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Propolis is a natural resinous substance made by bees through mixing various plant sources. Propolis has been widely recognized as a functional food due to its diverse range of beneficial bioactivities. However, the therapeutic effects of consuming propolis against atopic dermatitis (AD) remain largely unknown. The current study aimed to investigate the potential efficacy of propolis against AD and explore the active compound as well as the direct molecular target. In HaCaT keratinocytes, propolis inhibited TNF-α-induced interleukin (IL)-6 and IL-8 secretion. It also led to a reduction in chemokines such as monocyte chemoattractant protein-1 (MCP-1) and macrophage-derived chemokine (MDC), while restoring the levels of barrier proteins, filaggrin and involucrin. Propolis exhibited similar effects in AD-like human skin, leading to the suppression of AD markers and the restoration of barrier proteins. In DNCB-induced mice, oral administration of propolis attenuated AD symptoms, improved barrier function, and reduced scratching frequency and transepidermal water loss (TEWL). In addition, propolis reversed the mRNA levels of AD-related markers in mouse dorsal skin. These effects were attributed to caffeic acid phenethyl ester (CAPE), the active compound identified by comparing major components of propolis. Mechanistic studies revealed that CAPE as well as propolis could directly and selectively target MKK4. Collectively, these findings demonstrate that propolis may be used as a functional food agent for the treatment of AD.
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The mitogen-activated protein kinase kinase 4 (MKK4), a member of the MAP kinase kinase family, directly phosphorylates and activates the c-Jun NH2-terminal kinases (JNK), in response to proinflammatory cytokines and cellular stresses. Regulation of the MKK4 activity is considered to be a novel approach for the prevention and treatment of inflammation. The aim of this study was to identify whether fisetin, a potential anti-inflammatory compound, targets MKK4-JNK cascade to inhibit lipopolysaccharide (LPS)-stimulated inflammatory response. RAW264 macrophage pretreated with fisetin following LPS stimulation was used as a cell model to investigate the transactivation and expression of related-inflammatory genes by transient transfection assay, electrophoretic mobility shift assay (EMSA), or enzyme-linked immunosorbent assay (ELISA), and cellular signaling as well as binding of related-signal proteins by Western blot, pull-down assay and kinase assay, and molecular modeling. The transactivation and expression of cyclooxygenase-2 (COX-2) gene as well as prostaglandin E2 (PGE2) secretion induced by LPS were inhibited by fisetin in a dose-dependent manner. Signaling transduction analysis demonstrated that fisetin selectively inhibited MKK4-JNK1/2 signaling to suppress the phosphorylation of transcription factor AP-1 without affecting the NF-κB and Jak2-Stat3 signaling as well as the phosphorylation of Src, Syk, and TAK1. Furthermore, in vitro and ex vivo pull-down assay using cell lysate or purified protein demonstrated that fisetin could bind directly to MKK4. Molecular modeling using the Molecular Operating Environment™ software indicated that fisetin docked into the ATP-binding pocket of MKK4 with a binding energy of -71.75 kcal/mol and formed a 1.70 Å hydrogen bound with Asp247 residue of MKK4. The IC50 of fisetin against MKK4 was estimated as 2.899 µM in the kinase assay, and the ATP-competitive effect was confirmed by ATP titration. Taken together, our data revealed that fisetin is a potent selective ATP-competitive MKK4 inhibitor to suppress MKK4-JNK1/2-AP-1 cascade for inhibiting LPS-induced inflammation.
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Fallo Hepático , Trasplante de Hígado , Humanos , Fallo Hepático/prevención & control , AnimalesRESUMEN
Acute liver failure (ALF) is a critical condition that can lead to substantial liver dysfunction. It is characterized by complex clinical manifestations and rapid progression, presenting significant challenges in diagnosis and treatment. We investigated the protective effect of mefunidone (MFD), a novel antifibrosis pyridone agent, on ALF in mice, and explored its potential mechanism of action. MFD pretreatment can alleviate lipopolysaccharide (LPS) and d-galactosamine (D-GalN)-induced ALF, reduce hepatocyte apoptosis, and reduce inflammation and oxidative stress. Additionally, MFD alleviated LPS/D-GalN-stimulated reactive oxygen species (ROS) production and cell death in AML12 cells. RNA sequencing enrichment analysis showed that MFD significantly affected the Mitogen-Activated Protein Kinase (MAPK) pathway. In vivo and in vitro experiments showed that MFD inhibited MKK4 and JNK phosphorylation. JNK activation caused by MKK4 and JNK activators could eliminate the therapeutic effect of MFD on AML12. In addition, MFD pretreatment alleviated ConA-induced ALF, reduced inflammation and oxidative stress in mice, and reduced mouse mortality. These results suggest that MFD can potentially protect against ALF, partially by inhibiting the MKK4-JNK pathway, and is a promising new therapeutic drug for ALF.
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Fallo Hepático Agudo , MAP Quinasa Quinasa 4 , Piperazinas , Piridonas , Animales , Masculino , Ratones , Línea Celular , Galactosamina/toxicidad , Lipopolisacáridos/toxicidad , Fallo Hepático Agudo/tratamiento farmacológico , Fallo Hepático Agudo/inducido químicamente , Fallo Hepático Agudo/metabolismo , Fallo Hepático Agudo/patología , MAP Quinasa Quinasa 4/metabolismo , MAP Quinasa Quinasa 4/antagonistas & inhibidores , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/fisiología , Ratones Endogámicos C57BL , Estrés Oxidativo/efectos de los fármacos , Piridonas/farmacología , Piridonas/uso terapéutico , Piperazinas/farmacología , Piperazinas/uso terapéuticoRESUMEN
Diminished hepatocyte regeneration is a key feature of acute and chronic liver diseases and after extended liver resections, resulting in the inability to maintain or restore a sufficient functional liver mass. Therapies to restore hepatocyte regeneration are lacking, making liver transplantation the only curative option for end-stage liver disease. Here, we report on the structure-based development and characterization (nuclear magnetic resonance [NMR] spectroscopy) of first-in-class small molecule inhibitors of the dual-specificity kinase MKK4 (MKK4i). MKK4i increased liver regeneration upon hepatectomy in murine and porcine models, allowed for survival of pigs in a lethal 85% hepatectomy model, and showed antisteatotic and antifibrotic effects in liver disease mouse models. A first-in-human phase I trial (European Union Drug Regulating Authorities Clinical Trials [EudraCT] 2021-000193-28) with the clinical candidate HRX215 was conducted and revealed excellent safety and pharmacokinetics. Clinical trials to probe HRX215 for prevention/treatment of liver failure after extensive oncological liver resections or after transplantation of small grafts are warranted.
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Inhibidores Enzimáticos , Fallo Hepático , MAP Quinasa Quinasa 4 , Animales , Humanos , Ratones , Hepatectomía/métodos , Hepatocitos , Hígado , Hepatopatías/tratamiento farmacológico , Fallo Hepático/tratamiento farmacológico , Fallo Hepático/prevención & control , Regeneración Hepática , Porcinos , MAP Quinasa Quinasa 4/antagonistas & inhibidores , Inhibidores Enzimáticos/uso terapéuticoRESUMEN
Esophageal squamous cell carcinoma (ESCC) is a common malignancy worldwide with a low survival rate due to a lack of therapeutic targets. Here, our results showed that nuclear mitotic apparatus protein 1 (NUMA1) transcript and protein levels are significantly upregulated in ESCC patient samples and its high expression predicated poor prognosis. Knock-down of NUMA1 promoted cell apoptosis and suppressed cell proliferation and colony formation. By using cell-derived xenograft (CDX) and patient-derived xenograft (PDX) mice models, we found silencing the NUMA1 expression suppressed tumor progression. In addition, conditional knocking-out of NUMA1 reduced 4NQO-induced carcinogenesis in mice esophagus, which further confirmed the oncogenic role of NUMA1 in ESCC. Mechanistically, from the immunoprecipitation assay we revealed that NUMA1 interacted with GSTP1 and TRAF2, promoted the association of TRAF2 with GSTP1 while inhibited the interaction of TRAF2 and ASK1, thus to regulate sustained activation of JNK. In summary, our findings suggest that NUMA1 plays an important role during ESCC progression and it functions through regulating ASK1-MKK4-SAPK/JNK signaling pathway.
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Neoplasias Esofágicas , Carcinoma de Células Escamosas de Esófago , Humanos , Animales , Ratones , Carcinoma de Células Escamosas de Esófago/genética , Sistema de Señalización de MAP Quinasas , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patología , Factor 2 Asociado a Receptor de TNF/metabolismo , Línea Celular Tumoral , Proliferación Celular , Apoptosis , Regulación Neoplásica de la Expresión Génica , Movimiento Celular , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismoRESUMEN
Mitogen-activated protein kinase kinase 4 (MKK4), serves as a critical component of the mitogen-activated protein kinase signaling pathway, facilitating the direct phosphorylation and activation of the c-Jun N-terminal kinase (JNK) and p38 families of MAP kinases in response to environmental stresses. In the current research, we identified two MKK4 subtypes, namely SpMKK4-1 and SpMKK4-2, from Scylla paramamosain, followed by the analysis of their molecular characteristics and tissue distributions. The expression of SpMKK4s was induced upon WSSV and Vibrio alginolyticus challenges, and the bacteria clearance capacity and antimicrobial peptide (AMP) genes' expression upon bacterial infection were significantly decreased after knocking down SpMKK4s. Additionally, the overexpression of both SpMKK4s remarkably activated NF-κB reporter plasmid in HEK293T cells, suggesting the activation of the NF-κB signaling pathway. These results indicated the participation of SpMKK4s in the innate immunity of crabs, which shed light on a better understanding of the mechanisms through which MKK4s regulate innate immunity.
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Braquiuros , Virus del Síndrome de la Mancha Blanca 1 , Humanos , Animales , FN-kappa B , Vibrio alginolyticus/fisiología , Virus del Síndrome de la Mancha Blanca 1/fisiología , MAP Quinasa Quinasa 4/genética , Filogenia , Células HEK293 , Perfilación de la Expresión Génica , Inmunidad Innata , Proteínas Quinasas Activadas por Mitógenos , Proteínas de Artrópodos/metabolismoRESUMEN
AIMS: Acute liver failure (ALF) is a life-threatening disease characterized by abrupt and extensive hepatic necrosis and apoptosis, resulting in high mortality. The approved drug, N-acetylcysteine (NAC), is only effective for acetaminophen (APAP)-associated ALF at the early stage. Thus, we investigate whether fluorofenidone (AKF-PD), a novel antifibrosis pyridone agent, protects against ALF in mice and explore its underlying mechanisms. METHODS: ALF mouse models were established using APAP or lipopolysaccharide/D-galactosamine (LPS/D-Gal). Anisomycin and SP600125 were used as JNK activator and inhibitor, respectively, and NAC served as a positive control. Mouse hepatic cell line AML12 and primary mouse hepatocytes were used for in vitro studies. RESULTS: AKF-PD pretreatment alleviated APAP-induced ALF with decreased necrosis, apoptosis, reactive oxygen species (ROS) markers, and mitochondrial permeability transition in liver. Additionally, AKF-PD alleviated mitochondrial ROS stimulated by APAP in AML12 cells. RNA-sequencing in the liver and subsequent gene set enrichment analysis showed that AKF-PD significantly impacted MAPK and IL-17 pathway. In vitro and in vivo studies demonstrated that AKF-PD inhibited APAP-induced phosphorylation of MKK4/JNK, while SP600125 only inhibited JNK phosphorylation. The protective effect of AKF-PD was abolished by anisomycin. Similarly, AKF-PD pretreatment abolished hepatotoxicity caused by LPS/D-Gal, decreased ROS levels, and diminished inflammation. Furthermore, unlike NAC, AKF-PD, inhibited the phosphorylation of MKK4 and JNK upon pretreatment, and improved survival in cases of LPS/D-Gal-induced mortality with delayed dosing. CONCLUSIONS: In summary, AKF-PD can protect against ALF caused by APAP or LPS/D-Gal, in part, via regulating MKK4/JNK pathway. AKF-PD might be a novel candidate drug for ALF.
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Fallo Hepático Agudo , Sistema de Señalización de MAP Quinasas , Ratones , Animales , Especies Reactivas de Oxígeno/metabolismo , Acetaminofén/metabolismo , Lipopolisacáridos/farmacología , Anisomicina/metabolismo , Anisomicina/farmacología , Hígado , Fallo Hepático Agudo/inducido químicamente , Fallo Hepático Agudo/tratamiento farmacológico , Fallo Hepático Agudo/prevención & control , Piridonas/farmacología , Necrosis/metabolismo , Ratones Endogámicos C57BL , HepatocitosRESUMEN
MKK4 (mitogen-activated protein kinase kinase 4; also referred to as MEK4) is a dual-specificity protein kinase that phosphorylates and regulates both JNK (c-Jun N-terminal kinase) and p38 MAPK (p38 mitogen-activated protein kinase) signaling pathways and therefore has a great impact on cell proliferation, differentiation and apoptosis. Overexpression of MKK4 has been associated with aggressive cancer types, including metastatic prostate and ovarian cancer and triple-negative breast cancer. In addition, MKK4 has been identified as a key regulator in liver regeneration. Therefore, MKK4 is a promising target both for cancer therapeutics and for the treatment of liver-associated diseases, offering an alternative to liver transplantation. The recent reports on new inhibitors, as well as the formation of a startup company investigating an inhibitor in clinical trials, show the importance and interest of MKK4 in drug discovery. In this review, we highlight the significance of MKK4 in cancer development and other diseases, as well as its unique role in liver regeneration. Furthermore, we present the most recent progress in MKK4 drug discovery and future challenges in the development of MKK4-targeting drugs.
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Neoplasias Ováricas , Proteínas Quinasas p38 Activadas por Mitógenos , Femenino , Humanos , Masculino , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , MAP Quinasa Quinasa 4/metabolismo , Neoplasias Ováricas/patología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , FosforilaciónRESUMEN
Triple-negative breast cancer (TNBC) is one of the most malignant tumors with poor prognosis. Methuosis is a new type of nonapoptotic cell death characterized by the accumulation of cytoplasmic vacuoles. In this study, we synthesized and screened a series of N-phenyl-4-pyrimidinediamine derivatives in TNBC cells, finding that DZ-514 was the best compound with high toxicity independent of the inhibition of BCL6. DZ-514 decreased cell viability, inhibited cell cycle progression, and induced caspase-independent cell death in TNBC cells. Interestingly, DZ-514 induced cytoplasm vacuolation, which could be blocked by Baf A1, the V-ATPase inhibitor. Furthermore, we found that DZ-514-induced vacuoles were derived from macropinosomes rather than autophagosomes. Most importantly, methuosis induced by DZ-514 was partially mediated by activating the ROS-MKK4-p38 axis. Finally, we demonstrated that DZ-514 significantly inhibited tumor growth in an HCC1806 xenograft mouse model. These findings revealed that the novel methuosis inducer DZ-514 could be developed for TNBC treatment.
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Neoplasias de la Mama Triple Negativas , Animales , Humanos , Ratones , Apoptosis , Muerte Celular , Línea Celular Tumoral , Proliferación Celular , Endosomas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Ensayos Antitumor por Modelo de Xenoinjerto , MAP Quinasa Quinasa 4/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
BACKGROUND: Activation of blood stasis is a crucial aspect of stroke treatment, and the Tong-Qiao-Huo-Xue-Decoction (TQHXD) formula is commonly utilized for this purpose. However, the mechanism underlying the protective effects of TQHXD against cerebral ischemia-reperfusion (I/R) injury is unclear. PURPOSE: Identification of the TQHXD components responsible for its protective effects and determination of their mode of action against cerebral I/R injury. METHODS: Gas chromatography (GC) and high-performance liquid chromatography (HPLC) were carried out to determine the active aspects of TQHXD. The active components and targets of TQHXD were looked up in the TCMSP and HERB databases; the Genecards, OMIM, TTD, and DrugBank databases were used to identify targets related to cerebral infarction; and the intersecting targets were obtained. The drug-ingredient-target-disease network and PPI network were subsequently built using Cytoscape 3.7.1 and STRING websites. Autodock VINA was used to perform molecular docking between the core target ASK1 and the active components of TQHXD detected by HPLC and GC. After successfully creating a rat model of middle cerebral artery occlusion (MCAO), the therapeutic effect of TQHXD was observed using triphenyltetrazolium and hematoxylin-eosin staining. We used Tunel-NeuN staining and transmission electron microscopy (TEM) to quantify hippocampal apoptosis. RT-qPCR and western blotting were used to detect protein and mRNA expression, respectively. RESULTS: HPLC and GC identified six active ingredients. Network pharmacology analyses were performed to test 66 intersection targets, including ASK1, MKK4, and JNK. Ferulic acid, HSYA, ligustilide, paeoniflorin, and muscone all displayed high binding affinity with ASK1 in molecular docking studies. The neuroprotective effects of TQHXD in I/R rats were demonstrated in the experimental models. In comparison with the model group, TQHXD decreased the apoptosis rate and reduced the protein levels of p-ASK1, caspase 3, p-MKK4, CytC, p-c-Jun, Bax/Bcl-2, and p-JNK, while considerably increasing the mRNA levels of Bcl-2 and decreasing those of Bax. CONCLUSION: By controlling the ASK1/MKK4/JNK pathway, TQHXD protects neurons from I/R damage and prevents apoptosis. Thus, TQHXD may be effective for the treatment of ischemic stroke. And the mechanism behind these therapeutic actions of TQHXD is supported by this research.
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Fármacos Neuroprotectores , Daño por Reperfusión , Animales , Apoptosis , Caspasa 3/metabolismo , Medicamentos Herbarios Chinos , Eosina Amarillenta-(YS)/farmacología , Eosina Amarillenta-(YS)/uso terapéutico , Hematoxilina/farmacología , Hematoxilina/uso terapéutico , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Sistema de Señalización de MAP Quinasas , Simulación del Acoplamiento Molecular , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , ARN Mensajero/metabolismo , Ratas , Daño por Reperfusión/tratamiento farmacológico , Proteína X Asociada a bcl-2/metabolismoRESUMEN
The mitogen-activated protein kinase kinase 4 (MKK4) has recently been identified as druggable target for the treatment of acute liver failure in RNAi experiments. In these experiments MKK4 was identified to be a major regulator in hepatocyte regeneration. Inhibitors thereof may serve as medication to promote liver regeneration or reducing hepatocyte death. Just a small number of potent inhibitors with acceptable selectivity towards relevant off-targets are known up to date. Among the known potent inhibitors, selectivity is highly sensitive towards minor modifications of the molecule, which makes it necessary to carefully balance between potency and selectivity. In the herein presented study, a new class of Vemurafenib-derived inhibitors was investigated with α-carbolines as new scaffold. This new scaffold showed a remarkable intrinsic selectivity towards the chosen off-targets, without affecting potency towards MKK4 on a broad range of structural modifications.
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Hepatocitos , MAP Quinasa Quinasa 4 , Hepatocitos/metabolismo , Vemurafenib/farmacologíaRESUMEN
Chemotherapy-induced side effects affect the quality of life and efficacy of treatment of cancer patients. Current approaches for treating the side effects of chemotherapy are poorly effective and may cause numerous harmful side effects. Therefore, developing new and effective drugs derived from natural non-toxic compounds for the treatment of chemotherapy-induced side effects is necessary. Experiments in vivo and in vitro indicate that Panax ginseng (PG) and its ginsenosides are undoubtedly non-toxic and effective options for the treatment of chemotherapy-induced side effects, such as nephrotoxicity, hepatotoxicity, cardiotoxicity, immunotoxicity, and hematopoietic inhibition. The mechanism focus on anti-oxidation, anti-inflammation, and anti-apoptosis, as well as the modulation of signaling pathways, such as nuclear factor erythroid-2 related factor 2 (Nrf2)/heme oxygenase-1 (HO-1), P62/keap1/Nrf2, c-jun N-terminal kinase (JNK)/P53/caspase 3, mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinases (ERK), AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR), mitogen-activated protein kinase kinase 4 (MKK4)/JNK, and phosphatidylinositol 3-kinase (PI3K)/AKT. Since a systemic review of the effect and mechanism of PG and its ginsenosides on chemotherapy-induced side effects has not yet been published, we provide a comprehensive summarization with this aim and shed light on the future research of PG.
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OBJECTIVES: The mitogen-activated protein kinase kinase 4 (MKK4) plays a key role in several processes like inflammation, apoptosis, and tumorigenesis. Several authors have proposed that genetic variations in these genes may alter their expression with subsequent cancer risk. This study aimed to examine the possible association of MKK4 rs3826392 and rs3809728 variants in Mexican patients with colorectal cancer (CRC). These variants were also compared with clinical features as sex, age, TNM stage, and tumor location. MATERIALS AND METHODS: The study included genomic DNA from 218 control subjects and 250 patients. Genotyping of the MKK4 variants was performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) procedure. RESULTS: Individuals with A/T and T/T genotypes for the rs3809728 (-1044 A>T) variant showed a significantly increased risk for CRC (P=0.012 and 0.007, respectively); while individuals with the G/G genotype for the rs3826392 (-1304 T>G) variant showed a decreased risk for CRC (P=0.012). Genotypes of the MKK4 rs3809728 variant were also significantly related to colon localization and advanced TNM stage in CRC patients. T-T haplotype (rs3826392 and rs3809728) of the MKK4 gene was associated with risk in patients with CRC. CONCLUSION: The rs3826392 variant in the MKK4 gene could be a cancer protective factor, while the rs3809728 variant could be a risk factor. These variants play a significant role in CRC risk.
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Agrobacterium tumefaciens is a specialized plant pathogen that causes crown gall disease and is commonly used for Agrobacterium-mediated transformation. As a pathogen, Agrobacterium triggers plant immunity, which affects transformation. However, the signaling components and pathways in plant immunity to Agrobacterium remain elusive. We demonstrate that two Arabidopsis mitogen-activated protein kinase kinases (MAPKKs) MKK4/MKK5 and their downstream mitogen-activated protein kinases (MAPKs) MPK3/MPK6 play major roles in both Agrobacterium-triggered immunity and Agrobacterium-mediated transformation. Agrobacteria induce MPK3/MPK6 activity and the expression of plant defense response genes at a very early stage. This process is dependent on the MKK4/MKK5 function. The loss of the function of MKK4 and MKK5 or their downstream MPK3 and MPK6 abolishes plant immunity to agrobacteria and increases transformation frequency, whereas the activation of MKK4 and MKK5 enhances plant immunity and represses transformation. Global transcriptome analysis indicates that agrobacteria induce various plant defense pathways, including reactive oxygen species (ROS) production, ethylene (ET), and salicylic acid- (SA-) mediated defense responses, and that MKK4/MKK5 is essential for the induction of these pathways. The activation of MKK4 and MKK5 promotes ROS production and cell death during agrobacteria infection. Based on these results, we propose that the MKK4/5-MPK3/6 cascade is an essential signaling pathway regulating Agrobacterium-mediated transformation through the modulation of Agrobacterium-triggered plant immunity.
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(1) Background: The c-Jun-NH2-terminal protein kinase (JNK) is a mitogen-activated protein kinase involved in regulating physiological processes in the central nervous system. However, the dual genetic deletion of Mkk4 and Mkk7 (upstream activators of JNK) in adult mice is not reported. The aim of this study was to induce the genetic deletion of Mkk4/Mkk7 in adult mice and analyze their effect in hippocampal neurogenesis. (2) Methods: To achieve this goal, Actin-CreERT2 (Cre+/-), Mkk4flox/flox, Mkk7flox/flox mice were created. The administration of tamoxifen in these 2-month-old mice induced the gene deletion (Actin-CreERT2 (Cre+/-), Mkk4∆/∆, Mkk7∆/∆ genotype), which was verified by PCR, Western blot, and immunohistochemistry techniques. (3) Results: The levels of MKK4/MKK7 at 7 and 14 days after tamoxifen administration were not eliminated totally in CNS, unlike what happens in the liver and heart. These data could be correlated with the high levels of these proteins in CNS. In the hippocampus, the deletion of Mkk4/Mkk7 induced a misalignment position of immature hippocampal neurons together with alterations in their dendritic architecture pattern and maturation process jointly to the diminution of JNK phosphorylation. (4) Conclusion: All these data supported that the MKK4/MKK7-JNK pathway has a role in adult neurogenic activity.
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Hipocampo/fisiología , MAP Quinasa Quinasa 4/fisiología , MAP Quinasa Quinasa 7/fisiología , Sistema de Señalización de MAP Quinasas , Neurogénesis , Animales , Proteína Doblecortina , Eliminación de Gen , Ratones TransgénicosRESUMEN
Currently, the therapeutic options for treatment of liver failure are very limited. As mitogen-activated protein kinase kinase 4 (MKK4) has recently been identified by in vivo RNAi experiments to be a major regulator in hepatocyte regeneration, we pursued the development of a small molecule targeting this protein kinase. Starting from the approved BRAFV600E inhibitor vemurafenib (8), that showed a high off-target affinity to MKK4 in an initial screening, we followed a scaffold-hopping approach, changing the core heterocycle from 1H-pyrrolo[2,3-b]pyridine to 1H-pyrazolo[2,3-b]pyridine (10). Affinity to MKK4 could be conserved while the selectivity against off-target protein kinases was slightly improved. Further modifications led to 58 and 59 showing high affinity to MKK4 in the low nanomolar range and excellent selectivity profile from mandatory multiparameter-optimization for the essential anti-targets (MKK7, JNK1) and off-targets (BRAF, MAP4K5, ZAK) in the MKK4 pathway. Herein we report the first selective MKK4 inhibitors in this class.
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Diseño de Fármacos , Regeneración Hepática/efectos de los fármacos , MAP Quinasa Quinasa 4/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Pirazoles/farmacología , Piridinas/farmacología , Relación Dosis-Respuesta a Droga , Humanos , MAP Quinasa Quinasa 4/metabolismo , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Pirazoles/síntesis química , Pirazoles/química , Piridinas/síntesis química , Piridinas/química , Relación Estructura-ActividadRESUMEN
Mitogen-activated protein kinase 4, MKK4, is a key upstream kinase in the JNK/p38 MAPK pathway that has been reported to participate in multiple immune responses. In this study, the gene that encodes ApMKK4 was isolated and identified from Artemia parthenogenetica. It was found to contain a 1134 bp open reading frame encoding 378 amino acids. The predicted protein contains D domain, DVD domain and kinase domain. Homology analysis revealed that ApMKK4 shares 38-69% identity with MKK4 homologs from other species. Results revealed that ApMKK4 was mainly expressed during early development of which highest at the gastrula stage. After challenged by Vibrio harveyi and Micrococcus lysodeikticus, ApMKK4 was remarkably upregulated at 10 and 103 cfu/mL bacterial concentrations, respectively. Through siRNAi, the transcript level of ApMKK4 was significantly decreased by 46-67%. Intriguingly, when the ApMKK4-knockdown nauplii faced with bacterial stimulation, the expression of ApMKK4 was completely restored in a short time. Moreover, this phenomenon also occurred in related antimicrobial peptide genes, ABF-1 and ABF-2. Our research reveals that ApMKK4 plays a pivotal role during early development and immune responses against bacterial infections.
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Artemia/genética , Artemia/inmunología , Proteínas de Artrópodos/genética , Proteínas de Artrópodos/inmunología , Regulación de la Expresión Génica/inmunología , Inmunidad Innata/genética , Secuencia de Aminoácidos , Animales , Proteínas de Artrópodos/química , Perfilación de la Expresión Génica , MAP Quinasa Quinasa 4/química , MAP Quinasa Quinasa 4/genética , MAP Quinasa Quinasa 4/inmunología , Micrococcus/fisiología , Alineación de Secuencia , Vibrio/fisiologíaRESUMEN
Mitogen-activated protein kinase 4 (MKK4), a member of the MAP kinase family, play important roles in response to many environmental and cellular stresses in mammals. In this study, three MKK4 subtypes, EcMKK4-1, EcMKK4-2 and EcMKK4-3, were obtained from grouper Epinephelus coioides. The open reading frame (ORF) of EcMKK4s are obtained and the EcMKK4s proteins contain highly conserved domains: a S_TKc domain, a canonical diphosphorylation group and two conserved MKKK ATP binding motifs, Asp-Phe-Gly (DFG) and Ala-Pro-Glu (APE). EcMKK4s could be found both in the cytoplasmic and nuclear. The EcMKK4s mRNA were detected in all E. coioides tissues examined with the different expression levels, and the expression were up-regulated during SGIV (Singapore grouper iridescent virus) or Vibrio alginolyticus infection. EcMKK4 could significantly reduce the activation of AP-1 reporter gene. The results suggested that EcMKK4s might play important roles in pathogen-caused inflammation.