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1.
Cell Prolif ; 53(10): e12901, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32960500

RESUMO

OBJECTIVES: To assess the expression, prognostic value, and functionality of T-lymphokine-activated killer (T-LAK) cell-originated protein kinase (TOPK) in chordoma pathogenesis. MATERIALS AND METHODS: TOPK expression in chordoma was assessed via immunohistochemical staining of a tissue microarray (TMA) and correlated with patient clinicopathology. TOPK expression in chordoma cell lines and fresh patient tissues was then evaluated by Western blot. TOPK small interfering RNA (siRNA) and the specific inhibitor OTS514 were applied to determine the roles of TOPK in chordoma pathogenicity. The effect of TOPK expression on chordoma cell clonogenicity was also investigated using clonogenic assays. A 3D cell culture model was utilized to mimic in vivo environment to validate the effect of TOPK inhibition on chordoma cells. RESULTS: TOPK was highly expressed in 78.2% of the chordoma specimens in the TMA and all chordoma cell lines. High TOPK expression significantly correlated with metastasis, recurrence, disease status and shorter overall survival. Knockdown of TOPK with specific siRNA resulted in significantly decrease chordoma cell viability. Inhibition of TOPK with OTS514 significantly inhibited chordoma cell growth and proliferation, colony-forming capacity and ex vivo spheroid growth. CONCLUSIONS: High expression of TOPK is an important predictor of poor prognosis in chordoma. Inhibition of TOPK resulted in significantly decrease chordoma cell proliferation and increase apoptosis. Our results indicate TOPK as a novel prognostic biomarker and therapeutic target for chordoma.


Assuntos
Neoplasias Ósseas/patologia , Cordoma/patologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/mortalidade , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cordoma/metabolismo , Cordoma/mortalidade , Feminino , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Metástase Neoplásica , Prognóstico , Inibidores de Proteínas Quinases/farmacologia , Quinolonas/farmacologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Tiofenos/farmacologia
2.
PLoS Negl Trop Dis ; 14(8): e0008660, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32866199

RESUMO

Aedes mosquitoes can transmit dengue and several other severe vector-borne viral diseases, thereby influencing millions of people worldwide. Insects primarily control and clear the viral infections via their innate immune systems. Mitogen-Activated Protein Kinases (MAPKs) and antimicrobial peptides (AMPs) are both evolutionarily conserved components of the innate immune systems. In this study, we investigated the role of MAPKs in Aedes mosquitoes following DENV infection by using genetic and pharmacological approaches. We demonstrated that knockdown of ERK, but not of JNK or p38, significantly enhances the viral replication in Aedes mosquito cells. The Ras/ERK signaling is activated in both the cells and midguts of Aedes mosquitoes following DENV infection, and thus plays a role in restricting the viral infection, as both genetic and pharmacological activation of the Ras/ERK pathway significantly decreases the viral titers. In contrast, inhibition of the Ras/ERK pathway enhances DENV infection. In addition, we identified a signaling crosstalk between the Ras/ERK pathway and DENV-induced AMPs in which defensin C participates in restricting DENV infection in Aedes mosquitoes. Our results reveal that the Ras/ERK signaling pathway couples AMPs to mediate the resistance of Aedes mosquitoes to DENV infection, which provides a new insight into understanding the crosstalk between MAPKs and AMPs in the innate immunity of mosquito vectors during the viral infection.


Assuntos
Aedes/virologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Vírus da Dengue/imunologia , Quinases de Proteína Quinase Ativadas por Mitógeno/farmacologia , Mosquitos Vetores/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Animais , Anti-Infecciosos/farmacologia , Linhagem Celular , Sistema Digestório/virologia , Feminino , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Imunidade Inata , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Mosquitos Vetores/virologia , Carga Viral , Replicação Viral/efeitos dos fármacos
3.
Sci Rep ; 10(1): 11574, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32665693

RESUMO

Drug combinations have been proposed to combat drug resistance, but putative treatments are challenged by low bench-to-bed translational efficiency. To explore the effect of cell culture format and readout methods on identification of synergistic drug combinations in vitro, we studied response to 21 clinically relevant drug combinations in standard planar (2D) layouts and physiologically more relevant spheroid (3D) cultures of HCT-116, HT-29 and SW-620 cells. By assessing changes in viability, confluency and spheroid size, we were able to identify readout- and culture format-independent synergies, as well as synergies specific to either culture format or readout method. In particular, we found that spheroids, compared to 2D cultures, were generally both more sensitive and showed greater synergistic response to combinations involving a MEK inhibitor. These results further shed light on the importance of including more complex culture models in order to increase the efficiency of drug discovery pipelines.


Assuntos
Neoplasias do Colo/tratamento farmacológico , Detecção Precoce de Câncer , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Inibidores de Proteínas Quinases/farmacologia , Antineoplásicos/farmacologia , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Células HT29 , Ensaios de Triagem em Larga Escala , Humanos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Esferoides Celulares/efeitos dos fármacos
4.
Sci Rep ; 10(1): 10282, 2020 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-32581305

RESUMO

Kidney stone disease (KSD) is a prevalent disorder that causes human morbidity worldwide. The etiology of KSD is heterogeneous, ranging from monogenic defect to complex interaction between genetic and environmental factors. Since mutations of genes responsible for KSD in a majority of families are still unknown, our group is identifying mutations of these genes by means of genomic and genetic analyses. In this study, we identified a novel loss-of-function mutation of PBK, encoding the PDZ binding kinase, that was found to be associated with KSD in an affected Thai family. Glycine (Gly) substituted by arginine (Arg) at position 43 (p.Gly43Arg) in PBK cosegregated with the disease in affected members of this family, but was absent in 180 normal control subjects from the same local population. Gly43 is highly evolutionarily conserved in vertebrates, and its substitution affects protein structure by alterations in H-bond forming patterns. This p.Gly43Arg substitution results in instability of the variant PBK protein as examined in HEK293T cells. The variant PBK protein (p.Gly43Arg) demonstrated decreased kinase activity to phosphorylate p38 MAPK as analyzed by immunoblotting and antibody microarray techniques. Taken together, these findings suggest a possible new mechanism of KSD associated with pathogenic PBK variation.


Assuntos
Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Substituição de Aminoácidos , Análise Mutacional de DNA , Feminino , Células HEK293 , Humanos , Cálculos Renais/genética , Mutação com Perda de Função , Masculino , Pessoa de Meia-Idade , Linhagem , Estabilidade Proteica , Tailândia
5.
Cancer Res ; 80(16): 3413-3423, 2020 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-32586982

RESUMO

Survival for high-risk neuroblastoma remains poor and treatment for relapsed disease rarely leads to long-term cures. Large sequencing studies of neuroblastoma tumors from diagnosis have not identified common targetable driver mutations other than the 10% of tumors that harbor mutations in the anaplastic lymphoma kinase (ALK) gene. However, at neuroblastoma recurrence, more frequent mutations in genes in the RAS-MAPK pathway have been detected. The PTPN11-encoded tyrosine phosphatase SHP2 is an activator of the RAS pathway, and we and others have shown that pharmacologic inhibition of SHP2 suppresses the growth of various tumor types harboring KRAS mutations such as pancreatic and lung cancers. Here we report inhibition of growth and downstream RAS-MAPK signaling in neuroblastoma cells in response to treatment with the SHP2 inhibitors SHP099, II-B08, and RMC-4550. However, neuroblastoma cell lines harboring endogenous NRAS Q61K mutation (which is commonly detected at relapse) or isogenic neuroblastoma cells engineered to overexpress NRASQ61K were distinctly resistant to SHP2 inhibitors. Combinations of SHP2 inhibitors with other RAS pathway inhibitors such as trametinib, vemurafenib, and ulixertinib were synergistic and reversed resistance to SHP2 inhibition in neuroblastoma in vitro and in vivo. These results suggest for the first time that combination therapies targeting SHP2 and other components of the RAS-MAPK pathway may be effective against conventional therapy-resistant relapsed neuroblastoma, including those that have acquired NRAS mutations. SIGNIFICANCE: These findings suggest that conventional therapy-resistant, relapsed neuroblastoma may be effectively treated via combined inhibition of SHP2 and MEK or ERK of the RAS-MAPK pathway.


Assuntos
Genes ras , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Recidiva Local de Neoplasia/tratamento farmacológico , Neuroblastoma/tratamento farmacológico , Proteína Tirosina Fosfatase não Receptora Tipo 11/antagonistas & inibidores , Aminopiridinas/uso terapêutico , Animais , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Sinergismo Farmacológico , Xenoenxertos , Humanos , Indóis/uso terapêutico , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Camundongos Endogâmicos NOD , Camundongos SCID , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Mutação , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Neuroblastoma/genética , Neuroblastoma/patologia , Piperidinas/uso terapêutico , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Piridonas/uso terapêutico , Pirimidinas/uso terapêutico , Pirimidinonas/uso terapêutico , Pirróis/uso terapêutico , Triazóis/uso terapêutico , Vemurafenib/uso terapêutico
6.
BMC Plant Biol ; 20(1): 194, 2020 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-32381024

RESUMO

BACKGROUND: The mitogen-activated protein kinases (MAPKs), as a part of the MAPKKK-MAPKK-MAPK cascade, play crucial roles in plant development as an intracellular signal transduction pathway to respond various environmental signals. However, few MAPKK have been functionally characterized in grapevine. RESULTS: In the study, five MAPKK (MKK) members were identified in grapevine (cultivar 'Pinot Noir'), cloned and designated as VvMKK1-VvMKK5. A phylogenetic analysis grouped them into four sub-families based on the similarity of their conserved motifs and gene structure to Arabidopsis MAPKK members. qRT-PCR results indicated that the expression of VvMKK1, VvMKK2, VvMKK4, and VvMKK5 were up-regulated in mature leaf and young blades, and roots, but exhibited low expression in leaf petioles. VvMKK2, VvMKK3, and VvMKK5 genes were differentially up-regulated when grapevine leaves were inoculated with spores of Erisyphe necator, or treated with salicylic acid (SA), ethylene (ETH), H2O2, or exposed to drought, indicating that these genes may be involved in a variety of signaling pathways. Over expression of VvMKK2 and VvMKK4 genes in transgenic Arabidopsis plants resulted in the production of seeds with a significantly higher germination and survival rate, and better seedling growth under stress conditions than wild-type plants. Overexpression of VvMKK2 in Arabidopsis improved salt and drought stress tolerance while overexpression of VvMKK4 only improved salt stress tolerance. CONCLUSIONS: Results of the present investigation provide a better understanding of the interaction and function of MAPKKK-MAPKK-MAPK genes at the transcriptional level in grapevine and led to the identification of candidate genes for drought and salt stress in grapes.


Assuntos
Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Proteínas de Plantas/genética , Vitis/enzimologia , Vitis/genética , Arabidopsis/genética , Clonagem Molecular , Genes de Plantas , Plantas Geneticamente Modificadas , Estresse Fisiológico
7.
Dev Biol ; 463(1): 63-76, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32360193

RESUMO

Capturing stable embryonic stem cell (ESC) lines from domesticated animals still remains one of the challenges of non-rodent embryology. The stake is high, as stable ESCs derived from species such as cattle present high economic and scientific value. Understanding of the processes leading to the embryonic lineage segregation is crucial to provide species-orientated molecular environment capable of supporting self-renewal and pluripotency. Therefore, the aim of this study was to validate the action of the two core regulatory pathways (WNT and MEK/ERK) during bovine embryo development. In vitro produced bovine embryos were obtained in the presence of inhibitors (i), which enable activation of the WNT pathway (via GSK3i, CHIR99021) and suppression of MEK signalling by PD0325901 in the 2i system and PD184325 and SU5402 in the 3i system. We have followed the changes in the distribution of the key lineage specific markers both at the transcript and protein level. Our results showed that WNT signalling promotes the expression of key inner cell mass (ICM) specific markers in bovine embryos, regardless of the MEK/ERK inhibitor cocktail used. MEK/ERK downregulation is crucial to maintain OCT4 and NANOG expression within the ICM and to prevent their exclusion from the trophectoderm (TE). At the same time, the classical TE marker (CDX2) was downregulated at the mRNA and protein level. As a follow up for the observed pluripotency stimulating effect of the inhibitors, we have tested the potential of the 2i and the 3i culture conditions (supported by LIF) to derive primary bovine ESC lines. As a result, we propose a model in which all of the primary signalling pathways determining embryonic cell fate are active in bovine embryos, yet the requirement for pluripotency maintenance in cattle may differ from the described standards. WNT activation leads to the formation (and stabilisation of the ICM) and MEK/ERK signalling is maintained at low levels. Unlike in the mouse, GATA6 is expressed in both ICM and TE. MEK/ERK signalling affects HP formation in cattle, but this process is activated at the post-blastocyst stage. With regard to self-renewal, 2i is preferable, as 3i also blocks the FGF receptor, what may prevent PI3K signalling, important for pluripotency and self-renewal.


Assuntos
Blastocisto/metabolismo , Células-Tronco Pluripotentes/metabolismo , Via de Sinalização Wnt/fisiologia , Animais , Benzamidas/farmacologia , Bovinos , Diferenciação Celular , Difenilamina/análogos & derivados , Difenilamina/farmacologia , Técnicas de Cultura Embrionária , Implantação do Embrião/fisiologia , Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento/genética , Camadas Germinativas/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Células-Tronco Pluripotentes/fisiologia , Inibidores de Proteínas Quinases/farmacologia
8.
Oncogene ; 39(21): 4170-4182, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32277233

RESUMO

Nonmelanoma skin cancer (NMSC) such as cutaneous squamous cell carcinoma (cSCC) is caused by solar ultraviolet (SUV) exposure and is the most common cancer in the United States. T-LAK cell-originated protein kinase (TOPK), a serine-threonine kinase is activated by SUV irradiation and involved in skin carcinogenesis. Strategies with research focusing on the TOPK signaling pathway and targeted therapy in skin carcinogenesis may helpful for the discovery of additional treatments against skin cancer. In this study, we found that TOPK can directly bind to and phosphorylate c-Jun (as one of the core member of AP-1) at Ser63 and Ser73 after SSL exposure in a JNKs-independent manner. TOPK knocking down, or HI-TOPK-032 (TOPK specific inhibitor) attenuated colony formation and cell proliferation of skin cancer cells. Phosphorylated levels of c-Jun were overexpressed in human AK and cSCC compared with normal skin tissues, and HI-TOPK-032 inhibited the phosphorylation of c-Jun in SCC cell line in a dose-dependent manner. Furthermore, HI-TOPK-032 decreased SSL-induced AP-1 transactivation activity. Moreover, acute SSL-induced inflammation was attenuated by the topical application of HI-TOPK-032 in SKH1 hairless mice. Importantly, HI-TOPK-032 suppressed chronic SSL-induced skin carcinogenesis and c-Jun phosphorylation levels in SKH1 hairless mice. Our results demonstrate that TOPK can phosphorylate and activate c-Jun at Ser63 and Ser73 in the process of skin carcinogenesis and HI-TOPK-032 could be used as a potential chemopreventive drug against cSCC development.


Assuntos
Carcinogênese , Indolizinas/farmacologia , Quinases de Proteína Quinase Ativadas por Mitógeno , Proteínas de Neoplasias , Inibidores de Proteínas Quinases/farmacologia , Quinoxalinas/farmacologia , Neoplasias Cutâneas , Luz Solar/efeitos adversos , Raios Ultravioleta/efeitos adversos , Animais , Carcinogênese/efeitos dos fármacos , Carcinogênese/genética , Carcinogênese/metabolismo , Carcinogênese/efeitos da radiação , Humanos , Camundongos , Camundongos Pelados , Camundongos Knockout , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/enzimologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia
9.
Yi Chuan ; 42(4): 403-421, 2020 Apr 20.
Artigo em Chinês | MEDLINE | ID: mdl-32312709

RESUMO

Mitogen-activated protein kinase kinase (MAPKK or MKK) is an important component of the MAPK cascade, which plays important roles in plant growth and development as well as in various stress responses. At present, the MKK gene family has been identified in a variety of plants, but there has been no systematic study in Cruciferous plant Arabidopsis pumila. To explore the evolution and function of the MKK gene family in Arabidopsis pumila, 16 ApMKK genes were identified from the Arabidopsis pumila genome by genome-wide analysis, and they were distributed on 10 chromosomes of Arabidopsis pumila. According to phylogenetic analysis and multiple sequence alignment, these putative genes were divided into five known subfamilies, i.e, Groups A, B, C, D, and E, which includes 5, 2, 4, 3, 2 members, respectively. Evolutionary and syntenic analysis showed that there are seven pairs of duplication genes in Arabidopsis pumila: ApMKK1-1/1-2, ApMKK2-1/2-2, ApMKK3-1/3-2, ApMKK4-1/4-2, ApMKK5-1/5-2, ApMKK9-1/9-2, and ApMKK10-1/10-2. Ka/Ks and Tajima analysis indicated that evolution of ApMKK1-1/1-2 was accelerated after the duplication event. Combining the distribution of cis-element in the promoter region of ApMKKs and the expression profile of ApMKKs in mature leaves, stems, flowers and fruits as well as under salt stress, we found that the expressions of paralogous genes (duplication genes) were tissue-specific and their functions were diversified. The expression patterns of some duplicated genes in tissues were different, but the expression patterns under salt stress were basically the same. These results lay the foundation for analyzing the complex mechanisms of MKK-mediated growth and development and abiotic stress signal transduction pathways in Arabidopsis pumila.


Assuntos
Arabidopsis/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Família Multigênica , Filogenia , Proteínas de Plantas/genética , Regulação da Expressão Gênica de Plantas , Transdução de Sinais , Estresse Fisiológico
10.
Plant Mol Biol ; 103(4-5): 391-407, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32193788

RESUMO

Mitogen-activated protein kinases (MAPKs) are important in regulating plant development as well as stress response. In this study, we genome-widely identified 56 MAPK genes in upland cotton. These MAPK genes unequally distribute on 22 chromosomes of cotton genome, but no MAPK gene is located on At_Chr6, Dt_Chr6, At_Chr13 and Dt_Chr13. The exons and introns in GhMAPK gene family vary widely at the position, number and length. Furthermore, GhMAPK family can be divided into 4 groups (A, B, C and D), and the TEY type of T-loop exists in three groups (A, B and C), but the TDY type of T-loop is only in group D. Further study revealed that some GhMAPK genes (including GhMPK6) are preferentially expressed in elongating fibers. GhMPK6 maintains a high phosphorylation level in elongating fibers, and its phosphorylation was enhanced in fibers by phytohormones brassinosteroid (BR), ethylene and indole-3-acetic acid (IAA). Additionally, GhMPK6 could interact with GhMKK2-2 and GhMKK4, suggesting that GhMKK2-2/4-GhMPK6 module may be involved in phosphorylation of its downstream proteins for regulating fiber elongation of cotton.


Assuntos
Regulação da Expressão Gênica de Plantas/fisiologia , Genoma de Planta , Gossypium/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas de Plantas/metabolismo , Fibra de Algodão , Regulação Enzimológica da Expressão Gênica , Estudo de Associação Genômica Ampla , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Proteínas de Plantas/genética
11.
EMBO J ; 39(5): e103444, 2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-32011004

RESUMO

The MAP kinase (MAPK) Hog1 is the central regulator of osmoadaptation in yeast. When cells are exposed to high osmolarity, the functionally redundant Sho1 and Sln1 osmosensors, respectively, activate the Ste11-Pbs2-Hog1 MAPK cascade and the Ssk2/Ssk22-Pbs2-Hog1 MAPK cascade. In a canonical MAPK cascade, a MAPK kinase kinase (MAP3K) activates a MAPK kinase (MAP2K) by phosphorylating two conserved Ser/Thr residues in the activation loop. Here, we report that the MAP3K Ste11 phosphorylates only one activating phosphorylation site (Thr-518) in Pbs2, whereas the MAP3Ks Ssk2/Ssk22 can phosphorylate both Ser-514 and Thr-518 under optimal osmostress conditions. Mono-phosphorylated Pbs2 cannot phosphorylate Hog1 unless the reaction between Pbs2 and Hog1 is enhanced by osmostress. The lack of the osmotic enhancement of the Pbs2-Hog1 reaction suppresses Hog1 activation by basal MAP3K activities and prevents pheromone-to-Hog1 crosstalk in the absence of osmostress. We also report that the rapid-and-transient Hog1 activation kinetics at mildly high osmolarities and the slow and prolonged activation kinetics at severely high osmolarities are both caused by a common feedback mechanism.


Assuntos
Sistema de Sinalização das MAP Quinases/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Feromônios/metabolismo , Saccharomyces cerevisiae/enzimologia , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , MAP Quinase Quinase Quinases , Proteínas de Membrana , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Concentração Osmolar , Fosforilação , Proteínas Quinases , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Estresse Fisiológico
12.
Int J Biol Macromol ; 149: 600-608, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-32004612

RESUMO

Fucoidan is a fucose-rich polysaccharide that has gained attention for its various anticancer properties. However, the effect and underlying mechanism of fucoidan on triple-negative breast cancer (TNBC) are still unknown. Herein, we investigated the anticancer potential of fucoidan from Laminaria japonica. We found that fucoidan showed modest antiproliferative activity against TNBC cells, while it effectively reduced migratory and invasive capacities. Mechanistically, fucoidan suppressed activation of MAPK and PI3K followed by inhibition of AP-1 and NF-κB signaling in TNBC. Additionally, fucoidan downregulated expressions of proangiogenic factors in TNBC cells, and fucoidan blocked tumor-elicited tube formation by human umbilical vascular endothelial cells (HUVECs). We also observed that fucoidan blocked tumor adhesion and invasion towards HUVECs. Surprisingly, fucoidan robustly suppressed tube formation on HUVECs. Moreover, fucoidan inhibited in vivo angiogenesis and micrometastasis in a transgenic zebrafish model. Together, L. japonica fucoidan exhibits potent antitumor effects by its attenuation of invasiveness and proangiogenesis in TNBC.


Assuntos
Laminaria/química , Neovascularização Patológica/tratamento farmacológico , Polissacarídeos/farmacologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , NF-kappa B/genética , Micrometástase de Neoplasia , Neovascularização Patológica/patologia , Polissacarídeos/química , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição AP-1/genética , Neoplasias de Mama Triplo Negativas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
13.
J Immunol ; 204(5): 1310-1321, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31969384

RESUMO

Mechanical cell-matrix interactions can drive the innate immune responses to infection; however, the molecular underpinnings of these responses remain elusive. This study was undertaken to understand the molecular mechanism by which the mechanosensitive cation channel, transient receptor potential vanilloid 4 (TRPV4), alters the in vivo response to lung infection. For the first time, to our knowledge, we show that TRPV4 protects the lung from injury upon intratracheal Pseudomonas aeruginosa in mice. TRPV4 functions to enhance macrophage bacterial clearance and downregulate proinflammatory cytokine secretion. TRPV4 mediates these effects through a novel mechanism of molecular switching of LPS signaling from predominant activation of the MAPK, JNK, to that of p38. This is accomplished through the activation of the master regulator of inflammation, dual-specificity phosphatase 1. Further, TRPV4's modulation of the LPS signal is mechanosensitive in that both upstream activation of p38 and its downstream biological consequences depend on pathophysiological range extracellular matrix stiffness. We further show the importance of TRPV4 on LPS-induced activation of macrophages from healthy human controls. These data are the first, to our knowledge, to demonstrate new roles for macrophage TRPV4 in regulating innate immunity in a mechanosensitive manner through the modulation of dual-specificity phosphatase 1 expression to mediate MAPK activation switching.


Assuntos
Pulmão , Sistema de Sinalização das MAP Quinases , Ativação de Macrófagos , Macrófagos/imunologia , Pneumonia Bacteriana , Infecções por Pseudomonas , Pseudomonas aeruginosa/imunologia , Canais de Cátion TRPV/imunologia , Animais , Feminino , Humanos , Inflamação/genética , Inflamação/imunologia , Inflamação/microbiologia , Lipopolissacarídeos/imunologia , Pulmão/imunologia , Pulmão/microbiologia , Pulmão/patologia , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/imunologia , Macrófagos/patologia , Camundongos , Camundongos Mutantes , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/imunologia , Pneumonia Bacteriana/genética , Pneumonia Bacteriana/imunologia , Pneumonia Bacteriana/microbiologia , Pneumonia Bacteriana/prevenção & controle , Infecções por Pseudomonas/genética , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/prevenção & controle , Canais de Cátion TRPV/genética
14.
FASEB J ; 34(1): 95-106, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31914697

RESUMO

Diabetic nephropathy (DN) is one of the leading causes of mortality in diabetic patients, but its pathogenesis is unclear. We aimed to study the role of the pro-ANP convertase Corin in the pathogenesis of DN. Corin and ANP expression in DN rat kidneys and high-glucose-treated HK-2 cells was analyzed by real-time PCR, western blotting, and immunohistochemical staining. The effect of Corin-siRNA or ANP-siRNA HK-2 cells on EA.hy926 cell migration was determined by scratch-wound healing assay. The expression of mitogen-activated protein kinase (MAPK) and endothelial NO synthase (eNOS) in EA.hy926 cells treated with conditioned medium from Corin-siRNA- or ANP-siRNA-transfected HK-2 cells was determined by western blotting. We found a significant reduction in Corin and ANP expression in DN rat kidneys. These results were recapitulated in HK-2 cells treated with high glucose. EA.hy926 cells treated with conditioned medium from Corin-deficient HK-2 cells had inhibited migration, increased MAPK activity, and decreased eNOS activity. Similar effects were observed with ANP-siRNA transfection. Finally, adding ANP to the Corin-deficient HK-2 conditioned medium rescued the above defects, indicating that Corin mediates its effects through ANP. In conclusion, Corin plays a renoprotective role through pro-ANP processing, and defects in Corin cause endothelial dysfunction through MAPK and eNOS signaling in DN.


Assuntos
Nefropatias Diabéticas/metabolismo , Endotélio/patologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Serina Endopeptidases/metabolismo , Animais , Linhagem Celular , Proliferação de Células , Sobrevivência Celular , Diabetes Mellitus Experimental , Endotélio/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Glucose/toxicidade , Células Endoteliais da Veia Umbilical Humana , Humanos , Túbulos Renais Proximais/citologia , Masculino , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Óxido Nítrico Sintase Tipo III/genética , Interferência de RNA , RNA Interferente Pequeno , Ratos Sprague-Dawley , Serina Endopeptidases/genética , Serina Endopeptidases/urina
15.
Mol Cell Biol ; 40(7)2020 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-31932483

RESUMO

Oxidation of a highly conserved cysteine (Cys) residue located in the kinase activation loop of mitogen-activated protein kinase kinases (MAPKK) inactivates mammalian MKK6. This residue is conserved in the fission yeast Schizosaccharomyces pombe MAPKK Wis1, which belongs to the H2O2-responsive MAPK Sty1 pathway. Here, we show that H2O2 reversibly inactivates Wis1 through this residue (C458) in vitro We found that C458 is oxidized in vivo and that serine replacement of this residue significantly enhances Wis1 activation upon addition of H2O2 The allosteric MAPKK inhibitor INR119, which binds in a pocket next to the activation loop and C458, prevented the inhibition of Wis1 by H2O2 in vitro and significantly increased Wis1 activation by low levels of H2O2 in vivo We propose that oxidation of C458 inhibits Wis1 and that INR119 cancels out this inhibitory effect by binding close to this residue. Kinase inhibition through the oxidation of a conserved Cys residue in MKK6 (C196) is thus conserved in the S. pombe MAPKK Wis1.


Assuntos
Peróxido de Hidrogênio/farmacologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Cisteína/química , Regulação Fúngica da Expressão Gênica/fisiologia , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Oxirredução , Inibidores de Proteínas Quinases/farmacologia , Proteínas de Schizosaccharomyces pombe/antagonistas & inibidores , Proteínas de Schizosaccharomyces pombe/genética , Alinhamento de Sequência
16.
J Mol Neurosci ; 70(1): 56-64, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31617063

RESUMO

Glioblastoma (GBM) is the most aggressive intracranial tumors. Despite the comprehensive treatments, the median survival of GBM patients is still dismal. Consequently, it is critical to explore potential biomarkers and underlying molecular mechanisms of GBM. We integrated two datasets (GSE19728 and GSE 4290) to identify differentially expressed genes (DEGs) of GBM. Eighty-two GBM samples and 31 brain normal samples were screened by using GEO2R and Draw Venn Diagram. We carried out Database for Annotation, Visualization and Integrated Discovery (DAVID) to analyze gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) to analyze the DEGs. To further screen hub genes, protein-protein interaction (PPI) of these DEGs was visualized by Cytoscape with Search Tool for the Retrieval of Interacting Genes (STRING). GEPIA and UALCAN website were utilized to identify the hub genes expression and survival data. In total, 568 common DEGs were determined, including 141 upregulated genes and 427 downregulated genes. Thirty-five hub genes were identified in the highest module consisting of 35 nodes and 535 edges, which are mainly associated with cell cycle, p53 signaling pathway. According to the further analysis results of hub genes, we found that the PDZ-binding kinase (PBK) gene had a high expression and significantly worse survival in GBM contrasted to brain normal samples (P < 0.05). PBK could be a potential prognostic factor and therapeutic target for GBM treatment. In the future, the potential biomarkers for significant prognostic information can be preliminarily assessed using this method, although further experimentations are needed to verify the results.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias Encefálicas/genética , Glioblastoma/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Mapas de Interação de Proteínas
17.
Biochem Biophys Res Commun ; 522(1): 270-277, 2020 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-31757421

RESUMO

TOPK has been suggested to contribute to invasion of lung, prostate, gastric, pancreatic or breast cancer cells. However, how TOPK mediates TGF-ß1/Smad signaling leading to epithelial-mesenchymal transition (EMT) and invasion of breast cancer cells remains unknown. Here we report that TOPK upregulates T-box transcription factor TBX3 to enhance TGF-ß1-induced EMT and invasion of MDA-MB-231 breast cancer cells. Expression of endogenous TOPK was promoted by TGF-ß1 treatment of MDA-MB-231 cells time-dependently. In addition, knockdown of TOPK attenuated TGF-ß1-induced phosphorylation or transcriptional activity of Smad3. Meanwhile, levels of both mRNA and protein of TBX3 induced by TGF-ß1 were abolished by TOPK depletion. Also, knockdown of TBX3 inhibited TGF-ß1 induction of EMT-related genes Snail, Slug or Fibronectin. Furthermore, ablation of TOPK or TBX3 suppressed TGF-ß1-induced MDA-MB-231 cell invasion. Collectively, we conclude that TOPK positively regulates TBX3 in TGF-ß1/Smad signaling pathway, thereby enhancing EMT and invasion of breast cancer cells, implying a mechanistic role of TOPK in TGF-ß1/Smad signaling.


Assuntos
Neoplasias da Mama/metabolismo , Transição Epitelial-Mesenquimal , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Smad/metabolismo , Proteínas com Domínio T/genética , Fator de Crescimento Transformador beta1/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Transdução de Sinais , Proteínas com Domínio T/metabolismo , Regulação para Cima
18.
Am J Med Genet A ; 182(1): 195-200, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31697451

RESUMO

RASopathies are a group of phenotypically overlapping disorders that arise from dysregulation of the RAS/MAPK pathway. These disorders include Noonan syndrome, Costello syndrome, cardiofaciocutaneous syndrome, and neurofibromatosis-Type 1. While somatic mutations in the three human Ras genes (KRAS, HRAS, and NRAS) are a common finding in a variety of cancers, germline mutations in each of the these genes cause developmental RASopathy phenotypes with mutations in specific genes typically correlating with specific phenotypes. We present the case of a germline heterozygous NRAS mutation producing a severe phenotype involving embryonal rhabdomyosarcoma, severe intellectual disability, and numerous melanocytic nevi in addition to more typical manifestations of Noonan syndrome. Additionally, the specific p.G12R NRAS mutation in this case is a common somatic mutation in cancer cells, and analysis of previously reported NRAS-RASopathy cases suggests that mutations at traditionally oncogenic codons are associated with elevated cancer risk not present with mutations at other sites.


Assuntos
GTP Fosfo-Hidrolases/genética , Predisposição Genética para Doença , Proteínas de Membrana/genética , Rabdomiossarcoma Embrionário/genética , Proteínas ras/genética , Adolescente , Criança , Síndrome de Costello/genética , Síndrome de Costello/patologia , Displasia Ectodérmica/genética , Displasia Ectodérmica/patologia , Facies , Insuficiência de Crescimento/genética , Insuficiência de Crescimento/patologia , Feminino , Mutação em Linhagem Germinativa/genética , Cardiopatias Congênitas/genética , Cardiopatias Congênitas/patologia , Humanos , Deficiência Intelectual/genética , Deficiência Intelectual/patologia , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Neurofibromatose 1/genética , Neurofibromatose 1/patologia , Síndrome de Noonan/genética , Síndrome de Noonan/patologia , Fenótipo , Rabdomiossarcoma Embrionário/patologia , Adulto Jovem
19.
Aging (Albany NY) ; 11(23): 11084-11110, 2019 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-31806859

RESUMO

Low-grade chronic adipose tissue inflammation contributes to the onset and development of aging-related insulin resistance and type 2 diabetes. In the current study, α-mangostin, a xanthone isolated from mangosteen (Garcinia mangostana), was identified to ameliorate lipopolysaccharides-induced acute adipose tissue inflammation in mice, by reducing the expression of pro-inflammatory cytokines and chemokines. In a cohort of young (3 months) and old (18-20 months) mice, α-mangostin mitigated aging-associated adiposity, hyperlipidemia, and insulin resistance. Further study showed that α-mangostin alleviated aging-related adipose tissue inflammation by reducing macrophage content and shifting pro-inflammatory macrophage polarization. Moreover, α-mangostin protected the old mice against liver injury through suppressing the secretion of microRNA-155-5p from macrophages. The above results demonstrated that α-mangostin represents a new scaffold to alleviate adipose tissue inflammation, which might be a novel candidate to treat aging-related metabolic disorders.


Assuntos
Tecido Adiposo/efeitos dos fármacos , Envelhecimento , Inflamação/tratamento farmacológico , Doenças Metabólicas/prevenção & controle , Xantonas/farmacologia , Tecido Adiposo/metabolismo , Animais , Citocinas/genética , Citocinas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , NF-kappa B , Inibidores de Proteínas Quinases/farmacologia , Sirtuína 3/genética , Sirtuína 3/metabolismo
20.
Aging (Albany NY) ; 11(23): 11659-11672, 2019 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-31822637

RESUMO

Nickel (Ni), an environmental hazard, widely causes allergic contact hypersensitivity worldwide. Despite that Ni-stimulated pro-inflammatory response is vital in allergy, the underlying molecular mechanisms remain largely unclear. Here, we demonstrated that NiCl2 activated nuclear factor kappa B (NF-κB), mitogen-activated protein kinases (MAPKs) and interferon regulatory factor 3 (IRF3) signaling pathways in primary bone marrow-derived macrophages (BMDMs), leading to the altered transcription levels of interleukin-1ß (IL-1ß), -6, -8, -18, tumor necrosis factor-α (TNF-α) and interferon ß (INF-ß). We also found that nickel chloride (NiCl2) activated Nod-like receptor 3 (NLRP3) inflammasome pathway, resulting in the proteolytic cleavage and release of IL-1ß. NiCl2 induced the accumulation of mitochondrial reactive oxygen species (mtROS) and the release of mitochondrial DNA (mtDNA), thus activating NLRP3 inflammasome pathway. Additionally, NiCl2-induced apoptosis was dependent on the generation of mtROS, and caspase-1 activation might also partly contribute to the apoptotic process. Altogether, abovementioned results indicate that NiCl2 induces inflammatory activation in BMDMs via NF-κB, MAPKs, IRF3 signaling pathways as well as NLRP3 inflammasome pathway, which provides a mechanism to improve the efficiency of treatment against Ni-induced allergic reactions.


Assuntos
Inflamação/induzido quimicamente , Fator Regulador 3 de Interferon/metabolismo , Macrófagos/efeitos dos fármacos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Níquel/farmacologia , Animais , Apoptose , Sobrevivência Celular/efeitos dos fármacos , Poluentes Ambientais , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Inflamação/metabolismo , Fator Regulador 3 de Interferon/genética , Macrófagos/metabolismo , Potencial da Membrana Mitocondrial , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/fisiologia , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Espécies Reativas de Oxigênio
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