Glutamine up-regulates MAPK phosphatase-1 induction via activation of Ca2+→ ERK cascade pathway

The non-essential amino acid L-glutamine (Gln) displays potent anti-inflammatory activity by deactivating p38 mitogen activating protein kinase and cytosolic phospholipase A₂ via induction of MAPK phosphatase-1 (MKP-1) in an extracellular signal-regulated kinase (ERK)-dependent way. In this study, the mechanism of Gln-mediated ERK-dependency in MKP-1 induction was investigated. Gln increased ERK phosphorylation and activity, and phosphorylations of Ras, c-Raf, and MEK, located in the upstream pathway of ERK, in response to lipopolysaccharidein vitro and in vivo. Gln-induced dose-dependent transient increases in intracellular calcium ([Ca²⁺]_i) in MHS macrophage cells. Ionomycin increased [Ca²⁺]_i and activation of Ras → ERK pathway, and MKP-1 induction, in the presence, but not in the absence, of LPS. The Gln-induced pathways involving Ca²⁺→ MKP-1 induction were abrogated by a calcium blocker. Besides Gln, other amino acids including L-phenylalanine and l-cysteine (Cys) also induced Ca²⁺ response, activation of Ras → ERK, and MKP-1 induction, albeit to a lesser degree. Gln and Cys were comparable in suppression against 2, 4-dinitrofluorobenzene-induced contact dermatitis. Gln-mediated, but not Cys-mediated, suppression was abolished by MKP-1 small interfering RNA. These data indicate that Gln induces MKP-1 by activating Ca²⁺→ ERK pathway, which plays a key role in suppression of inflammatory reactions.     

Identification of mitogen-activated protein kinase kinase gene family and MKK–MAPK interaction network in maize

Plant mitogen-activated protein kinases (MAPK) are involved in important processes, including stress signaling and development. MAPK kinases (MAPKK, MKK) have been investigated in several plant species including Arabidopsis thaliana, Oryza sativa, Populus trichocarpa, and Brachypodium distachyon. In the present study, nine putative maize MKK genes have been identified. Analysis of the conserved protein motifs, exon–intron junctions and intron phase has revealed high levels of conservation within the phylogenetic groups. Next, we defined four new ZmMKK–ZmMPK interactions using yeast two-hybrid. Finally, we examined the biological functions of the ZmMKK4 gene. Overexpression of ZmMKK4 in Arabidopsis conferred tolerance to oxidative stress by increased germination rate and early seedling growth compared with WT plants. Taken together, we provide a comprehensive bioinformatics analysis of the MKK gene family in maize genome and our data provide an important foundation for further functional study of MAPK and MKK families in maize.     

Shedding of the Mer tyrosine kinase receptor is mediated by ADAM17 protein through a pathway involving reactive oxygen species, protein kinase Cδ, and p38 mitogen-activated protein kinase (MAPK)

Mer tyrosine kinase (MerTK) is an integral membrane protein that is preferentially expressed by phagocytic cells, where it promotes efferocytosis and inhibits inflammatory signaling. Proteolytic cleavage of MerTK at an unidentified site leads to shedding of its soluble ectodomain (soluble MER; sMER), which can inhibit thrombosis in mice and efferocytosis in vitro. Herein, we show that MerTK is cleaved at proline 485 in murine macrophages. Site-directed deletion of 6 amino acids spanning proline 485 rendered MerTK resistant to proteolysis and suppression of efferocytosis by cleavage-inducing stimuli. LPS is a known inducer of MerTK cleavage, and the intracellular signaling pathways required for this action are unknown. LPS/TLR4-mediated generation of sMER required disintegrin and metalloproteinase ADAM17 and was independent of Myd88, instead requiring TRIF adaptor signaling. LPS-induced cleavage was suppressed by deficiency of NADPH oxidase 2 (Nox2) and PKCδ. The addition of the antioxidant N-acetyl cysteine inhibited PKCδ, and silencing of PKCδ inhibited MAPK p38, which was also required. In a mouse model of endotoxemia, we discovered that LPS induced plasma sMER, and this was suppressed by Adam17 deficiency. Thus, a TRIF-mediated pattern recognition receptor signaling cascade requires NADPH oxidase to activate PKCδ and then p38, culminating in ADAM17-mediated proteolysis of MerTK. These findings link innate pattern recognition receptor signaling to proteolytic inactivation of MerTK and generation of sMER and uncover targets to test how MerTK cleavage affects efferocytosis efficiency and inflammation resolution in vivo.     

Activation of macrophage nuclear factor-κB and induction of inducible nitric oxide synthase by LPS

Background  

Chronic lung disease (CLD) of prematurity is a major problem of neonatal care. Bacterial infection and inflammatory response have been thought to play an important role in the development of CLD and steroids have been given, with some benefit, to neonates with this disease. In the present study, we assessed the ability of lipopolysaccharide (LPS) to stimulate rat alveolar macrophages to produce nitric oxide (NO), express inducible nitric oxide synthase (iNOS) and activate nuclear factor-κB (NF-κB) in vitro. In addition, we investigated the impact of dexamethasone and budesonide on these processes.     

Activation of the AMP-activated protein kinase–p38 MAP kinase pathway mediates apoptosis induced by conjugated linoleic acid in p53-mutant mouse mammary tumor cells

Conjugated linoleic acid (CLA) inhibits tumorigenesis and tumor growth in most model systems, an effect mediated in part by its pro-apoptotic activity. We previously showed that trans-10,cis-12 CLA induced apoptosis of p53-mutant TM4t mouse mammary tumor cells through both mitochondrial and endoplasmic reticulum stress pathways. In the current study, we investigated the role of AMP-activated protein kinase (AMPK), a key player in fatty acid metabolism, in CLA-induced apoptosis in TM4t cells. We found that t10,c12-CLA increased phosphorylation of AMPK, and that CLA-induced apoptosis was enhanced by the AMPK agonist 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) and inhibited by the AMPK inhibitor compound C. The increased AMPK activity was not due to nutrient/energy depletion since ATP levels did not change in CLA-treated cells, and knockdown of the upstream kinase LKB1 did not affect its activity. Furthermore, our data do not demonstrate a role for the AMPK-modulated mTOR pathway in CLA-induced apoptosis. Although CLA decreased mTOR levels, activity was only modestly decreased. Moreover, rapamycin, which completely blocked the activity of mTORC1 and mTORC2, did not induce apoptosis, and attenuated rather than enhanced CLA-induced apoptosis. Instead, the data suggest that CLA-induced apoptosis is mediated by the AMPK–p38 MAPK–Bim pathway: CLA-induced phosphorylation of AMPK and p38 MAPK, and increased expression of Bim, occurred with a similar time course as apoptosis; phosphorylation of p38 MAPK was blocked by compound C; the increased Bim expression was blocked by p38 MAPK siRNA; CLA-induced apoptosis was attenuated by the p38 inhibitor SB-203580 and by siRNAs directed against p38 MAPK or Bim.     

Phosphorylation of KIBRA by the extracellular signal-regulated kinase (ERK)–ribosomal S6 kinase (RSK) cascade modulates cell proliferation and migration

In mammals, KIBRA is defined as a memory performance-associated protein. The physiological function and regulation of KIBRA in non-neuronal cells are much less understood. Recent studies have identified KIBRA as a novel regulator of the Hippo signaling pathway, which plays a critical role in tumorigenesis by inhibiting cell proliferation and promoting apoptosis. We recently reported that KIBRA is phosphorylated by the mitotic kinases Aurora and cyclin-dependent kinase 1 during mitosis. In this current study, we show that KIBRA is also phosphorylated by the ERK (extracellular signal-regulated kinases)–RSK (p90 ribosomal S6 kinases) cascade. We demonstrated that ERK1/2 phosphorylate KIBRA at Ser⁵⁴⁸ in cells as well as in vitro. Moreover, we found that RSK1/2 specifically phosphorylates KIBRA at two highly conserved sites (Thr⁹²⁹ and Ser⁹⁴⁷) in vitro and in cells. RSK-mediated phosphorylation is required for KIBRA binding to RSK1, but not RSK2. Surprisingly, KIBRA knockdown impaired cell migration and proliferation in breast cancer cells. By using inducible-expression cell lines, we further show that phospho-regulation of KIBRA by ERK1/2 and RSK1/2 is required for proper cell proliferation and RSK-mediated phosphorylation also modulates KIBRA's migratory activity in MDA-MB-231 breast cancer cells. Our findings uncover unexpected results and a new mechanism through which KIBRA regulates cell migration and proliferation.     

Arabidopsis S6 kinase mutants display chromosome instability and altered RBR1�CE2F pathway activity

The 40S ribosomal protein S6 kinase (S6K) is a conserved component of signalling pathways controlling growth in eukaryotes. To study S6K function in plants, we isolated single‐ and double‐knockout mutations and RNA‐interference (RNAi)‐silencing lines in the linked Arabidopsis S6K1 and S6K2 genes. Hemizygous s6k1s6k2/++ mutant and S6K1 RNAi lines show high phenotypic instability with variation in size, increased trichome branching, produce non‐viable pollen and high levels of aborted seeds. Analysis of their DNA content by flow cytometry, as well as chromosome counting using DAPI staining and fluorescence in situ hybridization, revealed an increase in ploidy and aneuploidy. In agreement with this data, we found that S6K1 associates with the Retinoblastoma‐related 1 (RBR1)–E2FB complex and this is partly mediated by its N‐terminal LVxCxE motif. Moreover, the S6K1–RBR1 association regulates RBR1 nuclear localization, as well as E2F‐dependent expression of cell cycle genes. Arabidopsis cells grown under nutrient‐limiting conditions require S6K for repression of cell proliferation. The data suggest a new function for plant S6K as a repressor of cell proliferation and required for maintenance of chromosome stability and ploidy levels.     

Modulation of L-α-lysophosphatidylinositol/GPR55 mitogen-activated protein kinase (MAPK) signaling by cannabinoids

GPR55 is activated by l-α-lysophosphatidylinositol (LPI) but also by certain cannabinoids. In this study, we investigated the GPR55 pharmacology of various cannabinoids, including analogues of the CB₁ receptor antagonist Rimonabant®, CB₂ receptor agonists, and Cannabis sativa constituents. To test ERK1/2 phosphorylation, a primary downstream signaling pathway that conveys LPI-induced activation of GPR55, a high throughput system, was established using the AlphaScreen® SureFire® assay. Here, we show that CB₁ receptor antagonists can act both as agonists alone and as inhibitors of LPI signaling under the same assay conditions. This study clarifies the controversy surrounding the GPR55-mediated actions of SR141716A; some reports indicate the compound to be an agonist and some report antagonism. In contrast, we report that the CB₂ ligand {"type":"entrez-nucleotide","attrs":{"text":"GW405833","term_id":"288331434","term_text":"GW405833"}}GW405833 behaves as a partial agonist of GPR55 alone and enhances LPI signaling. GPR55 has been implicated in pain transmission, and thus our results suggest that this receptor may be responsible for some of the antinociceptive actions of certain CB₂ receptor ligands. The phytocannabinoids Δ⁹-tetrahydrocannabivarin, cannabidivarin, and cannabigerovarin are also potent inhibitors of LPI. These Cannabis sativa constituents may represent novel therapeutics targeting GPR55.     

Activation of the integrins α5β1 and αvβ3 and focal adhesion kinase (FAK) during arteriogenesis

Migration and proliferation of smooth muscle cells (SMC) are important events during arteriogenesis, but the underlying mechanism is still only partially understood. The present study investigates the expression of integrins alpha 5 beta 1 and v beta 3 as well as focal adhesion kinase (FAK) and phosphorylated FAK (pY397), key mediators for cell migration and proliferation, in collateral vessels (CV) in rabbit hind limbs induced by femoral ligation or an arteriovenous (AV) shunt created between the distal femoral artery stump and the accompanying femoral vein by confocal immunofluorescence. In addition, the effect of the extracellular matrix components fibronectin (FN), laminin (LN), and Matrigel on expression of these focal adhesion molecules proliferation was studied in cultured SMCs. We found that: (1) in normal vessels (NV), both integrins alpha 5 beta 1 and alpha v beta 3 were mainly expressed in endothelial cells, very weak in smooth muscle cells (SMC); (2) in CVs, both alpha 5 beta 1 and alpha v beta 3 were significantly upregulated (P < 0.05); this was more evident in the shunt-side CVs, 1.5 and 1.3 times higher than that in the ligation side, respectively; (3) FAK and FAK(py397) were expressed in NVs and CVs in a similar profile as was alpha 5 beta 1 and alpha v beta 3; (4) in vitro SMCs cultured on fibronectin (overexpressed in collaterals) expressed higher levels of FAK, FAK (pY397), alpha 5 beta 1, and alpha v beta 3 than on laminin, whereas SMCs growing inside Matrigel expressed little of these proteins and showed no proliferation. In conclusion, our data demonstrate for the first time that the integrin-FAK signaling axis is activated in collateral vessels and that altered expression of FN and LN may play a crucial role in mediating the integrin-FAK signaling pathway activation. These findings explain a large part of the positive remodeling that collateral vessels undergo under the influence of high fluid shear stress.     

Activation of protein kinase C-α is essential for stimulation of cell proliferation by ceramide 1-phosphate

We previously demonstrated that ceramide-1-phosphate (C1P) stimulates fibroblast and macrophage proliferation, but the mechanisms involved in this action have only been partially described. Here we demonstrate that C1P induces translocation of protein kinase C-alpha (PKC-α) from the soluble to the membrane fraction of bone marrow-derived macrophages. Translocation of this enzyme was accompanied by its phosphorylation on Ser 657 residue. Activation of PKC-α was independent of prior stimulation of phosphatidylinositol-dependent or phosphatidylcholine-dependent phospholipase C activities, but required activation of sphingomyelin synthesis. Inhibition of PKC-α activation also blocked C1P-stimulated macrophage proliferation indicating that this enzyme is essential for the mitogenic effect of C1P.     

Activation of multiple MAPK pathways (ERKs,JNKs, p38‐MAPK) by diverse stimuli in the amphibian heart

We investigated the expression and activation of three MAPK subfamilies in the isolated perfused amphibian heart. ERK was detected as a 43 kDa band; p38MAPK was detected as a band corresponding to 38 kDa and JNKs were detected as two bands corresponding to 46 and 52 kDa, respectively. PMA induced the activation of the ERK pathway as assessed by determining the phosphorylation state of ERK and the upstream component MEK1/2. PD98059 abolished this activation. p38MAPK was phosphorylated by sorbitol (almost 12fold, maximal within 10–15 min) and JNKs were phosphorylated and activated by sorbitol or anoxia/reoxygenation (approximately 4 and 2.5fold, respectively). SB203580 completely blocked the activation of p38MAPK by sorbitol. These results indicate that the MAPK pathways activated by phorbol esters, hyperosmotic stress or anoxia/reoxygenation in the amphibian heart may have an important role in this experimental system.     

Requirements of the cytosolic iron–sulfur cluster assembly pathway in Arabidopsis

The assembly of iron–sulfur (Fe–S) clusters requires dedicated protein factors inside the living cell. Striking similarities between prokaryotic and eukaryotic assembly proteins suggest that plant cells inherited two different pathways through endosymbiosis: the ISC pathway in mitochondria and the SUF pathway in plastids. Fe–S proteins are also found in the cytosol and nucleus, but little is known about how they are assembled in plant cells. Here, we show that neither plastid assembly proteins nor the cytosolic cysteine desulfurase ABA3 are required for the activity of cytosolic aconitase, which depends on a [4Fe–4S] cluster. In contrast, cytosolic aconitase activity depended on the mitochondrial cysteine desulfurase NFS1 and the mitochondrial transporter ATM3. In addition, we were able to complement a yeast mutant in the cytosolic Fe–S cluster assembly pathway, dre2, with the Arabidopsis homologue AtDRE2, but only when expressed together with the diflavin reductase AtTAH18. Spectroscopic characterization showed that purified AtDRE2 could bind up to two Fe–S clusters. Purified AtTAH18 bound one flavin per molecule and was able to accept electrons from NAD(P)H. These results suggest that the proteins involved in cytosolic Fe–S cluster assembly are highly conserved, and that dependence on the mitochondria arose before the second endosymbiosis event leading to plastids.     

Activation of brain hexokinase by magnesium ions and by magnesium ion–adenosine triphosphate complex

1. An alternative explanation for the kinetic data obtained by Bachelard (1971) for the brain hexokinase reaction is presented. 2. Apparently sigmoidal saturation curves for MgATP²⁻ based upon Bachelard's (1971) studies can be corrected to hyperbolic curves by use of a stability constant for MgATP²⁻ complex formation. 3. A number of other effects related to the concentration-dependent stability of the MgATP²⁻ complex and to the presence of the inhibitory free uncomplexed ATP⁴⁻ concentration are also explained in terms of a non-allosteric role for either Mg²⁺ or MgATP²⁻ fully consistent with a number of previous reports on this enzyme. 4. A brief discussion of the validity of Hill plots in studies of multisubstrate co-operative enzymes is presented. 5. A simple model is presented that demonstrates how enzymes obeying Michaelis–Menten kinetics can demonstrate sigmoidal velocity responses if the true substrate of the reaction is the metal–substrate complex.     

The modulation of radiation-induced cell death by genistein in K562 cells：Activation of thymidine kinase 1

Ionizing radiation is one of the most effective tools in cancer therapy. In a previous study, we reported that protein tyrosine kinase (PTK) inhibitors modulate the radiation responses in the human chronic myelogenous leukemia (CML) cell line K562. The receptor tyrosine kinase inhibitor, genistein, delayed radiation-induced cell death, while non-recepter tyrosine kinase inhibitor, herbimycin A (HMA) enhances radiation-induced apoptosis. In this study, we focused on the modulation of radiation-induced cell death by genistein and performed PCR-select suppression subtractive hybridization (SSH) to understand its molecular mechanism. We identified human thymidine kinase 1 (TK1), which is cell cycle regulatory gene and confirmed expression of TK1 mRNA by Northern blot analysis. Expression ofTK1 mRNA and TK 1 enzymatic activity were parallel in their increase and decrease. TK1 is involved in G1-S phase transition of cell cycle progression. In cell cycle analysis, we showed that radiation induced G2 arrest in K562 cells but it was not able to sustain. However, the addition of genistein to irradiated cells sustained a prolonged G2 arrest up to 120 h. In addition, the expression of cell cycle-related proteins, cyclin A and cyclin B 1, provided the evidences of G I/S progression and G2-arrest, and their relationship with TKI in cells treated with radiation and genistein. These results suggest that the activation of TK1 may be critical to modulate the radiation-induced cell death and cell cycle progression in irradiated K562 cells.