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1.
Cell Rep ; 38(2): 110223, 2022 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-35021072

RESUMO

MEK1 and MEK2, the only known activators of ERK, are attractive therapeutic candidates for both cancer and autoimmune diseases. However, how MEK signaling finely regulates immune cell activation is only partially understood. To address this question, we specifically delete Mek1 in hematopoietic cells in the Mek2 null background. Characterization of an allelic series of Mek mutants reveals the presence of distinct degrees of spontaneous B cell activation, which are inversely proportional to the levels of MEK proteins and ERK activation. While Mek1 and Mek2 null mutants have a normal lifespan, 1Mek1 and 1Mek2 mutants retaining only one functional Mek1 or Mek2 allele in hematopoietic cell lineages die from glomerulonephritis and lymphoproliferative disorders, respectively. This establishes that the fine-tuning of the ERK/MAPK pathway is critical to regulate B and T cell activation and function and that each MEK isoform plays distinct roles during lymphocyte activation and disease development.


Assuntos
Ativação Linfocitária/fisiologia , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/metabolismo , Alelos , Animais , Linfócitos B/metabolismo , Feminino , Humanos , Ativação Linfocitária/genética , MAP Quinase Quinase 1/fisiologia , MAP Quinase Quinase 2/genética , MAP Quinase Quinase 2/fisiologia , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Camundongos , Camundongos da Linhagem 129 , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Fosforilação , Transdução de Sinais/fisiologia , Linfócitos T/metabolismo
2.
Cancer Res ; 81(12): 3174-3186, 2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-33863779

RESUMO

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.


Assuntos
Carcinoma de Células Renais/patologia , Inibidor p16 de Quinase Dependente de Ciclina/fisiologia , MAP Quinase Quinase 1/fisiologia , Proteína Quinase 14 Ativada por Mitógeno/fisiologia , Serina-Treonina Quinases TOR/metabolismo , Proteína 1 do Complexo Esclerose Tuberosa/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Animais , Apoptose , Carcinoma de Células Renais/etiologia , Carcinoma de Células Renais/metabolismo , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Neoplasias Renais/etiologia , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Serina-Treonina Quinases TOR/genética , Células Tumorais Cultivadas
3.
Cancer Cell ; 34(2): 315-330.e7, 2018 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-30033091

RESUMO

Platinum-based chemotherapeutics represent a mainstay of cancer therapy, but resistance limits their curative potential. Through a kinome RNAi screen, we identified microtubule-associated serine/threonine kinase 1 (MAST1) as a main driver of cisplatin resistance in human cancers. Mechanistically, cisplatin but no other DNA-damaging agents inhibit the MAPK pathway by dissociating cRaf from MEK1, while MAST1 replaces cRaf to reactivate the MAPK pathway in a cRaf-independent manner. We show clinical evidence that expression of MAST1, both initial and cisplatin-induced, contributes to platinum resistance and worse clinical outcome. Targeting MAST1 with lestaurtinib, a recently identified MAST1 inhibitor, restores cisplatin sensitivity, leading to the synergistic attenuation of cancer cell proliferation and tumor growth in human cancer cells and patient-derived xenograft models.


Assuntos
Antineoplásicos/farmacologia , Cisplatino/farmacologia , MAP Quinase Quinase 1/fisiologia , Proteínas Associadas aos Microtúbulos/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Proto-Oncogênicas c-raf/fisiologia , Animais , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Ativação Enzimática , Feminino , Humanos , Camundongos
4.
Chembiochem ; 18(16): 1593-1598, 2017 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-28561913

RESUMO

Interference from endogenous signaling enzymes represents a major hurdle for building orthogonal signaling cascades inside cells, particularly among closely related isoforms within an enzyme family. Here, we employed a genetically encoded chemical decaging strategy to build orthogonally activated kinase isoforms, with the endogenous counterparts temporally disabled by an extracellularly delivered bacterial effector. This approach eliminated any potential interference from other kinase isoforms as well as endogenous kinases, which allowed the specific, gain-of-function report of mitogen-activated protein kinase kinase 1 (MEK1) activity as opposed to MEK2 with high temporal resolution. Our study dissected the distinct enzymatic activity, feedback regulation and signal outputs between these closely related kinase isoforms.


Assuntos
MAP Quinase Quinase 1/fisiologia , MAP Quinase Quinase 2/fisiologia , Reação de Cicloadição , Retroalimentação Fisiológica , Células HEK293 , Compostos Heterocíclicos com 1 Anel/química , Humanos , Interleucina-8/metabolismo , Isoenzimas/antagonistas & inibidores , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/fisiologia , Lisina/análogos & derivados , Lisina/química , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 1/química , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 2/antagonistas & inibidores , MAP Quinase Quinase 2/química , MAP Quinase Quinase 2/genética , Redes e Vias Metabólicas , Fosforilação , Engenharia de Proteínas , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais
5.
Proc Natl Acad Sci U S A ; 113(11): 3030-5, 2016 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-26929372

RESUMO

Loss of the tumor suppressor gene PTEN is implicated in breast cancer progression and resistance to targeted therapies, and is thought to promote tumorigenesis by activating PI3K signaling. In a transgenic model of breast cancer, Pten suppression using a tetracycline-regulatable short hairpin (sh)RNA cooperates with human epidermal growth factor receptor 2 (HER2/neu), leading to aggressive and metastatic disease with elevated signaling through PI3K and, surprisingly, the mitogen-activated protein kinase (MAPK) pathway. Restoring Pten function is sufficient to down-regulate both PI3K and MAPK signaling and triggers dramatic tumor regression. Pharmacologic inhibition of MAPK signaling produces similar effects to Pten restoration, suggesting that the MAPK pathway contributes to the maintenance of advanced breast cancers harboring Pten loss.


Assuntos
Sistema de Sinalização das MAP Quinases/fisiologia , Neoplasias Mamárias Experimentais/fisiopatologia , Proteínas de Neoplasias/fisiologia , PTEN Fosfo-Hidrolase/deficiência , Receptor ErbB-2/fisiologia , Animais , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Genes erbB-2 , Humanos , MAP Quinase Quinase 1/fisiologia , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/patologia , Vírus do Tumor Mamário do Camundongo/fisiologia , Camundongos , Camundongos Nus , Camundongos Transgênicos , Metástase Neoplásica , Proteínas de Neoplasias/deficiência , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/fisiologia , Fosfatidilinositol 3-Quinases/fisiologia , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-akt/fisiologia , Interferência de RNA , RNA Interferente Pequeno/genética , Transdução de Sinais/fisiologia
6.
Tumour Biol ; 37(5): 5837-46, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26586395

RESUMO

Glabridin, an isoflavone isolated from licorice, owns a variety of pharmacological effects. Several reports have demonstrated that glabridin could regulate multiple cellular signaling pathways to inhibit the progression of cancer. However, the target proteins have not been elucidated yet. We used shape screening and induced fit docking to screen the protein data bank against glabridin. Braf and MEK1/2, important intermediate molecules of the braf/MEK cascade, were identified as the potential targets of glabridin. The experimental data showed that glabridin could inhibit the phosphorylation of MEK1/2 and the phosphorylation levels of downstream molecules including ERK1/2 and transcription factors ATF1 and CREB, but had no effect on the phosphorylation of braf. In particular, the in vitro pull-down assay indicated that glabridin selectively bound to braf and MEK1/2. What is more, exposure to glabridin significantly suppressed the proliferation of hepatocellular carcinoma HepG2 cell line. In addition, glabridin might arrest cell cycle in G1 through downregulation of cyclinD3, CDK2, and CDK4. In conclusion, glabridin is a potential multi-molecule-targeting inhibitor in the field of clinical prevention or treatment of cancer.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Carcinoma Hepatocelular/patologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Isoflavonas/farmacologia , Neoplasias Hepáticas/patologia , MAP Quinase Quinase 1/antagonistas & inibidores , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fenóis/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Divisão Celular/efeitos dos fármacos , Biologia Computacional , Desenho de Fármacos , Células Hep G2 , Humanos , MAP Quinase Quinase 1/fisiologia , Modelos Moleculares , Fosforilação/efeitos dos fármacos , Ligação Proteica , Conformação Proteica , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Proto-Oncogênicas B-raf/fisiologia , Ensaio Tumoral de Célula-Tronco
7.
Respir Res ; 16: 93, 2015 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-26242183

RESUMO

BACKGROUND: Polo-like kinase 1 (Plk1) is a serine/threonine protein kinase that has been implicated in the regulation of mitosis. In addition, the activation of mitogen-activated protein kinase (MAPK) is a key event in the early stage of the growth factor response. The role of Plk1 in MAPK phosphorylation in cells has not been investigated. METHODS: Immunoblot analysis was used to evaluate Plk1 and MAPK phosphorylation in cells upon stimulation with platelet-derived growth factor (PDGF). We also generated stable Plk1 knockdown (KD) cells to assess the role of Plk1 in MAPK activation and cell proliferation. Furthermore, we used a non-phosphorylatable Plk1 mutant to determine the function of Plk1 phosphorylation in these processes. RESULTS: Treatment with PDGF increased Plk1 phosphorylation at Thr-210 (an indication of Plk1 activation) in human airway smooth muscle cells. Plk1 KD attenuated the PDGF-induced phosphorylation of MEK1/2 and ERK1/2 as well as cell proliferation. However, phosphorylation of Raf-1 and AKT upon stimulation with PDGF was not reduced in Plk1 KD cells. Furthermore, the expression of T210A Plk1 (alanine substitution at Thr-210) inhibited the PDGF-stimulated MEK1/2 phosphorylation, ERK1/2 phosphorylation and cell proliferation. CONCLUSIONS: Together, these findings suggest that Plk1 is activated upon growth factor stimulation, which may control the activation of MEK1/2 and ERK1/2, and smooth muscle cell proliferation.


Assuntos
Proteínas de Ciclo Celular/fisiologia , Proliferação de Células/fisiologia , MAP Quinase Quinase 1/fisiologia , MAP Quinase Quinase 2/fisiologia , Miócitos de Músculo Liso/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Brônquios/citologia , Brônquios/fisiologia , Humanos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/fisiologia , Traqueia/citologia , Traqueia/fisiologia , Quinase 1 Polo-Like
8.
Cardiovasc Res ; 108(1): 50-61, 2015 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-26249805

RESUMO

AIMS: Activin receptor-like kinase 7 (ALK7), one of the type I transforming growth factor-ß receptors, is expressed in various tissues, including the heart. However, the participation of ALK7 in the regulation of cardiac hypertrophy has not yet been studied. Here, we sought to determine the regulatory role and underlying mechanisms of ALK7 in cardiac hypertrophy. METHODS AND RESULTS: We performed aortic banding (AB) in ALK7-knockout mice, cardiac-specific ALK7-transgenic mice, and the wild-type littermates of these mice. Cardiac hypertrophy was evaluated using pathological analysis, echocardiographic measurement, haemodynamic measurement, and molecular analysis. Our results revealed that ALK7 disruption led to an aggravated cardiac hypertrophic response that was accompanied by increased cardiac fibrosis and reduced contractile function, whereas cardiac-specific ALK7 overexpression exhibited the opposite phenotype in response to pressure overload. Similarly, ALK7 protected against angiotensin II-induced cardiomyocyte hypertrophy in vitro. Mechanistically, we demonstrated that ALK7-dependent cardioprotection was mediated largely through inhibition of the MEK-ERK1/2 signalling pathway. CONCLUSION: Our data suggest that ALK7 acts as a novel regulator of pathological cardiac hypertrophy via the negative regulation of MEK-ERK1/2 signalling and may serve as a potential therapeutic target for pathological cardiac hypertrophy.


Assuntos
Receptores de Ativinas Tipo I/fisiologia , Cardiomegalia/prevenção & controle , Miocárdio/patologia , Animais , Apoptose , Humanos , MAP Quinase Quinase 1/fisiologia , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ratos Sprague-Dawley , Proteínas Smad/fisiologia
9.
Cell Signal ; 27(10): 2068-76, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26208884

RESUMO

The mitogen activated protein kinases ERK1/2 play an important role in response to toll like receptor (TLR) activation and cytokine production, including IL-10 and IL-12. Here, we examined the role of MEK1 in ERK1/2 activation in response to TLR4 agonist by using bone marrow-derived macrophages (BMDMs) from wild type (WT) and Mek1(d/d)Sox2(Cre) mice. Our data demonstrates that MEK1 is essential for ERK1/2 activation in response to LPS. Furthermore, stimulation of the TLR4 receptor of BMDMs derived from Mek1(d/d)Sox2(Cre) mice showed enhanced STAT4 phosphorylation and increased IL-12 secretion, but exhibited a significantly lower IL-10 production as compared to WT macrophages. Most interestingly, TLR ligation in the presence of recombinant IL-10 (rIL-10) or retinoic acid (RA) led to ERK1/2 activation independent of MEK1 in BMDMs derived from Mek1(d/d)Sox2(Cre) mice and led to inhibition of STAT4 and decreased IL-12 levels. Collectively, these data suggest that MEK1 is required for TLR4 mediated ERK activation and in turn regulates the production of IL-10 and IL-12. It also indicates that ERK1/2 can be activated independent of MEK1 in the presence of IL-10 and RA and this activation negatively regulates IL-12, but positively regulates IL-10 production. These findings may have significant implications for the development of drugs that modulate MEK1 activity in the treatment of inflammatory, autoimmune and proliferative diseases such as cancer.


Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Interleucina-10/biossíntese , Interleucina-12/biossíntese , MAP Quinase Quinase 1/fisiologia , Macrófagos/enzimologia , Animais , Ativação Enzimática , Lipopolissacarídeos/farmacologia , Sistema de Sinalização das MAP Quinases , Macrófagos/imunologia , Camundongos da Linhagem 129 , Fosforilação , Processamento de Proteína Pós-Traducional , Fator de Transcrição STAT4/metabolismo , Receptor 4 Toll-Like/metabolismo , Tretinoína/farmacologia
10.
Arch Pharm Res ; 38(12): 2153-62, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26119076

RESUMO

It has been reported that alkaloids derived from Coptis chinensis exert anti-adipogenic activity on 3T3-L1 adipocytes by downregulating peroxisome proliferation-activity receptor-γ (PPAR-γ) and CCAAT/enhancer binding protein-α (C/EBP-α). However, the signaling-based mechanism of the inhibitory role of epiberberine in the early stages of 3T3-L1 adipocyte differentiation is uncharacterized. Here, we show that epiberberine had inhibitory effects on adipocyte differentiation and significantly decreased lipid accumulation by downregulating an adipocyte-specific transcription factor, sterol regulatory element-binding protein-1 (SREBP-1). Furthermore, we observed that epiberberine markedly suppressed the differentiation-mediated phosphorylation of components of both the Raf/mitogen-activated protein kinase 1 (MEK1)/extracellular signal-regulated protein kinase 1/2 (ERK1/2) and AMP-activated protein kinase-α1 (AMPKα)/Akt pathways. In addition, gene expression of fatty acid synthase (FAS) was significantly inhibited by treatment with epiberberine during adipogenesis. These results indicate that the anti-adipogenic mechanism of epiberberine is associated with inhibition of phosphorylation of Raf/MEK1/ERK1/2 and AMPKα/Akt, followed by downregulation of the major transcription factors of adipogenesis, such as PPAR-γ, C/EBP-α, and SREBP-1, and FAS. Taken together, this study suggests that the anti-adipogenic effect of epiberberine is mediated by downregulation of the Raf/MEK1/ERK1/2 and AMPKα/Akt pathways during 3T3-L1 adipocyte differentiation. Moreover, the anti-adipogenic effects of epiberberine were not accompanied by modulation of ß-catenin.


Assuntos
Proteínas Quinases Ativadas por AMP/fisiologia , Adipogenia/fisiologia , Berberina/análogos & derivados , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Proteína Oncogênica v-akt/fisiologia , Quinases raf/fisiologia , Células 3T3-L1 , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Adipogenia/efeitos dos fármacos , Animais , Fármacos Antiobesidade/farmacologia , Berberina/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Relação Dose-Resposta a Droga , Medicamentos de Ervas Chinesas/farmacologia , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 1/fisiologia , MAP Quinase Quinase 2/antagonistas & inibidores , MAP Quinase Quinase 2/fisiologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteína Oncogênica v-akt/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Quinases raf/antagonistas & inibidores
11.
Development ; 141(16): 3197-211, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25100655

RESUMO

The mammalian genome contains two ERK/MAP kinase genes, Mek1 and Mek2, which encode dual-specificity kinases responsible for ERK/MAP kinase activation. In order to define the function of the ERK/MAPK pathway in the lung development in mice, we performed tissue-specific deletions of Mek1 function on a Mek2 null background. Inactivation of both Mek genes in mesenchyme resulted in several phenotypes, including giant omphalocele, kyphosis, pulmonary hypoplasia, defective tracheal cartilage and death at birth. The absence of tracheal cartilage rings establishes the crucial role of intracellular signaling molecules in tracheal chondrogenesis and provides a putative mouse model for tracheomalacia. In vitro, the loss of Mek function in lung mesenchyme did not interfere with lung growth and branching, suggesting that both the reduced intrathoracic space due to the dysmorphic rib cage and the omphalocele impaired lung development in vivo. Conversely, Mek mutation in the respiratory epithelium caused lung agenesis, a phenotype resulting from the direct impact of the ERK/MAPK pathway on cell proliferation and survival. No tracheal epithelial cell differentiation occurred and no SOX2-positive progenitor cells were detected in mutants, implying a role for the ERK/MAPK pathway in trachea progenitor cell maintenance and differentiation. Moreover, these anomalies were phenocopied when the Erk1 and Erk2 genes were mutated in airway epithelium. Thus, the ERK/MAPK pathway is required for the integration of mesenchymal and epithelial signals essential for the development of the entire respiratory tract.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , MAP Quinase Quinase 1/fisiologia , MAP Quinase Quinase 2/fisiologia , Sistema de Sinalização das MAP Quinases , Sistema Respiratório/embriologia , Animais , Apoptose , Sequência de Bases , Cartilagem/embriologia , Diferenciação Celular , Proliferação de Células , Perfilação da Expressão Gênica , Pulmão/embriologia , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 2/genética , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Mutação , Fenótipo , Células-Tronco/citologia , Traqueia/embriologia
12.
PLoS One ; 9(6): e100862, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24959884

RESUMO

Uncontrolled adipogenesis and adipocyte proliferation have been connected to human comorbidities. Retinoic acid (RA) is known to inhibit adipocyte differentiation, however the underlying mechanisms have not been adequately understood. This study reports that RA acting as a ligand to RA receptors (RARs and RXRs) is not a sine qua non to the inhibition of adipogenesis. Our intriguing observation of a negative correlation between increased retinoylation and adipogenesis led us to explore retinoylated proteins in adipocytes. Exportin (CRM1) was found to be retinoylated, which in turn can affect the spatio-temporal regulation of the important signaling molecule mitogen-activated protein kinase kinase 1 (MEK1), likely by disrupting its export from the nucleus. Nuclear enrichment of MEK1 physically sequesters peroxisome proliferator-activated receptor gamma (PPARγ), the master regulator of adipogenesis, from its target genes and thus inhibits adipogenesis while also disrupting the MEK1-extracellular-signal regulated kinase (ERK) signaling cascade. This study is first to report the inhibition of adipocyte differentiation by retinoylation.


Assuntos
Adipócitos/citologia , MAP Quinase Quinase 1/fisiologia , PPAR gama/metabolismo , Tretinoína/fisiologia , Células 3T3-L1 , Adipócitos/metabolismo , Adipogenia , Animais , Diferenciação Celular/genética , Carioferinas/metabolismo , MAP Quinase Quinase 1/metabolismo , Sistema de Sinalização das MAP Quinases , Camundongos , PPAR gama/fisiologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Tretinoína/metabolismo , Proteína Exportina 1
13.
J Leukoc Biol ; 95(6): 917-30, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24532642

RESUMO

Whereas the main function of APN is to enhance insulin activity, it is also involved in modulating the macrophage phenotype. Here, we demonstrate that at physiological concentrations, APN activates Erk1/2 via the IKKß-p105/NF-κΒ1-Cot/tpl2 intracellular signal transduction cassette in macrophages. In peritoneal macrophages stimulated with APN, Cot/tpl2 influences the ability to phagocytose beads. However, Cot/tpl2 did not modulate the known capacity of APN to decrease lipid content in peritoneal macrophages in response to treatment with oxLDL or acLDL. A microarray analysis of gene-expression profiles in BMDMs exposed to APN revealed that APN modulated the expression of ∼3300 genes; the most significantly affected biological functions were the inflammatory and the infectious disease responses. qRT-PCR analysis of WT and Cot/tpl2 KO macrophages stimulated with APN for 0, 3, and 18 h revealed that Cot/tpl2 participated in the up-regulation of APN target inflammatory mediators included in the cytokine-cytokine receptor interaction pathway (KEGG ID 4060). In accordance with these data, macrophages stimulated with APN increased secretion of cytokines and chemokines, including IL-1ß, IL-1α, TNF-α, IL-10, IL-12, IL-6, and CCL2. Moreover, Cot/tpl2 also played an important role in the production of these inflammatory mediators upon stimulation of macrophages with APN. It has been reported that different types of signals that stimulate TLRs, IL-1R, TNFR, FcγR, and proteinase-activated receptor-1 activate Cot/tpl2. Here, we demonstrate that APN is a new signal that activates the IKKß-p105/NF-κΒ1-Cot/tpl2-MKK1/2-Erk1/2 axis in macrophages. Furthermore, this signaling cassette modulates the biological functions triggered by APN in macrophages.


Assuntos
Adiponectina/farmacologia , MAP Quinase Quinase Quinases/fisiologia , Ativação de Macrófagos/efeitos dos fármacos , Proteínas Proto-Oncogênicas/fisiologia , Animais , Quimiocinas/biossíntese , Citocinas/biossíntese , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Quinase I-kappa B/fisiologia , Interleucina-10/fisiologia , MAP Quinase Quinase 1/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/fisiologia , Fagocitose/efeitos dos fármacos
14.
Gynecol Oncol ; 133(1): 128-37, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24434059

RESUMO

Mitogen-activated protein kinases (MAPKs) are a family of ubiquitous eukaryotic signal transduction enzymes which link extracellular stimuli to intracellular gene expression pathways. While several three-tiered MAPK cascades have been elucidated in mammals, the prototypical pathway involves a network of proteins and kinases including the Rat sarcoma protein (Ras), mitogen-activated protein kinase kinase kinase (Raf or MAP3K), mitogen-activated protein kinase kinase (MEK or MAP2K), and extracellular signal regulated protein kinase (ERK or MAPK). This MAPK cascade (the Ras/Raf/MEK/ERK pathway) is a receptor tyrosine kinase mediated signaling pathway that regulates cell proliferation, cell cycle progression, and cell migration. There are multiple molecular mechanisms of interaction and activation between the upstream nodes of the Ras/Raf/MEK/ERK cascade and other cell signaling pathways, all ultimately leading to the activation of the nuclear transcription factor ERK. Important downstream targets include MEK1/2, which comprise the final step leading to ERK transcription factor activation. While multiple conduits exist to activate ERK upstream of MEK, there is little redundancy downstream. Located at this pivotal intersection between a limited number of upstream activators and its exclusive downstream targets, MEK is an appealing molecular target of novel cancer therapies. MEK inhibitors are small molecules that inhibit MEK phosphorylation by binding to a pocket adjacent to the ATP binding site, decreasing both the amount of MEK activity, and the quantity of activated ERK in the cell. Unique allosteric noncompetitive binding sites of MEK inhibitors allow specific targeting of MEK enzymes and prevent cross-activation of other serine/threonine protein kinases through the conserved ATP binding site. This paper reviews the translational evidence in favor of MEK inhibitors in cancer, their role in gynecologic malignancies, and details regarding the status of the fourteen MEK inhibitors currently being clinically tested: trametinib, selumetinib, pimasertib, refametinib, PD-0325901, MEK162, TAK733, RO5126766, WX-554, RO4987655, cobimetinib, AZD8330, MSC2015103B, and ARRY-300.


Assuntos
Carcinoma/tratamento farmacológico , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 2/antagonistas & inibidores , Neoplasias Ovarianas/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Carcinoma/enzimologia , Neoplasias Colorretais/tratamento farmacológico , Feminino , Humanos , MAP Quinase Quinase 1/fisiologia , MAP Quinase Quinase 2/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Neoplasias Ovarianas/enzimologia
15.
Gastroenterology ; 146(3): 822-834.e7, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24315826

RESUMO

BACKGROUND & AIMS: Kras signaling via mitogen-activated protein kinase (MAPK) is highly up-regulated in pancreatic cancer cells. We investigated whether MAPK signaling is required for the initiation and maintenance of pancreatic carcinogenesis in mice. METHODS: We studied the formation and maintenance of pancreatic intraepithelial neoplasia (PanINs) in p48Cre; TetO-KrasG12D; Rosa26(rtTa-IRES-EGFP) (iKras*) mice and LSL-KrasG12D mice bred with p48Cre mice (KC). Mice were given oral PD325901, a small-molecule inhibitor of MEK1 and MEK2 (factors in the MAPK signaling pathway), along with injections of cerulein to induce pancreatitis. Other mice were given PD325901 only after PanINs developed. Pancreatic tissues were collected and evaluated using histologic, immunohistochemical, immunofluorescence, and electron microscopy analyses. Acinar cells were isolated from the tissues and the effects of MEK1 and 2 inhibitors were assessed. RESULTS: PD325901 prevented PanIN formation, but not pancreatitis, in iKras* and KC mice. In iKras* or KC mice given PD325901 at 5 weeks after PanINs developed, PanINs regressed and acinar tissue regenerated. The regression occurred through differentiation of the PanIN cells to acini, accompanied by re-expression of the acinar transcription factor Mist1. CONCLUSIONS: In iKras* and KC mice, MAPK signaling is required for the initiation and maintenance of pancreatic cancer precursor lesions. MAPK signaling promotes formation of PanINs by enabling dedifferentiation of acinar cells into duct-like cells that are susceptible to transformation.


Assuntos
Células Acinares/patologia , Carcinoma in Situ/fisiopatologia , Desdiferenciação Celular/fisiologia , Quinases de Proteína Quinase Ativadas por Mitógeno/fisiologia , Neoplasias Pancreáticas/fisiopatologia , Transdução de Sinais/fisiologia , Células Acinares/fisiologia , Animais , Carcinoma in Situ/patologia , Modelos Animais de Doenças , Feminino , MAP Quinase Quinase 1/fisiologia , MAP Quinase Quinase 2/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Camundongos , Camundongos Mutantes , Neoplasias Pancreáticas/patologia , Pancreatite/fisiopatologia , Regulação para Cima/fisiologia
16.
FEBS Lett ; 587(24): 3921-7, 2013 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-24211253

RESUMO

Mitogen-activated protein kinase kinase 1 (MAP2K1/MEK1) as well as Yes-associated protein (YAP), the downstream effector of Hippo signaling pathway, is linked to hepatocarcinogenesis. However, little is known about whether and how MEK1 interacts with YAP. In this study, we find that MEK1-YAP interaction is critical for liver cancer cell proliferation and maintenance of transformed phenotypes both in vitro and in vivo. Moreover, MEK1 and YAP proteins are closely correlated in human liver cancer samples. Mechanistically, inhibition of MEK1 by both PD98059 and U0126 as well as RNAi reduces beta-transducin repeat containing E3 ubiquitin protein ligase (BTRC), which acts as a potential endogenous YAP protector.


Assuntos
Carcinogênese/metabolismo , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , MAP Quinase Quinase 1/fisiologia , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional , Animais , Carcinogênese/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Proteínas de Ciclo Celular , Proliferação de Células , Células HEK293 , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Nus , Ligação Proteica/fisiologia , Células Tumorais Cultivadas , Regulação para Cima
17.
J Immunol ; 191(4): 1976-83, 2013 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-23842750

RESUMO

Regulatory T cells (Tregs) counteract anticancer immune responses through a number of mechanisms, limiting dendritic cell (DC)-based anticancer immunotherapy. In this study, we investigated the influence of various DC activation stimuli on the Treg functionality. We compared DCs activated by electroporation with mRNA encoding constitutively active TLR4 (caTLR4) and CD40 ligand (DiMix-DCs), or these factors together with mRNA encoding the costimulatory molecule CD70 (TriMix-DCs) with DCs maturated in the presence of a mixture of inflammatory cytokines (DCs maturated with a combination of the cytokines IL-1ß, IL-6, TNF-α, and PGE2) for their ability to counteract Tregs on different levels. We first demonstrated that there was no difference in the extent of Treg induction starting from CD4(+)CD25(-) T cells under the influence of the different DC maturation stimuli. Second, we showed that both DiMix- and TriMix-DCs could partly alleviate Treg inhibition of CD8(+) T cells. Third, we observed that CD8(+) T cells that had been precultured with DiMix-DCs or TriMix-DCs were partially protected against subsequent Treg suppression. Finally, we showed that Tregs cocultured in the presence of TriMix-DCs, but not DiMix-DCs, partially lost their suppressive capacity. This was accompanied by a decrease in CD27 and CD25 expression on Tregs, as well as an increase in the expression of T-bet and secretion of IFN-γ, TNF-α, and IL-10, suggesting a shift of the Treg phenotype toward a Th1 phenotype. In conclusion, these data suggest that TriMix-DCs are not only able to suppress Treg functions, but moreover could be able to reprogram Tregs to Th1 cells under certain circumstances.


Assuntos
Ligante CD27/fisiologia , Ligante de CD40/fisiologia , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Tolerância Imunológica/imunologia , Linfopoese/fisiologia , Linfócitos T Reguladores/imunologia , Receptor 4 Toll-Like/fisiologia , Ligante CD27/genética , Linfócitos T CD4-Positivos/citologia , Ligante de CD40/genética , Diferenciação Celular/efeitos dos fármacos , Divisão Celular , Células Cultivadas , Técnicas de Cocultura , Citocinas/farmacologia , Células Dendríticas/metabolismo , Eletroporação , Humanos , Imunofenotipagem , Ativação Linfocitária , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 1/fisiologia , Monócitos/citologia , Monócitos/efeitos dos fármacos , RNA Mensageiro/administração & dosagem , RNA Mensageiro/genética , Proteínas Recombinantes de Fusão/metabolismo , Linfócitos T Reguladores/citologia , Células Th1/imunologia , Receptor 4 Toll-Like/genética
18.
Am J Pathol ; 183(2): 617-25, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23747947

RESUMO

A number of inhibitors have been used to dissect the functional relevance of Jak2 in endothelial homeostasis, with disparate results. Given that Jak2 deficiency leads to embryonic lethality, the exact role of Jak2 in the regulation of postnatal endothelial function is yet to be fully elucidated. We generated a model in which Jak2 deficiency can be induced by tamoxifen in adult mice. Loss of Jak2 significantly impaired endothelium-dependent response capacity for vasodilators. Matrigel plug assays indicated a notable decrease in endothelial angiogenic function in Jak2-deficient mice. Studies in a hindlimb ischemic model indicated that Jak2 activity is likely to be a prerequisite for prompt perfusion recovery, based on the concordance of temporal changes in Jak2 expression during the course of ischemic injury and perfusion recovery. A remarkable delay in perfusion recovery, along with reduced capillary and arteriole formation, was observed in Jak2-deficient mice. Antibody array studies indicated that loss of Jak2 led to repressed eNOS expression. In mechanistic studies, Jak2 deficiency attenuated Raf-1/MEK1 signaling, which then reduced activity of Sp-1, an essential transcription factor responsible for eNOS expression. These data are important not only for understanding the exact role that Jak2 plays in endothelial homeostasis, but also for assessing Jak2-based therapeutic strategies in a variety of clinical settings.


Assuntos
Janus Quinase 2/deficiência , MAP Quinase Quinase 1/fisiologia , Óxido Nítrico Sintase Tipo III/metabolismo , Proteínas Quinases/fisiologia , Proteínas Proto-Oncogênicas c-raf/fisiologia , Animais , Aorta/efeitos dos fármacos , Aorta/enzimologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/enzimologia , Endotélio Vascular/fisiologia , Inibidores Enzimáticos/farmacologia , Membro Posterior/irrigação sanguínea , Isquemia/enzimologia , Janus Quinase 2/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Tamoxifeno/farmacologia , Vasodilatadores/farmacologia
19.
APMIS ; 121(4): 299-310, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23030595

RESUMO

Interaction between the immune system and cancer cells allows for the use of biological response modifiers, like OK-432, in cancer therapy. We have studied the involvement of monocytes (MOs) in the immune response to OK-432 by examining MCP-1, MIP-1α and MIP-1ß secretion, in vitro. OK-432-induced IL-6/TNF-α secretion has previously been shown to depend on mitogen-activated protein kinases (MAPKs) ERK1/2 and p38, and we therefore investigated the role of these MAPKs in OK-432-induced chemokine secretion. Here we demonstrate that pharmacological MEK1/2 kinase inhibition generally impaired chemokine secretion from MOs, whereas p38 MAPK inhibition in particular reduced MIP-1α production. Furthermore, simultaneous inhibition of MEK1/2 and Syk kinase was seen to have an additive impact on reduced MCP-1, MIP-1α and MIP-1ß secretion. Based on single cell flow cytometry analyses, OK-432, lipoteichoic acid (LTA) and lipopolysaccharide (LPS) were seen to induce p38 MAPK and NF-κB phosphorylation in MOs with different time kinetics. LTA and LPS have been shown to induce ERK1/2 phosphorylation, whereas the levels of phosphorylated ERK1/2 remained constant following OK-432 treatment at the time points tested. Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns, and we demonstrate increased TLR2 cell surface levels on the MO population, most profoundly following stimulation with LTA and OK-432. Together these results indicate that modulation of MEK1/2 and p38 MAPK signalling could affect the response to OK-432 treatment, having the potential to improve its therapeutic potential within cancer and lymphangioma treatment.


Assuntos
Quimiocinas/metabolismo , Fatores Imunológicos/farmacologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Monócitos/efeitos dos fármacos , NF-kappa B/metabolismo , Picibanil/farmacologia , Butadienos/farmacologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 1/fisiologia , MAP Quinase Quinase 2/antagonistas & inibidores , MAP Quinase Quinase 2/fisiologia , Monócitos/imunologia , Monócitos/metabolismo , Nitrilas/farmacologia , Fosforilação/efeitos dos fármacos , Proteínas Tirosina Quinases/antagonistas & inibidores , Quinase Syk , Ácidos Teicoicos/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
20.
J Immunol ; 190(1): 159-67, 2013 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-23225884

RESUMO

MEK1 phosphorylates ERK1/2 and regulates T cell generation, differentiation, and function. MEK1 has recently been shown to translocate to the nucleus. Its nuclear function is largely unknown. By studying human CD4 T cells, we demonstrate that a low level of MEK1 is present in the nucleus of CD4 T cells under basal conditions. T cell activation further increases the nuclear translocation of MEK1. MEK1 interacts with the nuclear receptor corepressor silencing mediator of retinoid and thyroid hormone receptor (SMRT). MEK1 reduces the nuclear level of SMRT in an activation-dependent manner. MEK1 is recruited to the promoter of c-Fos upon TCR stimulation. Conversely, SMRT is bound to the c-Fos promoter under basal conditions and is removed upon TCR stimulation. We examined the role of SMRT in regulation of T cell function. Small interfering RNA-mediated knockdown of SMRT results in a biphasic effect on cytokine production. The production of the cytokines IL-2, IL-4, IL-10, and IFN-γ increases in the early phase (8 h) and then decreases in the late phase (48 h). The late-phase decrease is associated with inhibition of T cell proliferation. The late-phase inhibition of T cell activation is, in part, mediated by IL-10 that is produced in the early phase and, in part, by ß-catenin signaling. Thus, we have identified a novel nuclear function of MEK1. MEK1 triggers a complex pattern of early T cell activation, followed by a late inhibition through its interaction with SMRT. This biphasic dual effect most likely reflects a homeostatic regulation of T cell function by MEK1.


Assuntos
Transporte Ativo do Núcleo Celular/imunologia , Linfócitos T CD4-Positivos/imunologia , MAP Quinase Quinase 1/fisiologia , Correpressor 1 de Receptor Nuclear/fisiologia , Correpressor 2 de Receptor Nuclear/antagonistas & inibidores , Correpressor 2 de Receptor Nuclear/fisiologia , Transporte Ativo do Núcleo Celular/genética , Linfócitos T CD4-Positivos/enzimologia , Linfócitos T CD4-Positivos/metabolismo , Regulação para Baixo/genética , Regulação para Baixo/imunologia , Inativação Gênica/imunologia , Inibidores do Crescimento/genética , Inibidores do Crescimento/metabolismo , Inibidores do Crescimento/fisiologia , Humanos , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 1/metabolismo , Correpressor 2 de Receptor Nuclear/genética , Regiões Promotoras Genéticas/imunologia , Ligação Proteica/genética , Ligação Proteica/imunologia , Proteínas Proto-Oncogênicas c-fos/metabolismo
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