MAP Kinase Inhibitor Research Articles

An Optimized Chromatographic Strategy for Multiplexing In Parallel Reaction Monitoring Mass Spectrometry: Insights from Quantitation of Activated Kinases

Reliable quantitation of protein abundances in defined sets of cellular proteins is critical to numerous biological applications. Traditional immunodetection-based methods are limited by the quality and availability of specific antibodies, especially for site-specific post-translational modifications. Targeted proteomic methods, including the recently developed parallel reaction monitoring (PRM) mass spectrometry, have enabled accurate quantitative measurements of up to a few hundred specific target peptides. However, the degree of practical multiplexing in label-free PRM workflows remains a significant limitation for the technique. Here we present a strategy for significantly increasing multiplexing in label-free PRM that takes advantage of the superior separation characteristics and retention time stability of meter-scale monolithic silica-C18 column-based chromatography. We show the utility of the approach in quantifying kinase abundances downstream of previously developed active kinase enrichment methodology based on multidrug inhibitor beads. We examine kinase activation dynamics in response to three different MAP kinase inhibitors in colorectal carcinoma cells and demonstrate reliable quantitation of over 800 target peptides from over 150 kinases in a single label-free PRM run. The kinase activity profiles obtained from these analyses reveal compensatory activation of TGF-β family receptors as a response to MAPK blockade. The gains achieved using this label-free PRM multiplexing strategy will benefit a wide array of biological applications.

KRAS Allelic Imbalance Enhances Fitness and Modulates MAP Kinase Dependence in Cancer

Investigating therapeutic "outliers" that show exceptional responses to anti-cancer treatment can uncover biomarkers of drug sensitivity. We performed preclinical trials investigating primary murine acute myeloid leukemias (AMLs) generated by retroviral insertional mutagenesis in KrasG12D "knockin" mice with the MEK inhibitor PD0325901 (PD901). One outlier AML responded and exhibited intrinsic drug resistance at relapse. Loss of wild-type (WT) Kras enhanced the fitness of the dominant clone and rendered it sensitive to MEK inhibition. Similarly, human colorectal cancer cell lines with increased KRAS mutant allele frequency were more sensitive to MAP kinase inhibition, and CRISPR-Cas9-mediated replacement of WT KRAS with a mutant allele sensitized heterozygous mutant HCT116 cells to treatment. In a prospectively characterized cohort of patients with advanced cancer, 642 of 1,168 (55%) with KRAS mutations exhibited allelic imbalance. These studies demonstrate that serial genetic changes at the Kras/KRAS locus are frequent in cancer and modulate competitive fitness and MEK dependency.

Cycloartenyl Ferulate downregulates lipopolysaccharide stimulated iNOS mRNA via NF-kB suppression in RAW 264.7 macrophages

Cycloartenyl ferulate (CAF) is a major bioactive phytosteryl ferulate purified from rice bran ?- oryzanol. Previously we reported that CAF ameliorates DSS-induced colitis in mice. The present study was undertaken to investigate the effects of CAF on LPS (lipopolysaccharide) stimulated murine RAW 264.7 macrophages. Immunohistochemistry analysis demonstrated that LPS (10ng/mL) treatment exhibited nuclear translocation of NF-?B-p65 in RAW macrophages, which was markedly inhibited CAF (30?M). LPS (10ng/mL) stimulation for 1-4 hours significantly upregulated iNOS and COX-2 mRNA in RAW 264.7 macrophages, but COX-2 mRNA was faster than that of iNOS mRNA. Macrophages pretreated with CAF greatly inhibited the LPS stimulated iNOS mRNA in a dose dependent manner (1-30?M), but CAF weakly inhibited COX-2 mRNA. Interestingly, CAY 10404 (COX-2 inhibitor) inhibited LPS stimulated iNOS mRNA, but not COX-2. In addition, PGE2 (1?M) upregulated iNOS mRNA but did not show any remarkable effects on NF-?B-p65 nuclear translocation in RAW macrophages. PD98059 (p44/42 MAP kinase inhibitor) inhibited iNOS mRNA, but not COX-2. On the other hand, PD169316 (p38 MAP kinase inhibitor) neither inhibited iNOS mRNA nor COX-2. Our results suggest that iNOS mRNA expression by LPS is mediated via p44/42 MAP kinase pathway in RAW 264.7 macrophages, which depends on the preceding expression of COX-2 expression. CAF downregulates iNOS mRNA via an inhibition of nuclear translocation of NF-?B with different mode of action on COX-2 gene expression.Asian J. Med. Biol. Res. December 2016, 2(4): 523-531

Resveratrol Inhibits the Epidermal Growth Factor-Induced Migration of Osteoblasts: the Suppression of SAPK/JNK and Akt

Background/Aims: Resveratrol is a polyphenol enriched in the skins of grapes and berries, that shows various beneficial effects for human health. In the present study, we investigated the mechanism behind the epidermal growth factor (EGF)-induced migration of osteoblast-like MC3T3-E1 cells, and the effect of resveratrol on this cell migration. Methods: The cell migration was examined using Boyden chamber, and phosphorylation of each kinase was analyzed by Western blotting. Results: The EGF-induced migration was suppressed by PD98059, an inhibitor of MEK1/2, as well as SB203580, an inhibitor of p38 MAP kinase, SP600125, an inhibitor of SAPK/JNK, and deguelin, an inhibitor of Akt. In contrast, rapamycin, an inhibitor of upstream kinase of p70 S6 kinase, and fasudil, an inhibitor of Rho-kinase, hardly affected the migration. Resveratrol significantly reduced the EGF-induced migration in a dose-dependent manner. SRT1720, an SIRT1 activator, suppressed the migration by EGF. In addition, resveratrol markedly attenuated the EGF-induced phosphorylation of SAPK/JNK and Akt without affecting the phosphorylation of p44/p42 MAP kinase or p38 MAP kinase. The phosphorylation of SAPK/JNK and Akt induced by EGF was down-regulated by SRT1720. Conclusion: Our results strongly suggest that resveratrol reduces the EGF-stimulated migration of osteoblasts via suppression of SAPK and Akt, and that the inhibitory effect of resveratrol is mediated in part via SIRT1.

Sustained Contraction in Vascular Smooth Muscle by Activation of L-type Ca2+ Channels Does Not Involve Ca2+ Sensitization or Caldesmon.

Vascular smooth muscle (VSM) is unique in its ability to maintain an intrinsic level of contractile force, known as tone. Vascular tone is believed to arise from the constitutive activity of membrane-bound L-type Ca2+ channels (LTCC). This study used a pharmacological agonist of LTCC, Bay K8644, to elicit a sustained, sub-maximal contraction in VSM that mimics tone. Downstream signaling was investigated in order to determine what molecules are responsible for tone. Medial strips of swine carotid artery were stimulated with 100 nM Bay K8644 to induce a sustained level of force. Force and phosphorylation levels of myosin light chain (MLC), MAP kinase, MYPT1, CPI-17, and caldesmon were measured during Bay K8644 stimulation in the presence and absence of nifedipine, ML-7, U0126, bisindolylmaleimide (Bis), and H-1152. Nifedipine and ML-7 inhibited force and MLC phosphorylation in response to Bay K8644. Inhibition of Rho kinase (H-1152) but not PKC (Bis) inhibited Bay K8644 induced force. U0126 significantly increased Bay K8644-dependent force with no effect on MLC phosphorylation. Neither CPI-17 nor caldesmon phosphorylation were increased during the maintenance of sustained force. Our results suggest that force due to the influx of calcium through LTCCs is partially MLC phosphorylation-dependent but does not involve PKC or caldesmon. Interestingly, inhibition of MLC kinase (MLCK) and PKC significantly increased MAP kinase phosphorylation suggesting that MLCK and PKC may directly or indirectly inhibit MAP kinase activity during prolonged contractions induced by Bay K8544.

13-Ethylberberine reduces HMGB1 release through AMPK activation in LPS-activated RAW264.7 cells and protects endotoxemic mice from organ damage

High mobility group box 1 (HMGB1), a highly conserved non-histone DNA-binding protein, plays an important role in the pathogenesis of sepsis. Previously, the authors reported 13-ethylberberine (13-EBR) has anti-inflammatory and antibacterial effects. However, the effect of 13-EBR on HMGB1 release was not investigated. In the present study, it was hypothesized 13-EBR might reduce HMGB1 release by activating AMPK under septic conditions. The results obtained showed 13-EBR significantly reduced HMGB1 release from LPS-activated RAW264.7 cells, and that this reduction was reversed by silencing p38, or AMPK, or by co-treating cells with p38 MAPKinase inhibitor. 13-EBR increased the phosphorylations of p38 and AMPK, and the phosphorylation of p38 by 13-EBR was inhibited by AMPK-siRNA, indicating AMPK acted upstream of p38. In the lung tissues of LPS-treated mice, 13-EBR administration significantly increased p-AMPK but reduced inducible nitric oxide synthase (iNOS) protein levels. Hematoxylin and eosin staining revealed 13-EBR significantly reduced LPS-induced lung and liver damage. In addition, 13-EBR inhibited NF-kB in LPS-activated RAW264.7 cells, and in LPS-treated mice, 13-EBR administration significantly increased survival. Furthermore, co-administration of 13-EBR plus LPS prevented LPS-induced aortic rings hypocontractile response to phenylephrine in vitro. Taken together, these results indicate 13-EBR might offer a means of treating sepsis through AMPK activation.

The Novel Translocation t(4;15)(q31;q22) in AML Is Associated with a Proliferative Phenotype in the Presence of All-Trans Retinoic Acid (ATRA)

Introduction: We describe for the first time, a novel chromosomal translocation in AML. The cells bearing this translocation proliferated rapidly following stimulation with ATRA in vitro. A 30-year old woman presented with monocytic AML. Karyotype revealed: 46,XX,t(9;11)(p22;q23)[13]/46,XX[7]. Mutational analysis showed an activating mutation in NRAS (c.34G>A; p.G12S). She achieved remission with standard induction therapy followed by cytarabine consolidation in the absence of an optimal donor. Following a disease free interval of 8 months, she relapsed. Karyotype at relapse: 46,XX,t(4;15)(q31;q22),t(9;11)(p22;q23)[20] (Figure 1). Mutational analysis revealed no new mutations. In the setting of chemotherapy refractory disease, she was enrolled on a phase I clinical trial combining escalating doses of TCP (tranylcypromine, Parnate¨) with fixed doses of ATRA (NCT02273102). The patient died from rapid disease progression shortly after her first cycle of therapy. Unexpectedly, we observed a rapid proliferation in the patient’s blasts following treatment with ATRA in vitro. We hypothesized that the novel t(4;15) translocation was involved in the regulation of retinoic acid (RA) signaling, and may have contributed to the rapid disease progression observed in this patient when she was treated with ATRA. To this end, we fully characterized the fusion gene and created a cell line bearing the translocation. Using these cells, we will further elucidate the mechanisms accounting for a rare and potentially clinically relevant effect of ATRA, in vitro.Methods: Cytogenetics and molecular-cytogenetic techniques, next generation sequencing (WES), RNA-seq, RT-PCR, and direct Sanger sequencing were used to map the chromosomal translocation. Cell proliferation and Annexin V apoptosis assays were used to test the effects of ATRA and various retinoic acid receptor (RAR) agonists and antagonists in vitro. Cells were treated with ATRA, RARα, and RARγ agonists and antagonists for 72 h and cell proliferation and apoptosis were measured with CellTiter-Glow (Promega)¨ luminescence viability assay and Annexin V/PI staining (FACS), respectively.Results: We mapped the t(4;15)(q31;q22) at the single nucleotide level, and discovered a novel fusion of TMEM154 (4q31.3) and RASGRF1 (15q24.2) genes at both the DNA and RNA level. The fusion protein included exons 1-6 from TMEM154 and exons 15-24 from RASGRF1. RASGRF1 (Ras protein specific nucleotide releasing factor 1) activates Ras by catalyzing the exchange of Ras-bound GDP for GTP. We determined that TMEM154 (transmembrane protein 154) is regulated by RA based on gene expression profiling. This particular translocation appears to result in a proliferative advantage in AML cells treated with RAR agonists such as ATRA, even at physiological levels (10 nM) (Figure 2). In MLL-rearranged cell lines without t(4;15), ATRA has a neutral or anti-proliferative effect, suggesting that the t(4;15) product interacts with the RA pathway to induce proliferation of AML blasts. We hypothesize that by increasing expression of TMEM154-RASGRF1, ATRA drives proliferation through Ras signaling. Of note, RAR antagonists inhibited proliferation and induced cell death (Figure 2). Also, treating the cells with MAP Kinase and Ras inhibitors consistently diminished the proliferative effect of ATRA. Updated studies shedding light on the mechanism of interaction between RA signaling and the TMEM154-RASGRF1 fusion protein will be presented.Conclusions: Our study for the first time identifies t(4;15) as a novel and pathogenic translocation in AML. This rare event may confer a proliferative advantage in the presence of ATRA and caution is advised should patients with this lesion be considered for treatment with ATRA. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

352 - Heme-Driven ROS Production Leads to Increased P38 MAP Kinase Activity and Endothelial Barrier Disruption

Heme is involved in a wide number of biological processes including gas exchange, antioxidant defense, signal transduction, as well as energy production within the mitochondria. It’s concentration within the cell is tightly regulated, and the consequences of underproduction of heme (anemia) are widely recognized. However, the pathophysiological role of high heme concentrations is less studied, but is already implicated in a number of cardiopulmonary diseases. An overabundance of intracellular heme precedes an over production of mitochondrial reactive oxygen species (ROS). ROS imbalance in the cell overwhelms basal antioxidant defenses and leads to various stress responses. Primarily, in this study, we focus on heme-driven effects of cellular signaling on human lung micro vascular endothelial cells (HLMVECs) permeability. We show that the p38 mitogen activated protein kinase (MAP kinase) pathway is activated with a specific response over a time course of heme treatment and that heme driven ROS production and cellular responses leads to endothelial barrier disruptions. In addition, we focus on a few therapeutic targets of heme pathway with potential to alleviate the harmful and cascading effects of ROS: 1) Inhibition of Heme Carrier Protein 1 (HCP-1) that mediates free heme transition into the cell; 2) Inhibition of heme mediated mitochondrial ROS production to study the downstream effects of lowering mitochondrial ROS on HLMVEC permeability; 3) Inhibition of p38 MAP Kinase to study the downstream effects of lowering activity of this pathway counteracting the response of ROS activity. These potential strategies in combating the pathophysiology of high levels of free heme are preventive and solidify our understanding of intracellular heme activity. However, the specific mechanism by which heme/ROS activates stress signaling pathways is a topic for future study and of high therapeutic value.

Estimation of relative free energies of binding using pre-computed ensembles based on the single-step free energy perturbation and the site-identification by Ligand competitive saturation approaches.

Accurate and rapid estimation of relative binding affinities of ligand-protein complexes is a requirement of computational methods for their effective use in rational ligand design. Of the approaches commonly used, free energy perturbation (FEP) methods are considered one of the most accurate, although they require significant computational resources. Accordingly, it is desirable to have alternative methods of similar accuracy but greater computational efficiency to facilitate ligand design. In the present study relative free energies of binding are estimated for one or two non-hydrogen atom changes in compounds targeting the proteins ACK1 and p38 MAP kinase using three methods. The methods include standard FEP, single-step free energy perturbation (SSFEP) and the site-identification by ligand competitive saturation (SILCS) ligand grid free energy (LGFE) approach. Results show the SSFEP and SILCS LGFE methods to be competitive with or better than the FEP results for the studied systems, with SILCS LGFE giving the best agreement with experimental results. This is supported by additional comparisons with published FEP data on p38 MAP kinase inhibitors. While both the SSFEP and SILCS LGFE approaches require a significant upfront computational investment, they offer a 1000-fold computational savings over FEP for calculating the relative affinities of ligand modifications once those pre-computations are complete. An illustrative example of the potential application of these methods in the context of screening large numbers of transformations is presented. Thus, the SSFEP and SILCS LGFE approaches represent viable alternatives for actively driving ligand design during drug discovery and development. © 2016 Wiley Periodicals, Inc.

Valproate inhibits MAP kinase signalling and cell cycle progression in S. cerevisiae.

The mechanism of action of valproate (VPA), a widely prescribed short chain fatty acid with anticonvulsant and anticancer properties, remains poorly understood. Here, the yeast Saccharomyces cerevisiae was used as model to investigate the biological consequences of VPA exposure. We found that low pH strongly potentiates VPA-induced growth inhibition. Transcriptional profiling revealed that under these conditions, VPA modulates the expression of genes involved in diverse cellular processes including protein folding, cell wall organisation, sexual reproduction, and cell cycle progression. We further investigated the impact of VPA on selected processes and found that this drug: i) activates markers of the unfolded protein stress response such as Hac1 mRNA splicing; ii) modulates the cell wall integrity pathway by inhibiting the activation of the Slt2 MAP kinase, and synergizes with cell wall stressors such as micafungin and calcofluor white in preventing yeast growth; iii) prevents activation of the Kss1 and Fus3 MAP kinases of the mating pheromone pathway, which in turn abolishes cellular responses to alpha factor; and iv) blocks cell cycle progression and DNA replication. Overall, our data identify heretofore unknown biological responses to VPA in budding yeast, and highlight the broad spectrum of cellular pathways influenced by this chemical in eukaryotes.

Type-7 metabotropic glutamate receptors negatively regulate α1-adrenergic receptor signalling

We studied the interaction between mGlu7 and α1-adrenergic receptors in heterologous expression systems, brain slices, and living animals. L-2-Amino-4-phosphonobutanoate (L-AP4), and l-serine-O-phosphate (L-SOP), which activate group III mGlu receptors, restrained the stimulation of polyphosphoinositide (PI) hydrolysis induced by the α1-adrenergic receptor agonist, phenylephrine, in HEK 293 cells co-expressing α1-adrenergic and mGlu7 receptors. The inibitory action of L-AP4 was abrogated by (i) the mGlu7 receptor antagonist, XAP044; (ii) the C-terminal portion of type-2 G protein coupled receptor kinase; and (iii) the MAP kinase inhibitors, UO126 and PD98059. This suggests that the functional interaction between mGlu7 and α1-adrenergic receptors was mediated by the βγ-subunits of the Gi protein and required the activation of the MAP kinase pathway. Remarkably, activation of neither mGlu2 nor mGlu4 receptors reduced α1-adrenergic receptor-mediated PI hydrolysis. In mouse cortical slices, both L-AP4 and L-SOP were able to attenuate norepinephrine- and phenylephrine-stimulated PI hydrolysis at concentrations consistent with the activation of mGlu7 receptors. L-AP4 failed to affect norepinephrine-stimulated PI hydrolysis in cortical slices from mGlu7−/− mice, but retained its inhibitory activity in slices from mGlu4−/− mice. At behavioural level, i.c.v. injection of phenylephrine produced antidepressant-like effects in the forced swim test. The action of phenylephrine was attenuated by L-SOP, which was inactive per se. Finally, both phenylephrine and L-SOP increased corticosterone levels in mice, but the increase was halved when the two drugs were administered in combination. Our data demonstrate that α1-adrenergic and mGlu7 receptors functionally interact and suggest that this interaction might be targeted in the treatment of stress-related disorders.

Differential transcriptional responses to Ebola and Marburg virus infection in bat and human cells

The unprecedented outbreak of Ebola in West Africa resulted in over 28,000 cases and 11,000 deaths, underlining the need for a better understanding of the biology of this highly pathogenic virus to develop specific counter strategies. Two filoviruses, the Ebola and Marburg viruses, result in a severe and often fatal infection in humans. However, bats are natural hosts and survive filovirus infections without obvious symptoms. The molecular basis of this striking difference in the response to filovirus infections is not well understood. We report a systematic overview of differentially expressed genes, activity motifs and pathways in human and bat cells infected with the Ebola and Marburg viruses, and we demonstrate that the replication of filoviruses is more rapid in human cells than in bat cells. We also found that the most strongly regulated genes upon filovirus infection are chemokine ligands and transcription factors. We observed a strong induction of the JAK/STAT pathway, of several genes encoding inhibitors of MAP kinases (DUSP genes) and of PPP1R15A, which is involved in ER stress-induced cell death. We used comparative transcriptomics to provide a data resource that can be used to identify cellular responses that might allow bats to survive filovirus infections.

CXCL13/CXCR5 enhances sodium channel Nav1.8 current density via p38 MAP kinase in primary sensory neurons following inflammatory pain.

CXCL13 is a B lymphocyte chemoattractant and activates CXCR5 receptor in the immune system. Here we investigated whether CXCL13/CXCR5 mediates inflammatory pain in dorsal root ganglia (DRG) and the underlying mechanisms. Peripheral injection of complete Freund’s Adjuvant (CFA) increased the expression of CXCL13 and CXCR5 in DRG neurons. In Cxcr5−/− mice, CFA-induced pain hypersensitivity were attenuated. Whole-cell patch-clamp recording showed that the excitability of dissociated DRG neurons was increased after CFA injection or CXCL13 incubation from wild-type (WT) mice, but not from Cxcr5−/− mice. Additionally, sodium channel Nav1.8 was co-expressed with CXCR5 in dissociated DRG neurons, and the increased neuronal excitability induced by CFA or CXCL13 was reduced by Nav1.8 blocker. Intrathecal injection of Nav1.8 blocker also attenuated intrathecal injection of CXCL13-induced pain hypersensitivity. Furthermore, CXCL13 increased Nav1.8 current density in DRG neurons, which was inhibited by p38 MAP kinase inhibitor. CFA and CXCL13 increased p38 phosphorylation in the DRG of WT mice but not Cxcr5−/− mice. Finally, intrathecal p38 inhibitor alleviated CXCL13-induced pain hypersensitivity. Taken together, these results demonstrated that CXCL13, upregulated by peripheral inflammation, acts on CXCR5 on DRG neurons and activates p38, which increases Nav1.8 current density and further contributes to the maintenance of inflammatory pain.

Cross talk of c-Jun N-terminal kinase and p38 map kinase modulate insulin and TNFα-stimulated VCAM-1 expression in rat aorta endothelial cells

<p>VCAM-1 cell surface expression was determined by flow cytometry in rat aorta endothelial cells stimulated with insulin and/or Tumor Necrosis Factor-a in the absence or presence of short-hairpin RNA inhibitors of c-Jun N-terminal Kinase (JNK) and p38 MAP Kinase. Cells transfected with insulin alone exhibited moderate increases in cell surface VCAM-1 expression, whereas cells stimulated with TNFα alone or in combination with insulin, exhibited a significant (P, 0.05) increase in VCAM-1 surface expression. Cells transfected with shJNK alone showed increased VCAM-1 expression at the cell surface as compared to mock-transfected positive controls. In contrast, cells transfected with shp38 exhibited significant decreased insulin-stimulated VCAM-1 expression at the cell surface, but only moderate decreased VCAM-1 expression in cells stimulated by TNFα alone in combination with insulin. Interestingly, in cells first tranfected with shJNK and then shp38, VCAM-1 expression appeared to increase in an additive fashion. In contrast, cells transfected with shp38 first then shJNK, VCAM-1 expression exhibited increased VCAM-1 expression even in the presence of shp38. One can conclude that JNK is a potent negative regulator of insulin- and TNFα-stimulated cell surface expression of VCAM-1, whereas p38 is mild positive regulator of insulin-, but not TNFα-stimulated VCAM-1 expression. JNK appears to be a more potent mediator of insulin- and TNFα-stimulated cells surface VCAM-1 expression than p38 MAP Kinase. Thus, it could be a therapeutic target for amelioration of inflammation-associated VCAM-1 expression and its sequelae of events that lead to atherosclerosis.</p>

Cross talk of c-Jun N-terminal kinase and p38 map kinase modulate insulin and TNFα-stimulated VCAM-1 expression in rat aorta endothelial cells

VCAM-1 cell surface expression was determined by flow cytometry in rat aorta endothelial cells stimulated with insulin and/or Tumor Necrosis Factor-a in the absence or presence of short-hairpin RNA inhibitors of c-Jun N-terminal Kinase (JNK) and p38 MAP Kinase. Cells transfected with insulin alone exhibited moderate increases in cell surface VCAM-1 expression, whereas cells stimulated with TNFα alone or in combination with insulin, exhibited a significant (P, 0.05) increase in VCAM-1 surface expression. Cells transfected with shJNK alone showed increased VCAM-1 expression at the cell surface as compared to mock-transfected positive controls. In contrast, cells transfected with shp38 exhibited significant decreased insulin-stimulated VCAM-1 expression at the cell surface, but only moderate decreased VCAM-1 expression in cells stimulated by TNFα alone in combination with insulin. Interestingly, in cells first tranfected with shJNK and then shp38, VCAM-1 expression appeared to increase in an additive fashion. In contrast, cells transfected with shp38 first then shJNK, VCAM-1 expression exhibited increased VCAM-1 expression even in the presence of shp38. One can conclude that JNK is a potent negative regulator of insulin- and TNFα-stimulated cell surface expression of VCAM-1, whereas p38 is mild positive regulator of insulin-, but not TNFα-stimulated VCAM-1 expression. JNK appears to be a more potent mediator of insulin- and TNFα-stimulated cells surface VCAM-1 expression than p38 MAP Kinase. Thus, it could be a therapeutic target for amelioration of inflammation-associated VCAM-1 expression and its sequelae of events that lead to atherosclerosis.

13-Methylberberine reduces HMGB1 release in LPS-activated RAW264.7 cells and increases the survival of septic mice through AMPK/P38 MAPK activation

High mobility group box 1 (HMGB1), a late phase cytokine of sepsis, is viewed as a potential target for the treatment of sepsis. The authors considered that 13-methylberberine (13-MB) might reduce circulating HMGB1 levels and increase survival in a mouse model of sepsis by activating AMP-activated protein kinase (AMPK). Western blot analysis and vascular contraction testing were performed using RAW264.7 cells and rat thoracic aorta, respectively. The mechanisms responsible were investigated using various signal inhibitors and small interfering RNA techniques. 13-MB significantly reduced HMGB1 release by LPS-activated RAW264.7 cells, and this was prevented by silencing AMPK or p38, or by pretreating cells with p38 MAPKinase inhibitor, suggesting that the activations of p38 and AMPK were responsible for the observed reduction in HMGB1 release. As was expected, 13-MB increased the phosphorylations of p38 and AMPK. Interestingly, phosphorylations of p38 by 13-MB were inhibited by AMPKsiRNA, indicating that AMPK lies upstream of p38. Furthermore, 13-MB concentration-dependently inhibited IκB phosphorylation in LPS-activated RAW264.7 cells, and in aortic rings, co-treatment with 13-MB and LPS for 8h, in vitro, significantly restored the isometric contraction induced by phenylephrine. Importantly, 13-MB significantly increased the survival rate of LPS-induced endotoxemic mice. These results suggest 13-MB may be useful for treating diseases in which HMGB1 is viewed as a target.

A.I.5 - Laminopathies: Why make it simple when it can be complex?

Laminopathies are a diverse and complex group of rare genetic conditions due to mutations in the LMNA gene encoding Lamin A and C, constituents of the nuclear lamina, a meshwork of proteins underneath the nuclear envelope. Striated muscle laminopathies (SML) are the most frequent type of laminopathies (≈60% of all laminopathies published cases), that affect skeletal and/or cardiac muscle. SMLcomprise LMNA related congenital muscular dystrophy (L-CMD), Emery–Dreifuss muscular dystrophy (EDMD), limb-girdle muscular dystrophy, type 1B (LGMD1B) and dilated cardiomyopathy with conduction system disease (DCM-CD). Associated with this wide clinical heterogeneity, there is also a large genetic variability as more than 400 different LMNA mutations have been reported so far (www.umd.be/LMNA/ and unpublished data). In order to get insights in phenotype/genotype relations as well as better knowledge of the natural history of these different clinical entities, we set up a patient registry, OPALE, and initiate an international retrospective natural history study of the early onset SML. Over the years, numerous studies have reported that lamin A/C provide structural support to the nucleus, maintenance of nuclear architecture, nuclear migration, and apoptosis, and also take part in chromatin organization and epigenetics, transcription, cell cycle regulation, cell development and differentiation. To study the role of lamin A/C in skeletal and cardiac muscles, and to understand the pathophysiological processes induced by LMNA mutations, we created knock-in mouse models that reproduced LMNA mutation identified in SML patients. We demonstrated an aberrant increase in MAP kinases in hearts from Lmna H222P knock-in mice, providing proof of principle for MAP kinase inhibition as a therapeutic option to prevent or delay the onset of the contractile dysfunction and cardiomyopathy in SML. Recent insights of the pathophysiological mechanisms of LMNA mutations leading to skeletal and/or cardiac dysfunction in these mouse models will be presented.

Staphylococcus aureus PSM peptides induce tolerogenic dendritic cells upon treatment with ligands of extracellular and intracellular TLRs

Dendritic cells (DCs) are key players of the immune system and thus a target for immune evasion by pathogens. We recently showed that the virulence factor phenol-soluble modulin (PSM) produced by community-associated methicillin-resistant Staphylococcus aureus strains induces tolerogenic DCs upon Toll-like receptor (TLR) 2 activation via the p38-CREB-IL-10 pathway. Here, we addressed the question whether this tolerogenic phenotype of DCs induced by PSMs is specific for TLR2 activation. Therefore, bone marrow-derived DCs were treated with various ligands for extracellular and intracellular TLRs simultaneously with PSMα3. We show that PSMα3 modulates antigen uptake, maturation and cytokine production of DCs activated by TLR1/2, TLR2/6, TLR4, TLR7, and TLR9. Pre-incubation of DCs with a p38 MAP kinase inhibitor prevented the PSMα3-induced IL-10 secretion, as well as MHC class II up-regulation upon TLR activation. In consequence, the tolerogenic DCs induced by PSMα3 in response to several TLR ligands promoted priming of regulatory T cells. Thus, PSMs could be useful as inducers of tolerogenic DCs upon TLR ligand stimulation for therapeutic applications.

MPS 07-05 ROLE OF THE NATRIURETIC PEPTIDE-A RECEPTOR ON PODOCYTES IN ALDOSTERONE-INDUCED GLOMERULAR INJURY

Objective: Aldosterone is a key molecule in glomerular injury and tubulointerstitial fibrosis. Natriuretic peptide receptor/guanylyl cyclase-A (GC-A) signaling exerts renoprotective effects by eliciting natriuresis, reducing blood pressure, and inhibiting fibrosis. We demonstrated that uninephrectomized GC-A knockout (KO) mice with aldosterone and sodium overload exhibited accelerated hypertension with massive proteinuria. In this model, inhibition of p38 MAP kinase (MAPK) ameliorated renal injury. However, local effect of natriuretic peptide receptor/GC-A signaling in podocyte is not fully elucidated. Design and Method: To explore the role of GC-A signaling in podocyte, we generated podocytes-specific GC-A KO mice (pod-GC-A cKO). Pod-GC-A cKO mice or wild-type mice were uninephrectomized and then treated with high salt orally and aldosterone subcutaneously using an osmotic minipump for 4 weeks. Results: Although systolic blood pressure was not different between control and pod-GC-A cKO mice, pod-GC-A cKO mice showed significantly increased urinary albumin excretion by 13-fold (n = 7, p < 0.01). Glomerular hypertrophy, mesangial expansion and apoptosis determined by TUNEL assay were increased in pod-GC-A cKO mice. Urinary potassium excretion was not affected between two groups. Administration of aldosterone increased phosphorylation of p38 MAPK mainly in podocytes. In vitro study, overexpression of MKK3 in cultured human podocytes upregulated the phosphorylation of p38 MAPK and BAX mRNA expression. Such induction was significantly inhibited by the administration of ANP. Conclusions: These results suggest that endogenous natriuretic peptide/GC-A system on podocytes acts renoprotective and anti-apoptotic roles in aldosterone-induced renal injury by inhibiting phosphorylation of p38 MAPK.

Hyperbaric Oxygen Reduces Production of Reactive Oxygen Species in Neutrophils from Polytraumatized Patients Yielding in the Inhibition of p38 MAP Kinase and Downstream Pathways.

Trauma represents the leading cause of death among young people in western countries. Among the beneficial role of neutrophils in host defence, excessive priming and activation of neutrophils after major trauma lead to an overwhelming inflammatory response and secondary host tissue injury due to the release of toxic metabolites and enzymes. Hyperbaric oxygen (HBO) therapy has been proposed to possess antiinflammatory effects and might represent an appropriate therapeutic option to lower inflammation in a broad range of patients. Here, we studied the effects of HBO on the activity of neutrophils isolated from severely injured patients (days 1–2 after trauma), in fact on the production of reactive oxygen species (ROS) and release of neutrophil extracellular traps (NETs). We found exposure to HBO therapy to significantly diminish phorbol-12-myristate-13-acetate (PMA)-induced ROS production in neutrophils isolated from patients and healthy volunteers. At the same time, marked decrease in NETs release was found in control cells and a less pronounced reduction in patient neutrophils. Impaired ability to produce ROS following exposure to HBO was demonstrated to be linked to a strong downregulation of the activity of p38 MAPK. Only slight suppression of ERK activity could be found. In addition, HBO did not influence neutrophil chemotaxis or apoptosis, respectively. Collectively, this study shows for the first time that HBO therapy suppresses ROS production in inflammatory human neutrophils, and thus might impair ROS-dependent pathways, e.g. kinases activation and NETs release. Thus, HBO might represent a feasible therapy for patients suffering from systemic inflammation, including those with multiple trauma.