Bacillus Anthracis Endospores Regulate Ornithine Decarboxylase and Inducible Nitric Oxide Synthase Through ERK1/2 and p38 Mitogen-Activated Protein Kinases

Interactions between Bacillus anthracis (B. anthracis) and host cells are of particular interest given the implications of anthrax as a biological weapon. Inhaled B. anthracis endospores encounter alveolar macrophages as the first line of defense in the innate immune response. Yet, the consequences of this interaction remain unclear. We have demonstrated that B. anthracis uses arginase, inherent in the endospores, to reduce the ability of macrophages to produce nitric oxide (^?NO) from inducible nitric oxide synthase (NOS2) by competing for l-arginine, producing l-ornithine at the expense of ^?NO. In the current study, we used genetically engineered B. anthracis endospores to evaluate the contribution of germination and the lethal toxin (LT) in mediating signaling pathways responsible for the induction of NOS2 and ornithine decarboxylase (ODC), which is the rate-limiting enzyme in the conversion of l-ornithine into polyamines. We found that induction of NOS2 and ODC expression in macrophages exposed to B. anthracis occurs through the activation of p38 and ERK1/2 MAP kinases, respectively. Optimal induction of NOS2 was observed following exposure to germination-competent endospores, whereas ODC induction occurred irrespective of the endospores’ germination capabilities and was more prominent in macrophages exposed to endospores lacking LT. Our findings suggest that activation of kinase signaling cascades that determine macrophage defense responses against B. anthracis infection occurs through distinct mechanisms.     

ERK and p38 MAPK-Activated Protein Kinases: a Family of Protein Kinases with Diverse Biological Functions

Conserved signaling pathways that activate the mitogen-activated protein kinases (MAPKs) are involved in relaying extracellular stimulations to intracellular responses. The MAPKs coordinately regulate cell proliferation, differentiation, motility, and survival, which are functions also known to be mediated by members of a growing family of MAPK-activated protein kinases (MKs; formerly known as MAPKAP kinases). The MKs are related serine/threonine kinases that respond to mitogenic and stress stimuli through proline-directed phosphorylation and activation of the kinase domain by extracellular signal-regulated kinases 1 and 2 and p38 MAPKs. There are currently 11 vertebrate MKs in five subfamilies based on primary sequence homology: the ribosomal S6 kinases, the mitogen- and stress-activated kinases, the MAPK-interacting kinases, MAPK-activated protein kinases 2 and 3, and MK5. In the last 5 years, several MK substrates have been identified, which has helped tremendously to identify the biological role of the members of this family. Together with data from the study of MK-knockout mice, the identities of the MK substrates indicate that they play important roles in diverse biological processes, including mRNA translation, cell proliferation and survival, and the nuclear genomic response to mitogens and cellular stresses. In this article, we review the existing data on the MKs and discuss their physiological functions based on recent discoveries.     

Opposing Effects of Jun Kinase and p38 Mitogen-Activated Protein Kinases on Cardiomyocyte Hypertrophy

c-Jun N-terminal protein kinase (JNK) and p38, two distinct members of the mitogen-activated protein (MAP) kinase family, regulate gene expression in response to various extracellular stimuli, yet their physiological functions are not completely understood. In this report we show that JNK and p38 exerted opposing effects on the development of myocyte hypertrophy, which is an adaptive physiological process characterized by expression of embryonic genes and unique morphological changes. In rat neonatal ventricular myocytes, both JNK and p38 were stimulated by hypertrophic agonists like endothelin-1, phenylephrine, and leukemia inhibitory factor. Expression of MAP kinase kinase 6b (EE), a constitutive activator of p38, stimulated the expression of atrial natriuretic factor (ANF), which is a genetic marker of in vivo cardiac hypertrophy. Activation of p38 was required for ANF expression induced by the hypertrophic agonists. Furthermore, a specific p38 inhibitor, SB202190, significantly changed hypertrophic morphology induced by the agonists. Surprisingly, activation of JNK led to inhibition of ANF expression induced by MEK kinase 1 (MEKK1) and the hypertrophic agonists. MEKK1-induced ANF expression was also negatively regulated by expression of c-Jun. Our results demonstrate that p38 mediates, but JNK suppresses, the development of myocyte hypertrophy.     

Activation and Tyrosine Phosphorylation of 44-kDa Mitogen-Activated Protein Kinase (MAPK) Induced by Electroconvulsive Shock in Rat Hippocampus

Abstract: Electroconvulsive shock (ECS) has been reported to induce the phosphorylation and activation of 42-kDa, but not 44-kDa, mitogen-activated protein kinase (MAPK) in rat hippocampus. We studied the activation and tyrosine phosphorylation of MAPKs in rat brain after ECS. We observed the increase of the activities of both 42- and 44-kDa MAPKs in rat hippocampus after ECS. The activities reached peak at 2 min and returned to basal levels by 15 min after ECS. We also observed the increased phsophorylation on the tyrosine residue of 42-kDa MAPK in rat hippocampus after ECS, but not on that of 44-kDa MAPK. However, when we examined the immunoprecipitated 44-kDa MAPK, we could demonstrate that the tyrosine phosphorylation of 44-kDa MAPK at 2 min after ECS was markedly increased, in accordance with the increase of kinase activity. These results indicate that ECS induces the transient activation and tyrosine phosphorylation of 44-kDa MAPK, as well as 42-kDa MAPK, in rat hippocampus, although the amount of tyrosine phosphorylation is far less and the kinase activity is lower in 44-kDa MAPK than in 42-kDa MAPK.     

Persistent Activation of Mitogen-Activated Protein Kinases p42 and p44 and ets-2 Phosphorylation in Response to Colony-Stimulating Factor 1/c-fms Signaling

An antibody that specifically recognized phosphothreonine 72 in ets-2 was used to determine the phosphorylation status of endogenous ets-2 in response to colony-stimulating factor 1 (CSF-1)/c-fms signaling. Phosphorylation of ets-2 was detected in primary macrophages, cells that normally express c-fms, and in fibroblasts engineered to express human c-fms. In the former cells, ets-2 was a CSF-1 immediate-early response gene, and phosphorylated ets-2 was detected after 2 to 4 h, coincident with expression of ets-2 protein. In fibroblasts, ets-2 was constitutively expressed and rapidly became phosphorylated in response to CSF-1. In both cell systems, ets-2 phosphorylation was persistent, with maximal phosphorylation detected 8 to 24 h after CSF-1 stimulation, and was correlated with activation of the CSF-1 target urokinase plasminogen activator (uPA) gene. Kinase assays that used recombinant ets-2 protein as a substrate demonstrated that mitogen-activated protein (MAP) kinases p42 and p44 were constitutively activated in both cell types in response to CSF-1. Immune depletion experiments and the use of the MAP kinase kinase inhibitor PD98059 indicate that these two MAP kinases are the major ets-2 kinases activated in response to CSF-1/c-fms signaling. In the macrophage cell line RAW264, conditional expression of raf kinase induced ets-2 expression and phosphorylation, as well as uPA mRNA expression. Transient assays mapped ets/AP-1 response elements as critical for basal and CSF-1-stimulated uPA reporter gene activity. These results indicate that persistent activation of the raf/MAP kinase pathway by CSF-1 is necessary for both ets-2 expression and posttranslational activation in macrophages.     

Stimulation of p42 and p44 Mitogen-Activated Protein Kinases by Reactive Oxygen Species and Nitric Oxide in Hippocampus

Abstract: Reactive oxygen species (ROS) have been suggested to act as cellular messengers that mediate signal transduction cascades in various cell types. However, little is known about their role in this capacity in the nervous system. We have begun to investigate the role of ROS, and that of nitric oxide (NO), in mediating mitogen-activated protein kinase (MAPK) signaling in rat hippocampal slices. Our studies have revealed that direct exposure of hippocampal slices to hydrogen peroxide, xanthine/xanthine oxidase (a superoxide-generating system), sodium nitroprusside (an NO donor compound), S -nitroso- N -acetylpenicillamine (an NO donor compound), or 3-morpholinosydnonimine (a compound that produces NO and superoxide) results in an enhancement in tyrosine phosphorylation of several proteins, including proteins with apparent molecular masses of 42 and 44 kDa. We investigated the possibility that these proteins correspond to the active forms of p42 MAPK and p44 MAPK. Hippocampal slices exposed to various ROS and NO donors resulted in increases in levels of the active forms of both p42 MAPK and p44 MAPK. The ROS- and NO-enhanced tyrosine phosphorylation and activation of p42 MAPK and p44 MAPK were inhibited by pretreatment with the antioxidant N -acetyl- l -cysteine. Our observations indicate that ROS and NO can mediate protein tyrosine phosphorylation and MAPK signaling in the hippocampus via a redox-sensitive mechanism and suggest a potential cellular mechanism for their effects in the nervous system.     

High-Resolution Protein Interaction Map of the Drosophila melanogaster p38 Mitogen-Activated Protein Kinases Reveals Limited Functional Redundancy

Functional redundancy is a pivotal mechanism that supports the robustness of biological systems at a molecular, cellular, and organismal level. The extensive prevalence of redundancy in molecular networks has been highlighted by recent systems biology studies; however, a detailed mechanistic understanding of redundant functions in specific signaling modules is often missing. We used affinity purification of protein complexes coupled to tandem mass spectrometry to generate a high-resolution protein interaction map of the three homologous p38 mitogen-activated protein kinases (MAPKs) in Drosophila and assessed the utility of such a map in defining the extent of common and unique functions. We found a correlation between the depth of integration of individual p38 kinases into the protein interaction network and their functional significance in cultured cells and in vivo. Based on these data, we propose a central role of p38b in the Drosophila p38 signaling module, with p38a and p38c playing more peripheral, auxiliary roles. We also present the first in vivo evidence demonstrating that an evolutionarily conserved complex of p38b with glycogen synthase links stress sensing to metabolic adaptation.     

Protein phosphatase 2Calpha inhibits the human stress-responsive p38 and JNK MAPK pathways.

MAPK (mitogen-activated protein kinase) cascades are common eukaryotic signaling modules that consist of a MAPK, a MAPK kinase (MAPKK) and a MAPKK kinase (MAPKKK). Because phosphorylation is essential for the activation of both MAPKKs and MAPKs, protein phosphatases are likely to be important regulators of signaling through MAPK cascades. To identify protein phosphatases that negatively regulate the stress-responsive p38 and JNK MAPK cascades, we screened human cDNA libraries for genes that down-regulated the yeast HOG1 MAPK pathway, which shares similarities with the p38 and JNK pathways, using a hyperactivating yeast mutant. In this screen, the human protein phosphatase type 2Calpha (PP2Calpha) was found to negatively regulate the HOG1 pathway in yeast. Moreover, when expressed in mammalian cells, PP2Calpha inhibited the activation of the p38 and JNK cascades induced by environmental stresses. Both in vivo and in vitro observations indicated that PP2Calpha dephosphorylated and inactivated MAPKKs (MKK6 and SEK1) and a MAPK (p38) in the stress-responsive MAPK cascades. Furthermore, a direct interaction of PP2Calpha and p38 was demonstrated by a co-immunoprecipitation assay. This interaction was observed only when cells were stimulated with stresses or when a catalytically inactive PP2Calpha mutant was used, suggesting that only the phosphorylated form of p38 interacts with PP2Calpha.     

Hispolon from Phellinus linteus possesses mediate caspases activation and induces human nasopharyngeal carcinomas cells apoptosis through ERK1/2, JNK1/2 and p38 MAPK pathway

Hispolon, a phenol compound isolated from Phellinus linteus (PL), possesses anti-inflammatory, antiproliferative, and antioxidant effects. However, the effects of hispolon on human nasopharyngeal carcinomas have yet to be evaluated. Here, the molecular mechanism by which hispolon anticancer effects in human nasopharyngeal carcinomas cells was investigated. The results showed that hispolon significantly inhibited cell proliferation of HONE-1 and NP-039 cell lines. Furthermore, hispolon induced apoptosis through caspases-3, -8, and -9 activations and PARP cleavage in dose- and time-dependent manner in HONE-1 and NP-039 cells. Moreover, hispolon also showed that increase phosphorylation of ERK1/2, p38 MAPK and JNK1/2 in dose- and time-dependent manner by western blot analysis. However, hispolon-induced activation of the caspase-3, -8 and -9 significantly abolished by inhibition of p38 MAPK and JNK1/2 specific inhibitors. In this study, we determine that the effects of hispolon on the apoptosis and related regulation mechanism in HONE-1 and NPC-039 cells takes place. Our findings revealed that hispolon may be a useful candidate as a chemotherapeutic agent for NPC therapy.     

Negative Regulation of Protein Translation by Mitogen-Activated Protein Kinase-Interacting Kinases 1 and 2

Eukaryotic initiation factor 4E (eIF4E) is a key component of the translational machinery and an important modulator of cell growth and proliferation. The activity of eIF4E is thought to be regulated by interaction with inhibitory binding proteins (4E-BPs) and phosphorylation by mitogen-activated protein (MAP) kinase-interacting kinase (MNK) on Ser209 in response to mitogens and cellular stress. Here we demonstrate that phosphorylation of eIF4E via MNK1 is mediated via the activation of either the Erk or p38 pathway. We further show that expression of active mutants of MNK1 and MNK2 in 293 cells diminishes cap-dependent translation relative to cap-independent translation in a transient reporter assay. The same effect on cap-dependent translation was observed when MNK1 was activated by the Erk or p38 pathway. In line with these findings, addition of recombinant active MNK1 to rabbit reticulocyte lysate resulted in a reduced protein synthesis in vitro, and overexpression of MNK2 caused a decreased rate of protein synthesis in 293 cells. By using CGP 57380, a novel low-molecular-weight kinase inhibitor of MNK1, we demonstrate that eIF4E phosphorylation is not crucial to the formation of the initiation complex, mitogen-stimulated increase in cap-dependent translation, and cell proliferation. Our results imply that activation of MNK by MAP kinase pathways does not constitute a positive regulatory mechanism to cap-dependent translation. Instead, we propose that the kinase activity of MNKs, eventually through phosphorylation of eIF4E, may serve to limit cap-dependent translation under physiological conditions.     

Cry1Ac toxin induces macrophage activation via ERK1/2, JNK and p38 mitogen-activated protein kinases

The Cry1Ac toxin from Bacillus thuringiensis is used commercially as a bio-insecticide and is expressed in transgenic plants that are used for human and animal consumption. Although it was originally considered innocuous for mammals, the Cry1Ac toxin is not inert and has the ability to induce mucosal and systemic immunogenicity. Herein, we examined whether the Cry1Ac toxin promotes macrophage activation and explored the signalling pathways that may mediate this effect. Treatment of primary and RAW264.7 macrophages with the Cry1Ac toxin resulted in upregulation of the costimulatory molecules CD80, CD86 and ICOS-L and enhanced production of nitric oxide, the chemokine MCP-1 and the proinflammatory cytokines TNF-α and IL-6. Remarkably, the Cry1Ac toxin induced phosphorylation of the mitogen-activated protein kinases (MAPKs) ERK1/2, JNK and p38 and promoted nuclear translocation of nuclear factor-kappa B (NF-κB) p50 and p65. p38 and ERK1/2 MAPKs were involved in this effect, as indicated by the Cry1Ac-induced upregulation of CD80 and IL-6 and TNF-α abrogation by the p38 MAPK inhibitor SB203580. Furthermore, treatment the MEK1/2 kinase inhibitor PD98059 blocked increases in MCP-1 secretion and augmented Cry1Ac-induced ICOS-L upregulation. These data demonstrate the capacity of the Cry1Ac toxin to induce macrophage activation via the MAPK and NF-κB pathways.     

Activation by 2-arachidonoylglycerol of platelet p38MAPK/cPLA2 pathway

The endogenous cannabinoid 2-arachidonoylglycerol (2-AG) is described as a platelet agonist able to induce aggregation and to increase intracellular calcium. In the present report we have confirmed these data and demonstrated that the inhibitor of p38MAPK SB203580 and the inhibitor of cPLA(2) metabolism ETYA affect both these parameters. Thus, we aimed to define the role of p38MAPK/cytosolic phospholipase A(2) (cPLA(2)) pathway in 2-AG-induced human platelet activation. p38MAPK activation was assayed by phosphorylation. cPLA(2) activation was assayed by phosphorylation and as arachidonic acid release and thromboxane B(2) formation. It was shown that 2-AG in a dose- and time-dependent manner activates p38MAPK peaking at 10 μM after 1 min of incubation. The 2-AG effect on p38MAPK was not impaired by apyrase, indomethacin or RGDS peptide but it was significantly reduced by SR141716, specific inhibitor of type-1 cannabinoid receptor and unaffected by the specific inhibitor of type-2 cannabinoid receptor SR144528. Moreover, the incubation of platelets with 2-AG led to the phosphorylation of cPLA(2) and its activation. Platelet pretreatment with SB203580, inhibitor of p38MAPK, abolished both cPLA(2) phosphorylation and activation. In addition SR141716 strongly impaired cPLA(2) phosphorylation, arachidonic acid release and thromboxane B(2) formation, whereas SR144528 did not change these parameters. Finally platelet stimulation with 2-AG led to an increase in free oxygen radical species. In conclusion, data provide insight into the mechanisms involved in platelet activation by 2-AG, indicating that p38MAPK/cPLA(2) pathway could play a relevant role in this complicated process.     

Protein phosphatase 2A-mediated cross-talk between p38 MAPK and ERK in apoptosis of cardiac myocytes

Mitogen-activated protein kinases (MAPKs) play different regulatory roles in signaling oxidative stress-induced apoptosis in cardiac ventricular myocytes. The regulation and functional role of cross-talk between p38 MAPK and extracellular signal-regulated kinase (ERK) pathways were investigated in cardiac ventricular myocytes in the present study. We demonstrated that inhibition of p38 MAPK with SB-203580 and SB-239063 enhanced H(2)O(2)-stimulated ERK phosphorylation, whereas preactivation of p38 MAPK with sodium arsenite reduced H(2)O(2)-stimulated ERK phosphorylation. In addition, pretreatment of cells with the protein phosphatase 2A (PP2A) inhibitors okadaic acid and fostriecin increased basal and H(2)O(2)-stimulated ERK phosphorylation. We also found that PP2A coimmunoprecipitated with ERK and MAPK/ERK (MEK) in cardiac ventricular myocytes, and H(2)O(2) increased the ERK-associated PP2A activity that was blocked by inhibition of p38 MAPK. Finally, H(2)O(2)-induced apoptosis was attenuated by p38 MAPK or PP2A inhibition, whereas it was enhanced by MEK inhibition. Thus the present study demonstrated that p38 MAPK activation decreases H(2)O(2)-induced ERK activation through a PP2A-dependent mechanism in cardiac ventricular myocytes. This represents a novel cellular mechanism that allows for interaction of two opposing MAPK pathways and fine modulation of apoptosis during oxidative stress.     

B7-H4 Treatment of T Cells Inhibits ERK, JNK, p38, and AKT Activation

B7-H4 is a newly identified B7 homolog that plays an important role in maintaining T-cell homeostasis by inhibiting T-cell proliferation and lymphokine-secretion. In this study, we investigated the signal transduction pathways inhibited by B7-H4 engagement in mouse T cells. We found that treatment of CD3(+) T cells with a B7-H4.Ig fusion protein inhibits anti-CD3 elicited T-cell receptor (TCR)/CD28 signaling events, including phosphorylation of the MAP kinases, ERK, p38, and JNK. B7-H4.Ig treatment also inhibited the phosphorylation of AKT kinase and impaired its kinase activity as assessed by the phosphorylation of its endogenous substrate GSK-3. Expression of IL-2 is also reduced by B7-H4. In contrast, the phosphorylation state of the TCR proximal tyrosine kinases ZAP70 and lymphocyte-specific protein tyrosine kinase (LCK) are not affected by B7-H4 ligation. These results indicate that B7-H4 inhibits T-cell proliferation and IL-2 production through interfering with activation of ERK, JNK, and AKT, but not of ZAP70 or LCK.     

Differential role of the JNK and p38 MAPK pathway in c-Myc- and s-Myc-mediated apoptosis

The s-Myc is similar to c-Myc in its ability to induce apoptosis requiring caspase activation. However, s-Myc is distinct from c-Myc in that it has activity to suppress tumor growth and does not require wild-type p53 to induce apoptosis. These facts suggest differential regulation between s-Myc and c-Myc. Here we showed that s-Myc-mediated apoptosis triggered by UV was not inhibited by the inactive form mutant JNK (APF), though c-Myc-mediated apoptosis was. Moreover, we found that JNK did not affect the transactivation activity of s-Myc, but stimulated that of c-Myc. In contrast, both Myc-mediated apoptosis and caspase-3-like protease activation were suppressed by kinase-negative MKK6 and an inactive form mutant p38(AGF). Our results indicate that s-Myc does not require the JNK signaling unlike c-Myc during UV-triggered apoptosis, but the MKK6/p38MAPK pathway might regulate common apoptotic machinery for both s-Myc and c-Myc upstream of caspase.     

Involvement of p42/p44 MAPK, p38 MAPK, JNK, and NF-kappaB in IL-1beta-induced VCAM-1 expression in human tracheal smooth muscle cells

Interleukin-1beta (IL-1beta) has been shown to induce the expression of adhesion molecules on airway epithelial and smooth cells and contributes to inflammatory responses. Here, the roles of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappaB (NF-kappaB) pathways for IL-1beta-induced vascular cell adhesion molecule (VCAM)-1 expression were investigated in human tracheal smooth muscle cells (HTSMC). IL-1beta induced expression of VCAM-1 protein and mRNA in a time-dependent manner, which was significantly inhibited by inhibitors of MEK1/2 (U0126 and PD-98059), p38 (SB-202190), and c-Jun NH(2)-terminal kinase (JNK; SP-600125). Consistently, IL-1beta-stimulated phosphorylation of p42/p44 MAPK, p38, and JNK was attenuated by pretreatment with U0126, SB-202190, or SP-600125, respectively. IL-1beta-induced VCAM-1 expression was significantly blocked by the specific NF-kappaB inhibitors helenalin and pyrrolidine dithiocarbamate. As expected, IL-1beta-stimulated translocation of NF-kappaB into the nucleus and degradation of IkappaB-alpha were blocked by helenalin but not by U0126, SB-202190, or SP-600125. Moreover, the resultant enhancement of VCAM-1 expression increased the adhesion of polymorphonuclear cells to a monolayer of HTSMC, which was blocked by pretreatment with helenalin, U0126, SB-202190, or SP-600125 before IL-1beta exposure or by anti-VCAM-1 antibody. Together, these results suggest that in HTSMC, activation of p42/p44 MAPK, p38, JNK, and NF-kappaB pathways is essential for IL-1beta-induced VCAM-1 gene expression. These results provide new insight into the mechanisms of IL-1beta action that cytokines may promote inflammatory responses in airway disease.