Mitogen-activated Protein Kinase Activation In Response Research Articles

Anti-inflammatory effects of Malus toringoides extract in lipopolysaccharide-induced human umbilical vein endothelial cells

Background: Malus toringoides (Rehd.) Hughes is a traditional Tibetan medicine. It demonstrates significant hypoglycemic and hypolipidemic potential. However, the protective effects of M. toringoides extracts on endothelial cells and the mechanisms that underlie their activity have not yet been reported. Objectives: The aim of the study was to explore the anti-inflammatory effects and cellular mechanisms of extracts of M. toringoides (CBTM-E375) in lipopolysaccharide (LPS)-induced human umbilical vein endothelial cells (HUVECs). Materials and Methods: HUVECs were exposed to LPS, and the level of proinflammatory mediators was measured by enzyme-linked immunosorbent assay. Furthermore, the activation of heme oxygenase-1 (HO-1), nuclear factor erythroid 2-related factor 2 (Nrf2), and mitogen-activated protein kinase (MAPK) was examined by Western blot and immunofluorescence analysis. Results: CBTM-E375 significantly downregulated the levels of inflammatory mediators and upregulated the expression of HO-1 by modulating Nrf2 translocation in HUVECs. The transfection of HO-1 small interfering RNA into HUVECs actively reversed the effects of CBTM-E375 in suppressing the expression of proinflammatory cytokines. Furthermore, MAPK activation in response to LPS was also blocked by CBTM-E375. Conclusion: CBTM-E375 exerts anti-inflammatory effects, possibly by modulating the translocation of Nrf2 and expression of HO-1, and inhibiting the phosphorylation of MAPK signaling pathway.

Regulation of cotton (Gossypiumhirsutum) drought responses by mitogen-activated protein (MAP) kinase cascade-mediated phosphorylation of GhWRKY59.

Drought is a key limiting factor for cotton (Gossypium spp.) production, as more than half of the global cotton supply is grown in regions with high water shortage. However, the underlying mechanism of the response of cotton to drought stress remains elusive. By combining genome-wide transcriptome profiling and a loss-of-function screen using virus-induced gene silencing, we identified Gossypiumhirsutum GhWRKY59 as an important transcription factor that regulates the drought stress response in cotton. Biochemical and genetic analyses revealed a drought stress-activated mitogen-activated protein (MAP) kinase cascade consisting of GhMAP3K15-Mitogen-activated Protein Kinase Kinase 4 (GhMKK4)-Mitogen-activated Protein Kinase 6 (GhMPK6) that directly phosphorylates GhWRKY59 at residue serine 221. Interestingly, GhWRKY59 is required for dehydration-induced expression of GhMAPK3K15, constituting a positive feedback loop of GhWRKY59-regulated MAP kinase activation in response to drought stress. Moreover, GhWRKY59 directly binds to the W-boxes of DEHYDRATION-RESPONSIVE ELEMENT-BINDING PROTEIN 2 (GhDREB2), which encodes a dehydration-inducible transcription factor regulating the plant hormone abscisic acid (ABA)-independent drought response. Our study identified a complete MAP kinase cascade that phosphorylates and activates a key WRKY transcription factor, and elucidated a regulatory module, consisting of GhMAP3K15-GhMKK4-GhMPK6-GhWRKY59-GhDREB2, that is involved in controlling the cotton drought response.

Differential toll-like receptor activation in lung ischemia reperfusion injury

ObjectiveThe requirement for toll-like receptors (TLRs) in lung ischemia reperfusion injury (LIRI) has been demonstrated but not fully characterized. Previously, we reported that TLR-4 is required by alveolar macrophages but not pulmonary endothelial or epithelial cells for development of LIRI. Additionally, we demonstrated differential patterns of mitogen-activated protein kinase (MAPK) activation and cytokine release in these cell types during LIRI. Here, we sought to determine whether these differences in activation responses are related to cell-specific TLR activation requirements. MethodsPrimary cultures of alveolar macrophages, pulmonary endothelial, and immortalized epithelial cells were pretreated with TLR-2 or TLR-4 short interference RNA (ribonucleic acid) before hypoxia and reoxygenation. Cell lysates and media were analyzed for receptor knockdown, MAPK activation, and cytokine production. Rats were pretreated with TLR-2 or TLR-4 short interference RNA before lung ischemia reperfusion and changes in lung vascular permeability were assessed. ResultsKnockdown of TLR-2 in alveolar macrophages did not affect MAPK phosphorylation or cytokine secretion. Conversely, TLR-2 knockdown in pulmonary endothelial and epithelial cells demonstrated significant reductions in extracellular signal-regulated kinase 1/2 activation and cytokine secretion. The lung permeability index in LIRI was decreased by TLR-4 but not TLR-2. ConclusionsDifferential TLR signaling and MAPK activation in response to LIRI seem to be cell specific. Short interference RNA provides an outstanding tool for examination of the underlying mechanism.

Is Cryptochrome a Primary Sensor of Extremely Low Frequency Magnetic Fields in Childhood Leukemia?

Extremely low frequency magnetic fields (ELF MF) are classified as possibly carcinogenic to humans, but the biophysical mechanisms of a causal relationship remain unclear. A cryptochrome-based radical pair mechanism (RPM) has been invoked as the primary MF sensor in animal magnetoreception to explain effects from MF strengths in the nT range. Model studies of the RPM in aprotic solution require cryogenic temperatures and MFs in the µT range to elicit marginal responses, implying that physiological responses evoked by radical pairs in biological milieu are unlikely. We explore ideas about how signals from transient radical pairs in cryptochrome might be transduced and amplified in mutagenic responses. The cryptochrome-based RPM involves blue light activation of the flavin adenine dinucleotide (FAD) cofactor followed by electron transfer (ET) from a conserved triad of tryptophan residues. However, intramolecular ET involving additional conserved aromatic residues in cryptochrome likely extends beyond the canonical triad. Further, ascorbate, which is present at millimolar concentrations in leukocytes, is likely to transfer an electron to the ultimate amino acid radical formed during one-electron reduction of FAD. The ascorbyl radical has been proposed as a potential radical pair partner in the RPM. The flavin semiquinone radical may be reoxidized to the resting state via ET to O2 to form O2•−. Increased levels of the ascorbyl radical and O2•− would contribute to oxidative stress pathways in the cell. Oxidative stress responses in cancer are thought to be mediated in part by the mitogen activated protein kinase (MAPK) signaling pathway. We have presented evidence for alteration of MAPK activation in response to ELF MF. Acknowledgement: This project has received funding from the European Union's Seventh Programme for research, technological development, and demonstration under grant agreement No. 282891.

Modeling of the MAPK machinery activation in response to various abiotic and biotic stresses in plants by a system biology approach.

Mitogen-Activated Protein Kinases (MAPKs) cascade plays an important role in regulating plant growth and development, generating cellular responses to the extracellular stimuli. MAPKs cascade mainly consist of three sub-families i.e. mitogen-activated protein kinase kinase kinase (MAPKKK), mitogen-activated protein kinase kinase (MAPKK) and mitogen activated protein kinase (MAPK), several cascades of which are activated by various abiotic and biotic stresses. In this work we have modeled the holistic molecular mechanisms essential to MAPKs activation in response to several abiotic and biotic stresses through a system biology approach and performed its simulation studies. As extent of abiotic and biotic stresses goes on increasing, the process of cell division, cell growth and cell differentiation slow down in time dependent manner. The models developed depict the combinatorial and multicomponent signaling triggered in response to several abiotic and biotic factors. These models can be used to predict behavior of cells in event of various stresses depending on their time and exposure through activation of complex signaling cascades.

The MAPK pathway functions as a redundant survival signal that reinforces the PI3K cascade in c-Kit mutant melanoma

Stimulation of the c-Kit receptor tyrosine kinase has a critical role in the development and migration of melanocytes, and oncogenic c-Kit mutants contribute to the progression of some melanomas. c-Kit signalling activates the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways and their relative contribution to the activities of oncogenic and ligand-dependent c-Kit remains uncertain. We show that PI3K is a major regulator of MAPK activation in response to c-Kit activity and the dominant effector of c-Kit-driven melanocyte proliferation and melanoma survival. Nevertheless, inhibition of the PI3K pathway in c-Kit mutant melanoma cells did not replicate the apoptotic efficacy of the c-Kit inhibitor, imatinib mesylate. Instead, the simultaneous suppression of the PI3K and MAPK pathways promoted a strong synergistic apoptotic effect. These data indicate that MAPK functions as a redundant survival signal that reinforces the PI3K cascade in c-Kit mutant melanoma. Thus, the concurrent inhibition of PI3K and MAPK signalling is required to suppress oncogenic c-Kit activity and may provide an effective therapeutic strategy in c-Kit mutant melanomas.

Therapeutic Levels of the Hydroxmethylglutaryl-Coenzyme A Reductase Inhibitor Lovastatin Activate Ras Signaling via Phospholipase D2

Hydroxmethylglutaryl (HMG)-coenzyme A (CoA) reductase inhibitors (statins) lower serum cholesterol but exhibit pleiotropic biological effects that are difficult to ascribe solely to cholesterol depletion. Here, we investigated the effect of lovastatin on protein prenylation and cell signaling. We show that high concentrations (50 μM) of lovastatin inhibit Ras, Rho, and Rap prenylation but that therapeutic levels of lovastatin (50 nM to 500 nM) do not. In contrast, depletion of cellular cholesterol by therapeutic levels of lovastatin increased Ras GTP loading and mitogen-activated protein kinase (MAPK) activation in human umbilical vein endothelial cells and rodent fibroblasts. Elevated Ras signaling was not seen in statin-treated cells if cholesterol levels were maintained by supplementation. Activation of Ras-MAPK signaling was a consequence of, and dependent on, activation of phospholipase D2 (PLD2). Expression of dominant interfering PLD2 or biochemical inhibition of PLD2 abrogated Ras and MAPK activation induced by lovastatin. In contrast, ectopic expression of wild-type PLD2 enhanced Ras and MAPK activation in response to therapeutic levels of lovastatin. Statin-induced cholesterol depletion also modestly activated the epidermal growth factor receptor (EGFR), resulting in downregulation of EGFR expression. These results suggest that statins modulate key cell signaling pathways as a direct consequence of cholesterol depletion and identify the EGFR-PLD2-Ras-MAPK axis as an important statin target.

Dendritic cells from humans with hypomorphic mutations in IKBKG/NEMO have impaired mitogen-activated protein kinase activity

The covalent attachment of lysine 63-linked polyubiquitin to the zinc-finger domain of IKBKG/NEMO (also known as IKKγ) is necessary for full activation of NF-κB. Impairments of this biochemical mechanism explain the deleterious effects of hypomorphic NEMO mutations on NF-κB signaling function in humans suffering from X-linked ectodermal dysplasia and immunodeficiency. Nevertheless, the biological function of the NEMO zinc-finger domain in the regulation of mitogen-activated protein kinase (MAPK) activity is poorly understood. Here we show that dendritic cells from patients with EDI caused by a C-terminal E391X deletion of the zinc finger of NEMO exhibit impaired MAPK activation in response to lipopolysaccharide (LPS) stimulation. Interestingly, DCs from patients with a C417R missense mutation within the zinc finger domain of NEMO in which ubiquitination of NEMO is preserved are also defective in JNK and ERK activity following LPS stimulation. Our findings indicate that the structural integrity of the NEMO ZF domain is more important than its polyubiquitination for full activation of the MAPK. Furthermore, phosphorylation and polyubiquitination of upstream TAK1 were significantly reduced in the E391X zinc-finger deleted patients, indicating that the NEMO zinc finger may play an important role in assembling the proximal signaling complex for MAPK activation.

Dexamethasone Arterializes Venous Endothelial Cells by Inducing Mitogen-Activated Protein Kinase Phosphatase-1

Background— Vein grafting in coronary artery surgery is complicated by a high restenosis rate resulting from the development of vascular inflammation, intimal hyperplasia, and accelerated atherosclerosis. In contrast, arterial grafts are relatively resistant to these processes. Vascular inflammation is regulated by signaling intermediaries, including p38 mitogen-activated protein (MAP) kinase, that trigger endothelial cell (EC) expression of chemokines (eg, interleukin-8, monocyte chemotactic protein-1) and other proinflammatory molecules. Here, we have tested the hypothesis that p38 MAP kinase activation in response to arterial shear stress (flow) may occur more readily in venous ECs, leading to greater proinflammatory activation. Methods and Results— Comparative reverse-transcriptase polymerase chain reaction and Western blotting revealed that arterial shear stress induced p38-dependent expression of monocyte chemotactic protein-1 and interleukin-8 in porcine jugular vein ECs. In contrast, porcine aortic ECs were protected from shear stress–induced expression of p38-dependent chemokines as a result of rapid induction of MAP kinase phosphatase-1. However, we observed with both cultured porcine jugular vein ECs and perfused veins that venous ECs can be protected by brief treatment with dexamethasone, which induced MAP kinase phosphatase-1 to suppress proinflammatory activation. Conclusions— Arterial but not venous ECs are protected from proinflammatory activation in response to short-term exposure to high shear stress by the induction of MAP kinase phosphatase-1. Dexamethasone pretreatment arterializes venous ECs by inducing MAP kinase phosphatase-1 and may protect veins from inflammation.

Protein Kinase PKR-Dependent Activation of Mitogen-Activated Protein Kinases Occurs through Mitochondrial Adapter IPS-1 and Is Antagonized by Vaccinia Virus E3L

The p38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinases (MAPKs) play important roles in the host innate immune response. The protein kinase regulated by RNA (PKR) is implicated in p38 MAPK activation in response to proinflammatory signals in mouse embryonic fibroblasts. To test the role of PKR in the activation of p38 and JNK MAPKs in human cells following viral infection, HeLa cells made stably deficient in PKR by using an RNA interference strategy were compared to cells with sufficient PKR. The phosphorylation of both p38 and JNK in cells with sufficient PKR was activated following either infection with an E3L deletion (DeltaE3L) mutant of vaccinia virus or transfection with double-stranded RNA (dsRNA) in the absence of infection with wild-type vaccinia virus. The depletion of PKR by stable knockdown impaired the phosphorylation of both p38 and JNK induced by either the DeltaE3L mutant virus or dsRNA but not that induced by tumor necrosis factor alpha. The PKR-dependent activation of MAPKs in DeltaE3L mutant-infected cells was abolished by treatment with cytosine beta-d-arabinoside. The complementation of PKR-deficient cells with the human PKR wild-type protein, but not with the PKR catalytic mutant (K296R) protein, restored p38 and JNK phosphorylation following DeltaE3L mutant virus infection. Transient small interfering RNA knockdown established that the p38 and JNK kinase activation following DeltaE3L infection was dependent upon RIG-I-like receptor signal transduction pathway components, including the mitochondrial adapter IPS-1 protein.

Abscisic acid and hydrogen peroxide induce a novel maize group C MAP kinase gene, ZmMPK7, which is responsible for the removal of reactive oxygen species

Mitogen-activated protein kinase (MAPK) cascades are involved in biotic and abiotic stress responses. In plants, MAPKs are classified into four groups, designated A-D. Information about group C MAPKs is limited, and, in particular, no data from maize are available. In this article, we isolated a novel group C MAPK gene, ZmMPK7, from Zea mays. Exogenous abscisic acid (ABA) and hydrogen peroxide (H(2)O(2)) induced calcium-dependant transcription of ZmMPK7. Induction of this gene in response to ABA was blocked by several reactive oxygen species (ROS) manipulators such as imidazole, Tiron, and dimethylthiourea (DMTU). This result indicates that endogenous H(2)O(2) may be required for ZmMPK7-mediated ABA signaling. Expression of ZmMPK7 in Nicotonia tobaccum caused less H(2)O(2) to accumulate and alleviated ROS-mediated injuries following submission of the plants to osmotic stress. The enhanced total peroxidase (POD) activity in transgenic tobacco plants may contribute to removal of ROS. Finally, we have shown that the ZmMPK7 protein localizes in the nucleus. These results broaden our knowledge regarding plant group C MAPK activity in response to stress signals.

Lrig1 Is an Endogenous Inhibitor of Ret Receptor Tyrosine Kinase Activation, Downstream Signaling, and Biological Responses to GDNF

Glial cell line-derived neurotrophic factor (GDNF)/Ret signaling has potent trophic effects on ventral midbrain dopaminergic, motor, sensory, and sympathetic neurons. The molecular mechanisms that restrict Ret receptor tyrosine kinase activation are not well understood. Here, we show that Lrig1, a transmembrane protein containing leucine-rich repeats and Ig-like domains in its extracellular region, acts in a negative feedback loop to regulate the activity of Ret receptor tyrosine kinase. In particular, we demonstrate that Lrig1 is capable of physically interacting with Ret and that Lrig1/Ret association inhibits GDNF binding, recruitment of Ret to lipid rafts, receptor autophosphorylation, and mitogen-activated protein kinase (MAPK) activation in response to GDNF. In neuronal cells, Lrig1 overexpression also inhibits GDNF/Ret-induced neurite outgrowth in a cell-autonomous manner. Downregulation of Lrig1 using small interference RNA knock-down experiments potentiates both neuronal differentiation and MAPK activation in response to GDNF. Together, these results provide an insight into Lrig1 function and establish a new physiological mechanism to restrict signaling and biological responses induced by GDNF and Ret in neuronal cells.

Integrated activation of MAP3Ks balances cell fate in response to stress

In vivo, tissues and organs are exposed to numerous stressors that require cells to respond appropriately for viability and homeostasis. Cells respond to these stressors, which range from UV irradiation, heat shock, chemicals, and changes in osmolality, to oxidative stress and inflammatory cytokines, by activating pathways that protect cells from damage. If the stress is too great, cells commit to undergo apoptosis. Such cell fate decisions involve the stress-mediated activation of mitogen-activated protein kinase (MAPK) networks, ultimately under the control of MAPK kinase kinases, or MAP3Ks. It is the MAP3Ks that coordinate the localization, duration and magnitude of MAPK activation in response to cell stress. A single stressor may activate several MAP3Ks, each of which impacts the balance between survival and apoptotic signaling. In this prospect article, we review the specific MAP3Ks that integrate the physiological response to cell stressors. The interrelationships among different stressors are discussed, with an emphasis on how the balance of signaling through MAP3Ks controls the MAPK response to determine cell fate.

Expression and function of mixed lineage kinases in dendritic cells

Dendritic cells (DCs) sense the presence of conserved microbial structures in their local microenvironment via specific pattern recognition receptors (PRRs). This leads to a programme of changes, which include migration and activation, and enables them to induce adaptive T cell immunity. Mitogen-activated protein kinases (MAPKs) are implicated in this response, but the pathways leading from PRR ligation to MAPK activation, and hence DC activation, are not fully understood. Recent studies in the nervous system have suggested that the mixed lineage kinase (MLK) family of MAPK kinase kinase proteins may be involved as an intermediary step between PRRs and MAPKs. Therefore, in this study, we have used a well-established DC model to explore the role of MLKs in these cells. Messenger RNA for MLKs 2, 3, 4 and DLK and protein for MLKs 2, 3 and DLK are found in DC. DC activation in response to model PRR ligands, such as LPS or poly (I:C), is accompanied by phosphorylation of MLK3. In contrast, another known PRR ligand, zymosan, induces little MLK3 phosphorylation. Inhibition of MLK activity using a pharmacological inhibitor, CEP11004, blocks p38 and Jun N-terminal kinase (JNK) MAPK activation in response to LPS and poly (I:C), but not zymosan. The inhibition is associated with a block in DC activation as measured by cell-surface marker expression and cytokine secretion. Thus, MLKs are expressed in DC, and are implicated in DC activation, and the involvement of MLKs appears to be selective, depending on the nature of the DC stimulus.

Role of mitochondria in angiotensin II-induced reactive oxygen species and mitogen-activated protein kinase activation

Peptide hormone Angiotensin II (Ang II) activates NAD(P)H oxidase, via AT1 receptors leading to increased generation of reactive oxygen species (ROS), such as the superoxide anion (O(2)(-)). As an important intracellular second messenger, ROS can activate many downstream signaling molecules, including mitogen-activated protein kinases (MAPK), protein tyrosine phosphatases, protein tyrosine kinases, and transcriptional factors. Activation of these signaling cascades is highly related to risk for cardiovascular diseases. Accumulating evidence reveals that membrane-bound NAD(P)H oxidase is the main source responsible for Ang II-induced ROS generation. However, recent novel findings suggest that Ang II stimulation induces opening of mitochondrial K(ATP) channels, depolarizes mitochondrial potential (DeltaPsi(M)), and further amplifies ROS generation from mitochondria, resulting in redox-sensitive activation of MAPK. In this review, we discuss the possible mechanisms of Ang II-induced cardiac pharmacological preconditioning (PC), and focus on the role of mitochondrial K(ATP) channels, mitochondrial ROS production, and MAPK activation in response to Ang II stimulation.

Cytokine activation of p38 mitogen-activated protein kinase and apoptosis is opposed by alpha-4 targeting of protein phosphatase 2A for site-specific dephosphorylation of MEK3.

alpha-4 is an essential gene and is a dominant antiapoptotic factor in various tissues that is a regulatory subunit for type 2A protein phosphatases. A multiplexed phosphorylation site screen revealed that knockdown of alpha-4 by small interfering RNA (siRNA) increased p38 mitogen-activated protein kinase (MAPK) and c-Jun phosphorylation without changes in JNK or ERK. FLAG-alpha-4 coprecipitated hemagglutinin-MEK3 plus endogenous protein phosphatase 2A (PP2A) and selectively enhanced dephosphorylation of Thr193, but not Ser189, in the activation loop of MEK3. Overexpression of alpha-4 suppressed p38 MAPK activation in response to tumor necrosis factor alpha (TNF-alpha). The alpha-4 dominant-negative domain (DND) (residues 220 to 340) associated with MEK3, but not PP2A, and its overexpression sensitized cells to activation of p38 MAPK by TNF-alpha and interleukin-1beta, but not by ansiomycin or sorbitol. The response was diminished by nocodazole or by siRNA knockdown of the Opitz syndrome protein Mid1 that binds alpha-4 to microtubules. Interference by alpha-4 DND or alpha-4 siRNA increased caspase 3/7 activation in response to TNF-alpha. Growth of transformed cells in soft agar was enhanced by alpha-4 and suppressed by alpha-4 DND. The results show that alpha-4 targets PP2A activity to MEK3 to suppress p38 MAPK activation by cytokines, thereby inhibiting apoptosis and anoikis.

Stretch-induced MAP kinase activation in cardiac myocytes: Differential regulation through β 1-integrin and focal adhesion kinase

Mitogen-activated protein (MAP) kinases have been implicated in hemodynamic load induced heart failure. Both angiotensin II (Ang II) and mechanical stretch activate MAP kinases in cardiac myocytes. In this study, we used a neonatal rat ventricular myocyte (NRVM) model to determine the role of focal-adhesion kinase (FAK) in β 1 integrin mediated MAP kinase activation in response to mechanical stretch in presence and absence of Ang II receptor blockade (ATB). NRVM plated on deformable membranes coated with collagen IV were exposed to 20% equiaxial static-stretch. β 1 integrin signaling was blocked by adenovirus-mediated expression of a dominant-negative form of β 1D integrin (tac-β 1D). FAK signaling was disrupted by infecting NRVM with adenovirus expressing FAK-related non-kinase (FRNK). Western blot analysis was used to assess the phosphorylation of MAP kinases. In the presence and absence of ATB, mechanical stretch caused maximal phosphorylation of ERK, p38 and JNK at 5 min, which was significantly attenuated in NRVM expressing tac-β 1D. In the presence of ATB, FRNK overexpression significantly increased basal phosphorylation of ERK (40.2 ± 8.6% P < 0.05), p38 (39.5 ± 11.7%, P < 0.05), JNK (86 ± 29.4%, P < 0.05) and stretch-induced p38 (48.1 ± 8.7%, P < 0.05) and JNK (85.0 ± 19.4%, P < 0.05) phosphorylation. However, in the absence of ATB, FRNK overexpression significantly reduced basal and stretch-induced phosphorylation of only ERK. Examination of FAK activation revealed that β 1 integrin was required for stretch-induced phosphorylation of FAK at Y 397 and Y 925, but not Y 861. In summary, mechanical stretch-activated ERK1/2, p38 and JNK through FAK independent and dependent mechanisms. β 1 integrin was required for FAK independent activation of all three MAP kinases, whereas cross-talk between β 1 integrin and Ang II receptors mediated FAK dependent regulation of ERK1/2.

Mouse preimplantation embryo responses to culture medium osmolarity include increased expression of CCM2 and p38 MAPK activation

BackgroundMechanisms that confer an ability to respond positively to environmental osmolarity are fundamental to ensuring embryo survival during the preimplantation period. Activation of p38 mitogen-activated protein kinase (MAPK) occurs following exposure to hyperosmotic treatment. Recently, a novel scaffolding protein called Osmosensing Scaffold for MEKK3 (OSM) was linked to p38 MAPK activation in response to sorbitol-induced hypertonicity. The human ortholog of OSM is cerebral cavernous malformation 2 (CCM2). The present study was conducted to investigate whether CCM2 is expressed during mouse preimplantation development and to determine whether this scaffolding protein is associated with p38 MAPK activation following exposure of preimplantation embryos to hyperosmotic environments.ResultsOur results indicate that Ccm2 along with upstream p38 MAPK pathway constituents (Map3k3, Map2k3, Map2k6, and Map2k4) are expressed throughout mouse preimplantation development. CCM2, MAP3K3 and the phosphorylated forms of MAP2K3/MAP2K6 and MAP2K4 were also detected throughout preimplantation development. Embryo culture in hyperosmotic media increased p38 MAPK activity in conjunction with elevated CCM2 levels.ConclusionThese results define the expression of upstream activators of p38 MAPK during preimplantation development and indicate that embryo responses to hyperosmotic environments include elevation of CCM2 and activation of p38 MAPK.

Diacylglycerol Kinase in T Cell Anergy

Defective Ras signaling contributes to T cell anergy, which occurs when T cells are presented with antigens in the absence of costimulatory signals and leads to T cells’ unresponsiveness to subsequent stimulation. T cell anergy is a key mechanism by which inappropriate immune responses are prevented. Previous work had shown that diacylglycerol (DAG)-mediated signaling was defective in anergic T cells and that abundance of the transcript encoding diacylglycerol kinase α (DGK-α) was decreased in activated T cells and increased in anergic T cells. Two groups (Zha et al . and Olenchock et al .) extend these observations and show that DGK-α, an enzyme that converts DAG to phosphatic acid, contributes to T cell anergy. Zha et al . showed that introduction of constitutively active Ras, one of the components downstream of DAG, restored T cell responsiveness [interleukin-2 (IL-2) production and mitogen-activated protein kinase (MAPK) phosphorylation] to cells previously rendered anergic. Expression of the Dgka gene was increased in anergic T cells in the system used by Zha et al ., and overexpression of DGK-α in resting T cells diminished recruitment of the Ras guanine nucleotide exchange factor RasGRP1 to the plasma membrane, decreased IL-2 production, and decreased MAPK activation in response to stimulation. Olenchock et al . used cells from Dgka −/− mice to show that T cells from these animals failed to undergo anergy in response to T cell receptor activation in the absence of costimulatory signals. Together, these two reports demonstrate a key role in DAG metabolism in promoting T cell anergy (see Mueller). What remains to be determined is how DGK activity is regulated to prevent excessive activity in cells that receive an activating signal and to promote activity in cells that receive a signal that produces anergy. Y. Zha, R. Marks, A. W. Ho, A. C. Peterson, S. Janardhan, I. Brown, K. Praveen, S. Stang, J. C. Stone, T. F. Gajewski, T cell anergy is reversed by active Ras and is regulated by diacylglycerol kinase-α. Nat. Immunol. 7 , 1166-1173 (2006). [PubMed] B. A. Olenchock, R. Guo, J. H. Carpenter, M. Jordan, M. K. Topham, G. A. Koretzky, X.-P. Zhong, Disruption of diacylglycerol metabolism impairs the induction of T cell anergy. Nat. Immunol. 7 , 1174-1181 (2006). [PubMed] D. L. Mueller, Linking diacylglycerol kinase to T cell anergy. Nat. Immunol. 7 , 1132-1134 (2006). [PubMed]

Concentration‐dependent stimulation by tissue inhibitor of metalloproteinases(TIMP)‐2 of two signaling pathways in human osteosarcoma (MG‐63) Cells

AbstractWe reported previously that tyrosine kinase (TYK) and mitogen‐activated protein kinase (MAPK) play a role in TIMP‐dependent growth signaling. Here we demonstrate that the activation of MAPK stimulated by TIMP‐2 was sharply inhibited in MG‐63 cells by PP1, a selective inhibitor of Src‐family tyrosine kinases, suggesting that Src activation is possibly required for the MAPK activity in response to 1 ng/mL TIMP‐2. A specific cell‐permeable inhibitor of MAPK kinase (MEK), PD 98058 had an inhibitory effect on MAPK activity, indicating that MEK is an upstream effector of MAPK. By transfecting MG‐63 cells with dominant‐negative Ras, RasN17, and furthermore, by using a selective farnesyltransferase inhibitor, FTI‐277, we found that MAPK activation stimulated by TIMP‐2 was independent of Ras. We also demonstrated that the activation of MAPK in response to 1 ng/mL TIMP‐2 was inhibited by a cAMP agonist, 8‐bromo‐cAMP. On the contrary, [3H]thymidine incorporation and the activation of MAPK, all of which were heavily suppressed by increasing the TIMP‐2 concentration up to 100 ng/mL, were recovered by the addition of a cell‐permeable specific PKA inhibitor, H‐89. These results strongly suggest the presence of a negative crosstalk from the cAMP/PKA pathway to the TYK/MAPK one.