Mitogen-activated Protein Kinase Module Research Articles

Preferential Protection of Cerebral Autoregulation and Reduction of Hippocampal Necrosis With Norepinephrine After Traumatic Brain Injury in Female Piglets.

Traumatic brain injury contributes to morbidity in children and boys is disproportionately represented. Cerebral autoregulation is impaired after traumatic brain injury, contributing to poor outcome. Cerebral perfusion pressure is often normalized by the use of vasopressors to increase mean arterial pressure. In prior studies, we observed that phenylephrine prevented impairment of autoregulation in female but exacerbated in male piglets after fluid percussion injury. In contrast, dopamine prevented impairment of autoregulation in both sexes after fluid percussion injury, suggesting that pressor choice impacts outcome. The extracellular signal-regulated kinase isoform of mitogen-activated protein kinase produces hemodynamic impairment after fluid percussion injury, but the role of the cytokine interleukin-6 is unknown. We investigated whether norepinephrine sex-dependently protects autoregulation and limits histopathology after fluid percussion injury and the role of extracellular signal-regulated kinase and interleukin-6 in that outcome. Prospective, randomized animal study. University laboratory. Newborn (1-5 d old) pigs. Cerebral perfusion pressure, cerebral blood flow, and pial artery diameter were determined before and after fluid percussion injury in piglets equipped with a closed cranial window and post-treated with norepinephrine. Cerebrospinal fluid extracellular-signal-regulated kinase mitogen-activated protein kinase was determined by enzyme-linked immunosorbent assay. Norepinephrine does not protect autoregulation or prevent reduction in cerebral blood flow in male but fully protects autoregulation in female piglets after fluid percussion injury. Papaverine-induced dilation was unchanged by fluid percussion injury and norepinephrine. Norepinephrine increased extracellular signal-regulated kinase mitogen-activated protein kinase up-regulation in male but blocked such up-regulation in female piglets after fluid percussion injury. Norepinephrine aggravated interleukin-6 upregulation in males in an extracellular signal-regulated kinase mitogen-activated protein kinase-dependent mechanism but blocked interleukin-6 up-regulation in females after fluid percussion injury. Norepinephrine augments loss of neurons in CA1 and CA3 hippocampus of male piglets after fluid percussion injury in an extracellular signal-regulated kinase mitogen-activated protein kinase-dependent and interleukin-6-dependent manner but prevents loss of neurons in females after fluid percussion injury. Norepinephrine protects autoregulation and limits hippocampal neuronal cell necrosis via modulation of extracellular signal-regulated kinase mitogen-activated protein kinase and interleukin-6 after fluid percussion injury in a sex-dependent manner.

Cell therapy centered on IL-1Ra is neuroprotective in experimental stroke.

Cell-based therapies are emerging as new promising treatments in stroke. However, their functional mechanism and therapeutic potential during early infarct maturation has so far received little attention. Here, we asked if cell-based delivery of the interleukin-1 receptor antagonist (IL-1Ra), a known neuroprotectant in stroke, can promote neuroprotection, by modulating the detrimental inflammatory response in the tissue at risk. We show by the use of IL-1Ra-overexpressing and IL-1Ra-deficient mice that IL-1Ra is neuroprotective in stroke. Characterization of the cellular and spatiotemporal production of IL-1Ra and IL-1α/β identifies microglia, not infiltrating leukocytes, as the major sources of IL-1Ra after experimental stroke, and shows IL-1Ra and IL-1β to be produced by segregated subsets of microglia with a small proportion of these cells co-expressing IL-1α. Reconstitution of whole body irradiated mice with IL-1Ra-producing bone marrow cells is associated with neuroprotection and recruitment of IL-1Ra-producing leukocytes after stroke. Neuroprotection is also achieved by therapeutic injection of IL-1Ra-producing bone marrow cells 30 min after stroke onset, additionally improving the functional outcome in two different stroke models. The IL-1Ra-producing bone marrow cells increase the number of IL-1Ra-producing microglia, reduce the availability of IL-1β, and modulate mitogen-activated protein kinase (MAPK) signaling in the ischemic cortex. The importance of these results is underlined by demonstration of IL-1Ra-producing cells in the human cortex early after ischemic stroke. Taken together, our results attribute distinct neuroprotective or neurotoxic functions to segregated subsets of microglia and suggest that treatment strategies increasing the production of IL-1Ra by infiltrating leukocytes or microglia may also be neuroprotective if applied early after stroke onset in patients.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-016-1541-5) contains supplementary material, which is available to authorized users.

Methods to Study Protein Tyrosine Phosphatases Acting on Yeast MAPKs.

Mitogen activated protein kinases (MAPK) pathways play a key role in orchestrating the eukaryotic cellular response to different stimuli. In this process, phosphorylation of both conserved threonine and tyrosine residues of MAPKs is essential for their activation. Identification of tyrosine and dual specificity protein phosphatases capable of dephosphorylating these phosphosites is thus critical to gain insight into their regulation. Due to the conservation of pivotal elements in eukaryotic signaling, yeast has turned into a valuable tool to increase the knowledge of MAPK signaling in other cell types. Here we describe an in vivo method to evaluate the capacity of a protein, from yeast or other origin, to act as a MAPK phosphatase. It relies on the ability of the phosphatase to reduce, when overexpressed, both the amount of activated MAPK and the transcription from a specific promoter regulated by the corresponding pathway. To this end, the pathway has to be previously activated, preferentially through overexpression of a hyperactive allele of an upstream component within the MAPK module. Additionally, the ability of an overexpressed "trapping" inactive phosphatase version to modify these readouts is also analyzed. Western blotting analysis with specific anti-phospho MAPK antibodies and flow cytometry-based determination of fluorescence produced by GFP whose expression is driven by MAPK-regulated promoters are the selected techniques for monitoring these readouts.

Changes in BDNF and MAPK Signaling Pathways in Experimental Glaucoma

Glaucoma is currently recognized to be a multifactorial, progressive, neurodegenerative disorder. It is characterized by the retinal ganglion cells (RGCs) loss of axons as well as optic nerve atrophy, progressive degeneration of RGCs till cell death. In this work, we used DBA/2J mice as a model of spontaneous glaucoma and we investigated the involvement of BDNF and Mitogen-Activated Protein Kinases (MAPK) pathways in correlation with IOP elevation and progression of neurodegenerative processes in the retina of DBA/2J mice. In particular, we performed western blot analysis to study retinal levels of the BDNF and its receptor, TrkB, and to better understand possible modulation of p38 MAPK and ERK1/2 activation at different stages of retinal degeneration in DBA/2J mice. We showed that BDNF starts to decrease already at an early stage in correspondence to IOP elevation (7 months of age). MAPKs, in particular p38 MAPK and ERK1/2, appeared maximally affected at more advanced stages of neurodegeneration (10-12 and 18 months of age) characterized by RGC degeneration and death, optic nerve atrophy. Thus, BDNF signaling and MAPKs are differentially activated at different stages of retinal degeneration in DBA/2J mice, a murine model of glaucoma.

Tangeretin inhibits IL-1β induced proliferation of rheumatoid synovial fibroblasts and the production of COX-2, PGE2 and MMPs via modulation of p38 MAPK/ERK/JNK pathways

<p>The study aimed to determine the effect of tangeretin in inhibiting the interleukin-1β (IL-1β)-induced proliferation of RASFs and suppression of the production of inflammatory mediators. RASFs were isolated from synovial tissue obtained from patients with RA during knee arthroscopy. The cells were exposed to IL‑1β (1.0 ng/mL) and/ or tangeretin (50 or 100 µM). Cell viability was assessed following treatments. Expressions of MMPs and COX-2 were analysed by real-time PCR and western blotting. Production of prostaglandin E2 (PGE2) by RASFs were analysed by ELISA. Expressions of mitogen activated protein kinases (MAPKs) and nuclear factor-kB (NF-kB) were assessed by western blotting. Tangeretin significantly inhibited the proliferation of RASFs, as well as down-regulated the expression of MMP-1, MMP-3 and COX-2 mRNA and protein and also the phosphorylation of ERK, p38 and JNK. Raised level of expression of NF-κB and PGE2 induced by IL-1β was reduced by tangeretin. Results indicate that tangeretin was effective in inhibiting the synovial fibroblast proliferation, as well regulated MMPs, COX-2, PGE2 via modulation of p38 MAPK, ERK and JNK pathways. </p><p> </p>

The yeast cell wall integrity pathway signals from recycling endosomes upon elimination of phosphatidylinositol (4,5)-bisphosphate by mammalian phosphatidylinositol 3-kinase

Phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P2] is essential for recognition of the plasma membrane inner leaf by protein complexes. We expressed mammalian class I phosphoinositide 3-kinase (PI3K) in Saccharomyces cerevisiae to eliminate PtdIns(4,5)P2 by its conversion into PtdIns(3,4,5)P3, a lipid naturally missing in this yeast. This led to loss of actin function and endocytosis defects, causing a blockage in polarized secretion. Also, the cell wall integrity (CWI) mitogen-activated protein kinase (MAPK) pathway was activated, triggering a typical transcriptional response. In the absence of PtdIns(4,5)P2 at the plasma membrane, the Pkc1 protein kinase upstream the CWI MAPK module localized to post-Golgi endosomes marked by SNARE Snc1 and Rab GTPases Ypt31 and Ypt32. Other components at the head of the pathway, like the mechanosensor Wsc1, the GTPase Rho1 and its activator the GDP/GTP exchange factor Rom2, co-localized with Pkc1 in these compartments. Chemical inhibition of PI3K proved that both CWI activation and Pkc1 relocation to endosomes are reversible. These results suggest that the CWI pathway is able to respond to loss of plasma membrane identity from recycling endosomes.

Pharmacological Induction of Human Fetal Globin Gene in Hydroxyurea-Resistant Primary Adult Erythroid Cells.

Pharmacological induction of the fetal γ globin gene and the consequent formation of HbF (α2/γ2) in adult erythroid cells are one feasible therapeutic strategy for sickle cell disease (SCD) and severe β-thalassemias. Hydroxyurea (HU) is the current drug of choice for SCD, but serious side effects limit its clinical use. Moreover, 30 to 50% of patients are irresponsive to HU treatment. We have used high-throughput screening to identify benzo[de]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one and its derivatives (compounds I to VI) as potent γ globin inducers. Of the compounds, I to V exert superior γ globin induction and have better therapeutic potential than HU, likely because of their activation of the p38 mitogen-activated protein kinase (MAPK) signaling pathway and modulation of expression levels and/or chromosome binding of γ globin gene regulators, including BCL11A, and chromatin structure over the γ globin promoter. Unlike sodium butyrate (NaB), the global levels of acetylated histones H3 and H4 are not changed by compound II treatment. Remarkably, compound II induces the γ globin gene in HU-resistant primary human adult erythroid cells, the p38 signaling pathway of which appears to be irresponsive to HU and NaB as well as compound II. This study provides a new framework for the development of new and superior compounds for treating SCD and severe β-thalassemias.

Involvement of 14-3-3 Proteins in Regulating Tumor Progression of Hepatocellular Carcinoma.

There are seven mammalian isoforms of the 14-3-3 protein, which regulate multiple cellular functions via interactions with phosphorylated partners. Increased expression of 14-3-3 proteins contributes to tumor progression of various malignancies. Several isoforms of 14-3-3 are overexpressed and associate with higher metastatic risks and poorer survival rates of hepatocellular carcinoma (HCC). 14-3-3β and 14-3-3ζ regulate HCC cell proliferation, tumor growth and chemosensitivity via modulating mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK) and p38 signal pathways. Moreover, 14-3-3ε suppresses E-cadherin and induces focal adhesion kinase (FAK) expression, thereby enhancing epithelial-mesenchymal transition (EMT) and HCC cell migration. 14-3-3ζ forms complexes with αB-crystallin, which induces EMT and is the cause of sorafenib resistance in HCC. Finally, a recent study has indicated that 14-3-3σ induces heat shock protein 70 (HSP70) expression, which increases HCC cell migration. These results suggest that selective 14-3-3 isoforms contribute to cell proliferation, EMT and cell migration of HCC by regulating distinct targets and signal pathways. Targeting 14-3-3 proteins together with specific downstream effectors therefore has potential to be therapeutic and prognostic factors of HCC. In this article, we will overview 14-3-3’s regulation of its downstream factors and contributions to HCC EMT, cell migration and proliferation.

Advantages and limitations of shot-gun proteomic analyses on Arabidopsis plants with altered MAPK signaling.

The molecular mechanism of signal transduction by mitogen-activated protein kinases (MAPKs) in plants is an intensive research field of contemporary plant biology. Signaling in plants involves perception of changing environment and the intracellular transduction of such generated information leading to the activation of adaptive mechanisms or programmed cell death, as it is in the case of hypersensitive response to some biotrophic pathogen attack. At the molecular level, distinct signaling pathways, such as MAPK modules, involve reversible phosphorylation, enzymatic activation, protein-protein interaction and finally transcriptional activation via nuclear-resident transcription factors or cell reorganization via regulation of cytoplasmic or plasma membrane-associated proteins. All these processes are strictly regulated and subcellularly compartmentalized. Modern OMICS technologies encompassing transcriptomic (Krysan et al., 2002; Frei dit Frey et al., 2014), metabolomic (Lassowskat et al., 2014), proteomic (Miles et al., 2009; Conroy et al., 2013; Lassowskat et al., 2014; Takac et al., 2014) and phosphoproteomic (Hoehenwarter et al., 2013; Lassowskat et al., 2014) approaches have been employed to gain functional information on plant signaling. The main challenges of advanced (phospho)proteomic studies are: reliable identification of all signaling constituents of a particular MAPK pathway as well as identification and validation of respective target proteins. In this opinion article, we aim to briefly evaluate benefits and pitfalls of various proteomic approaches that are used to study MAPK signaling in plants. We emphasize on the advantages of comparative shot-gun proteomic analyses of Arabidopsis mutants and transgenic plants with altered MAPK signaling. The importance of critical data validation and interpretation is also discussed.

Arctigenin Protects against Lipopolysaccharide-Induced Pulmonary Oxidative Stress and Inflammation in a Mouse Model via Suppression of MAPK, HO-1, and iNOS Signaling.

Arctigenin, a bioactive component of Arctium lappa (Nubang), has anti-inflammatory activity. Here, we investigated the effects of arctigenin on lipopolysaccharide (LPS)-induced acute lung injury. Mice were divided into four groups: control, LPS, LPS + DMSO, and LPS + Arctigenin. Mice in the LPS + Arctigenin group were injected intraperitoneally with 50 mg/kg of arctigenin 1 h before an intratracheal administration of LPS (5 mg/kg). Lung tissues and bronchoalveolar lavage fluids (BALFs) were collected. Histological changes of the lung were analyzed by hematoxylin and eosin staining. Arctigenin decreased LPS-induced acute lung inflammation, infiltration of inflammatory cells into BALF, and production of pro-inflammatory cytokines. Moreover, arctigenin pretreatment reduced the malondialdehyde level and increased superoxide dismutase and catalase activities and glutathione peroxidase/glutathione disulfide ratio in the lung. Mechanically, arctigenin significantly reduced the production of nitric oxygen and inducible nitric oxygen synthase (iNOS) expression, enhanced the expression of heme oxygenase-1, and decreased the phosphorylation of mitogen-activated protein kinases (MAPKs). Arctigenin has anti-inflammatory and antioxidative effects on LPS-induced acute lung injury, which are associated with modulation of MAPK, HO-1, and iNOS signaling.

Pennogenyl saponins induce cell cycle arrest and apoptosis in human hepatocellular carcinoma HepG2 cells

Ethnopharmacological relevancePennogenyl saponins, the characterized components of Rhizoma Paridis, have been reported to have anticancer activity through induction of apoptosis or anti-metastasis in cultured cells or animal models. The aim of the study was to evaluate the anticancer properties of four pennogenyl saponins (PS1–PS4) on a panel of human cancer and normal cell lines, and explore the potential mechanisms underlying the selective anticancer effects of the steroidal saponins in cancer cells. Materials and methodsDifferences in the anticancer activity of pennogenyl saponins were examined by MTT assay in human cancer cell lines (HepG2 hepatocellular carcinoma cells, UACC-257 melanoma cells, MCF-7 breast and PC-3 prostate cancer cells) and normal human cell lines (L-02 liver cells and HEK293 kidney cells). Flow cytometry analysis, JC-1 staining and western blot analysis were applied to detect the effects of anticancer pennogenyl saponins on apoptosis, cell cycle, and expression and/or activation of main effectors involved in the potential signaling pathways. ResultsAmong the tested four saponins, only PS1 and PS2 selectively inhibited cell growth in HepG2, MCF-7 and PC-3 cells. Moreover, PS1 and PS2 could significantly induce apoptosis and cell cycle G2/M arrest in HepG2 cells, which were at least associated with activation of mitochondrial caspase-dependent and -independent apoptotic cascades, inhibition of cyclin-dependent kinase 1 and PI3K/Akt signaling pathway, and modulation of mitogen-activated protein kinases. ConclusionsPS1 and PS2 had potent and selective anticancer activity to breast, liver and prostate cancer cells. Furthermore, the anticancer effects of PS1 and PS2 were associated with induction of apoptosis and blockage of cell cycle progression through multiple targets in HepG2 cells. These findings suggest that PS1 and PS2 can be considered as potential agents for the treatment of some cancers such as hepatoma.

Antiglycative activity of sulforaphane: a new avenue to counteract neurodegeneration?

Antiglycative activity of sulforaphane: a new avenue to counteract neurodegeneration?

Bergenin Plays an Anti-Inflammatory Role via the Modulation of MAPK and NF-κB Signaling Pathways in a Mouse Model of LPS-Induced Mastitis.

Mastitis is a major disease in humans and other animals and is characterized by mammary gland inflammation. It is a major disease of the dairy industry. Bergenin is an active constituent of the plants of genus Bergenia. Research indicates that bergenin has multiple biological activities, including anti-inflammatory and immunomodulatory properties. The objective of this study was to evaluate the protective effects and mechanism of bergenin on the mammary glands during lipopolysaccharide (LPS)-induced mastitis. In this study, mice were treated with LPS to induce mammary gland mastitis as a model for the disease. Bergenin treatment was initiated after LPS stimulation for 24h. The results indicated that bergenin attenuated inflammatory cell infiltration and decreased the concentration of NO, TNF-α, IL-1β, and IL-6, which were increased in LPS-induced mouse mastitis. Furthermore, bergenin downregulated the phosphorylation of nuclear factor-kappaB (NF-κB) and mitogen-activated protein kinases (MAPK) signaling pathway proteins in mammary glands with mastitis. In conclusion, bergenin reduced the expression of NO, TNF-α, IL-1β, and IL-6 proinflammatory cytokines by inhibiting the activation of the NF-κB and MAPKs signaling pathways, and it may represent a novel treatment strategy for mastitis.

UDP-glucose dehydrogenase modulates proteoglycan synthesis in articular chondrocytes: its possible involvement and regulation in osteoarthritis.

IntroductionThe objective of this study was to investigate the possible role of UDP-glucose dehydrogenase (UGDH) in osteoarthritis (OA) and uncover whether, furthermore how interleukin-1beta (IL-1β) affects UGDH gene expression.MethodsUGDH specific siRNAs were applied to determine the role of UGDH in proteoglycan (PG) synthesis in human articular chondrocytes. Protein levels of UGDH and Sp1 in human and rat OA cartilage were detected. Then, human primary chondrocytes were treated with IL-1β to find out whether and how IL-1β could regulate the gene expression of UGDH and its trans-regulators, that is Sp1, Sp3 and c-Krox. Finally, p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 and stress-activated protein kinase/c-Jun N-terminal kinase (SAP/JNK) inhibitor SP600125 were used to pick out the pathway that mediated the IL-1β-modulated PGs synthesis and gene expression of UGDH, Sp1, Sp3 and c-Krox.ResultsUGDH specific siRNAs markedly inhibited UGDH mRNA and protein expression, and thus led to an obvious suppression of PGs synthesis in human articular chondrocytes. UGDH protein level in human and rat OA cartilage were much lower than the corresponding controls and negatively correlated to the degree of OA. Decrease in Sp1 protein level was also observed in human and rat OA cartilage respectively. Meanwhile, IL-1β suppressed UGDH gene expression in human articular chondrocytes in the late phase, which also modulated gene expression of Sp1, Sp3 and c-Krox and increased both Sp3/Sp1 and c-Krox/Sp1 ratio. Moreover, the inhibition of SAP/JNK and p38 MAPK pathways both resulted in an obvious attenuation of the IL-1β-induced suppression on the UGDH gene expression.ConclusionsUGDH is essential in the PGs synthesis of articular chondrocytes, while the suppressed expression of UGDH might probably be involved in advanced OA, partly due to the modulation of p38 MAPK and SAP/JNK pathways and its trans-regulators by IL-1β.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-014-0484-2) contains supplementary material, which is available to authorized users.

The transmembrane protein FgSho1 regulates fungal development and pathogenicity via the MAPK module Ste50-Ste11-Ste7 in Fusarium graminearum.

The mitogen-activated protein kinase (MAPK) signaling pathways have been characterized in Fusarium graminearum. Currently, the upstream sensors of these pathways are unknown. Biological functions of a transmembrane protein FgSho1 were investigated using a target gene deletion strategy. The relationship between FgSho1 and the MAPK cassette FgSte50-Ste11-Ste7 was analyzed in depth. The transmembrane protein FgSho1 is required for conidiation, full virulence, and deoxynivalenol (DON) biosynthesis in F.graminearum. Furthermore, FgSho1 and FgSln1 have an additive effect on virulence of F.graminearum. The yeast two-hybrid, coimmunoprecipitation, colocalization and affinity capture-mass spectrometry analyses strongly indicated that FgSho1 physically interacts with the MAPK module FgSte50-Ste11-Ste7. Similar to the FgSho1 mutant, the mutants of FgSte50, FgSte11, and FgSte7 were defective in conidiation, pathogenicity, and DON biosynthesis. In addition, FgSho1 plays a minor role in the response to osmotic stress but it is involved in the cell wall integrity pathway, which is independent of the module FgSte50-Ste11-Ste7 in F.graminearum. Collectively, results of this study strongly indicate that FgSho1 regulates fungal development and pathogenicity via the MAPK module FgSte50-Ste11-Ste7 in F.graminearum, which is different from what is known in the budding yeast Saccharomyces cerevisiae.

A blueberry-enriched diet improves renal function and reduces oxidative stress in metabolic syndrome animals: potential mechanism of TLR4-MAPK signaling pathway.

BackgroundMetabolic syndrome (MetS) is characterized by a cluster of health factors that indicate a higher risk for cardio-renal diseases. Recent evidence indicates that antioxidants from berries are alternative to attenuate oxidative stress and inflammation. We tested the hypothesis that inflammation-induced renal damage is triggered by the activation of TLR4, and subsequent modulation of redox-sensitive molecules and mitogen-activated protein kinase (MAPK) pathway.MethodsFive-week old lean and obese Zucker rats (LZR and OZR) were fed a blueberry-enriched diet or an isocaloric control diet for 15 weeks. A glucose tolerance test and acute renal clearance experiments were performed. Gene and protein expression levels for TLR4, cytokines and phosphorylation of ERK and p38MAPK were measured. Kidney redox status and urinary albumin levels were quantified. Renal pathology was evaluated histologically.ResultsControl OZR exhibited lower glucose tolerance; exacerbated renal function parameters; increased oxidative stress. Gene and protein expression levels of TLR4 were higher and this was accompanied by increased renal pathology with extensive albuminuria and deterioration in antioxidant levels in OZR. In addition, OZR had increased phosphorylation of ERK and p38MAPK. Blueberry-fed OZR exhibited significant improvements in all these parameters compared to OZR.ConclusionTLR4-MAPK signaling pathway is a key to the renal structural injury and dysfunction in MetS and blueberry (BB) protect against this damage by inhibiting TLR4.SignificanceThis is the first study to put forth a potential mechanism of TLR4-induced kidney damage in a model of MetS and to elucidate a downstream mechanism by which blueberry exert their reno-protective effects.

Timing Is Everything: Highly Specific and Transient Expression of a MAP Kinase Determines Auxin-Induced Leaf Venation Patterns in Arabidopsis

Mitogen-activated protein kinase (MAPK) cascades are universal signal transduction modules present in all eukaryotes. In plants, MAPK cascades were shown to regulate cell division, developmental processes, stress responses, and hormone pathways. The subgroup A of Arabidopsis MAPKs consists of AtMPK3, AtMPK6, and AtMPK10. AtMPK3 and AtMPK6 are activated by their upstream MAP kinase kinases (MKKs) AtMKK4 and AtMKK5 in response to biotic and abiotic stress. In addition, they were identified as key regulators of stomatal development and patterning. AtMPK10 has long been considered as a pseudo-gene, derived from a gene duplication of AtMPK6. Here we show that AtMPK10 is expressed highly but very transiently in seedlings and at sites of local auxin maxima leaves. MPK10 encodes a functional kinase and interacts with the upstream MAP kinase kinase (MAPKK) AtMKK2. mpk10 mutants are delayed in flowering in long-day conditions and in continuous light. Moreover, cotyledons of mpk10 and mkk2 mutants have reduced vein complexity, which can be reversed by inhibiting polar auxin transport (PAT). Auxin does not affect AtMPK10 expression while treatment with the PAT inhibitor HFCA extends the expression in leaves and reverses the mpk10 mutant phenotype. These results suggest that the AtMKK2-AtMPK10 MAPK module regulates venation complexity by altering PAT efficiency.

Characterization of the Neurospora crassa cell fusion proteins, HAM-6, HAM-7, HAM-8, HAM-9, HAM-10, AMPH-1 and WHI-2.

Intercellular communication of vegetative cells and their subsequent cell fusion is vital for different aspects of growth, fitness, and differentiation of filamentous fungi. Cell fusion between germinating spores is important for early colony establishment, while hyphal fusion in the mature colony facilitates the movement of resources and organelles throughout an established colony. Approximately 50 proteins have been shown to be important for somatic cell-cell communication and fusion in the model filamentous fungus Neurospora crassa. Genetic, biochemical, and microscopic techniques were used to characterize the functions of seven previously poorly characterized cell fusion proteins. HAM-6, HAM-7 and HAM-8 share functional characteristics and are proposed to function in the same signaling network. Our data suggest that these proteins may form a sensor complex at the cell wall/plasma membrane for the MAK-1 cell wall integrity mitogen-activated protein kinase (MAPK) pathway. We also demonstrate that HAM-9, HAM-10, AMPH-1 and WHI-2 have more general functions and are required for normal growth and development. The activation status of the MAK-1 and MAK-2 MAPK pathways are altered in mutants lacking these proteins. We propose that these proteins may function to coordinate the activities of the two MAPK modules with other signaling pathways during cell fusion.

Neuroprotective effects of bilobalide on cerebral ischemia and reperfusion injury are associated with inhibition of pro-inflammatory mediator production and down-regulation of JNK1/2 and p38 MAPK activation.

BackgroundMitogen-activated protein kinase (MAPK) signaling pathways are implicated in inflammatory and apoptotic processes of cerebral ischemia and reperfusion (I/R) injury. Hence, MAPK pathways represent a promising therapeutic target. Exploring the full potential of inhibitors of MAPK pathways is a useful therapeutic strategy for ischemic stroke. Bilobalide, a predominant sesquiterpene trilactone constituent of Ginkgo biloba leaves, has been shown to exert powerful neuroprotective properties, which are closely related to both anti-inflammatory and anti-apoptotic pathways. We investigated the neuroprotective roles of bilobalide in the models of middle cerebral artery occlusion and reperfusion (MCAO/R) and oxygen-glucose deprivation and reoxygenation (OGD/R) of cerebral I/R injury. Moreover, we attempted to confirm the hypothesis that its protection effect is via modulation of pro-inflammatory mediators and MAPK pathways.MethodsMale Sprague-Dawley rats were subjected to MCAO for 2 h followed by reperfusion for 24 h. Bilobalide was administered intraperitoneally 60 min before induction of middle cerebral artery occlusion (MCAO). After reperfusion, neurological deficit scores, infarct volume, infarct weight, and brain edema were assessed. Ischemic penumbrae of the cerebral cortex were harvested to determine superoxide dismutase (SOD), malondialdehyde (MDA), nitric oxide, TNF-α, interleukin 1β (IL-1β), p-ERK1/2, p-JNK1/2, and p-p38 MAPK concentration. Similarly, the influence of bilobalide on the expression of nitric oxide, TNF-α, IL-1β, p-ERK1/2, p-JNK1/2, and p-p38 MAPK was also observed in an OGD/R in vitro model of I/R injury.ResultsPretreatment with bilobalide (5, 10 mg/kg) significantly decreased neurological deficit scores, infarct volume, infarct weight, brain edema, and concentrations of MDA, nitric oxide, TNF-α, IL-1β, and increased SOD activity. Furthermore, bilobalide (5, 10 mg/kg) pretreatment significantly down-regulated both p-JNK1/2 and p-p38 MAPK expression, whereas they had no effect on p-ERK1/2 expression in the ischemic penumbra. Supporting these observations in vivo, pretreatment with bilobalide (50, 100 μM) significantly down-regulated nitric oxide, TNF-α, IL-1β, p-JNK1/2, and p-p38 MAPK expression, but did not change p-ERK1/2 expression in rat cortical neurons after OGD/R injury.ConclusionsThese data indicate that the neuroprotective effects of bilobalide on cerebral I/R injury are associated with its inhibition of pro-inflammatory mediator production and down-regulation of JNK1/2 and p38 MAPK activation.

PRO40 is a scaffold protein of the cell wall integrity pathway, linking the MAP kinase module to the upstream activator protein kinase C.

Mitogen-activated protein kinase (MAPK) pathways are crucial signaling instruments in eukaryotes. Most ascomycetes possess three MAPK modules that are involved in key developmental processes like sexual propagation or pathogenesis. However, the regulation of these modules by adapters or scaffolds is largely unknown. Here, we studied the function of the cell wall integrity (CWI) MAPK module in the model fungus Sordaria macrospora. Using a forward genetic approach, we found that sterile mutant pro30 has a mutated mik1 gene that encodes the MAPK kinase kinase (MAPKKK) of the proposed CWI pathway. We generated single deletion mutants lacking MAPKKK MIK1, MAPK kinase (MAPKK) MEK1, or MAPK MAK1 and found them all to be sterile, cell fusion-deficient and highly impaired in vegetative growth and cell wall stress response. By searching for MEK1 interaction partners via tandem affinity purification and mass spectrometry, we identified previously characterized developmental protein PRO40 as a MEK1 interaction partner. Although fungal PRO40 homologs have been implicated in diverse developmental processes, their molecular function is currently unknown. Extensive affinity purification, mass spectrometry, and yeast two-hybrid experiments showed that PRO40 is able to bind MIK1, MEK1, and the upstream activator protein kinase C (PKC1). We further found that the PRO40 N-terminal disordered region and the central region encompassing a WW interaction domain are sufficient to govern interaction with MEK1. Most importantly, time- and stress-dependent phosphorylation studies showed that PRO40 is required for MAK1 activity. The sum of our results implies that PRO40 is a scaffold protein for the CWI pathway, linking the MAPK module to the upstream activator PKC1. Our data provide important insights into the mechanistic role of a protein that has been implicated in sexual and asexual development, cell fusion, symbiosis, and pathogenicity in different fungal systems.