ABSTRACT
The timing of seed germination is controlled by the combination of internal dormancy and external factors. Temperature is a major environmental factor for seed germination. The permissive temperature range for germination is narrow in dormant seeds and expands during after-ripening (AR) (dormancy release). Quantitative trait loci analyses of preharvest sprouting in cereals have revealed that MKK3, a mitogen-activated protein kinase (MAPK) cascade protein, is a negative regulator of grain dormancy. Here, we show that the MAPKKK19/20-MKK3-MPK1/2/7/14 cascade modulates the germination temperature range in Arabidopsis seeds by elevating the germinability of the seeds at sub- and supraoptimal temperatures. The expression of MAPKKK19 and MAPKKK20 is induced around optimal temperature for germination in after-ripened seeds but repressed in dormant seeds. MPK7 activation depends on the expression levels of MAPKKK19/20, with expression occurring under conditions permissive for germination. Abscisic acid (ABA) and gibberellin (GA) are two major phytohormones which are involved in germination control. Activation of the MKK3 cascade represses ABA biosynthesis enzyme gene expression and induces expression of ABA catabolic enzyme and GA biosynthesis enzyme genes, resulting in expansion of the germinable temperature range. Our data demonstrate that the MKK3 cascade integrates temperature and AR signals to phytohormone metabolism and seed germination.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Gene Expression Regulation, Plant , Germination , Seeds , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis/growth & development , Arabidopsis Proteins/metabolism , Arabidopsis Proteins/genetics , MAP Kinase Kinase 3/metabolism , MAP Kinase Kinase 3/genetics , MAP Kinase Signaling System/physiology , Mitogen-Activated Protein Kinases/metabolism , Mitogen-Activated Protein Kinases/genetics , Plant Dormancy/genetics , Plant Dormancy/physiology , Seeds/growth & development , Seeds/metabolism , Seeds/genetics , Signal Transduction , TemperatureABSTRACT
BACKGROUND: Traumatic hemorrhagic shock (THS) is a complex pathophysiological process resulting in multiple organ failure. Intestinal barrier dysfunction is one of the mechanisms implicated in multiple organ failure. The present study aimed to explore the regulatory role of mitogen-activated protein kinase kinase 3 (MKK3) in THS-induced intestinal injury and to elucidate its potential mechanism. METHODS: Rats were subjected to trauma and hemorrhage to establish a THS animal model. MKK3-targeted lentiviral vectors were injected via the tail vein 72 h before modeling. Twelve hours post-modeling, the mean arterial pressure (MAP) and heart rate (HR) were monitored, and histological injury to the intestine was assessed via H&E staining and transmission electron microscopy. Mitochondrial function and mitochondrial reactive oxygen species (ROS) were evaluated. IEC-6 cells were exposed to hypoxia to mimic intestinal injury following THS in vitro. RESULTS: MKK3 deficiency alleviated intestinal injury and restored mitochondrial function in intestinal tissues from THS-induced rats and hypoxia-treated IEC-6 cells. In addition, MKK3 deficiency promoted Sirt1/PGC-1α-mediated mitochondrial biogenesis and restricted Pink1/Parkin-mediated mitophagy in the injured intestine and IEC-6 cells. Furthermore, the protective effect of MKK3 knockdown against hypoxia-induced mitochondrial damage was strengthened upon simultaneous LC3B/Pink1/Parkin knockdown or weakened upon simultaneous Sirt1 knockdown. CONCLUSION: MKK3 deficiency protected against intestinal injury induced by THS by promoting mitochondrial biogenesis and restricting excessive mitophagy.
Subject(s)
Intestines , MAP Kinase Kinase 3 , Mitochondria , Reactive Oxygen Species , Shock, Hemorrhagic , Animals , Male , Rats , Cell Line , Disease Models, Animal , Intestinal Mucosa/metabolism , Intestinal Mucosa/pathology , Intestines/pathology , MAP Kinase Kinase 3/metabolism , MAP Kinase Kinase 3/genetics , Mitochondria/metabolism , Mitophagy , Rats, Sprague-Dawley , Reactive Oxygen Species/metabolism , Shock, Hemorrhagic/complications , Shock, Hemorrhagic/metabolism , Shock, Hemorrhagic/genetics , Shock, Traumatic/metabolism , Shock, Traumatic/complications , Shock, Traumatic/geneticsABSTRACT
Tuberculosis, caused by Mycobacterium tuberculosis (Mtb), still ranks among the leading causes of annual human death by infectious disease. Mtb has developed several strategies to survive for years at a time within the host despite the presence of a robust immune response, including manipulating the progression of the inflammatory response and forming granulomatous lesions. Here we demonstrate that IQGAP1, a highly conserved scaffolding protein, compartmentalizes and coordinates multiple signaling pathways in macrophages infected with Mycobacterium marinum (Mm or M.marinum), the closest relative of Mtb. Upregulated IQGAP1 ultimately suppresses TNF-α production by repressing the MKK3 signal and reducing NF-κBp65 translocation, deactivating the p38MAPK pathway. Accordingly, IQGAP1 silencing and overexpression significantly alter p38MAPK activity by modulating the production of phosphorylated MKK3 during mycobacterial infection. Pharmacological inhibition of IQGAP1-associated microtubule assembly not only alleviates tissue damage caused by M.marinum infection but also significantly decreases the production of VEGF-a critical player for granuloma-associated angiogenesis during pathogenic mycobacterial infection. Similarly, IQGAP1 silencing in Mm-infected macrophages diminishes VEGF production, while IQGAP1 overexpression upregulates VEGF. Our data indicate that mycobacteria induce IQGAP1 to hijack NF-κBp65 activation, preventing the expression of proinflammatory cytokines as well as promoting VEGF production during infection and granuloma formation. Thus, therapies targeting host IQGAP1 may be a promising strategy for treating tuberculosis, particularly in drug-resistant diseases.
Subject(s)
Macrophages , NF-kappa B , Signal Transduction , Vascular Endothelial Growth Factor A , ras GTPase-Activating Proteins , Animals , Mice , Macrophages/immunology , Macrophages/metabolism , Macrophages/microbiology , MAP Kinase Kinase 3/metabolism , MAP Kinase Kinase 3/genetics , Mice, Inbred C57BL , Mycobacterium Infections, Nontuberculous/immunology , Mycobacterium Infections, Nontuberculous/microbiology , Mycobacterium Infections, Nontuberculous/metabolism , NF-kappa B/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism , ras GTPase-Activating Proteins/metabolism , ras GTPase-Activating Proteins/genetics , RAW 264.7 Cells , Transcription Factor RelA/metabolism , Tumor Necrosis Factor-alpha/metabolism , Vascular Endothelial Growth Factor A/metabolism , Vascular Endothelial Growth Factor A/geneticsABSTRACT
Myricetin is a typical flavonol with various pharmacological effects which shows favorable biological activities in cancer. However, the underlying mechanisms and potential targets of myricetin in NSCLC (non-small cell lung cancer) cells remain unclear. First, we demonstrated that myricetin not only inhibited the proliferation, migration and invasion, but also induced apoptosis in A549 and H1299 cells in a dose-dependent manner. Then, we confirmed myricetin may play an anti-NSCLC effect through modulating MAPK-related functions and signaling pathway by Network pharmacology. Furthermore, MKK3 (MAP Kinase Kinase 3) was identified and confirmed as a potential target of myricetin by biolayer interferometry (BLI) and molecular docking, revealing that myricetin directly bound to MKK3. Moreover, three mutations (D208, L240, and Y245) of key amino acids predicted by molecular docking obviously decreased the affinity between myricetin and MKK3. Finally, enzyme activity assay was utilized to determine the effect of myricetin on MKK3 activity in vitro, and the result showed that myricetin attenuated MKK3 activity. Subsequently, myricetin decreased the phosphorylation of p38 MAPK. Furthermore, knockdown of MKK3 reduced the susceptibility of A549 and H1299 cells to myricetin. These results suggested that myricetin inhibited the growth of NSCLC cells via targeting MKK3 and influencing the downstream p38 MAPK signaling pathway. The findings revealed that MKK3 is a potential target of myricetin in the NSCLC and myricetin is considered to be a small-molecular inhibitor of MKK3, which can improve comprehension of the molecular mechanisms of myricetin pharmacological effects in cancer and further development of MKK3 inhibitors.
Subject(s)
Carcinoma, Non-Small-Cell Lung , Lung Neoplasms , Humans , Carcinoma, Non-Small-Cell Lung/drug therapy , Lung Neoplasms/drug therapy , MAP Kinase Kinase 3/genetics , MAP Kinase Kinase 3/metabolism , Molecular Docking Simulation , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
BACKGROUND: Apart from the current understanding of enzyme function, the mechanism of ectonucleotide pyrophosphatase/phosphodiesterase 1 (Enpp1) deficiency-associated osteoporosis is unknown. We aimed to explore the changes in the expression of signaling pathways of bone tissues involved in Enpp1 deficiency. METHODS: The body weights and morphology and histology of the bones of male Enpp1 knockout (KO) and wild-type (WT) mice were assessed. The humeri of WT and Enpp1 KO mice at 12 weeks of age were subjected to high-throughput quantitative molecular measurements, and bioinformatics analysis was performed. Proteins from humeri and calvarial pre-osteoblasts (Pobs) were used to verify the differentially expressed signaling pathways and to explain the mechanism of Enpp1 deficiency-associated osteoporosis. RESULTS: Enpp1 KO mice had significantly lower body weight and trabecular bone mass in the hindlimbs than WT mice. Proteomics and immunoblotting showed that Enpp1 deletion downregulated the expression of the p38 mitogen-activated protein kinase (MAPK) signaling pathway in bones. Lysophosphatidic acid (LPA) was involved in activating the MKK3/p38 MAPK/PCNA pathway and proliferating Pobs in Enpp1 KO mice, whereas a p38 MAPK inhibitor suppressed the LPA-induced pro-proliferation phenotype (p < 0.05). CONCLUSION: The inhibition of MKK3/p38 MAPK/PCNA pathway plays an important role in the development of osteoporosis caused by Enpp1 deficiency, and LPA partially rescued the proliferation of pre-osteoblasts via the MKK3/p38 MAPK/PCNA pathway.
Subject(s)
Osteoporosis , p38 Mitogen-Activated Protein Kinases , Animals , Male , Mice , MAP Kinase Kinase 3/genetics , MAP Kinase Kinase 3/metabolism , Mice, Knockout , Osteoporosis/genetics , p38 Mitogen-Activated Protein Kinases/metabolism , Phosphoric Diester Hydrolases/genetics , Phosphoric Diester Hydrolases/metabolism , Proliferating Cell Nuclear Antigen/metabolism , Pyrophosphatases/genetics , Pyrophosphatases/metabolism , Signal Transduction/geneticsABSTRACT
In this study, we explored the precise mechanisms underlying the receptor for advanced glycation end products (RAGE)-mediated neuronal loss and behavioral dysfunction induced by hyperglycemia. We used immunoprecipitation (IP) and GST pull-down assays to assess the interaction between RAGE and mitogen-activated protein kinase kinase 3 (MKK3). Then, we investigated the effect of specific mutation of RAGE on plasticity at hippocampal synapses and behavioral deficits in db/db mice through electrophysiological recordings, morphological assays, and behavioral tests. We discovered that RAGE binds MKK3 and that this binding is required for assembly of the MEKK3-MKK3-p38 signaling module. Mechanistically, we found that activation of p38 mitogen-activated protein kinase (MAPK)/NF-κB signaling depends on mediation of the RAGE-MKK3 interaction by C-terminal RAGE (ctRAGE) amino acids (AAs) 2-5. We found that ctRAGE R2A-K3A-R4A-Q5A mutation suppressed neuronal damage, improved synaptic plasticity, and alleviated behavioral deficits in diabetic mice by disrupting the RAGE-MKK3 conjugation. High glucose induces direct binding of RAGE and MKK3 via ctRAGE AAs 2-5, which leads to assembly of the MEKK3-MKK3-p38 signaling module and subsequent activation of the p38MAPK/NF-κB pathway, and ultimately results in diabetic encephalopathy (DE).
Subject(s)
Diabetes Mellitus, Experimental , Diabetes Mellitus, Type 2 , MAP Kinase Kinase 3 , MAP Kinase Kinase Kinase 3 , Receptor for Advanced Glycation End Products , p38 Mitogen-Activated Protein Kinases , Animals , Cognition , Diabetes Mellitus, Experimental/metabolism , Diabetes Mellitus, Type 2/metabolism , Glycation End Products, Advanced/metabolism , MAP Kinase Kinase 3/genetics , MAP Kinase Kinase 3/metabolism , MAP Kinase Kinase Kinase 3/metabolism , Mice , Receptor for Advanced Glycation End Products/metabolism , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
KEY MESSAGE: HvMKK3 alleles are temperature sensitive and are major contributors to environmental stability of preharvest sprouting in barley. Preharvest sprouting (PHS) can severely damage barley (Hordeum vulgare L.) malting quality, but PHS resistance is often negatively correlated with malting quality. Seed dormancy is closely related to PHS. Increased temperature during grain fill can decrease seed dormancy in barley, but genetic components of seed dormancy temperature sensitivity are poorly understood. Six years of PHS data were used to fit quantitative trait locus (QTL) x environment mixed models incorporating marker data from seed dormancy genes HvAlaAT1, HvGA20ox1, and HvMKK3 and weather covariates in spring and winter two-row malting barley. Variation in winter barley PHS was best modeled by average temperature range during grain fill and spring barley PHS by total precipitation during grain fill. Average high temperature during grain fill also accurately modeled PHS for both datasets. A highly non-dormant HvMKK3 allele determined baseline PHS susceptibility and HvAlaAT1 interactions with multiple HvMKK3 alleles conferred environmental sensitivity. Polygenic variation for PHS within haplotype was detected. Residual genotype and QTL by environment interaction variance indicated additional environmental and genetic factors involved in PHS. These models provide insight into genotype and environmental regulation of barley seed dormancy, a method for PHS forecasting, and a tool for breeders to improve PHS resistance.
Subject(s)
Hordeum/genetics , Models, Biological , Quantitative Trait Loci , Seedlings/growth & development , Alleles , Gene-Environment Interaction , Genes, Plant , Hordeum/enzymology , Hordeum/growth & development , MAP Kinase Kinase 3/genetics , MAP Kinase Kinase 3/metabolism , Plant Dormancy/genetics , Seedlings/geneticsABSTRACT
Compared to other ethnicities, Hispanic children incur the highest rates of leukemia, and most cases are diagnosed as Acute Lymphoblastic Leukemia (ALL). Despite improved treatment and survival for ALL, disproportionate health outcomes in Hispanics persist. Thus, it is essential to identify oncogenic mutations within this demographic to aid in the development of new strategies to diagnose and treat ALL. Using whole-exome sequencing, five single nucleotide polymorphisms within mitogen-activated protein kinase 3 (MAP2K3) were identified in an ALL cancer patient library from the U.S./Mexico border. MAP2K3 R26T and P11T are located near the substrate-binding site, while R65L and R67W localized to the kinase domain. Truncated-MAP2K3 mutant Q73* was also identified. Transfection in HEK293 cells showed that the quadruple-MEK3 mutant (4M-MEK3) impacted protein stability, inducing degradation and reducing expression. The expression of 4M-MEK3 could be rescued by cysteine/serine protease inhibition, and proteasomal degradation of truncated-MEK3 occurred in a ubiquitin-independent manner. MEK3 mutants displayed reduced auto-phosphorylation and enzymatic activity, as seen by decreases in p38 phosphorylation. Furthermore, uncoupling of the MEK3/p38 signaling pathway resulted in less suppressive activity on HEK293 cell viability. Thus, disruption of MEK3 activation may promote proliferative signals in ALL. These findings suggest that MEK3 represents a potential therapeutic target for treating ALL.
Subject(s)
Cell Proliferation/genetics , MAP Kinase Kinase 3 , MAP Kinase Signaling System/genetics , Mutation , Neoplasm Proteins , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Proteolysis , HEK293 Cells , Hep G2 Cells , Humans , MAP Kinase Kinase 3/genetics , MAP Kinase Kinase 3/metabolism , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Precursor Cell Lymphoblastic Leukemia-Lymphoma/enzymology , Precursor Cell Lymphoblastic Leukemia-Lymphoma/geneticsABSTRACT
The skin typically tolerates exposure to various microbes and chemicals in the environment. Here, we investigated how the epidermis maintains this innate immune tolerance to stimuli that are recognized by Toll-like receptors (TLRs). Loss of tolerance to TLR ligands occurred after silencing of the histone deacetylases (HDACs) HDAC8 and HDAC9 in keratinocytes. Transcriptional analysis identified MAP2K3 as suppressed by HDAC8/9 activity and a potential key intermediary for establishing this tolerance. HDAC8/9 influenced acetylation at H3K9 and H3K27 marks in the MAP2K3 promoter. Proteomic analysis further identified SSRP1 and SUPT16H as associated with HDAC8/9 and responsible for transcriptional elongation of MAP2K3. Silencing of MAP2K3 blocked the capacity of HDAC8/9 to influence cytokine responses. Relevance in vivo was supported by observations of increased MAP2K3 in human inflammatory skin conditions and the capacity of keratinocyte HDAC8/9 to influence dendritic cell maturation and T cell proliferation. Keratinocyte-specific deletion of HDAC8/9 also increased inflammation in mice after exposure to ultraviolet radiation, imiquimod, or Staphylococcus aureus These findings define a mechanism for the epidermis to regulate inflammation in the presence of ubiquitous TLR ligands.
Subject(s)
Histone Deacetylases/immunology , MAP Kinase Kinase 3/immunology , Repressor Proteins/immunology , Skin/immunology , Animals , Cells, Cultured , Dendritic Cells/immunology , Epigenesis, Genetic , Histone Deacetylases/genetics , Humans , Imiquimod/pharmacology , Immune Tolerance , Immunity, Innate , Keratinocytes/immunology , MAP Kinase Kinase 3/genetics , Mice, Transgenic , Repressor Proteins/genetics , Staphylococcus aureus , T-Lymphocytes/immunology , Toll-Like Receptors/immunology , Ultraviolet RaysABSTRACT
OBJECTIVE: Von Hippel-Lindau (VHL) syndrome is a rare disease that occurs in an autosomal-dominant genetic pattern. Due to the high genetic variability of VHL diseases, current studies have limited clinical value. Moreover, casual genetic variations in patients with VHL syndrome are still unclear. METHODS: Here, we performed whole-exome sequencing of 25 individuals to identify reliable disease-related variations. Systemic computational analysis was performed for variant detection, and Sanger sequencing was used to validate detected mutations. RESULTS: Most of the known mutations in the VHL gene were observed in the studied population. In addition, a large fragment deletion in VHL exon 2 in the immediate family members of the last family was detected. This had not been reported earlier. Moreover, we identified 3 novel mutation sites in the MAP2K3 gene that may be involved in the occurrence and development of the VHL disease. CONCLUSIONS: These results demonstrated that the heterogeneous nature of VHL syndrome and novel mutational signatures may help to improve the diagnostic ability of VHL syndrome.
Subject(s)
MAP Kinase Kinase 3/genetics , Von Hippel-Lindau Tumor Suppressor Protein/genetics , von Hippel-Lindau Disease/genetics , DNA Mutational Analysis , Female , Humans , Male , Mutation , Pedigree , Exome SequencingABSTRACT
Esophageal squamous cell carcinoma (ESCC) is one of the most refractory malignancies worldwide. Mitogen-activated protein kinase 3 (MAP2K3) has a contradictory role in tumor progression, and the function and expression patterns of MAP2K3 in ESCC remain to be determined. We found that MAP2K3 expression to be downregulated in ESCC, and MAP2K3 downregulation correlated with clinically poor survival. MAP2K3 inhibited ESCC cell proliferation and invasion in vitro and in vivo. MAP2K3 suppressed STAT3 expression and activation. Mechanistically, MAPSK3 interacted with MDM2 to promote STAT3 degradation via the ubiquitin-proteasome pathway. Furthermore, exosomal miR-19b-3p derived from the plasma of patients with ESCC could suppress MAP2K3 expression to promote ESCC tumorigenesis. STAT3 was found to bind to the MIR19B promoter and increased the expression of miR-19b-3p in ESCC cells. In summary, our results demonstrated that the miR-19b-3p-MAP2K3-STAT3 feedback loop regulates ESCC tumorigenesis and elucidates the potential of therapeutically targeting this pathway in ESCC.
Subject(s)
Esophageal Neoplasms/pathology , Esophageal Squamous Cell Carcinoma/pathology , MAP Kinase Kinase 3/physiology , MicroRNAs/physiology , STAT3 Transcription Factor/physiology , Adult , Aged , Animals , Case-Control Studies , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation/genetics , Esophageal Neoplasms/genetics , Esophageal Squamous Cell Carcinoma/genetics , Feedback, Physiological/physiology , Female , Gene Expression Regulation, Neoplastic , Humans , MAP Kinase Kinase 3/genetics , Male , Mice , Mice, Inbred BALB C , Mice, Nude , MicroRNAs/genetics , Middle Aged , Neoplasm Invasiveness , STAT3 Transcription Factor/geneticsABSTRACT
Mitogen-activated protein kinases (MPKs) play essential roles in guard cell signaling, but whether MPK cascades participate in guard cell ethylene signaling and interact with hydrogen peroxide (H2 O2 ), nitric oxide (NO), and ethylene-signaling components remain unclear. Here, we report that ethylene activated MPK3 and MPK6 in the leaves of wild-type Arabidopsis thaliana as well as ethylene insensitive2 (ein2), ein3, nitrate reductase1 (nia1), and nia2 mutants, but this effect was impaired in ethylene response1 (etr1), nicotinamide adenine dinucleotide phosphate oxidase AtrbohF, mpk kinase1 (mkk1), and mkk3 mutants. By contrast, the constitutive triple response1 (ctr1) mutant had constitutively active MPK3 and MPK6. Yeast two-hybrid, bimolecular fluorescence complementation, and pull-down assays indicated that MPK3 and MPK6 physically interacted with MKK1, MKK3, and the C-terminal region of EIN2 (EIN2 CEND). mkk1, mkk3, mpk3, and mpk6 mutants had typical levels of ethylene-induced H2 O2 generation but impaired ethylene-induced EIN2 CEND cleavage and nuclear translocation, EIN3 protein accumulation, NO production in guard cells, and stomatal closure. These results show that the MKK1/3-MPK3/6 cascade mediates ethylene-induced stomatal closure by functioning downstream of ETR1, CTR1, and H2 O2 to interact with EIN2, thereby promoting EIN3 accumulation and EIN3-dependent NO production in guard cells.
Subject(s)
Arabidopsis Proteins/metabolism , DNA-Binding Proteins/metabolism , Ethylenes/pharmacology , MAP Kinase Kinase 1/metabolism , MAP Kinase Kinase 3/metabolism , Mitogen-Activated Protein Kinases/metabolism , Plant Stomata/drug effects , Plant Stomata/metabolism , Receptors, Cell Surface/metabolism , Transcription Factors/metabolism , Arabidopsis/drug effects , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , DNA-Binding Proteins/genetics , MAP Kinase Kinase 1/genetics , MAP Kinase Kinase 3/genetics , Mitogen-Activated Protein Kinases/genetics , Receptors, Cell Surface/genetics , Transcription Factors/geneticsABSTRACT
Esophageal cancer is one of the most common cancer with limited therapeutic strategies, thus it is important to develop more effective strategies to against it. Sulforaphene (SFE), an isothiocyanate isolated from radish seeds, was proved to inhibit esophageal cancer progression in the current study. Flow cytometric analysis showed SFE induced cell apoptosis and cycle arrest in G2/M phase. Also, scrape motility and transwell assays presented SFE reduced esophageal cancer cell metastasis. Microarray results showed the influence of SFE on esophageal cancer cells was related with stearoyl-CoA desaturase (SCD), cadherin 3 (CDH3), mitogen-activated protein kinase kinase 3 (MAP2K3) and growth arrest and DNA damage inducible beta (GADD45B). SCD and CDH3 could promote esophageal cancer metastasis via activating the Wnt pathway, while the latter one was involved in a positive feedback loop, GADD45B-MAP2K3-p38-p53, to suppress esophageal cancer growth. GADD45B was known to be the target gene of p53, and we proved in this study, it could increase the phosphorylation level of MAP2K3 in esophageal cancer cells, activating p38 and p53 in turn. SFE treatment elevated MAP2K3 and GADD45B expression and further stimulated this feedback loop to better exert antitumor effect. In summary, these results demonstrated that SFE had the potential for developing as a chemotherapeutic agent because of its inhibitory effects on esophageal cancer metastasis and proliferation.
Subject(s)
Cadherins/metabolism , Isothiocyanates/pharmacology , Stearoyl-CoA Desaturase/metabolism , Animals , Antigens, Differentiation/genetics , Antigens, Differentiation/metabolism , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Cadherins/physiology , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Disease Progression , Esophageal Neoplasms/drug therapy , Esophageal Neoplasms/metabolism , Female , Gene Expression/genetics , Gene Expression Regulation, Neoplastic/genetics , Humans , Isothiocyanates/metabolism , MAP Kinase Kinase 3/genetics , MAP Kinase Kinase 3/metabolism , MAP Kinase Signaling System/genetics , Mice , Mice, Nude , Stearoyl-CoA Desaturase/physiology , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Xenograft Model Antitumor Assays , p38 Mitogen-Activated Protein Kinases/metabolismSubject(s)
Colorectal Neoplasms/genetics , Intracellular Signaling Peptides and Proteins/genetics , MAP Kinase Kinase 3/genetics , Matrix Metalloproteinase 9/genetics , Cell Proliferation/genetics , Colorectal Neoplasms/pathology , HCT116 Cells , Humans , Matrix Metalloproteinase 1/genetics , NF-kappa B/genetics , Neoplasm Invasiveness/genetics , Neoplasm Invasiveness/pathology , Neoplasm Metastasis , Signal Transduction/genetics , p38 Mitogen-Activated Protein Kinases/geneticsABSTRACT
The transcriptional regulator YAP, which plays important roles in the development, regeneration, and tumorigenesis, is activated when released from inhibition by the Hippo kinase cascade. The regulatory mechanism of YAP in Hippo-low contexts is poorly understood. Here, we performed a genome-wide RNA interference screen to identify genes whose loss of function in a Hippo-null background affects YAP activity. We discovered that the coatomer protein complex I (COPI) is required for YAP nuclear enrichment and that COPI dependency of YAP confers an intrinsic vulnerability to COPI disruption in YAP-driven cancer cells. We identified MAP2K3 as a YAP regulator involved in inhibitory YAP phosphorylation induced by COPI subunit depletion. The endoplasmic reticulum stress response pathway activated by COPI malfunction appears to connect COPI and MAP2K3. In addition, we provide evidence that YAP inhibition by COPI disruption may contribute to transcriptional up-regulation of PTGS2 and proinflammatory cytokines. Our study offers a resource for investigating Hippo-independent YAP regulation as a therapeutic target for cancers and suggests a link between YAP and COPI-associated inflammatory diseases.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Coat Protein Complex I/metabolism , MAP Kinase Kinase 3/metabolism , Neoplasms/metabolism , RNA Interference , Transcription Factors/metabolism , Adaptor Proteins, Signal Transducing/genetics , Animals , Cell Line, Tumor , Coat Protein Complex I/genetics , Gene Expression Regulation, Neoplastic , Genome , Hippo Signaling Pathway , Humans , MAP Kinase Kinase 3/genetics , Mice , Neoplasms/genetics , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Signal Transduction , Transcription Factors/genetics , YAP-Signaling ProteinsABSTRACT
Background: Capsaicin is an active compound found in plants of the Capsicum genus; it has a range of therapeutic benefits, including anti-tumor effects. Here we aimed to delineate the inhibitory effects of capsaicin on nasopharyngeal carcinoma (NPC). Methods: The anti-cancer effects of capsaicin were confirmed in NPC cell lines and xenograft mouse models, using CCK-8, clonogenic, wound-healing, transwell migration and invasion assays. Co-immunoprecipitation, western blotting and pull-down assays were used to determine the effects of capsaicin on the MKK3-p38 axis. Cell proliferation and EMT marker expression were monitored in MKK3 knockdown (KD) or over-expression NPC cell lines treated with or without capsaicin. Finally, immunohistochemistry was performed on NPC specimens from NPC patients (n = 132) and the clinical relevance was analyzed. Results: Capsaicin inhibited cell proliferation, mobility and promoted apoptosis in NPC cells. Then we found that capsaicin directly targets p38 for dephosphorylation. As such, MKK3-induced p38 activation was inhibited by capsaicin. Furthermore, we found that capsaicin-induced inhibition of cell motility was mediated by fucokinase. Xenograft models demonstrated the inhibitory effects of capsaicin treatment on NPC tumor growth in vivo, and analysis of clinical NPC samples confirmed that MKK3 phosphorylation was associated with NPC tumor growth and lymphoid node metastasis. Conclusions: The MKK3-p38 axis represents a potential therapeutic target for capsaicin. MKK3 phosphorylation might serve as a biomarker to identify NPC patients most likely to benefit from adjunctive capsaicin treatment.
Subject(s)
Capsaicin/pharmacology , MAP Kinase Kinase 3/metabolism , MAP Kinase Signaling System/drug effects , Nasopharyngeal Carcinoma/drug therapy , Nasopharyngeal Neoplasms/drug therapy , Animals , Capsaicin/therapeutic use , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Epithelial-Mesenchymal Transition/drug effects , Female , Gene Knockdown Techniques , HEK293 Cells , Humans , MAP Kinase Kinase 3/genetics , MAP Kinase Signaling System/genetics , Male , Mice , Middle Aged , Nasopharyngeal Carcinoma/pathology , Nasopharyngeal Neoplasms/pathology , Neoplasm Invasiveness/pathology , Neoplasm Invasiveness/prevention & control , Phosphorylation/drug effects , Phosphotransferases (Alcohol Group Acceptor)/genetics , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Xenograft Model Antitumor AssaysABSTRACT
The activation of p38 mitogen-activated protein kinases (MAPKs) through a phosphorylation cascade is the canonical mode of regulation. Here, we report a novel activation mechanism for p38α. We show that Arg49 and Arg149 of p38α are methylated by protein arginine methyltransferase 1 (PRMT1). The non-methylation mutations of Lys49/Lys149 abolish the promotive effect of p38α on erythroid differentiation. MAPK kinase 3 (MKK3) is identified as the major p38α upstream kinase and MKK3-mediated activation of the R49/149K mutant p38α is greatly reduced. This is due to a profound reduction in the interaction of p38α and MKK3. PRMT1 can enhance both the methylation level of p38α and its interaction with MKK3. However, the phosphorylation of p38α by MKK3 is not a prerequisite for methylation. MAPK-activated protein kinase 2 (MAPKAPK2) is identified as a p38α downstream effector in the PRMT1-mediated promotion of erythroid differentiation. The interaction of MAPKAPK2 with p38α is also significantly reduced in the R49/149K mutant. Together, this study unveils a novel regulatory mechanism of p38α activation via protein arginine methylation on R49/R149 by PRMT1, which impacts partner interaction and thus promotes erythroid differentiation. This study provides a new insight into the complexity of the regulation of the versatile p38α signaling and suggests new directions in intervening p38α signaling.
Subject(s)
Arginine/metabolism , Erythropoiesis/genetics , MAP Kinase Kinase 3/metabolism , MAP Kinase Signaling System/genetics , Protein-Arginine N-Methyltransferases/metabolism , Repressor Proteins/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism , Cell Line, Tumor , Enzyme Activation/drug effects , Erythropoiesis/drug effects , Gene Knockdown Techniques , Humans , MAP Kinase Kinase 3/genetics , MAP Kinase Signaling System/drug effects , Mass Spectrometry , Methylation , Mutation , Phosphorylation , Protein Binding , Protein Processing, Post-Translational , Protein-Arginine N-Methyltransferases/genetics , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Repressor Proteins/genetics , p38 Mitogen-Activated Protein Kinases/geneticsABSTRACT
KEY MESSAGE: Using bulked segregant analysis of exome sequence, we fine-mapped the ABA-hypersensitive mutant ERA8 in a wheat backcross population to the TaMKK3-A locus of chromosome 4A. Preharvest sprouting (PHS) is the germination of mature grain on the mother plant when it rains before harvest. The ENHANCED RESPONSE TO ABA8 (ERA8) mutant increases seed dormancy and, consequently, PHS tolerance in soft white wheat 'Zak.' ERA8 was mapped to chromosome 4A in a Zak/'ZakERA8' backcross population using bulked segregant analysis of exome sequenced DNA (BSA-exome-seq). ERA8 was fine-mapped relative to mutagen-induced SNPs to a 4.6 Mb region containing 70 genes. In the backcross population, the ERA8 ABA-hypersensitive phenotype was strongly linked to a missense mutation in TaMKK3-A-G1093A (LOD 16.5), a gene associated with natural PHS tolerance in barley and wheat. The map position of ERA8 was confirmed in an 'Otis'/ZakERA8 but not in a 'Louise'/ZakERA8 mapping population. This is likely because Otis carries the same natural PHS susceptible MKK3-A-A660S allele as Zak, whereas Louise carries the PHS-tolerant MKK3-A-C660R allele. Thus, the variation for grain dormancy and PHS tolerance in the Louise/ZakERA8 population likely resulted from segregation of other loci rather than segregation for PHS tolerance at the MKK3 locus. This inadvertent complementation test suggests that the MKK3-A-G1093A mutation causes the ERA8 phenotype. Moreover, MKK3 was a known ABA signaling gene in the 70-gene 4.6 Mb ERA8 interval. None of these 70 genes showed the differential regulation in wild-type Zak versus ERA8 expected of a promoter mutation. Thus, the working model is that the ERA8 phenotype results from the MKK3-A-G1093A mutation.
Subject(s)
Genes, Plant , Germination , MAP Kinase Kinase 3/genetics , Triticum/genetics , Abscisic Acid , Alleles , Chromosome Mapping , Crosses, Genetic , Exome , Genetic Linkage , Mutation, Missense , Phenotype , Quantitative Trait Loci , Triticum/physiologyABSTRACT
miRNA mediated genetic regulation is widely involved in carcinogenesis of cervical cancer. In this study, miR-214 was confirmed to directly regulate MKK3 via imperfect base-pairing to its 3'UTR, resulting down-regulation of its expression level. Compared to normal tissues, a down-regulated level of miR-214 was observed in cervical cancer, while MKK3 was up-regulated. Next, we demonstrated that over-expression of miR-214 or knockdown of MKK3 can inhibit the growth, proliferation, invasion and migration of cervical cancer in vitro and in vivo. Moreover, the effects of miR-214 in HeLa cells were rescued by the restoration of MKK3. In conclusion, our results laid new foundations for investigating the pathogenesis and diagnosis of cervical cancer.