Mitogen-activated Protein Kinase Homologue Research Articles

The Phosphatase VdPtc3 Regulates Virulence in Verticillium dahliae by Interacting with VdAtg1.

Type 2C protein phosphatases regulate various biological processes in eukaryotes. However, their functions in Verticillium dahliae have not been characterized. In this study, homologs VdPtc1, VdPtc3, VdPtc5, VdPtc6, and VdPtc7 were identified in V. dahliae on the basis of homologous comparison with those in Saccharomyces cerevisiae. VdPtc2 and VdPtc4 are missing in the genome of the V. dahliae XJ592 strain. VdPtc3 is the homolog of Ptc2, Ptc3, and Ptc4 proteins in S. cerevisiae, implying that VdPtc3 may play versatile functions in V. dahliae. VdPtc3 promoted conidium development, melanin, and microsclerotium formation in V. dahliae. The ΔVdPtc3 strains showed increased sensitivity to NaCl and sorbitol and augmented the phosphorylation of p38 mitogen-activated protein kinase homolog Hog1 induced by osmotic stress. Besides, the ΔVdPtc3 strains also showed milder Verticillium wilt symptom on cotton. Furthermore, VdPtc3 interacts with VdAtg1, which modulates melanin and microsclerotium formation, as well as pathogenicity.

Fus3/Kss1-MAP kinase and Ste12-like control distinct biocontrol-traits besides regulation of insect cuticle penetration via phosphorylation cascade in a filamentous fungal pathogen.

Homolog of the yeast Fus3/Kss1 MAP kinase (MAPK) pathway and its target transcription factor, Ste12-like, are involved in penetration of host cuticle /pathogenicity in many ascomycete pathogens. However, details of their interaction during fungal infection, as well as their controlled other virulence-associated traits, are unclear. Ste12-like (BbSte12) and Fus3/Kss1 MAPK homolog (Bbmpk1) interacted in nucleus, and phosphorylation of BbSte12 by Bbmpk1 was essential for penetration of insect cuticle in an insect fungal pathogen, Beauveria bassiana. However, some distinct biocontrol-traits were found to be mediated by Ste12 and Bbmpk1. In contrast to ΔBbmpk1 colony that grew more rapid than wild type strain, inactivation of BbSte12 resulted in the opposite phenotype, which was consistent with their different proliferation rates in insect hemocoel after direct injection of conidia bypass the cuticle. Reduced conidial yield with decreased hydrophobicity was examined in the both mutants, however they displayed distinct conidiogenesis, accompanying with differently altered cell cycle, distinct hyphal branching and septum formation. Moreover, ΔBbmpk1 showed increased tolerance to oxidative agent, whereas the opposite phenotype was seen for ΔBbSte12 strain. RNA sequencing analysis revealed that Bbmpk1 controlled 356 genes depending on BbSte12 during cuticle penetration, but 1077 and 584 genes were independently controlled by Bbmpk1 and BbSte12. BbSte12 and Bbmpk1 separately participate additional pathways for control of conidiation, growth and hyphal differentiation, as well as oxidative stress response besides regulating cuticle penetration via phosphorylation cascade. This article is protected by copyright. All rights reserved.

A β-Cyclodextrin-Based Nanoparticle with Very High Transfection Efficiency Unveils siRNA-Activated TLR3 Responses in Human Prostate Cancer Cells

Synthetic double-stranded small interfering RNAs (siRNAs) mimic interference RNAs (RNAi) and can bind target mRNAs with a high degree of specificity, leading to selective knockdown of the proteins they encode. However, siRNAs are very labile and must be both protected and transported by nanoparticles to be efficiently delivered into cells. In this work, we used a Janus-type polycationic amphiphilic β-cyclodextrin derivative to efficiently transfect siRNAs targeting mRNAs encoding mitogen-activated protein kinase (p42-MAPK) or Ras homolog enriched in brain (Rheb) into different cancer cell lines as well as astrocytes. We took advantage of this high transfection efficiency to simultaneously knock down p42-MAPK and Rheb to boost docetaxel (DTX)-mediated toxicity in two human prostate cancer cell lines (LNCaP and PC3). We found that double knockdown of p42-MAPK and Rheb increased DTX-toxicity in LNCaP but not in PC3 cells. However, we also observed the same effect when scramble siRNA was used, therefore pointing to an off-target effect. Indeed, we found that the siRNA we used in this work induced toll-like receptor 3 activation, leading to β-interferon production and caspase activation. We believe that this mechanism could be very useful as a general strategy to elicit an immune response against prostate cancer cells.

MAPK CcSakA of the HOG Pathway Is Involved in Stipe Elongation during Fruiting Body Development in Coprinopsis cinerea.

Mitogen-activated protein kinase (MAPK) pathways, such as the high-osmolarity glycerol mitogen-activated protein kinase (HOG) pathway, are evolutionarily conserved signaling modules responsible for transmitting environmental stress signals in eukaryotic organisms. Here, we identified the MAPK homologue in the HOG pathway of Coprinopsis cinerea, which was named CcSakA. Furthermore, during the development of the fruiting body, CcSakA was phosphorylated in the fast elongating apical part of the stipe, which meant that CcSakA was activated in the apical elongating stipe region of the fruiting body. The knockdown of CcSakA resulted in a shorter stipe of the fruiting body compared to the control strain, and the expression of phosphomimicking mutant CcSakA led to a longer stipe of the fruiting body compared to the control strain. The chitinase CcChiE1, which plays a key role during stipe elongation, was downregulated in the CcSakA knockdown strains and upregulated in the CcSakA phosphomimicking mutant strains. The results indicated that CcSakA participated in the elongation of stipes in the fruiting body development of C. cinerea by regulating the expression of CcChiE1. Analysis of the H2O2 concentration in different parts of the stipe showed that the oxidative stress in the elongating part of the stipe was higher than those in the non-elongating part. The results indicated that CcSakA of the HOG pathway may be activated by oxidative stress. Our results demonstrated that the HOG pathway transmits stress signals and regulates the expression of CcChiE1 during fruiting body development in C. cinerea.

Mutational analysis of the phosphorylation sites of the Leishmania mexicana kinesin homologue LmxKin29

Kinesins are motor proteins, have been identified in a wide diversity of eukaryotes, from protists to mammals. The kinetoplastids’ genome sequences have also shown the presence of a large number of kinesins. The current study presents characterise a novel L.mexicana kinesin LmxM29.3.050, thought to be associated with flagellum formation. It was found express in both the amastigote and promastigote life stages. In this study we assigned this protein as a kinesin homologue to the unknown or orphan kinesins superfamily (1). The biochemical analysis showed the MAP kinase homologue LmxMPK3 can phosphorylate full length of LmxKin29 at serine 554. Localisation studies using GFP-tagged LmxKin29 revealed that it is predominantly found in between the nucleus and the flagellar pocket, while in dividing cells LmxKin29 was found at the anterior and posterior ends of the cells. Hence, LmxKin29 might play a role in cytokinesis. Double allele deletion was successfully generated. Morphological analysis of promastigotes with LmxKin29 tagged with GFP displayed no obvious phenotypic differences comparing the mutants with wild type cells.

Temozolomide and AZD7762 Induce Synergistic Cytotoxicity Effects on Human Glioma Cells.

This study investigated the effects of temozolomide (TMZ) and/or checkpoint kinase inhibitor AZD7762 in human glioma cells. Glioma cells were treated with TMZ and/or AZD7762 for 24 or 48 h, then the cellular survival was studied and the expression of various proteins was investigated. Both TMZ and AZD7762 induced concentration- and time-dependent cytotoxic effects, and combined TMZ and AZD7762 (TMZ+AZD) caused synergistic cytotoxic effects in glioma cells (p<0.05). AZD7762 suppressed the O6-methylguanine-DNA-methyltransferase (MGMT) expression. TMZ+AZD increased the expression of phospho-p53 (p-p53), p-p38 mitogen-activated protein kinase, and phosphatase and tensin homolog; and decreased the expression of p-extracellular signal-regulated kinase 1/2 and p-signal transducer and activator of transcription 3 in glioma cells. TMZ and AZD7762 combined induced synergistic cytotoxic effects on human glioma cells and such effects may be related to the AZD7762-induced suppression of MGMT expression and the modulation of multiple signaling pathways.

PdSlt2 Penicillium digitatum mitogen-activated-protein kinase controls sporulation and virulence during citrus fruit infection

The Slt2 mitogen-activated protein (MAP) kinase homologue of Penicillium digitatum, the most relevant pathogen-producing citrus green mould decay during postharvest, was identified and explored. The P. digitatum Slt2-MAPK coding gene (PdSlt2) was functionally characterized by homologous gene elimination and transcriptomic evaluation. The absence of PdSlt2 gene resulted in significantly reduced virulence during citrus infection. The ΔPdSlt2 mutants were also defective in asexual reproduction, showing impairment of sporulation during citrus infection. Gene expression analysis revealed that PdSlt2 was highly induced during citrus fruit infection at early stages (1 dpi). Moreover, PdSlt2 deletion altered gene expression profiles. The relative gene expression (RGE) of fungicide resistance- and fungal virulence-related genes showed that PdSlt2 acts as negative regulator of several transporter encoding genes (ABC and MFS transporters) and a positive regulator of two sterol demethylases. This study indicates that PdSlt2 MAPK is functionally preserved in P. digitatum and highlights the relevant role of the PdSlt2 MAP kinase-mediated signalling pathway in regulating diverse genes crucial for infection and asexual reproduction.

WNK lysine deficient protein kinase 1 regulates human endometrial stromal cell decidualization, proliferation, and migration in part through mitogen-activated protein kinase 7.

The differentiation of endometrial stromal cells into decidual cells, termed decidualization, is an integral step in the establishment of pregnancy. The mitogen-activated protein kinase homolog, WNK lysine deficient protein kinase 1 (WNK1), is activated downstream of epidermal growth factor receptor during decidualization. Primary human endometrial stromal cells (HESCs) were subjected to small interfering RNA knockdown of WNK1 followed by in vitro decidualization. This abrogated expression of the decidual marker genes, insulin like growth factor binding protein 1 (IGFBP1) and prolactin (PRL), and prevented adoption of decidual cell morphology. Analysis of the WNK1-dependent transcriptome by RNA-Seq demonstrated that WNK1 regulates the expression of 1858 genes during decidualization. Gene ontology and upstream regulator pathway analysis showed that WNK1 regulates cell migration, differentiation, and proliferation. WNK1 was required for many of the gene expression changes that drive decidualization, including the induction of the inflammatory cytokines, C-C motif chemokine ligand 8 (CCL8), interleukin 1 beta (IL1B), and interleukin 15 (IL15), and the repression of transforming growth factor-beta (TGF-beta) pathway genes, including early growth response 2 (EGR2), SMAD family member 3 (SMAD3), integrin subunit alpha 2 (ITGA2), integrin subunit alpha 4 (ITGA4), and integrin subunit beta 3 (ITGB3). In addition to abrogating decidualization, WNK1 knockdown decreased the migration and proliferation of HESCs. Furthermore, mitogen-activated protein kinase 7 (MAPK7), a known downstream target of WNK1, was activated during decidualization in a WNK1-dependent manner. Small interfering RNA knockdown of MAPK7 demonstrated that MAPK7 regulates a subset of WNK1-regulated genes and controls the migration and proliferation of HESCs. These results indicate that WNK1 and MAPK7 promote migration and proliferation during decidualization and regulate the expression of inflammatory cytokines and TGF-beta pathway genes in HESCs.

The citrus postharvest pathogen Penicillium digitatum depends on the PdMpkB kinase for developmental and virulence functions

The postharvest pathogen Penicillium digitatum causes green mold decay on citrus fruit, resulting in severe economic losses. To explore possible factors involved in fungal pathogenesis, phenotypic characterization of the budding yeast Fus3/Kiss1 mitogen-activated protein (MAP) kinase homolog was carried out. The P. digitatum MAP kinase B coding gene, designated PdMpkB, was functionally inactivated via homologous recombination. The fungal strain (∆PdMpkB) carrying a PdMpkBdeletion demonstrated altered gene expression profiles, reduced growth and conidiogenesis, elevated resistance to osmotic stress, and failed to induce green mold decay on citrus fruit. ∆PdMpkB was more resistant to CaCl2, NaCl and sorbitol than its progenitor strain, indicating a negative regulatory function of PdMpkB in osmotic stress adaptation. Fungal infection assays on citrus fruit revealed that ∆PdMpkB proliferated poorly within host tissues, induced water-soaking lesions, failed to break through host cuticle layers and thus, failed to produce aerial hyphae and conidia. Introduction of a functional copy of PdMpkB into a null mutant restored all defective phenotypes. Transcriptome analysis revealed that inactivation of PdMpkB impacted expression of the genes associated with cell wall-degrading enzyme activities, carbohydrate and amino acid metabolisms, conidial formation, and numerous metabolic processes. Our results define pivotal roles of the PdMpkB-mediated signaling pathway in developmental and pathological functions in the citrus postharvest pathogen P. digitatum.

Higher TRAF6 and Fbxo 3 Expression Is With Progressive Multiple Myeloma and Blockage Of TRAF6 Inhibits Tumor Growth and Bone Resorption

Tumor necrosis factor receptor-associated factor 6 (TRAF6) has been implicated in regulating the NF-kB and JNK signal transduction pathways; and, thus, is likely to promote tumor cell proliferation and osteoclast formation. F-box proteins such as the E3 ligase component Fbxo3 and another F-box family member Fbxl 2, regulate TRAF protein signaling.First, we investigated TRAF6 expression in bone marrow mononuclear cells (BMMCs) from multiple myeloma (MM) patients with progressive disease or in remission and healthy subjects. The results showed higher TRAF6 protein expression among MM patients with progressive disease than among those in remission or healthy subjects. Notably, changes in TRAF6 protein expression in MM BMMCs were found to correlate with response of individual patients to treatment with the proteasome inhibitors bortezomib or carfilzomib. We have previously reported inhibition of MM cell proliferation and increase of apoptosis through regulation of the NF-kB and JNK pathways using a silencing TRAF6 C-domain mRNA construct. In this study, we cloned a 167 amino acid (in residues 333 to 508) portion of the TRAF6 dominant negative (TRAF6dn) and synthesized decoy peptides of the TRAF6 interaction domain with CD40 and the TRAF6-RANK binding domain. Decreases in cell proliferation and increase in cell apoptosis in MM BMMCs treated with TRAF6dn occurred in a concentrationdependent fashion. We also found TRAF6dn markedly inhibited osteoclast cell formation of monocytes induced by RANKL and mCSF in a dose-dependent manner. Next, we examined the effect of TRAF6dn on NF-kB and JNK by determining phosphorylation of JUN kinase kinase (JNKK), which activates the MAP kinase homologues SAPK and JNK in response to IL-1 receptor stimulation. MM BMMCs exposed to the TRAF6dn fragment or TRAF6 decoy peptide showed reduced phosphoNF-kB protein and phosphorylation of JNKK. These studies suggest that the TRAF6dn or combined TRAF6 decoy and CD40 decoy peptide may be excellent agents to block both MM cell growth and osteoclast formation in MM.We further investigated the protein expression of Fbxo 3, Fbxl 2 and TRAF6 in fresh BMMCs from MM patients with progressive disease or in remission. Results of Western blot analysis showed protein expression of Fbxo 3 and TRAF6 was increased and Fbxl 2 was decreased among patients with progressive disease compared to patients in remission.Thus, these results may offer a new mechanism through which MM tumor cells are regulated and provide a new therapeutic approach to treat MM and reduce the clinical consequences resulting from enhanced bone loss that commonly occur in these patients. Disclosures:No relevant conflicts of interest to declare.

Distinct Roles of a Mitogen-Activated Protein Kinase in Cytokinesis between Different Life Cycle Forms of Trypanosoma brucei

Mitogen-activated protein kinase (MAPK) modules are evolutionarily conserved signaling cascades that function in response to the environment and play crucial roles in intracellular signal transduction in eukaryotes. The involvement of a MAP kinase in regulating cytokinesis in yeast, animals, and plants has been reported, but the requirement for a MAP kinase for cytokinesis in the early-branching protozoa is not documented. Here, we show that a MAP kinase homolog (TbMAPK6) from Trypanosoma brucei plays distinct roles in cytokinesis in two life cycle forms of T. brucei. TbMAPK6 is distributed throughout the cytosol in the procyclic form but is localized in both the cytosol and the nucleus in the bloodstream form. RNA interference (RNAi) of TbMAPK6 results in moderate growth inhibition in the procyclic form but severe growth defects and rapid cell death in the bloodstream form. Moreover, TbMAPK6 appears to be implicated in furrow ingression and cytokinesis completion in the procyclic form but is essential for cytokinesis initiation in the bloodstream form. Despite the distinct defects in cytokinesis in the two forms, RNAi of TbMAPK6 also caused defective basal body duplication/segregation in a small cell population in both life cycle forms. Altogether, our results demonstrate the involvement of the TbMAPK6-mediated pathway in regulating cytokinesis in trypanosomes and suggest distinct roles of TbMAPK6 in cytokinesis between different life cycle stages of T. brucei.

Impaired induction of allergic lung inflammation by Alternaria alternata mutant MAPK homologue Fus3

ABSTRACTThe fungal allergen Alternaria alternata is associated with development of asthma, though the mechanisms underlying the allergenicity of Alternaria are largely unknown. The aim of this study was to identify whether the MAP kinase homologue Fus3 of Alternaria contributed to allergic airway responses. Wild-type (WT) and Fus3 deficient Alternaria extracts were given intranasal to mice. Extracts from Fus3 deficient Alternaria that had a functional copy of Fus3 introduced were also administered (CpFus3). Mice were challenged once and levels of BAL eosinophils and innate cytokines IL-33, thymic stromal lymphopoeitin (TSLP), and IL-25 (IL-17E) were assessed. Alternaria extracts or protease-inhibited extract were administered with (OVA) during sensitization prior to ovalbumin only challenges to determine extract adjuvant activity. Levels of BAL inflammatory cells, Th2 cytokines, and OX40-expressing Th2 cells as well as airway infiltration and mucus production were measured. WT Alternaria induced innate airway eosinophilia within 3 days. Mice given Fus3 deficient Alternaria were significantly impaired in developing airway eosinophilia that was largely restored by CpFus3. Further, BAL IL-33, TSLP, and Eotaxin-1 levels were reduced after challenge with Fus3 mutant extract compared with WT and CpFus3 extracts. WT and CpFus3 extracts demonstrated strong adjuvant activity in vivo as levels of BAL eosinophils, Th2 cytokines, and OX40-expressing Th2 cells as well as peribronchial inflammation and mucus production were induced. In contrast, the adjuvant activity of Fus3 extract or protease-inhibited WT extract was largely impaired. Finally, protease activity and Alt a1 levels were reduced in Fus3 mutant extract. Thus, Fus3 contributes to the Th2-sensitizing properties of Alternaria.

C2-Phytoceramide Perturbs Lipid Rafts and Cell Integrity in Saccharomyces cerevisiae in a Sterol-Dependent Manner

Specific ceramides are key regulators of cell fate, and extensive studies aimed to develop therapies based on ceramide-induced cell death. However, the mechanisms regulating ceramide cytotoxicity are not yet fully elucidated. Since ceramides also regulate growth and stress responses in yeast, we studied how different exogenous ceramides affect yeast cells. C2-phytoceramide, a soluble form of phytoceramides, the yeast counterparts of mammalian ceramides, greatly reduced clonogenic survival, particularly in the G2/M phase, but did not induce autophagy nor increase apoptotic markers. Rather, the loss of clonogenic survival was associated with PI positive staining, disorganization of lipid rafts and cell wall weakening. Sensitivity to C2-phytoceramide was exacerbated in mutants lacking Hog1p, the MAP kinase homolog of human p38 kinase. Decreasing sterol membrane content reduced sensitivity to C2-phytoceramide, suggesting sterols are the targets of this compound. This study identified a new function of C2-phytoceramide through disorganization of lipid rafts and induction of a necrotic cell death under hypo-osmotic conditions. Since lipid rafts are important in mammalian cell signaling and adhesion, our findings further support pursuing the exploitation of yeast to understand the basis of synthetic ceramides’ cytotoxicity to provide novel strategies for therapeutic intervention in cancer and other diseases.

Prognosis of patients with multifocal glioblastoma: a case-control study

The prognosis of patients with glioblastoma who present with multifocal disease is not well documented. The objective of this study was to determine whether multifocal disease on initial presentation is associated with worse survival. The authors retrospectively reviewed records of 368 patients with newly diagnosed glioblastoma and identified 47 patients with multifocal tumors. Each patient with a multifocal tumor was then matched with a patient with a solitary glioblastoma on the basis of age, Karnofsky Performance Scale (KPS) score, and extent of resection, using a propensity score matching methodology. Radiation and temozolomide treatments were also well matched between the 2 cohorts. Kaplan-Meier estimates and log-rank tests were used to compare patient survival. The incidence of multifocal tumors was 12.8% (47/368). The median age of patients with multifocal tumors was 61 years, 76.6% had KPS scores ≥ 70, and 87.2% underwent either a biopsy or partial resection of their tumors. The 47 patients with multifocal tumors were almost perfectly matched on the basis of age (p = 0.97), extent of resection (p = 1.0), and KPS score (p = 0.80) compared with 47 patients with a solitary glioblastoma. Age (>65 years), partial resection or biopsy, and low KPS score (<70) were associated with worse median survival within the multifocal group. In the multifocal group, 19 patients experienced tumor progression on postradiation therapy MRI, compared with 11 patients (26.8%) with tumor progression in the unifocal group (p = 0.08). Patients with multifocal tumors experienced a significantly shorter median overall survival of 6 months (95% CI 4-10 months), compared with the 11-month median survival (95% CI 10-19 months) of the matched solitary glioblastoma group (p = 0.02, log-rank test). Two-year survival rates were 4.3% for patients with multifocal tumors and 29.0% for the unifocal cohort. Patients with newly diagnosed multifocal tumors were found to have an almost 2-fold increase in the hazard of death compared with patients with solitary glioblastoma (hazard ratio 1.8, 95% CI 1.1-3.1; p = 0.02). Tumor samples were analyzed for expression of phosphorylated mitogen-activated protein kinase, phosphatase and tensin homolog, O(6)-methylguanine-DNA methyltransferase, laminin β1 and β2, as well as epidermal growth factor receptor amplification, and no significant differences in expression profile between the multifocal and solitary glioblastoma groups was found. Patients with newly diagnosed multifocal glioblastoma on presentation experience significantly worse survival than patients with solitary glioblastoma. Patients with multifocal tumors continue to pose a therapeutic challenge in the temozolomide era and magnify the challenges faced while treating patients with malignant gliomas.

Induction of MAP kinase homologues during growth and morphogenetic development of Karnal bunt (Tilletia indica) under the influence of host factor(s) from wheat spikes.

Signaling pathways that activate different mitogen-activated protein kinases (MAPKs) in response to certain environmental conditions, play important role in mating type switching (Fus3) and pathogenicity (Pmk1) in many fungi. In order to determine the roles of such regulatory genes in Tilletia indica, the causal pathogen of Karnal bunt (KB) of wheat, semi-quantitative and quantitative RT-PCR was carried out to isolate and determine the expression of MAP kinase homologues during fungal growth and development under in vitro culture. Maximum expression of TiFus3 and TiPmk1 genes were observed at 14th and 21st days of culture and decreased thereafter. To investigate whether the fungus alters the expression levels of same kinases upon interaction with plants, cultures were treated with 1% of host factors (extracted from S-2 stage of wheat spikes). Such treatment induced the expression of MAPks in time dependent manner compared to the absence of host factors. These results suggest that host factor(s) provide certain signal(s) which activate TiFus3 and TiPmk1 during morphogenetic development of T. indica. The results also provides a clue about the role of host factors in enhancing the disease potential due to induction of MAP kinases involved in fungal development and pathogenecity.

TRAF6 Dominant Negative Peptides Block Intracellular Signaling Resulting in Anti-Myeloma and Anti-Bone Resorptive Effects in Multiple Myeloma

Abstract 2919Tumor necrosis factor receptor-associated factor 6 (TRAF6) has been implicated in regulating the NF-kB and JNK signal transduction pathways; and, thus, is likely to promote tumor cell proliferation and osteoclast formation. We have previously reported inhibition of cell proliferation and increase of apoptosis in multiple myeloma (MM) cells through regulation of these intracellular pathways through silencing of TRAF6 C-domain mRNA. To determine TRAF6 protein expression in fresh MM tumor cells, we performed an immunofluorescence assay (IFA). The results showed that expression of this factor in tumor cells from bone marrow (BM) from MM patients with progressive disease is higher than in cells from patients with monoclonal gammopathies without disease progression or normal controls. We further examined the effects of TRAF6 negative dominant peptides on intracellular signaling pathways. Briefly, cells from the RPMI8226 or MM1s MM cell lines or primary MM BM samples were treated with or without TRAF6 inhibition peptide for 24 hours and then stimulated with either IGF1 (30ng/ml) or IL1 β (20ng/ml) for 30 minutes. The cells were lysed and Western blot analysis performed to determine protein phosphorylation and RT-PCR for gene expression. TRAF6 has been found to be an E3 ligase for Akt ubiquitination. We found that IGF1 increased the phosphorylation of AKT and treatment with TRAF6 inhibition peptide markedly decreased its phosphorylation compared to treatment with a control peptide in RPMI8226 and primary MM tumor cells. Downstream of AKT, C-Raf phosphorylation was also significantly reduced with treatment with TRAF6 inhibition peptide. Notably, cyclin D gene expression in MM tumor cells treated with TRAF6 inhibition peptide was reduced as determined with an RT-PCR. In contrast, the gene expression of mTOR was increased in RPMI8226 cells treated with TRAF6 inhibition peptide whereas there was no change in its expression in MM1s and primary MM tumor cells. It is quite possible that the increase in mTOR expression in RPMI8226 cells may act as a negative feedback which results from blockage of the ubiquitination of TRAF6. We further examined the effect of the TRAF6 inhibition peptide on NF-kB and JNK signaling as determined through evaluation of JUN kinase kinase (JNKK), which activates the MAP kinase homologues SAPK and JNK in response to IL-1 receptor stimulation. Phospho-NF-kB protein was reduced and phosphorylation of JNKK was clearly decreased with exposure to the TRAF6 inhibition peptide. We examined c-Jun, a component of the transcription factor complex AP-1, which binds and activates transcription at TRE/AP-1 elements. Total endogenous c-Jun is reduced following exposure of RPMI8226 cells to the TRAF6 inhibition peptide. Consistent with our past findings, TRAF6 inhibition peptide significantly inhibited osteoclast formation from CD14+ induced by RANKL and M-CSF with in a concentration dependent fashion whereas control peptides showed no effects on osteoclast formation. In addition, inhibition of the TRAF6 signaling blocked not only myeloma cell proliferation induced by AKT and NF-kB activation but also osteoclast cell formation mediated through transcription at TRE/AP-1 elements. The study has been extended to our SCID-hu murine model of human myeloma. Disclosures:No relevant conflicts of interest to declare.

Inhibition of ubiquitin proteasome function suppresses proliferation of pulmonary artery smooth muscle cells

Inhibition of proteasome function has been shown to suppress several types of cells proliferation; this study investigates whether this also occurs in pulmonary artery smooth muscle cells (PASMCs) and its potential mechanisms. Serotonin induced 4.27-fold increase in DNA synthesis in PASMCs, and this effect was dose-dependently blocked by prior incubation of cells with MG132, a specific proteasome inhibitor. Inhibition of proteasome function did not modulate serotonin-triggered pro-proliferation signaling pathways, such as extracellular signal-regulated mitogen-activated protein kinase (ERK1/2 MAPK) and Ras homolog gene family member A (RhoA). Further study indicated that treatment of PASMCs with serotonin reduced p21(WAF1) protein level but not its transcription; this was reversed by inhibiting ERK1/2 MAPK or RhoA cascade equally. In addition, MG132 increased the protein level of p21(WAF1) in a dose-dependent manner in the presence of serotonin, 10μM MG132 led to a 4.2-fold increase in p21(WAF1) protein level, and this effect was not mediated by increasing p21(WAF1) mRNA level. More importantly, cell lacking p21(WAF1) by siRNA transfection abolished the inhibitive effect of MG132 on cells proliferation. Our study suggests that accumulation of p21(WAF1) protein level caused by proteasome inhibition particularly mediated its inhibitive effect on PASMCs proliferation, and inhibition of proteasome function might have potential value in the treatment of pulmonary hypertension.

Abstract LB-305: The tyrosine kinase inhibitor bafetinib (INNO-406) inhibits osteoclast formation and bone resorption

Abstract Lyn phosphorylation up-regulates several kinases including Syk, phospholipase Cγ2 and phosphatidyl inostitol-3 kinase through the immunoreceptor tyrosine-based activation motif (ITAM). Lyn plays critical roles in cell proliferation, Ca2+ mobilization and cell differentiation. This kinase also plays an essential role in transmission of inhibitory signals through phosphorylation of tyrosine residues within the immunoreceptor tyrosine-based inhibitory motifs (ITIM). ITAM-ITIM cross talk is important for macrophage differentiation and osteoclast formation. Bafetinib (INNO-406) is an inhibitor of Bcr-Abl, Fyn and Lyn kinase. No studies have evaluated potential anti-bone resorptive effects of this tyrosine kinase inhibitor. Osteoclasts are multinucleated bone-resorbing cells derived from monocytes that play critical roles in bone remodeling. To evaluate the effects of bafetinib on osteoclast formation and bone resorption, we isolated monocytes using immunomagnetic bead selection from normal or multiple myeloma (MM) patients' PBMCs. CD14+ cells (monocytes) were treated with 50ng/ml RANKL and 20 ng/ml MCSF at the beginning of the culture and during a medium change at 3 days. During the second day of culture, bafetinib or the nitrogen-containing bisphosphonate zoledronic acid, an inhibitor of osteoclast formation and bone resorption, was added into the cells. The cells were fixed and tartrate-resistant acid phosphatase staining performed on day 21. Bafetinib and zoledronic acid both markedly inhibited osteoclast cell formation of monocytes induced by RANKL and MCSF at similar concentrations in a concentration-dependent fashion in both monocytes derived from MM patients and normal subjects. Next, we assessed bone resorption using monocytes that were induced with M-CSF and RANKL and cultured on bone slides for 28 days. At that time, bone resorption was determined using toluidine blue staining. Resorption pits were measured and percentage of surface area with lacunar resorption on each bone slice was determined using an image analysis system. At a concentration as low as 5μM, bafetinib significantly inhibited bone resorption in a concentration-dependent fashion (P&amp;lt;0.001). We further examined the effects of bafetinib on NF-κB and JNK phosphorylation in human monocytes that were induced with RANKL and MCSF through assessment of JUN kinase kinase (JNKK), which activates the MAP kinase homologues SAPK and JNK in response to IL-1 receptor stimulation. Phospho-NF-κB protein levels were reduced and phosphorylation of JNKK was also decreased following exposure to bafetinib. These studies suggest that bafetinib may be a new therapeutic option to reduce skeletal complications through its ability to block osteoclast development and reduce bone resorption. We are evaluating the effects of this tyrosine kinase inhibitor on bone resorption in vivo using our SCID-hu murine models of human myeloma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-305. doi:10.1158/1538-7445.AM2011-LB-305

The SLT2 mitogen‐activated protein kinase‐mediated signalling pathway governs conidiation, morphogenesis, fungal virulence and production of toxin and melanin in the tangerine pathotype of Alternaria alternata

Fungi respond and adapt to different environmental stimuli via signal transduction systems. We determined the function of a yeast SLT2 mitogen-activated protein (MAP) kinase homologue (AaSLT2) in Alternaria alternata, the fungal pathogen of citrus. Analysis of the loss-of-function mutant indicated that AaSLT2 is required for the production of a host-selective toxin, and is crucial for fungal pathogenicity. Moreover, the A. alternata slt2 mutants displayed hypersensitivity to cell wall-degrading enzymes and chemicals such as Calcofluor white and Congo red. This implicates an important role of AaSLT2 in the maintenance of cell wall integrity in A. alternata. The A. alternata slt2 mutants were also hypersensitive to a heteroaromatic compound, 2-chloro-5-hydroxypyridine, and a plant growth regulator, 2,3,5-triiodobenzoic acid. Developmentally, the AaSLT2 gene product was shown to be critical for conidial formation and hyphal elongation. Compared with the wild-type, the mutants produced fewer but slightly larger conidia with less transverse septae. The mutants also accumulated lower levels of melanin and chitin. Unlike the wild-type progenitor, the A. alternata slt2 mutants produced globose, swollen hyphae that did not elongate in a straight radial direction. All defective phenotypes in the mutant were restored by transformation and expression of a wild-type copy of AaSLT2 under the control of its endogenous promoter. This study highlights an important role of the AaSLT2 MAP kinase-mediated signalling pathway, regulating diverse physiological, developmental and pathological functions, in the tangerine pathotype of A. alternata.

A Yeast STE11 Homologue CoMEKK1 Is Essential for Pathogenesis-Related Morphogenesis in Colletotrichum orbiculare

Several signal transduction pathways, including mitogen-activated protein kinase (MAPK) pathways, are involved in appressorium development in Colletotrichum orbiculare, the causal agent of cucumber anthracnose disease. In this study, CoMEKK1, a yeast MAPK kinases (MAPKK) kinase STE11 homolog, was identified as a disrupted gene in an Agrobacterium tumefaciens-mediated transformation mutant. The phenotype of comekk1 disruptant was similar to that of cmk1, a Saccharomyces cerevisiae Fus3/Kss1 MAPK homolog mutant. Moreover, comekk1 and cmk1 mutants were sensitive to high osmotic and salinity stresses, indicating that Comekk1p/Cmk1p signal transduction is involved in stress tolerance. The transformants of the wild type and the comekk1 mutant expressing a constitutively active form of the CoMEKK1 showed slower hyphal growth and abnormal appressorium formation, whereas those of the cmk1 disruptant did not. A Cmk1p-green fluorescent protein (GFP) intracellular localization experiment indicated that nuclear localization of the Cmk1p-GFP fusion protein induced by salt stress was diminished in comekk1 mutants. These results indicate that Comekk1p functions upstream of Cmk1p.