhibition and antiPD-1 therapy in vivo. CD8 depletion antibodies were used to assess the role of CD8+ T cells in combined CDK7 and PD-1 blockade. The associations among CDK7, p38α, MYC, PD-L1, infiltrating T cells, and survival outcomes were validated in two tissue microarrays and public transcriptomic data of NSCLC. RESULTS: High CDK7 mRNA and protein levels were identified to be associated with poor prognosis in NSCLC. CDK7 silencing and CDK7 inhibitor THZ1 elicited apoptosis and suppressed tumor growth. Moreover, CDK7 ablation specifically suppressed p38α/MYC-associated genes, and THZ1 inhibited MYC transcriptional activity through downregulating p38α. CDK7 inhibition sensitized NSCLC to p38α inhibitor. Further, THZ1 suppressed PD-L1 expression by inhibiting MYC activity. THZ1 boosted antitumor immunity by recruiting infiltrating CD8+ T cells and synergized with antiPD-1 therapy. The CDK7/MYC/PD-L1 signature and infiltrating T cell status collectively stratified NSCLC patients into different risk groups. CONCLUSION: These data suggest that the combined CDK7 inhibitor THZ1 and antiPD-1 therapy can be an effective treatment in NSCLC.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Imidazoles/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Terapia Molecular Dirigida , Proteínas de Neoplasias/antagonistas & inhibidores , Fenilendiaminas/uso terapéutico , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Piridinas/farmacología , Pirimidinas/uso terapéutico , Transducción de Señal/efectos de los fármacos , Animales , Antígeno B7-H1/biosíntesis , Antígeno B7-H1/genética , Antígeno B7-H1/fisiología , Linfocitos T CD8-positivos/inmunología , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Quinasas Ciclina-Dependientes/biosíntesis , Quinasas Ciclina-Dependientes/genética , Resistencia a Antineoplásicos , Sinergismo Farmacológico , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Desnudos , Proteína Quinasa 14 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 14 Activada por Mitógenos/fisiología , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Fenilendiaminas/administración & dosificación , Fenilendiaminas/farmacología , Pronóstico , Proteínas Proto-Oncogénicas c-myc/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-myc/fisiología , Pirimidinas/administración & dosificación , Pirimidinas/farmacología , Interferencia de ARN , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Neoplásico/biosíntesis , ARN Neoplásico/genética , Análisis de Matrices Tisulares , Transcriptoma , Ensayos Antitumor por Modelo de Xenoinjerto , Quinasa Activadora de Quinasas Ciclina-DependientesRESUMEN
Physiological or pathological muscle disuse/inactivity or loss of the neural-muscular junction cause muscle atrophy. Atrophy-inducing conditions cause metabolic oxidative stress in the muscle tissue, activation of the ubiquitin-proteasome and of the autophagosome-lysosome systems, enhanced removal of the damaged proteins and organelles, and loss of muscle mass and strength. The signaling pathways that control these catabolic processes are only partially known. In this study, we systematically analyzed the role of p38α mitogen-activated protein kinase (MAPK) in denervation-mediated atrophy. Mice with attenuated activity of p38α (p38AF ) are partially protected from muscle damage and atrophy. Denervated (Den) muscles of these mutant mice exhibit reduced signs of oxidative stress, decreased unfolded protein response and lower levels of ubiquitinated proteins relative to Den muscles of control mice. Further, whereas autopahagy flux is inhibited in Den muscles of control mice, Den muscles of p38AF mice maintain normal level of autophagy flux. Last, muscle denervation affects differently the energy metabolism of muscles in normal and mutant mice; whereas denervation appears to increase mitochondrial oxidative metabolism in control mice, it elevates anaerobic glycolytic metabolism in p38AF mice. Our results indicate, therefore, that attenuation of p38α activity in mice protects Den muscles by reducing oxidative stress, lowering protein damage and improving the clearance of damaged mitochondria by autophagy.
Asunto(s)
Autofagia , Redes y Vías Metabólicas , Proteína Quinasa 14 Activada por Mitógenos/fisiología , Desnervación Muscular/métodos , Músculo Esquelético/patología , Atrofia Muscular/patología , Estrés Oxidativo , Animales , Femenino , Lisosomas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/metabolismo , Mitocondrias/patología , Músculo Esquelético/inervación , Músculo Esquelético/metabolismo , Atrofia Muscular/etiología , Atrofia Muscular/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Transducción de Señal , Ubiquitina/metabolismoRESUMEN
Growing evidence suggests that hepatic macrophages play an important role in tissue repair after liver injury by coordinating the induction and resolution of inflammation, removing apoptotic cells, and promoting hepatocyte proliferation. Understanding the role of macrophages in the pathogenesis of liver injury will help pave the way to future therapeutics. Here, we investigated whether macrophage p38α plays a regulatory role in the tissue repair following d-galactosamine (GalN)/tumor necrosis factor-α (TNF-α)-induced acute liver injury. We found that macrophage p38α-deficient mice displayed decreased mortality and relieved liver injury as evident from less apoptosis, accelerated regeneration, decreased granulocytes recruitment, monocytes infiltration, and cytokine production after GalN/TNF-α treatment. Mechanistically, we found that p38 signaling was activated by lipopolysaccharide/interferon-γ treatment but not by inteleukin-4 stimulation, while pharmaceutical inhibition of p38α induced a shift in polarization from M1 macrophages to M2 macrophages. Together, our results indicated that macrophage p38α signaling is involved in the pathogenesis of liver injury induced by GalN/TNF-α, and inhibition of p38α signaling in macrophage could ameliorate liver injury and accelerate regeneration, probably by promoting the polarization of macrophages from the M1 phenotype to the M2 phenotype.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas/enzimología , Galactosamina/toxicidad , Regeneración Hepática/fisiología , Macrófagos/enzimología , Proteína Quinasa 14 Activada por Mitógenos/deficiencia , Factor de Necrosis Tumoral alfa/toxicidad , Animales , Antígenos de Diferenciación/biosíntesis , Antígenos de Diferenciación/genética , Proteínas de Ciclo Celular/biosíntesis , Proteínas de Ciclo Celular/genética , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Citocinas/biosíntesis , Citocinas/genética , Células Hep G2 , Hepatocitos/efectos de los fármacos , Humanos , Inmunofenotipificación , Interferón gamma/farmacología , Dosificación Letal Mediana , Lipopolisacáridos/farmacología , Regeneración Hepática/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Macrófagos/clasificación , Masculino , Ratones , Ratones Noqueados , Proteína Quinasa 14 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 14 Activada por Mitógenos/fisiología , Fosforilación/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Células RAW 264.7RESUMEN
An improved understanding of pathogenic pathways in AKI may identify novel therapeutic approaches. Previously, we conducted unbiased liquid chromatography-tandem mass spectrometry-based protein expression profiling of the renal proteome in mice with acute folate nephropathy. Here, analysis of the dataset identified enrichment of pathways involving NFκB in the kidney cortex, and a targeted data mining approach identified components of the noncanonical NFκB pathway, including the upstream kinase mitogen-activated protein kinase kinase kinase 14 (MAP3K14), the NFκB DNA binding heterodimer RelB/NFκB2, and proteins involved in NFκB2 p100 ubiquitination and proteasomal processing to p52, as upregulated. Immunohistochemistry localized MAP3K14 expression to tubular cells in acute folate nephropathy and human AKI. In vivo, kidney expression levels of NFκB2 p100 and p52 increased rapidly after folic acid injection, as did DNA binding of RelB and NFκB2, detected in nuclei isolated from the kidneys. Compared with wild-type mice, MAP3K14 activity-deficient aly/aly (MAP3K14aly/aly) mice had less kidney dysfunction, inflammation, and apoptosis in acute folate nephropathy and less kidney dysfunction and a lower mortality rate in cisplatin-induced AKI. The exchange of bone marrow between wild-type and MAP3K14aly/aly mice did not affect the survival rate of either group after folic acid injection. In cultured tubular cells, MAP3K14 small interfering RNA targeting decreased inflammation and cell death. Additionally, cell culture and in vivo studies identified the chemokines MCP-1, RANTES, and CXCL10 as MAP3K14 targets in tubular cells. In conclusion, MAP3K14 promotes kidney injury through promotion of inflammation and cell death and is a promising novel therapeutic target.
Asunto(s)
Lesión Renal Aguda/enzimología , Lesión Renal Aguda/etiología , Proteína Quinasa 14 Activada por Mitógenos/fisiología , Animales , Femenino , Ratones , Ratones Endogámicos C57BLRESUMEN
Myelination in the peripheral nervous system (PNS) is controlled by both positive and negative regulators within Schwann cells to ensure timely onset and correct myelin thickness for saltatory conduction by neurons. Transcription factors such as Sox10, octamer-binding transcription factor 6 (Oct6) and Krox20 form a positive regulatory network, whereas negative regulators such as cJun and Sox2 oppose myelination in Schwann cells. The role of the p38 MAPK pathway has been studied in PNS myelination, but its precise function remains unclear, with both positive and negative effects of p38 activity reported upon both myelination and processes of nerve repair. To clarify the role of p38 MAPK in the PNS, we have analysed mice with a Schwann cell-specific ablation of the major p38 isoform, p38alpha. In line with previous findings of an inhibitory role for p38 MAPK, we observe acceleration of post-natal myelination in p38alpha null nerves, a delay in myelin down-regulation following injury, together with a small increase in levels of re-myelination following injury. Finally we explored roles for p38alpha in controlling axonal regeneration and functional repair following PNS injury and observe that loss of p38alpha function in Schwann cells does not appear to affect these processes as previously reported. These studies therefore provide further proof for a role of p38 MAPK signalling in the control of myelination by Schwann cells in the PNS, but do not show an apparent role for signalling by this MAP kinase in Schwann cells controlling other elements of Wallerian degeneration and functional repair following injury. Cover Image for this issue: doi: 10.1111/jnc.13793.
Asunto(s)
Proteína Quinasa 14 Activada por Mitógenos/fisiología , Fibras Nerviosas Mielínicas/enzimología , Traumatismos de los Nervios Periféricos/enzimología , Nervios Periféricos/enzimología , Recuperación de la Función/fisiología , Células de Schwann/enzimología , Animales , Animales Recién Nacidos , Células Cultivadas , Femenino , Masculino , Ratones , Fibras Nerviosas Mielínicas/patología , Traumatismos de los Nervios Periféricos/patología , Nervios Periféricos/patología , Ratas , Células de Schwann/patologíaRESUMEN
Mechanical stretch is a major determinant that leads to heart failure, which is associated with a steady increase in myocardial angiotensinogen (Aogen) expression and formation of the biological peptide angiotensin II (Ang II). c-jun NH2-terminal kinase (JNK) and p38α have been found to have opposing roles on stretch-induced Aogen gene expression in neonatal rat ventricular myocytes (NRVM). JNK negatively regulated Aogen expression in NRVM following acute stretch, whereas with prolonged stretch, JNK phosphorylation was downregulated and p38α was found responsible for upregulation of Aogen expression. However, the mechanisms responsible for regulation of these kinases, especially the cross-talk between p38 and JNK1/2, remain to be determined. In this study, a combination of pharmacologic and molecular approaches (adenovirus-mediated gene transfer) were used to examine the mechanisms by which p38 regulates JNK phosphorylation in NRVM under stretch and non-stretch conditions. Pharmacologic inhibition of p38 significantly increased JNK phosphorylation in NRVM at 15 min, whereas overexpression of wild-type p38α significantly decreased JNK phosphorylation. While p38α overexpression prevented stretch-induced JNK phosphorylation, pharmacologic p38 inhibition abolished the JNK dephosphorylation during 15-60 min of stretch. Expression of constitutively-active MKK3 (MKK3CA), the upstream activator of p38, abolished JNK phosphorylation in both basal and stretched NRVM. Pharmacologic inhibition of MAP kinase phosphatase-1 (MKP-1) or protein phosphatase-1 (PP1) increased JNK phosphorylation in NRVM, suggesting the involvement of these phosphatases on reversing stretch-induced JNK activation. Inhibition of MKP-1, but not PP1, reduced JNK phosphorylation in NRVM overexpressing MKK3CA under basal conditions (no-stretch). Inhibition of MKP-1 also enhanced stretch-induced JNK phosphorylation in NRVM at 15 to 60 min. In summary, these results indicate that MKP-1 inhibits JNK phosphorylation in stretched NRVM through p38 dependent and independent mechanisms, whereas PP1 regulates JNK through a p38-independent mechanism.
Asunto(s)
Fosfatasa 1 de Especificidad Dual/fisiología , Proteínas Quinasas JNK Activadas por Mitógenos/fisiología , Proteína Quinasa 14 Activada por Mitógenos/fisiología , Miocitos Cardíacos/fisiología , Animales , Animales Recién Nacidos , Fenómenos Biomecánicos , Ratas , Ratas Sprague-DawleyRESUMEN
BACKGROUND AND OBJECTIVES: As the population ages, osteometabolic diseases and osteoporotic fractures emerge, resulting in substantial healthcare resource utilization and impaired quality of life. Many types of mechanical stimulation have the potential of being recognized by bone cells after a mechanical sign is transformed into a biological one (a process called mechanotransduction). The therapeutic ultrasound (TU) is one of several resources capable of promoting bone cell mechanical stimulation. Therefore, the main purpose of present study was to evaluate the effect of TU on the proliferation of pre-osteoblasts using in vitro bioassays. STUDY DESIGN/MATERIALS AND METHODS: We used MC3T3-E1 pre-osteoblast lineage cells kept in Alpha medium. Cells were treated using pulsed mode therapeutic ultrasound, with frequency of 1 MHz, intensity of 0.2 W/cm(2) (SATA), duty cycle of 20%, for 30 minutes. Nifedipine and rapamycin were used to further investigate the role of L-type Ca(2+) channels and mTOR pathway. Intracellular calcium, TGF-ß1, magnesium, and the mRNA levels of osteopontin, osteonectin, NF-κB1, p38α were evaluated. RESULTS: The results show that TU stimulates the growth of MC3T3-E1 cells and decreases the supernatant calcium and magnesium content. Also, it increases intracellular calcium, activates NF-κB1 and mTOR complex via p38α. Moreover, TU promoted a decrease in the TGF-ß1 synthesis, which is a cell growth inhibitor. CONCLUSIONS: Therapeutic ultrasound, with frequency of 1 MHz, intensity of 0.2 W/cm(2) (SATA) and pulsed mode, for 30 minutes, was able to increase the proliferation of preosteoblast-like bone cells. This effect was mediated by a calcium influx, with a consequent activation of the mTOR pathway, through increased NF-κB1 and p38α.
Asunto(s)
Proliferación Celular/efectos de la radiación , Proteína Quinasa 14 Activada por Mitógenos/fisiología , FN-kappa B/fisiología , Osteoblastos/efectos de la radiación , Serina-Treonina Quinasas TOR/fisiología , Terapia por Ultrasonido , Células 3T3 , Animales , Técnicas de Cultivo de Célula , Diferenciación Celular , Ratones , Osteoblastos/metabolismo , Osteoblastos/patologíaRESUMEN
p38α is a ubiquitous protein kinase strongly activated by stress signals, inflammatory cytokines, and many other stimuli, which has been implicated in the modulation of multiple cellular processes. There is good evidence in the literature that p38α plays an important tumor-suppressor role by interfering with malignant cell transformation. This is mainly based on the ability of the p38α pathway to regulate tissue homeostasis by integrating signals that balance cell proliferation and differentiation or induce apoptosis. However, recent reports have also illustrated protumorigenic functions for p38α. Thus, p38α signaling may facilitate the survival and proliferation of tumor cells contributing to the progression of some tumor types. In addition, p38α activation helps tumor cells to survive chemotherapeutic treatments. In all these cases, the inhibition of p38α has a potential therapeutic interest. Further elucidation of the context-dependent functions of p38α signaling in tumoral processes is of obvious importance for the use of inhibitors of this pathway in cancer therapy.
Asunto(s)
Antineoplásicos/uso terapéutico , Proteína Quinasa 14 Activada por Mitógenos/antagonistas & inhibidores , Terapia Molecular Dirigida , Proteínas de Neoplasias/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Animales , Antineoplásicos/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Transformación Celular Neoplásica/efectos de los fármacos , Colon/enzimología , Sinergismo Farmacológico , Activación Enzimática/efectos de los fármacos , Humanos , Hígado/enzimología , Pulmón/enzimología , Ratones , Proteína Quinasa 14 Activada por Mitógenos/fisiología , Metástasis de la Neoplasia , Proteínas de Neoplasias/fisiología , Neoplasias/enzimología , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/enzimología , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Microambiente Tumoral/efectos de los fármacosRESUMEN
p38 MAPKs regulate migration and invasion. However, the mechanisms involved are only partially known. We had previously identified fibulin 3, which plays a role in migration, invasion, and tumorigenesis, as a gene regulated by p38α. We have characterized in detail how p38 MAPK regulates fibulin 3 expression and its role. We describe here for the first time that p38α, p38γ, and p38δ down-regulate fibulin 3 expression. p38α has a stronger effect, and it does so through hypermethylation of CpG sites in the regulatory sequences of the gene. This would be mediated by the DNA methylase, DNMT3A, which is down-regulated in cells lacking p38α, but once re-introduced represses Fibulin 3 expression. p38α through HuR stabilizes dnmt3a mRNA leading to an increase in DNMT3A protein levels. Moreover, by knocking-down fibulin 3, we have found that Fibulin 3 inhibits migration and invasion in MEFs by mechanisms involving p38α/ß inhibition. Hence, p38α pro-migratory/invasive effect might be, at least in part, mediated by fibulin 3 down-regulation in MEFs. In contrast, in HCT116 cells, Fibulin 3 promotes migration and invasion through a mechanism dependent on p38α and/or p38ß activation. Furthermore, Fibulin 3 promotes in vitro and in vivo tumor growth of HCT116 cells through a mechanism dependent on p38α, which surprisingly acts as a potent inducer of tumor growth. At the same time, p38α limits fibulin 3 expression, which might represent a negative feed-back loop.
Asunto(s)
Movimiento Celular , Neoplasias del Colon/patología , Metilación de ADN , Embrión de Mamíferos/metabolismo , Proteínas de la Matriz Extracelular/genética , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Proteína Quinasa 14 Activada por Mitógenos/fisiología , Animales , Western Blotting , Adhesión Celular , Proliferación Celular , Células Cultivadas , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Regulación hacia Abajo , Embrión de Mamíferos/citología , Proteínas de la Matriz Extracelular/metabolismo , Fibroblastos/citología , Humanos , Masculino , Ratones , Ratones Noqueados , Ratones Desnudos , Invasividad Neoplásica , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Elementos de Respuesta/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Cicatrización de Heridas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
In CNS lesions, "reactive astrocytes" form a prominent cellular response. However, the nature of this astrocyte immune activity is not well understood. In order to study astrocytic immune responses to inflammation and injury, we generated mice with conditional deletion of p38α (MAPK14) in GFAP+ astrocytes. We studied the role of p38α signaling in astrocyte immune activation both in vitro and in vivo, and simultaneously examined the effects of astrocyte activation in CNS inflammation. Our results showed that specific subsets of cytokines (TNFα, IL-6) and chemokines (CCL2, CCL4, CXCL1, CXCL2, CXCL10) are critically regulated by p38α signaling in astrocytes. In an in vivo CNS inflammation model of intracerebral injection of LPS, we observed markedly attenuated astrogliosis in conditional GFAPcre p38α(-/-) mice. However, GFAPcre p38α(-/-) mice showed marked upregulation of CCL2, CCL3, CCL4, CXCL2, CXCL10, TNFα, and IL-1ß compared to p38αfl/fl cohorts, suggesting that in vivo responses to LPS after GFAPcre p38α deletion are complex and involve interactions between multiple cell types. This finding was supported by a prominent increase in macrophage/microglia and neutrophil recruitment in GFAPcre p38α(-/-) mice compared to p38αfl/fl controls. Together, these studies provide important insights into the critical role of p38α signaling in astrocyte immune activation.
Asunto(s)
Astrocitos/enzimología , Quimiocinas/biosíntesis , Proteína Quinasa 14 Activada por Mitógenos/fisiología , Animales , Astrocitos/inmunología , Encéfalo/inmunología , Encéfalo/metabolismo , Moléculas de Adhesión Celular/metabolismo , Células Cultivadas , Interleucina-1beta/fisiología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lipopolisacáridos/farmacología , Sistema de Señalización de MAP Quinasas , Ratones Noqueados , FN-kappa B/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
BACKGROUND: Berberine (BBR), a component from traditional Chinese medicine, has been shown to possess anti-tumor activity against a wide spectrum of cancer cells including human lung cancer, but the detailed mechanism underlining this has not been well elucidated. METHODS: In this study, the effect of berberine on cell growth and apoptosis were assessed by MTT, flow cytometry and Hoechst 33258 staining assays. The phosphorylation of p38 MAPK and ERK1/2, and expressions of p38 MAPK isoforms α and ß, total ERK1/2, p53, FOXO3a and p21 protein were evaluated by Western Blot analysis. Silencing of p38 MAPK isoform α and ß, p53, FOXO3a and p21 were performed by siRNA methods. Exogenous expression of FOXO3a was carried out by electroporated transfection assays. RESULTS: We showed that BBR significantly inhibited growth and induced cell cycle arrest of non small cell lung cancer (NSCLC) cells in the G0/G1 phase in a dose-dependent manner. Furthermore, we found that BBR increased phosphorylation of p38 MAPK and ERK1/2 in a time-dependent and induced protein expression of tumor suppressor p53 and transcription factor FOXO3a in a dose-dependent fashion. The specific inhibitor of p38 MAPK (SB203580), and silencing of p38α MAPK by small interfering RNAs (siRNAs), but not ERK1/2 inhibitor (PD98059) blocked the stimulatory effects of BBR on protein expression of p53 and FOXO3a. Interestingly, inhibition of p53 using one specific inhibitor (Pifithrin-α) and silencing of p53 using siRNAs overcome the inhibitory effect of BBR on cell growth. Silencing of FOXO3a appeared to attenuate the effect of BBR on p53 expression, cell proliferation and apoptosis. Furthermore, BBR induces the protein expression of cell cycle inhibitor p21 (CIP1/WAF1), which was not observed in cells silencing of p53 or FOXO3α gene. Intriguingly, exogenous expression of FOXO3a enhanced the expression of p21 (CIP1/WAF1) and strengthened BBR-induced apoptosis. CONCLUSION: Our results show that BBR inhibits proliferation and induces apoptosis of NSCLC cells through activation of p38α MAPK signaling pathway, followed by induction of the protein expression of p53 and FOXO3a. The latter contribute to the BBR-increased p21 (CIP1/WAF1) protein expression. The exogenous FOXO3a, interaction and mutually exclusive events of p53 and FOXO3a augment the overall response of BBR.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis , Berberina/farmacología , Factores de Transcripción Forkhead/metabolismo , Proteína Quinasa 14 Activada por Mitógenos/fisiología , Proteína p53 Supresora de Tumor/metabolismo , Adenocarcinoma , Adenocarcinoma del Pulmón , Línea Celular Tumoral , Proliferación Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Proteína Forkhead Box O3 , Factores de Transcripción Forkhead/genética , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Neoplasias Pulmonares , Sistema de Señalización de MAP Quinasas , Fosforilación , Procesamiento Proteico-Postraduccional , Activación Transcripcional , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Cardiac failure is a major cause of death in patients with type 2 diabetes, but the molecular mechanism that links diabetes to heart failure remains unclear. Insulin resistance is a hallmark of type 2 diabetes, and insulin receptor substrates 1 and 2 (IRS1 and IRS2) are the major insulin-signaling components regulating cellular metabolism and survival. To determine the role of IRS1 and IRS2 in the heart and examine whether hyperinsulinemia causes myocardial insulin resistance and cellular dysfunction via IRS1 and IRS2, we generated heart-specific IRS1 and IRS2 gene double-knockout (H-DKO) mice and liver-specific IRS1 and IRS2 double-knockout (L-DKO) mice. H-DKO mice had reduced ventricular mass; developed cardiac apoptosis, fibrosis, and failure; and showed diminished Aktâforkhead box class O-1 signaling that was accompanied by impaired cardiac metabolic gene expression and reduced ATP content. L-DKO mice had decreased cardiac IRS1 and IRS2 proteins and exhibited features of heart failure, with impaired cardiac energy metabolism gene expression and activation of p38α mitogen-activated protein kinase (p38). Using neonatal rat ventricular cardiomyocytes, we further found that chronic insulin exposure reduced IRS1 and IRS2 proteins and prevented insulin action through activation of p38, revealing a fundamental mechanism of cardiac dysfunction during insulin resistance and type 2 diabetes.
Asunto(s)
Insuficiencia Cardíaca/etiología , Hiperinsulinismo/fisiopatología , Proteínas Sustrato del Receptor de Insulina/deficiencia , Resistencia a la Insulina/fisiología , Proteína Quinasa 14 Activada por Mitógenos/fisiología , Transducción de Señal/efectos de los fármacos , Animales , Metabolismo Energético/genética , Insulina/fisiología , Ratones , Ratones Noqueados , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , RatasRESUMEN
The MAPK p38α senses environmental stressors and orchestrates inflammatory and immunomodulatory reactions. However, the molecular mechanism how p38α controls immunomodulatory responses in myeloid cells remains elusive. We found that in monocytes and macrophages, p38α activated the mechanistic target of rapamycin (mTOR) pathway in vitro and in vivo. p38α signaling in myeloid immune cells promoted IL-10 but inhibited IL-12 expression via mTOR and blocked the differentiation of proinflammatory CD4(+) Th1 cells. Cellular stress induced p38α-mediated mTOR activation that was independent of PI3K but dependent on the MAPK-activated protein kinase 2 and on the inhibition of tuberous sclerosis 1 and 2, a negative regulatory complex of mTOR signaling. Remarkably, p38α and PI3K concurrently modulated mTOR to balance IL-12 and IL-10 expression. Our data link p38α to mTOR signaling in myeloid immune cells that is decisive for tuning the immune response in dependence on the environmental milieu.
Asunto(s)
Exposición a Riesgos Ambientales , Inmunidad Innata , Proteína Quinasa 14 Activada por Mitógenos/fisiología , Serina-Treonina Quinasas TOR/fisiología , Animales , Línea Celular Transformada , Células Cultivadas , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Exposición a Riesgos Ambientales/efectos adversos , Humanos , Inmunidad Innata/genética , Interleucina-10/biosíntesis , Subunidad p40 de la Interleucina-12/biosíntesis , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Noqueados , Proteína Quinasa 14 Activada por Mitógenos/genética , Monocitos/inmunología , Monocitos/metabolismo , Transducción de Señal/genética , Transducción de Señal/inmunologíaRESUMEN
High NaCl activates the transcription factor nuclear factor of activated T cells 5 (NFAT5), leading to increased transcription of osmoprotective target genes. Kinases PKA, PI3K, AKT1, and p38α were known to contribute to the high NaCl-induced increase of NFAT5 activity. We now identify another kinase, GSK-3ß. siRNA-mediated knock-down of GSK-3ß increases NFAT5 transcriptional and transactivating activities without affecting high NaCl-induced nuclear localization of NFAT5 or NFAT5 protein expression. High NaCl increases phosphorylation of GSK-3ß-S9, which inhibits GSK-3ß. In GSK-3ß-null mouse embryonic fibroblasts transfection of GSK-3ß, in which serine 9 is mutated to alanine, so that it cannot be inhibited by phosphorylation at that site, inhibits high NaCl-induced NFAT5 transcriptional activity more than transfection of wild-type GSK-3ß. High NaCl-induced phosphorylation of GSK-3ß-S9 depends on PKA, PI3K, and AKT, but not p38α. Overexpression of PKA catalytic subunit α or of catalytically active AKT1 reduces inhibition of NFAT5 by GSK-3ß, but overexpression of p38α together with its catalytically active upstream kinase, MKK6, does not. Thus, GSK-3ß normally inhibits NFAT5 by suppressing its transactivating activity. When activated by high NaCl, PKA, PI3K, and AKT1, but not p38α, increase phosphorylation of GSK-3ß-S9, which reduces the inhibitory effect of GSK-3ß on NFAT5, and thus contributes to activation of NFAT5.
Asunto(s)
Glucógeno Sintasa Quinasa 3/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Factores de Transcripción/metabolismo , Transporte Activo de Núcleo Celular/fisiología , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3 beta , Células HEK293 , Humanos , Ratones , Proteína Quinasa 14 Activada por Mitógenos/fisiología , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Cloruro de Sodio/administración & dosificaciónRESUMEN
Tumor necrosis factor (TNF)-α can alter tissue repair functions in a variety of cells including endothelial cells. However, the mechanism by which TNF-α mediates these functional changes has not fully been studied. We investigated the role of mitogen-activated protein kinases (MAPKs) on mediating the regulatory effect of TNF-α on the tissue repair functions of human pulmonary artery endothelial cells (HPAECs). TNF-α protected HPAECs from undergoing apoptosis induced by serum and growth factor deprivation, augmented collagen gel contraction, and stimulated wound closure. TNF-α activated c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinases 1 and 2 (ERK1/2), and p38. Inhibitors of JNK (SP600125, 5 µM) or ERK1/2 (PD98059, 5 µM) significantly inhibited TNF-α-stimulated cell survival, contraction of collagen gels, and wound closure. In contrast, the p38 inhibitor SB203580 (5 µM) further amplified all of the TNF-α effects on HPAECs. TNF-α specifically activated p38α but not other p38 isoforms and suppression of p38α by an siRNA resulted in further amplification of the TNF-α effect. These results suggest that TNF-α stimulates tissue repair functions of HPAECs, and this may be mediated, at least in part, positively via JNK and ERK1/2, and negatively through p38α. MAPKs may play a role in endothelial cell-mediated tissue repair, especially in an inflammatory milieu where TNF-α is present.
Asunto(s)
Células Endoteliales/efectos de los fármacos , Endotelio Vascular/citología , Sistema de Señalización de MAP Quinasas/fisiología , Proteína Quinasa 14 Activada por Mitógenos/fisiología , Proteína Quinasa 1 Activada por Mitógenos/fisiología , Proteína Quinasa 3 Activada por Mitógenos/fisiología , Proteína Quinasa 8 Activada por Mitógenos/fisiología , Arteria Pulmonar/citología , Factor de Necrosis Tumoral alfa/farmacología , Cicatrización de Heridas/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/fisiología , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Células Cultivadas/efectos de los fármacos , Células Cultivadas/enzimología , Células Cultivadas/fisiología , Colágeno , Medio de Cultivo Libre de Suero/farmacología , Células Endoteliales/enzimología , Células Endoteliales/fisiología , Activación Enzimática/efectos de los fármacos , Geles , Humanos , Técnicas In Vitro , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 14 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 8 Activada por Mitógenos/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Interferencia de ARN , ARN Interferente Pequeño/farmacología , Proteínas Recombinantes/farmacología , Factor de Necrosis Tumoral alfa/fisiología , Vasculitis/enzimología , Vasculitis/fisiopatología , Cicatrización de Heridas/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresRESUMEN
To define signaling pathways that drive FSH- and epidermal growth factor (EGF)-like peptide-induced cumulus expansion and oocyte meiotic resumption, in vitro cultured pig cumulus-oocyte complexes were treated with specific protein kinase inhibitors. We found that FSH-induced maturation of oocytes was blocked in germinal vesicle (GV) stage by protein kinase A (PKA), MAPK14, MAPK3/1, and EGF receptor (EGFR) tyrosine kinase inhibitors (H89, SB203580, U0126, and AG1478 respectively) whereas phosphoinositide-3-kinase/v-akt murine thymoma viral oncogene homolog (PI3K/AKT) inhibitor (LY294002) blocked maturation of oocytes in metaphase I (MI). Amphiregulin (AREG)-induced maturation of oocytes was efficiently blocked in GV by U0126, AG1478, and low concentrations of LY294002; H89, SB203580, and high concentrations of LY294002 allowed the oocytes to undergo breakdown of GV and blocked maturation in MI. Both FSH- and AREG-induced cumulus expansion was incompletely inhibited by H89 and completely inhibited by SB203580, U0126, AG1478, and LY294002. The inhibitors partially or completely inhibited expression of expansion-related genes (HAS2, PTGS2, and TNFAIP6) with two exceptions: H89 inhibited only TNFAIP6 expression and LY294002 increased expression of PTGS2. The results of this study are consistent with the idea that PKA and MAPK14 pathways are essential for FSH-induced transactivation of the EGFR, and synthesis of EGF-like peptides in cumulus cells and MAPK3/1 is involved in regulation of transcriptional and posttranscriptional events in cumulus cells required for meiotic resumption and cumulus expansion. PI3K/AKT signaling is important for regulation of cumulus expansion, AREG-induced meiotic resumption, and oocyte MI/MII transition. The present data also indicate the existence of an FSH-activated and PKA-independent pathway involved in regulation of HAS2 and PTGS2 expression in cumulus cells.
Asunto(s)
Células del Cúmulo/fisiología , Hormona Folículo Estimulante/farmacología , Glicoproteínas/farmacología , Péptidos y Proteínas de Señalización Intercelular/farmacología , Meiosis/efectos de los fármacos , Transducción de Señal/fisiología , Sus scrofa/fisiología , Anfirregulina , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Ciclooxigenasa 2/genética , Receptores ErbB/fisiología , Femenino , Regulación de la Expresión Génica/fisiología , Glucuronosiltransferasa/genética , Meiosis/fisiología , Proteína Quinasa 14 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 14 Activada por Mitógenos/fisiología , Oocitos/efectos de los fármacos , Oocitos/fisiología , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal/efectos de los fármacosRESUMEN
Repeated stress releases dynorphins and causes subsequent activation of κ-opioid receptors (KORs) in limbic brain regions. The serotonergic dorsal raphe nucleus (DRN) has previously been found to be an important site of action for the dysphoric effects of dynorphin-κ-opioid receptor system activation during stress-evoked behaviors, and KOR-induced activation of p38α mitogen-activated protein kinase (MAPK) in serotonergic neurons was found to be a critical mediator of the aversive properties of stress. Yet, how dynorphins and KORs functionally regulate the excitability of serotonergic DRN neurons both in adaptive and pathological stress states is poorly understood. Here we report that acute KOR activation by the selective agonist U69,593 [(+)-(5α,7α,8ß)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4.5]dec-8-yl]benzeneacetamide] inhibits serotonergic neuronal excitability within the DRN through both presynaptic inhibition of excitatory synaptic transmission and postsynaptic activation of G-protein-gated inwardly rectifying potassium channels (GIRKs) electrophysiologically recorded in brain slices. C57BL/6 mice subjected to repeated swim, stress sessions had significantly reduced KOR-mediated GIRK currents recorded in serotonergic neurons in DRN postsynaptically, without significantly affecting presynaptic KOR-mediated regulation of excitatory transmission. This effect was blocked by genetic excision of p38α MAPK selectively from serotonergic neurons. An increase in phospho-immunoreactivity suggests that this functional dysregulation may be a consequence of tyrosine phosphorylation of GIRK (K(IR)3.1) channels. These data elucidate a mechanism for stress-induced dysregulation of the excitability of neurons in the DRN and identify a functional target of stress-induced p38α MAPK activation that may underlie some of the negative effects of pathological stress exposure.
Asunto(s)
Canales de Potasio Rectificados Internamente Asociados a la Proteína G/fisiología , Proteína Quinasa 14 Activada por Mitógenos/fisiología , Núcleos del Rafe/enzimología , Transducción de Señal/fisiología , Estrés Psicológico/enzimología , Animales , Bencenoacetamidas/farmacología , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Técnicas de Cultivo de Órganos , Fosforilación , Pirrolidinas/farmacología , Núcleos del Rafe/efectos de los fármacos , Tiempo de Reacción/fisiología , Serotonina/fisiología , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Tirosina/metabolismoRESUMEN
The Atlantic killifish (Fundulus heteroclitus) is an environmental sentinel organism used extensively for studies of environmental toxicants and osmoregulation. Previous research in our laboratory has shown that acute acclimation to seawater is mediated by an increase in SGK1. SGK1 promotes the trafficking of CFTR chloride channels from intracellular vesicles to the plasma membrane of the gill within the first hour in seawater resulting in increased chloride secretion. Although we have shown that the increase in gill SGK1 does not require activation of the glucocorticoid receptor, the mechanisms that mediate the rise SGK1 during acute acclimation is unknown. To test the hypothesis that mitogen activated protein kinase (MAPK14) is responsible for the rise in SGK1 we identified the coding sequence of killifish MAPK14-1 and designed a translational blocking vivo-morpholino targeting MAPK14-1. Injection of the MAPK14-1 vivo-morpholino resulted in a 30% reduction of MAPK14-1 and a 45% reduction in phosphorylated-MAPK14-1 protein in the gill of killifish transitioned from freshwater to seawater. Knock down of phosphorlyated-MAPK14-1 completely blocked the rise in SGK1 mRNA and protein in the killifish gill, providing the first direct and in vivo evidence that MAPK14-1 is necessary for acute seawater acclimation.