P38 Mitogen-associated Protein Kinase Research Articles

SVCAM1 in the Hippocampus Contributes to Postoperative Cognitive Dysfunction in Mice by Inducing Microglial Activation Through the VLA-4 Receptor.

Postoperative cognitive dysfunction (POCD) is a severe postsurgical complication, but its underlying mechanisms remain unclear. Neuroinflammation mediated by microglial activation plays a major role in POCD pathophysiology. Upregulation of vascular cell adhesion molecule 1 (VCAM1) on brain endothelial cells is closely correlated with microglial activation in the mouse hippocampus. However, the role of VCAM1 upregulation in microglial activation remains unknown. Soluble VCAM1 (sVCAM1) activates the very late antigen-4 (VLA-4) receptor under inflammatory conditions. Therefore, we hypothesized that sVCAM1 which is shed from VCAM1 contributes to POCD by triggering hippocampal microglial activation through the VLA-4 receptor. We found that VCAM1 and sVCAM1 expression in the mouse hippocampus was upregulated after surgery, and the upregulation was accompanied by hippocampal microglial activation. sVCAM1 levels in mouse and human serum were increased after surgery. Anti-VCAM1 treatment inhibited microglial activation, proinflammatory cytokine production, VLA-4 expression and P38 mitogen-associated protein kinase (MAPK) pathway activation and attenuated hippocampal-dependent cognitive dysfunction. In vitro, recombinant sVCAM1 promoted M1 polarization in BV2 cells, increased VLA-4 expression and activated the P38 MAPK pathway. These effects were reversed by VLA-4 receptor blockade. Anti-VLA-4 treatment ameliorated hippocampal-dependent cognitive dysfunction after surgery by inhibiting microglial activation, proinflammatory cytokine production and P38 pathway activation. In conclusion, increased sVCAM1 in the hippocampus is involved in microglial activation and cognitive dysfunction induced by surgery. Inhibiting the sVCAM1-VLA-4 interaction in microglia may be a therapeutic strategy for POCD.

A human pluripotent stem cell-based model of SARS-CoV-2 infection reveals an ACE2-independent inflammatory activation of vascular endothelial cells through TLR4.

SummaryTo date, the direct causative mechanism of SARS-CoV-2-induced endotheliitis remains unclear. Here, we report that human ECs barely express surface ACE2, and ECs express less intracellular ACE2 than non-ECs of the lungs. We ectopically expressed ACE2 in hESC-ECs to model SARS-CoV-2 infection. ACE2-deficient ECs are resistant to the infection but are more activated than ACE2-expressing ones. The virus directly induces endothelial activation by increasing monocyte adhesion, NO production, and enhanced phosphorylation of p38 mitogen-associated protein kinase (MAPK), NF-κB, and eNOS in ACE2-expressing and -deficient ECs. ACE2-deficient ECs respond to SARS-CoV-2 through TLR4 as treatment with its antagonist inhibits p38 MAPK/NF-κB/ interleukin-1β (IL-1β) activation after viral exposure. Genome-wide, single-cell RNA-seq analyses further confirm activation of the TLR4/MAPK14/RELA/IL-1β axis in circulating ECs of mild and severe COVID-19 patients. Circulating ECs could serve as biomarkers for indicating patients with endotheliitis. Together, our findings support a direct role for SARS-CoV-2 in mediating endothelial inflammation in an ACE2-dependent or -independent manner.

Abstract 14573: Association of Human Mesenchymal Stem Cell and Lodenafil in Hypoxia-induced Pulmonary Arterial Hypertension

Introduction: Right ventricle (RV) remodeling and vascular dysfunction are present in pulmonary arterial hypertension (PAH). Hypothesis: This work focuses on the benefits of the association of human umbilical cord mesenchymal stem cell (hMSC) with lodenafil, a PDE5 inhibitor, on PAH in rats. Methods: Experiments were in accordance with the Animal Care and Use Committee at the Federal University of Rio de Janeiro. PAH was induced in male Wistar rats by exposure to hypoxia (10% O 2 ) during 3 weeks plus i.p . injection of an antagonist of VEGF receptor (SU5416; 20 mg/kg/week). Animals were randomly divided (n=6): PAH + vehicle; PAH + lodenafil (gavage, 10 mg/kg); PAH + lodenafil + hMSC (5x10 5 , i.v.) for 14 days. Results: PAH induced the reduction of the ratio of pulmonary acceleration time and RV ejection time from 0.42 ± 0.01 to 0.24 ± 0.01, which was recovered to 0.31 ± 0.01 after treatment with lodenafil + hMSC. RV afterload confirmed by increased systolic pressure (mmHg) of 52.1 ± 8.8 (contro: 24.0 ± 3.1) was normalized to 29.6 ± 2.2 with association. Pulmonary arteries wall thickness (%) measured by immunohistochemistry for alpha-SMA was increased by HAP from 44.7 ± 1.4 to 64.2 ± 1.2 and was partial (57.7 ± 1.4) or totally recovered (47.3 ± 0.9) after treatment with lodenafil and association, respectively. Increased RV collagen fraction (%) of 7.4 ± 1.2 was induced by HAP when compared to control group (1.9 ± 0.1) and although lodenafil did not alter this parameter (6.6 ± 0.9) its association with hMSC restored to 2.7 ± 0.4. Increased RV wall thickness (mm) observed in PAH (from 0.60 ± 0.02 to 1.05 ± 0.13) recovered to 0.58 ± 0.04 after treatment with association. ERK1/2 expression evaluated by western blot analysis recovered from 0.79 ± 0.07 to 0.12 ± 0.06, which reinforced the reduction of cellular proliferation by treatment with lodenafil + hMSC. RV expression of p38 mitogen associated protein kinase was measured by stained area quantification using immunohistochemistry (%) was also increased in PAH group (from 9.4 ± 2.5 to 31.4 ± 2.2, p < 0.05) and the association of lodenafil + hMSC reduced to 11.4 ± 3.4. Conclusions: Therapy of hMSC and lodenafil improved cardiac remodeling and vascular dysfunction in SuHx-PAH rats, representing an important approach for the future treatment of PAH.

Immunotoxicity of titanium dioxide nanoparticles via simultaneous induction of apoptosis and multiple toll-like receptors signaling through ROS-dependent SAPK/JNK and p38 MAPK activation.

BackgroundTitanium dioxide nanoparticles (TiO2 NPs) represent a scientific breakthrough in the areas of biological and medicinal applications. Interaction of TiO2 NPs with components of innate immune system remains elusive.AimThis study explored in vitro immunotoxicity of murine macrophage RAW 264.7 to TiO2 NPs (20 nm, negative charge) and its underlying molecular mechanism by way of immunoredox profiling.Materials and methodsIn this study, chemically synthesized BSA-functionalized TiO2 NPs (20 nm, negative charge) were characterized and immunotoxicity was investigated on RAW 264.7 cells.ResultsWe found that reactive oxygen species levels significantly increased with increasing nitric oxide production, whereas depleting endogenous antioxidant super oxide dismutase as well as nuclear factor erythroid 2-related factor 2 (Nrf2) protein levels. Furthermore, NPs exposure increased the expression of apoptotic factors such as BAX, BIM, and PUMA with disruption of mitochondrial membrane potential (Δψm) that lead to decrease in immunocytes. Molecular immune profiling revealed the activation of multiple toll-like receptors (TLRs) 4/9/12/13 simultaneously with the phosphorylation of p-p38MAPK and p-SAPK/c-Jun N-terminal kinase (JNK) compared to untreated control.ConclusionCollectively, this study shows that the molecular nature of TiO2SA20(−) NP-induced immunotoxicity in RAW 264.7 macrophage is simultaneous induction of immunocyte apoptosis and multiple TLRs signaling through oxidative stress-dependent SAPK/JNK and p38 mitogen-associated protein kinase activation. This is the first study to address newer molecular mechanism of TiO2SA20(−) NP-induced immunotoxicity.

Ghrelin ameliorates nerve growth factor Dysmetabolism and inflammation in STZ-induced diabetic rats.

Diabetic encephalopathy is characterized by cognitive impairment and neuroinflammation, deficient neurotrophic support, and neuronal and synaptic loss. Ghrelin, a 28 amino acid peptide, is associated with neuromodulation and cognitive improvement, which has been considered as a potential protective agent for several neurodegenerative diseases. Here we sought to investigate the role of ghrelin in preventing diabetic-related neuropathology. We found that ghrelin attenuated astrocytic activation and reduced levels of interleukin-6 and tumor necrosis factor-α in streptozotocin-induced diabetic rats. In addition, ghrelin inhibited p38 mitogen-associated protein kinase activation. The upregulation of nerve growth factor (NGF) precursor and matrix metalloproteinase (MMP)-9 and downregulation of mature NGF and MMP-7 in the diabetic brain were reversed by ghrelin. Treatment with ghrelin elevated synaptophysin expression and synaptic density in diabetic rats. Taken together, our results demonstrate that ghrelin ameliorates diabetes-related neurodegeneration by preventing NGF dysmetabolism and synaptic degeneration through regulating MMP levels as well as inhibiting neuroinflammation.

Combination of microRNA-21 and microRNA-146a Attenuates Cardiac Dysfunction and Apoptosis During Acute Myocardial Infarction in Mice.

Recent studies have revealed the cytoprotective roles of microRNAs (miRNAs) miR-21 and miR-146a against ischemic cardiac injuries. While these studies investigated each of these miRNAs as an independent individual factor, our previous study has suggested the possible interaction between these two miRNAs. The present study was designed to investigate this possibility by evaluating the effects of miR-21 and miR-146a combination on cardiac ischemic injuries and the underlying mechanisms. MiR-21 and miR-146a synergistically decreased apoptosis under ischemia/hypoxic conditions in cardiomyocytes compared with either miR-21 or miR-146a alone. Mice coinjected with agomiR-21 and agomiR-146a had decreased infarct size, increased ejection fraction (EF), and fractional shortening (FS). These effects were greater than those induced by either of the two agomiRs. Furthermore, greater decreases in p38 mitogen-associated protein kinase phosphorylation (p-p38 MAPK) were observed with miR-21: miR-146a combination as compared to application of either of the miRNAs. These data suggest that combination of miR-21 and miR-146a has a greater protective effect against cardiac ischemia/hypoxia-induced apoptosis as compared to these miRNAs applied individually. This synergistic action is mediated by enhanced potency of inhibition of cardiomyocyte apoptosis by the miR-21—PTEN/AKT—p-p38—caspase-3 and miR-146a—TRAF6—p-p38—caspase-3 signal pathways.

Budesonide Inhibits Intracellular Infection with Non-Typeable Haemophilus influenzae despite Its Anti-Inflammatory Effects in Respiratory Cells and Human Lung Tissue: A Role for p38 MAP Kinase

Background: Inhaled corticosteroids (ICS) are widely used in the treatment of obstructive lung diseases. Recent data suggest a higher pneumonia risk in chronic obstructive pulmonary disease (COPD) patients treated with ICS. Objective: Since non-typeable Haemophilus influenzae (NTHi) is the most common pathogen associated with acute exacerbations of COPD, we investigated the effects of budesonide (BUD) on NTHi-induced inflammation and invasive infection. Methods: The alveolar epithelial cell line A549 and specimens of human lung tissue (HLT) were used in our experiments. Intracellular infection was determined by a lysis/culture assay of infected cells. Activated p38 mitogen-associated protein kinase (MAPK) was assessed using Western blotting and immunohistochemistry, expression of toll-like receptor 2 (TLR2) was determined by PCR, and CXCL-8 levels were measured using ELISA. Immunohistochemistry was used for detection of CXCL-8, platelet-activating factor receptor (PAF-R) and NTHi. Results: BUD significantly reduced CXCL-8 secretion in A549 cells and lung tissue infected with NTHi. Furthermore, BUD decreased the expression of PAF-R in HLT and A549 cells. In A549 cells and HLT, BUD inhibited intracellular infection and - synergistically with NTHi - increased the expression of TLR2 (in A549 cells). TLR2 stimulation did not influence the intracellular infection of A549 cells, but p38 MAPK inhibition resulted in a significant reduction of infection. Conclusion: The present study adds new insights into the effects of glucocorticoids on pulmonary host defence after NTHi infection. Although the inflammatory response to infection is suppressed by BUD, interestingly, the intracellular infection is also inhibited. This effect seems to depend on the inhibition of p38 MAPK - a key enzyme in many pro-inflammatory pathways - as well as of PAF-R expression.

Calcium ion-induced formation of β-sheet/-turn structure leading to alteration of osteogenic activity of bone morphogenetic protein-2

Preserving bioactivity of bone morphogenetic protein 2 (BMP-2) still remains a challenge in protein-based therapy. It is not known how Ca2+ released from extracellular matrix or existing in physiological environment influences bioactivity in situ till now. Here, effects of extracellular Ca2+ on conformation and osteogenic bioactivity of recombinant human BMP-2 (rhBMP-2) were investigated systematically. In vitro results indicated that Ca2+ could bind rhBMP-2 rapidly and had no obvious effect on cell behaviors. Low concentration of Ca2+ (0.18 mM) enhanced rhBMP-2-induced osteogenic differentiation, while high Ca2+ concentration (>1.80 mM) exerted negative effect. In vivo ectopic bone formation exhibited similar trend. Further studies by circular dichroism spectroscopy, fluorescence spectroscopy, together with cell culture experiments revealed at low concentration, weak interaction of Ca2+ and rhBMP-2 slightly increased β-sheet/-turn content and facilitated recognition of BMP-2 and BMPRIA. But, high Ca2+ concentration (>1.8 mM) induced formation of Ca-rhBMP-2 complex and markedly increased content of β-sheet/-turn, which led to inhibition binding of rhBMP-2 and BMPRIA and thus suppression of downstream Smad1/5/8, ERK1/2 and p38 mitogen-associated protein kinase signaling pathways. Our work suggests osteogenic bioactivity of BMP-2 can be adjusted via extracellular Ca2+, which should provide guide and assist for development of BMP-2-based materials for bone regeneration.

Elevation of Plasma High-Density Lipoproteins Inhibits Development of Experimental Abdominal Aortic Aneurysms

Patients with abdominal aortic aneurysms have lower concentrations of high-density lipoproteins (HDLs), leading us to investigate whether increasing plasma HDLs could influence aneurysm formation. Using the angiotensin II-induced hypercholesterolemic and the CaCl(2)-induced normocholesterolemic mouse model of AAA, we investigated the hypothesis that elevation of HDLs inhibits AAA. HDLs elevated before or at the time of AAA induction reduced AAA formation in both models but had no effect on early ruptures. Analysis of protein lysates from specific aortic segments demonstrated site-specific effects of HDLs on early signal transduction and cellular attrition. We found that HDLs reduced extracellular signal related kinases 1/2 activation in the suprarenal segment, while having no effect on p38 mitogen-associated protein kinase activation in any aortic segment and inhibiting c-Jun N-terminal kinase activation in all aortic segments. In addition, HDL elevation inhibited angiotensin II-induced apoptosis while inducing autophagy in the suprarenal segment of the aorta. Using Illumina gene array profiling we investigated the ability of HDL to modulate basal suprarenal aortic gene expression. Increasing plasma HDLs inhibit experimental AAA formation, independent of hypercholesterolemia via reduced extracellular signal related kinases 1/2 activation and alteration of the balance of cellular attrition. HDLs modulate genes involved in matrix remodelling, cell migration, and proliferation.

TLR 2 Induces Vascular Smooth Muscle Cell Migration Through cAMP Response Element−Binding Protein−Mediated Interleukin-6 Production

Migration of vascular smooth muscle cells (VSMCs) from the media into intima contributes to the development of atherosclerosis. Gene deletion experiments implicate a role for toll-like receptor 2 (TLR2) in atherogenesis. However, the underlying mechanisms remain unclear. We postulate that TLR2 promotes VSMC migration by enhancing interleukin (IL)-6 production. Migration assays revealed that TLR2 agonists promoted VSMC migration but not cell proliferation or viability. TLR2 deficiency or inhibition of TLR2 signaling with anti-TLR2 antibody suppressed TLR2 agonist-induced VSMC migration and IL-6 production, which was mediated via p38 mitogen-associated protein kinase and extracellular signal-regulated kinase 1/2 signaling pathways. Neutralizing anti-IL-6 antibodies impaired TLR2-mediated VSMC migration and formation of filamentous actin fiber and lamellipodia. Blockade of p38 mitogen-associated protein kinase or extracellular signal-regulated kinase 1/2 activation inhibited TLR2 agonist pam3CSK4-induced phosphorylation of cAMP response element-binding protein, which regulates IL-6 promoter activity through the cAMP response element site. Moreover, cAMP response element-binding protein small interfering RNA inhibited pam3CSK4-induced IL-6 production and VSMC migration. Additionally, Rac1 small interfering RNA inhibited pam3CSK4-induced VSMC migration but not IL-6 production. Our results suggest that on ligand binding, TLR2 activates p38 mitogen-associated protein kinase and extracellular signal-regulated kinase 1/2 signaling in VSMCs. These signaling pathways act in concert to activate cAMP response element-binding protein and subsequent IL-6 production, which in turn promotes VSMC migration via Rac1-mediated actin cytoskeletal reorganization.

P38 Mitogen-activated Protein Kinase is Not Activated in the Quadriceps of Patients with Stable Chronic Obstructive Pulmonary Disease

Quadriceps weakness is associated with a poor prognosis in COPD. Several data suggest that immobility and muscle wasting may be related through up-regulation of the p38 Mitogen associated protein kinase (MAPK) signalling pathway. We therefore hypothesised that this might occur in the quadriceps of COPD patients. We studied 105 stable COPD outpatients (mean FEV1 43.9% predicted) and 27 age and gender matched controls. We measured fat-free mass, quadriceps strength and daily physical activity using triaxial accelerometry. A quadriceps biopsy was obtained in which components of the p38-MAPK signalling pathway were examined. Patients had reduced fat-free mass index (16.1 (2.2) kg/m2 vs 17.2 (2.2) kg/m2, p = 0.02) and exhibited quadriceps weakness (mean (SD) maximal voluntary contraction force 72.1% (18.7) predicted and 82.3% (7.1) predicted for patients and controls respectively, p = 0.01). Physical activity was significantly reduced in the patient group; in particular mean (SD) locomotion time was 101 () minutes/12 hours in controls and 48 () minutes in patients (p < 0.0001). However, in biopsies obtained from these patients, no differences were observed for total or phosphorylated HSP27 or p38 MAPK protein or p38 MAPK mRNA (MAPK14); of the downstream products both GADD45β and c-jun mRNA were reduced in COPD patients while c-myc was not different from controls. No parameter correlated with physiological data within the patient group. We conclude that, despite the presence of reduced fat-free mass, quadriceps weakness and inactivity, p38 MAPK signalling was not up-regulated in skeletal muscle of stable out-patients with COPD.

Substance P Activates Both Contractile and Inflammatory Pathways in Lymphatics Through the Neurokinin Receptors NK1R and NK3R

The aim of this study was to elucidate the molecular signaling mechanisms by which substance P (SP) modulates lymphatic muscle contraction and to determine whether SP stimulates both contractile as well as inflammatory pathways in the lymphatics. A rat mesenteric lymphatic muscle cell culture model (RMLMCs) and known specific pharmacological inhibitors were utilized to delineate SP-mediated signaling pathways in lymphatics. We detected expression of neurokinin receptor 1 (NK1R) and neurokinin receptor 3 (NK3R) in RMLMCs. SP stimulation increased phosphorylation of myosin light chain 20 (MLC₂₀) as well as p38 mitogen associated protein kinase (p38-MAPK) and extracellular signal regulated kinase (ERK1/2) indicating activation of both a contractile and a pro-inflammatory MAPK pathway. Pharmacological inhibition of both NK1R and NK3R significantly affected the downstream SP signaling. We further examined whether there was any crosstalk between the two pathways upon SP stimulation. Inhibition of ERK1/2 decreased levels of p-MLC₂₀ after SP activation, in a PKC dependent manner, indicating a potential crosstalk between these two pathways. These data provide the first evidence that SP-mediated crosstalk between pro-inflammatory and contractile signaling mechanisms exists in the lymphatic system and may be an important bridge between lymphatic function modulation and inflammation.

Pentoxifylline attenuates leukoreduced stored blood-induced neutrophil activation through inhibition of mitogen-activated protein kinases

Context: Transfusion of packed red blood cells is an independent risk factor for postoperative bacterial infections and multiple organ failure.Materials and Methods: Whole blood was obtained from healthy volunteers to investigate the role of mitogen associated protein kinase (MAPK) pathways in PRBC-induced neutrophil respiratory burst, hypothesizing that the attenuating effects of pentoxifylline on this process are due to modulations in MAPK-associated signaling.Results: Pre-incubation of neutrophils with supernatant prior to fMLP stimulation increased p38 MAPK and ERK phosphorylation over fMLP alone pentoxifylline significantly reduced p38MAPK and ERK phosphorylation in similarly stimulated neutrophils.Conclusion: The addition of pentoxifylline stored red blood cells attenuates neutrophil activation.

Pentoxifylline attenuates leukoreduced stored blood-induced neutrophil activation through inhibition of mitogen-activated protein kinases

Transfusion of packed red blood cells is an independent risk factor for postoperative bacterial infections and multiple organ failure. Whole blood was obtained from healthy volunteers to investigate the role of mitogen associated protein kinase (MAPK) pathways in PRBC-induced neutrophil respiratory burst, hypothesizing that the attenuating effects of pentoxifylline on this process are due to modulations in MAPK-associated signaling. Pre-incubation of neutrophils with supernatant prior to fMLP stimulation increased p38 MAPK and ERK phosphorylation over fMLP alone pentoxifylline significantly reduced p38MAPK and ERK phosphorylation in similarly stimulated neutrophils. The addition of pentoxifylline stored red blood cells attenuates neutrophil activation.

Identification of CD36 molecular features required for its in vitro angiostatic activity

Thrombospondin-1 (TSP-1), a natural inhibitor of angiogenesis, acts directly on endothelial cells (EC) via CD36 to inhibit their migration and morphogenesis induced by basic fibroblast growth factor. Here we show that CD36 triggered by TSP-1 inhibits in vitro angiogenesis stimulated by vascular endothelial growth factor-A (VEGF-A). To demonstrate that the TSP-1 inhibitory signal was mediated by CD36, we transduced CD36 in CD36-deficient endothelial cells. Both TSP-1 and the agonist anti-CD36 mAb SMO, which mimics TSP-1 activity, reduced the VEGF-A165-induced migration and sprouting of CD36-ECs. To address the mechanisms by which CD36 may exert its angiostatic function, we investigated the functional components of the C-terminal cytoplasmic tail by site-directed mutagenesis. Our results indicate that C464, R467, and K469 of CD36 are required for the inhibitory activity of TSP-1. In contrast, point mutation of C466 did not alter TSP-1 ability to inhibit EC migration and sprouting. Moreover, we show that activation of CD36 by TSP-1 down-modulates the VEGF receptor-2 (VEGFR-2) and p38 mitogen-associated protein kinase phosphorylation induced by VEGF-A165, and this effect was specifically abolished by point mutation at C464. These results identify specific amino acids of the C-terminal cytoplasmic tail of CD36 crucial for the in vitro angiostatic activity of TSP-1 and extend our knowledge of regulation of VEGFR-2-mediated biological activities on ECs.

Mechanisms of Porphyromonas gingivalis-induced monocyte chemoattractant protein-1 expression in endothelial cells

Monocyte chemoattractant protein-1 (MCP-1) is expressed in vascular endothelial cells of inflamed gingival tissues and plays an important role in periodontal pathogenesis. Endothelial cells produce high levels of MCP-1 in response to Porphyromonas gingivalis, an important periodontal pathogen. The present study investigated the mechanisms involved in MCP-1 production by human umbilical vein endothelial cells (HUVEC) following infection with P. gingivalis. In contrast to P. gingivalis, Bacteroides forsythus only weakly stimulated MCP-1 production while Treponema denticola could not induce MCP-1 in HUVEC. The MCP-1 production was independent of endogenous interleukin (IL)-1α as IL-1 receptor antagonist treatment did not reduce MCP-1 production by P. gingivalis. Meanwhile, antioxidant treatment and inhibition of NAD(P)H oxidase significantly reduced MCP-1 production. Pharmacological inhibition of p38 mitogen-associated protein (MAP) kinase, c-Jun N-terminal kinase (JNK), nuclear factor-κB (NF-κB) or activator protein-1 (AP-1) also substantially attenuated P. gingivalis-induced MCP-1 expression by HUVEC. Indeed, activation of NF-κB and AP-1 was observed in P. gingivalis-infected HUVEC. These results suggest that MCP-1 expression is upregulated in P. gingivalis-infected endothelial cells via reactive oxygen species, p38 MAP kinase, JNK, NF-κB, and AP-1.

Does Ca 2+ Attenuate CaMKII-Dependent Phosphorylation at an NMDAR Complex?

Ca 2+ influx through hippocampal N -methyl-D-aspartate receptors (NMDARs) activates signaling pathways that affect α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR) trafficking, leading to long-lasting changes in synaptic function. Krapivinsky et al. investigated molecular interactions in a surprising pathway involving the synaptic Ras-small guanosine triphosphatase (GTPase)-activating protein (SynGAP). MUPP1, a multi-PDZ domain scaffolding protein that was enriched in postsynaptic densities, coimmunoprecipitated with the NMDAR. A yeast two-hybrid screen of a human brain library indicated that MUPP1's PDZ13 domain interacted with SynGAPα; this was confirmed by glutathione- S -transferase pull-down and coimmunoprecipitation. SynGAP immunoprecipitated from quiescent cultured hippocampal neurons was phosphorylated. Phosphorylation depended on Ca 2+ -calmodulin-dependent kinase II (CamKII) activity and, unexpectedly, synaptic stimulation or exposure to glutamate led to dephosphorylation. Disruption of the SynGAP-MUPP1 complex with membrane-permeant trans-activating transduction (TAT) fusion proteins containing the 111 C-terminal amino acids of SynGAP (TAT-SynGAP111) or PDZ13 (TAT-PDZ13) also promoted SynGAP dephosphorylation. CaMKII associated with the SynGAP-MUPP1 complex through MUPP1's second PDZ domain; however, exposure to Ca 2+ and calmodulin promoted CamKII dissociation. Disruption of SynGAP-MUPP1 interaction with TAT-PDZ13 or TAT-SynGAP111 attenuated p38 mitogen-associated protein kinase (MAPK) phosphorylation (as does synaptic activity). In an in vitro assay, SynGAP stimulated Rap GTPase activity. Finally, patch clamp analysis indicated that exposure to PDZ13 or SynGAP111 increased the amplitude and frequency of AMPAR-mediated miniature excitatory postsynaptic currents, whereas immunofluorescence analysis indicated that TAT-PDZ13 and TAT-SynGAP111 promoted AMPAR clustering. Thus, the authors propose a model in which some effects of Ca 2+ entry through the NMDAR are mediated through dissociation of CaMKII from the MUPP1-SynGAP complex, eliciting a decrease in SynGAP phosphorylation and thereby an increase in its Rap GAP activity and a consequent decrease in p38 MPAK activation. G. Krapivinsky, I. Medina, L. Krapivinsky, S. Gapon, D. E. Clapham, SynGAP-MUPP1-CaMKII synaptic complexes regulate p38 MAP kinase activity and NMDA receptor-dependent synaptic AMPA receptor potentiation. Neuron 43 , 563-574 (2004). [Online Journal]

Protein-bound polysaccharide isolated from basidiomycetes inhibits endotoxin-induced activation by blocking lipopolysaccharide-binding protein and CD14 functions

The protein-bound polysaccharide isolated from basidiomycetes (PSK) is a biological response modifier capable of exhibiting various biological activities, such as antitumor and antimicrobial effects. In the present study, we found that PSK suppressed interleukin (IL)-6 production in murine peritoneal macrophages stimulated with endotoxic lipopolysaccharide (LPS) and its synthetic lipid A (compound 506). Nitric oxide production and p38 mitogen-associated protein kinase phosphorylation induced in a murine macrophage cell line, J774-A1, by LPS and compound 506 were also inhibited by PSK. Further, PSK distinctly suppressed nuclear factor-κB activation in Ba/F3 cells expressing mouse Toll-like receptor 4 and MD-2, following stimulation with LPS and compound 506, however, not with Taxol. These PSK-induced inhibitory activities were caused by inhibition of the physical associations of LPS with LPS-binding protein (LBP) and CD14. PSK also protected mice from LPS-induced lethality, presumably by down-regulating IL-6 and tumor necrosis factor-α concentrations in serum. These findings indicate that PSK, which also has an ability to regulate LBP/CD14 functions, may be useful for clinical control of endotoxic sepsis.

The p38 mitogen-activated protein kinase inhibitor SB203580 antagonizes the inhibitory effects of interleukin-1β on long-term potentiation in the rat dentate gyrus in vitro

Levels of the pro-inflammatory cytokine interleukin-1β are known to be elevated in patients with chronic disorders such as Alzheimer's disease. We have investigated the effects of interleukin-1β on long-term potentiation and N-methyl- d-aspartate receptor-mediated field potentials in the rat dentate gyrus in vitro utilizing field extracellular recordings obtained from the middle third of the molecular layer of the dentate gyrus. Presynaptic stimulation was applied to the commissural/association pathway at a frequency of 0.05 Hz and at a distance of 50 μm from the granule cell body layer. As previously reported, interleukin-1β (1 ng/ml) caused an inhibition of long-term potentiation (108±2% of baseline 1 h following application of tetanic stimulation compared with 145±5% in vehicle control slices). This action of interleukin-1β on long-term potentiation, as well as an inhibition of N-methyl- d-aspartate receptor-mediated field potentials, was attenuated by pre-treatment of slices with the p38 mitogen-associated protein kinase inhibitor SB203580 (1 μM). SB203580 alone had no significant affect on long-term potentiation, but did cause an increase in baseline synaptic transmission [107±2% of baseline, 1 h after SB203580 (1 μM) treatment]. The p42/44 mitogen-activated protein kinase cascade inhibitor PD98059 (50 μM) did not inhibit the interleukin-1β-induced inhibition of N-methyl- d-aspartate receptor-mediated field potentials. The cyclooxygenase inhibitor indomethacin (50 μM) was found to attenuate the interleukin-1β-induced effects on both long-term potentiation and N-methyl- d-aspartate receptor-mediated field potentials. The lipid second messenger analogue C2 ceramide (20 μM) was found to attenuate the expression of long-term potentiation (108±3% of baseline 1 h following tetanic stimulation), and this effect was not blocked by pre-treatment with SB203580. To investigate a possible role for interleukin-1β in the normal expression of long-term potentiation, the interleukin-1 receptor antagonist (25 ng/ml) was applied during the maintenance phase of long-term potentiation. This was found to depress the sustained expression of long-term potentiation (116±6% of baseline 1 h following tetanic stimulation). Our results indicate possible signalling mechanisms by which interleukin-1β at pathophysiological concentrations may serve to inhibit long-term potentiation, and also suggests a role for IL-1β in the physiological expression of synaptic plasticity in the rat dentate gyrus in vitro.

Effect of Taxol and Taxotere on Gene Expression in Macrophages: Induction of the Prostaglandin H Synthase-2 Isoenzyme

Induction of genes encoding cytokines or other, unidentified proteins may contribute to the pharmacological effects of taxol. We hypothesized that prostaglandin H synthase-2 (PGHS-2) was one of the unidentified genes induced by taxol. Taxol alone or taxol plus IFN-gamma increased PGE2 formation, PGHS-2 protein expression, and PGHS-2 mRNA expression in RAW 264.7 murine macrophages. The kinetics for mRNA induction, protein expression, and catalysis were self-consistent. A selective inhibitor of PGHS-2 blocked PGE2 formation by cells incubated with taxol; a selective inhibitor of PGHS-1 had no effect. A glucocorticoid blocked the induction of mRNA, the expression of PGHS-2 protein, and the formation of PGE2. Neither taxol alone nor taxol plus IFN-gamma altered the expression of the PGHS-1 isoenzyme in RAW 264.7 cells. Taxotere, an analogue that stabilizes microtubules as potently as taxol, did not alter the expression of PGHS-2, implying that its induction in RAW 264.7 murine macrophages did not originate from microtubule stabilization. Taxol and taxotere each induced PGHS-2 expression in human monocytes suspended in 10% human serum. However, human monocytes suspended in 10% bovine serum responded only to LPS, not to taxol or taxotere, implying that they act independently of the LPS-mimetic process that is prominent in mice. Taxol induced PGHS-2 in human and murine monocytes via a p38 mitogen-associated protein kinase pathway. The inclusion of PGHS-2 among the early response genes induced in leukocytes may be relevant to the beneficial and adverse effects encountered during taxol administration.