A Non-Redundant Role for Drosophila Mkk4 and Hemipterous/Mkk7 in TAK1-Mediated Activation of JNK

Background

The JNK pathway is a mitogen-activated protein (MAP) kinase pathway involved in the regulation of numerous physiological processes during development and in response to environmental stress. JNK activity is controlled by two MAPK kinases (MAPKK), Mkk4 and Mkk7. Mkk7 plays a prominent role upon Tumor Necrosis Factor (TNF) stimulation. Eiger, the unique TNF-superfamily ligand in Drosophila, potently activates JNK signaling through the activation of the MAPKKK Tak1.

Methodology/Principal Findings

In a dominant suppressor screen for new components of the Eiger/JNK-pathway in Drosophila, we have identified an allelic series of the Mkk4 gene. Our genetic and biochemical results demonstrate that Mkk4 is dispensable for normal development and host resistance to systemic bacterial infection but plays a non-redundant role as a MAPKK acting in parallel to Hemipterous/Mkk7 in dTAK1-mediated JNK activation upon Eiger and Imd pathway activation.

Conclusions/Significance

In contrast to mammals, it seems that in Drosophila both MAPKKs, Hep/Mkk7 and Mkk4, are required to induce JNK upon TNF or pro-inflammatory stimulation.     

Stretch-Induced Stress Fiber Remodeling and the Activations of JNK and ERK Depend on Mechanical Strain Rate,but Not FAK

Background

Cells within tissues are subjected to mechanical forces caused by extracellular matrix deformation. Cells sense and dynamically respond to stretching of the matrix by reorienting their actin stress fibers and by activating intracellular signaling proteins, including focal adhesion kinase (FAK) and the mitogen-activated proteins kinases (MAPKs). Theoretical analyses predict that stress fibers can relax perturbations in tension depending on the rate of matrix strain. Thus, we hypothesized stress fiber organization and MAPK activities are altered to an extent dependent on stretch frequency.

Principal Findings

Bovine aortic endothelial cells and human osteosarcoma cells expressing GFP-actin were cultured on elastic membranes and subjected to various patterns of stretch. Cyclic stretching resulted in strain rate-dependent increases in stress fiber alignment, cell retraction, and the phosphorylation of the MAPKs JNK, ERK and p38. Transient step changes in strain rate caused proportional transient changes in the levels of JNK and ERK phosphorylations without affecting stress fiber organization. Disrupting stress fiber contractile function with cytochalasin D or Y27632 decreased the levels of JNK and ERK phosphorylation. Previous studies indicate that FAK is required for stretch-induced cell alignment and MAPK activations. However, cyclic uniaxial stretching induced stress fiber alignment and the phosphorylation of JNK, ERK and p38 to comparable levels in FAK-null and FAK-expressing mouse embryonic fibroblasts.

Conclusions

These results indicate that cyclic stretch-induced stress fiber alignment, cell retraction, and MAPK activations occur as a consequence of perturbations in fiber strain. These findings thus shed new light into the roles of stress fiber relaxation and reorganization in maintenance of tensional homeostasis in a dynamic mechanical environment.     

Insulin resistance in non-obese subjects is associated with activation of the JNK pathway and impaired insulin signaling in skeletal muscle

Background

The pathogenesis of insulin resistance in the absence of obesity is unknown. In obesity, multiple stress kinases have been identified that impair the insulin signaling pathway via serine phosphorylation of key second messenger proteins. These stress kinases are activated through various mechanisms related to lipid oversupply locally in insulin target tissues and in various adipose depots.

Methodology/Principal Findings

To explore whether specific stress kinases that have been implicated in the insulin resistance of obesity are potentially contributing to insulin resistance in non-obese individuals, twenty healthy, non-obese, normoglycemic subjects identified as insulin sensitive or resistant were studied. Vastus lateralis muscle biopsies obtained during euglycemic, hyperinsulinemic clamp were evaluated for insulin signaling and for activation of stress kinase pathways. Total and regional adipose stores and intramyocellular lipids (IMCL) were assessed by DXA, MRI and ¹H-MRS. In muscle of resistant subjects, phosphorylation of JNK was increased (1.36±0.23 vs. 0.78±0.10 OD units, P<0.05), while there was no evidence for activation of p38 MAPK or IKKβ. IRS-1 serine phosphorylation was increased (1.30±0.09 vs. 0.22±0.03 OD units, P<0.005) while insulin-stimulated tyrosine phosphorylation decreased (10.97±0.95 vs. 0.89±0.50 OD units, P<0.005). IMCL levels were twice as high in insulin resistant subjects (3.26±0.48 vs. 1.58±0.35% H₂O peak, P<0.05), who also displayed increased total fat and abdominal fat when compared to insulin sensitive controls.

Conclusions

This is the first report demonstrating that insulin resistance in non-obese, normoglycemic subjects is associated with activation of the JNK pathway related to increased IMCL and higher total body and abdominal adipose stores. While JNK activation is consistent with a primary impact of muscle lipid accumulation on metabolic stress, further work is necessary to determine the relative contributions of the various mediators of impaired insulin signaling in this population.     

Crosstalk between JNK and SUMO signaling pathways: deSUMOylation is protective against H2O2-induced cell injury

Background

Oxidative stress is a key feature in the pathogenesis of several neurological disorders. Following oxidative stress stimuli a wide range of pathways are activated and contribute to cellular death. The mechanism that couples c-Jun N-terminal kinase (JNK) signaling, a key pathway in stress conditions, to the small ubiquitin-related modifier (SUMO), an emerging protein in the field, is largely unknown.

Methodology/Principal Findings

With this study we investigated if SUMOylation participates in the regulation of JNK activation as well as cellular death in a model of H₂O₂ induced-oxidative stress. Our data show that H₂O₂ modulates JNK activation and induces cellular death in neuroblastoma SH-SY5Y cells. Inhibition of JNK''s action with the D-JNKI1 peptide rescued cells from death. Following H₂O₂, SUMO-1 over-expression increased phosphorylation of JNK and exacerbated cell death, although only in conditions of mild oxidative stress. Furthermore inhibition of SUMOylation, following transfection with SENP1, interfered with JNK activation and rescued cells from H₂O₂ induced death. Importantly, in our model, direct interaction between these proteins can occur.

Conclusions/Significance

Taken together our results show that SUMOylation may significantly contribute to modulation of JNK activation and contribute to cell death in oxidative stress conditions.     

Protective effect of a sesamin derivative, 3-bis (3-methoxybenzyl) butane-1, 4-diol on ischemic and hypoxic neuronal injury

Background

Stroke is one of the leading causes of neuronal death. Sesamin is known for neuroprotection by its antioxidant and anti-inflammatory properties but it lacks blood–brain barrier (BBB) activity. A panel of sesamin derivatives was screened and 3-bis (3-methoxybenzyl) butane-1,4-diol (BBD) was selected for high BBB activity and tested for its neuroprotective effect.

Methods

The focal cerebral ischemia of Sprague–Dawley rats and hypoxia models of murine BV-2 microglia or PC12 cells under oxygen/glucose deprivation were used for in vivo and in vitro test, respectively. Lipid peroxidation and superoxide dismutase (SOD) activity from the ischemic brain were tested and reactive oxygen species (ROS), cytokine production, prostaglandin (PGE₂) and related signaling pathways from hypoxic cells were examined by ELISA or Western blot assay, respectively.

Results

BBD showed a protective effect when given 90 min after the focal cerebral ischemia. It also reduced lipid peroxidation and preserved SOD activity from the ischemic brain. The mechanism of BBD was further confirmed by attenuating ROS, cytokine production, and PGE₂ release from hypoxic BV-2 or PC12 cells. BBD significantly reduced hypoxia-induced c-Jun N-terminal kinases (JNK) and modulated AKT-1 and caspase-3 (survival and apoptotic pathways) in BV-2 cells, and inhibited hypoxia-induced JNK and cyclooxygenase-2 activation in PC12 cells.

Conclusions

The neuroprotective effect of BBD on ischemia/hypoxia models was involved with antioxidant and anti-inflammatory effects. The result would help the development of new CNS drug for protection of ischemia/hypoxia injury.     

MST Kinases Monitor Actin Cytoskeletal Integrity and Signal via c-Jun N-Terminal Kinase Stress-Activated Kinase To Regulate p21Waf1/Cip1 Stability

As well as providing a structural framework, the actin cytoskeleton plays integral roles in cell death, survival, and proliferation. The disruption of the actin cytoskeleton results in the activation of the c-Jun N-terminal kinase (JNK) stress-activated protein kinase (SAPK) pathway; however, the sensor of actin integrity that couples to the JNK pathway has not been characterized in mammalian cells. We now report that the mammalian Ste20-like (MST) kinases mediate the activation of the JNK pathway in response to the disruption of the actin cytoskeleton. One consequence of actin disruption is the JNK-mediated stabilization of p21^Waf1/Cip1 (p21) via the phosphorylation of Thr57. The expression of MST1 or MST2 was sufficient to stabilize p21 in a JNK- and Thr57-dependent manner, while the stabilization of p21 by actin disruption required MST activity. These data indicate that, in addition to being components of the Salvador-Warts-Hippo tumor suppressor network and binding partners of c-Raf and the RASSF1A tumor suppressor, MST kinases serve to monitor cytoskeletal integrity and couple via the JNK SAPK pathway to the regulation of a key cell cycle regulatory protein.The actin cytoskeleton is a dynamic structure that determines cell morphology and motility. In addition, the cytoskeleton also influences other biological functions, such as proliferation, survival, and death, although the mechanistic details linking the cytoskeleton to these processes have not been fully elucidated. Considerable effort has focused on characterizing the signal transduction pathways that control cytoskeletal organization (33). The actin cytoskeleton itself also may regulate cell signaling; for example, mechanical stretching, shear stress, and cytoskeletal disruption each have been shown to activate stress-activated protein kinase (SAPK) pathways (34). Although in Saccharomyces cerevisiae an actin integrity-responsive pathway has been identified in which actin cytoskeleton disassembly results in the activation of the Ssk2p kinase that lies upstream of the Hog1 SAPK pathway (7, 56), an analogous pathway in mammalian cells has not been delineated.SAPK pathways are specific examples of mitogen-activated protein kinase (MAPK) cascades (43). At the bottom of archetypal MAPK pathways are signal-propagating kinases such as ERK1 and ERK2; in the case of SAPK signaling, the similarly positioned kinases are JNK and p38 family members. MAPK are phosphorylated and regulated by MAPK kinases (MAP2K); for c-Jun N-terminal kinase (JNK), the MAP2K are MKK4 and MKK7, while for p38 they are MKK3 and MKK6. Moving stepwise further upstream are MAP3K and MAP4K, although in some pathways there may be no need for a MAP4K, the Ras activation of the MAP3K Raf in the ERK MAPK pathway being one example.Although much recent interest has focused on their antiproliferative and proapoptotic functions as a component of the Salvador-Warts-Hippo tumor suppressor network (31) and as binding partners of the c-Raf MAP3K (42) and RASSF1A tumor suppressor (39), the mammalian Ste20-like kinases 1 and 2 (MST1 and MST2, respectively) were first identified (17) because of their homology with the Saccharomyces cerevisiae Ste20 MAP4K that acts upstream of three MAPK cascades, including the Ste11/Pbs2/Hog1 SAPK pathway (51). Although the MST kinase domains are very similar to those in Ste20 and mammalian p21-activated kinases (PAK), there is little homology outside this domain, and as a result MST1 and MST2 make up their own Ste20 subfamily without direct orthologues prior to the emergence of the bilaterian subregnum. Given the homology with Ste20, initial characterization focused on the possibility that MST kinases were involved in MAPK regulation, and indeed MST kinases were found to activate SAPK pathways (27), which was associated with the activation of MKK6 and MKK7 (27). It also was found that MST1 coexpression with a kinase-dead version of the MAP3K MEKK1 blocked JNK activation (26). Consistently with these results, MST1 could not activate JNK in cells deleted for both MAP2K enzymes MKK4 and MKK7 (53). Therefore, it appears that MST kinases work at the same level (MAP4K) as Ste20 in the regulation of the SAPK pathways. Although proapoptotic signaling has been shown to contribute to MST activation via caspase-mediated proteolysis, which removes an autoinhibitory domain (27), little is known about how other nonapoptotic stimuli regulate MST.There are several possible consequences resulting from the activation of SAPK pathways in response to modifications to actin cytoskeleton organization or integrity. Actin disruption and consequent JNK activation may induce cell cycle arrest (23) or apoptosis (11), or it may promote cell survival (2). We previously showed that one way JNK activation following cytoskeletal disruption might contribute to cell cycle arrest is through the stabilization of the cyclin-dependent kinase inhibitor (CDKI) p21^Waf1/Cip1 (p21) (14). The eventual outcome of SAPK activation following actin cytoskeleton modification may be influenced by signal intensity, duration, and cellular context. Further progress toward determining how cytoskeletal disruption generates these outcomes will be possible when the details describing how actin cytoskeletal changes activate SAPK signaling have been established.We wished to determine whether MST kinases sense the integrity of the actin cytoskeleton and link with SAPK signaling. We found that MST2 was colocalized with filamentous actin structures. The expression of MST1 or MST2 was sufficient to activate JNK1, and cytoskeletal disruption activated MST as well as JNK1 in an MST-dependent manner. One consequence of actin disruption is the JNK-mediated stabilization of p21, which was determined to be via phosphorylation of Thr57. The expression of MST1 or MST2 was sufficient to stabilize p21 in a JNK- and Thr57-dependent manner, while the stabilization of p21 by actin disruption required MST activity. These data indicate that MST kinases serve to monitor cytoskeletal integrity and couple via the JNK SAPK pathway to the regulation of a key cell cycle regulatory protein.     

The pheromone-induced nuclear accumulation of the Fus3 MAPK in yeast depends on its phosphorylation state and on Dig1 and Dig2

Background  

Like mammalian MAP kinases, the mating-specific Fus3 MAPK of yeast accumulates in the nuclei of stimulated cells. Because Fus3 does not appear to be subjected to active nucleo-cytoplasmic transport, it is not clear how its activation by mating pheromone effects the observed change in its localization. One possibility is that the activation of Fus3 changes its affinity for nuclear and cytoplasmic tethers.     

Syntheses and biological evaluation of 1-heteroaryl-2-aryl-1H-benzimidazole derivatives as c-Jun N-terminal kinase inhibitors with neuroprotective effects

1-Heteroaryl-2-aryl-1H-benzimidazole derivatives were synthesized as inhibitors of c-Jun N-terminal kinases, JNK3. Their activities were evaluated through measurement of K_d using SPR, JNK3 kinase assay, and cell-viability of human neuroblastoma cells. Most tested compounds showed high affinity (10 μM–46 nM) to JNK3. Among them, compound 16f exhibited potent activities (K_d = 46 nM). Especially, 16f was also found to present a potent cell protective effect (IC₅₀ = 1.09 μM) against toxicity induced by anisomycin, showing a possibility as protective therapeutics in neuronal cell apoptosis.     

Exploration of 1-(3-chloro-4-(4-oxo-4H-chromen-2-yl)phenyl)-3-phenylurea derivatives as selective dual inhibitors of Raf1 and JNK1 kinases for anti-tumor treatment

Based on the roles of Raf1 and JNK1 in hepatocarcinoma development, scaffold-based drug design was employed to produce a series of compounds, which subsequently were synthesized and explored as potential dual inhibitors Raf1 and JNK1 kinases for anti-tumor treatment. The compound 1-(3-chloro-4-(6-ethyl-4-oxo-4H-chromen-2-yl)phenyl)-3-(4-chloro-phenyl)urea (3d) showed 66%, 67% and 13% inhibition rate at 50 μM against Raf1, JNK1 and p38-alpha, respectively, but no effect on ERK1 and ERK2, and inhibited the expression of pERK1/2 markedly and HepG2 cells proliferation with IC₅₀ at 8.3 μM. Furthermore, 3d showed lower toxicity against normal liver cell-lines QSG7701 and HL7702. Molecular docking study further showed that 3d can fit into binding domain of JNK1 and Raf1. Our data suggested the activities of 3d were associated with dual inhibition of JNK1 and Raf1 kinases.     

Expression profile of genes regulated by activity of the Na-H exchanger NHE1

Background  

In mammalian cells changes in intracellular pH (pH_i), which are predominantly controlled by activity of plasma membrane ion exchangers, regulate a diverse range of normal and pathological cellular processes. How changes in pH_i affect distinct cellular processes has primarily been determined by evaluating protein activities and we know little about how pH_i regulates gene expression.     

Purinergic (ATP) signaling stimulates JNK1 but not JNK2 MAPK in osteoblast-like cells: contribution of intracellular Ca2+ release, stress activated and L-voltage-dependent calcium influx, PKC and Src kinases

This work shows that ATP activates JNK1, but not JNK2, in rat osteoblasts and ROS-A 17/2.8 osteoblast-like cells. In ROS-A 17/2.8 cells ATP induced JNK1 phosphorylation in a dose- and time-dependent manner. JNK1 phosphorylation also increased after osteoblast stimulation with ATPγS and UTP, but not with ADPβS. RT-PCR studies supported the expression of P2Y₂ receptor subtype. ATP-induced JNK1 activation was reduced by PI-PLC, IP₃ receptor, PKC and Src inhibitors and by gadolinium, nifedipine and verapamil or a Ca²⁺-free medium. ERK 1/2 or p38 MAPK inhibitors diminished JNK1 activation by ATP, suggesting a cross-talk between these pathways. ATP stimulated osteoblast-like cell proliferation consistent with the participation of P2Y₂ receptors. These results show that P2Y₂ receptor stimulation by ATP induces JNK1 phosphorylation in ROS-A 17/2.8 cells in a way dependent on PI-PLC/IP₃/intracellular Ca²⁺ release and Ca²⁺ influx through stress activated and L-type voltage-dependent calcium channels and involves PKC and Src kinases.     

PI3 kinase is important for Ras,MEK and Erk activation of Epo-stimulated human erythroid progenitors

Background  

Erythropoietin is a multifunctional cytokine which regulates the number of erythrocytes circulating in mammalian blood. This is crucial in order to maintain an appropriate oxygen supply throughout the body. Stimulation of primary human erythroid progenitors (PEPs) with erythropoietin (Epo) leads to the activation of the mitogenic kinases (MEKs and Erks). How this is accomplished mechanistically remained unclear.     

Hit-to-lead optimization and kinase selectivity of imidazo[1,2-a]quinoxalin-4-amine derived JNK1 inhibitors

As the result of a rhJNK1 HTS, the imidazo[1,2-a]quinoxaline 1 was identified as a 1.6 μM rhJNK1 inhibitor. Optimization of this compound lead to AX13587 (rhJNK1 IC₅₀ = 160 nM) which was co-crystallized with JNK1 to identify key molecular interactions. Kinase profiling against 125+ kinases revealed AX13587 was an inhibitor of JNK, MAST3, and MAST4 whereas its methylene homolog AX14373 (native JNK1 IC₅₀ = 47 nM) was a highly specific JNK inhibitor.     

Glucocorticoid evoked upregulation of RCAN1-1 in human leukemic CEM cells susceptible to apoptosis

Background

Estradiol (E₂) mediates various intracellular signaling cascades from the plasma membrane via several estrogen receptors (ERs). The pituitary is an estrogen-responsive tissue, and we have previously reported that E₂ can activate mitogen-activated protein kinases (MAPKs) such as ERK1/2 and JNK1/2/3 in the membrane ERα (mERα)-enriched GH₃/B₆/F₁₀ rat pituitary tumor cell line. Phytoestrogens are compounds found in plants and foods such as soybeans, alfalfa sprouts, and red grapes. They are structurally similar to E₂ and share a similar mechanism of action through their binding to ERs. Phytoestrogens bind to nuclear ERs with a much lower affinity and therefore are less potent in mediating genomic responses. However, little is known about their ability to act via mERs to mediate nongenomic effects.

Methods

To investigate the activation of different nongenomic pathways, and determine the involvement of mERα, we measured prolactin (PRL) release by radio-immunoassay, MAPK activations (ERK1/2 and JNK1/2/3) via a quantitative plate immunoassay, and intracellular [Ca²⁺] by Fura-2 fluorescence imaging in cells treated with E₂ or four different phytoestrogens (coumestrol, daidzein, genistein, and trans-resveratrol).

Results

Coumesterol and daidzein increased PRL release similar to E₂ in GH₃/B₆/F₁₀ cells, while genistein and trans-resveratrol had no effect. All of these compounds except genistein activated ERK1/2 signaling at 1–10 picomolar concentrations; JNK 1/2/3 was activated by all compounds at a 100 nanomolar concentration. All compounds also caused rapid Ca²⁺ uptake, though in unique dose-dependent Ca²⁺ response patterns for several aspects of this response. A subclone of GH₃ cells expressing low levels of mERα (GH₃/B₆/D₉) did not respond to any phytoestrogen treatments for any of these responses, suggesting that these nongenomic effects were mediated via mERα.

Conclusion

Phytoestrogens were much more potent in mediating these nongenomic responses (activation of MAPKs, PRL release, and increased intracellular [Ca²⁺]) via mERα than was previously reported for genomic responses. The unique non-monotonic dose responses and variant signaling patterns caused by E₂ and all tested phytoestrogens suggest that complex and multiple signaling pathways or binding partners could be involved. By activating these different nongenomic signaling pathways, phytoestrogens could have significant physiological consequences for pituitary cell functions.     

Development of indole/indazole-aminopyrimidines as inhibitors of c-Jun N-terminal kinase (JNK): Optimization for JNK potency and physicochemical properties

A novel series of indole/indazole-aminopyrimidines was designed and synthesized with an aim to achieve optimal potency and selectivity for the c-Jun kinase family or JNKs. Structure guided design was used to optimize the series resulting in a significant potency improvement. The best compound (17) has IC₅₀ of 3 nM for JNK1 and 20 nM for JNK2, with greater than 40-fold selectivity against other kinases with good physicochemical and pharmacokinetic properties.     

Osteopontin enhances the expression and activity of MMP-2 via the SDF-1/CXCR4 axis in hepatocellular carcinoma cell lines

Background and Aims

Osteopontin, SDF-1α, and MMP-2 are important secreted molecules involved in the pathophysiology of human hepatocellular carcinoma (HCC). This study investigates the effect of the SDF-1α/CXCR4 axis on expression and activity of MMP-2 induced by osteopontin.

Methods

The expression of CXCR4, SDF-1α, MMP-2 and their associated cellular signaling cascades, involving Akt and MAP Kinases, were determined by Western blotting. The activities of MMP-2 and MMP-9 were assayed by gel zymography. The role of the osteopontin receptors integrin α_vβ₃ and CD44v6 was evaluated using neutralizing antibodies. We also established CXCR4-deficient SMMC7721 cell lines by transfection with miRNA-CXCR4 plasmids and determined cell invasion activity in a transwell assay.

Results

In comparison with untreated cells, recombinant human osteopontin (rhOPN) up-regulated CXCR4, SDF-1α, and MMP-2 expression about 5-, 4-, and 6-fold on the protein levels through binding to integrin α_vβ₃ and CD44v6 in hepatocellular carcinoma cells (SMMC7721 and HepG2). Inhibition of the SDF-1α/CXCR4 axis down-regulated the rhOPN-induced MMP-2 expression and activity. rhOPN also activated Akt, p38 and JNK. Down-regulation of CXCR4 decreased the rhOPN-induced invasion in SMMC7721 cells.

Conclusion

These results indicate that rhOPN up-regulates MMP-2 through the SDF-1α/CXCR4 axis, mediated by binding to integrin α_vβ₃ and CD44v6 and activating the PI-3K/Akt and JNK pathways in HepG2 and SMMC7721 cells. Therefore, the osteopontin-SDF-1α/CXCR4-MMP-2 system may be a new therapeutic target for treating HCC progression.     

Differential Activation of Mitogen-Activated Protein Kinases,ERK 1/2, p38MAPK and JNK p54/p46 During Postnatal Development of Rat Hippocampus

Mitogen-activated protein kinases (MAPKs) are a group of serine-threonine kinases, including p38^MAPK, ERK 1/2 and JNK p54/p46, activated by phosphorylation in response to extracellular stimuli. The early postnatal period is characterized by significant changes in brain structure as well as intracellular signaling. In the hippocampus MAPKs have been involved in the modulation of development and neural plasticity. However, the temporal profile of MAPK activation throughout the early postnatal development is incomplete. An understanding of this profile is important since slight changes in the activity of these enzymes, in response to environmental stress in specific developmental windows, might alter the course of development. The present study was undertaken to investigate the hippocampal differential activation of MAPK during postnatal period. MAPK activation and total content were evaluated by Western blotting of hippocampal tissue obtained from male Wistar rats at postnatal days (P) 1, 4, 7, 10, 14, 21, 30 and 60. The total content and phosphorylation of each MAPK was expressed as mean ± SEM and then calculates as a percentile compared to P1 (set at 100 %). The results showed: (1) phosphorylation peaks of p38^MAPK at PN4 (p = 0.036) and PN10 to PN60; (2) phosphorylation of ERK1 and ERK2 were increased with age (ERK1 p = 0.0000005 and ERK2 p = 0.003); (3) phosphorylation profile of JNK p54/p46 was not changed during the period analyzed (JNKp56 p = 0.716 and JNKp46 p = 0.192). Therefore, the activity profile of ERK 1/2 and p38^MAPK during postnatal development of rat hippocampus are differentially regulated. Our results demonstrate that ERK 1/2 and p38^MAPK are dynamically regulated during postnatal neurodevelopment, suggesting temporal correlation of MAPK activity with critical periods when programmed cell death and synaptogenesis are occurring. This suggests an important role for these MAPKs in postnatal development of rat hippocampus.

     

Human cord blood stem cell paracrine factors activate the survival protein kinase Akt and inhibit death protein kinases JNK and p38 in injured cardiomyocytes

Background aimsWe hypothesized that paracrine factors from human umbilical cord blood mononuclear cells (hUCBC) activate in injured cardiomyocytes the survival protein kinase Akt and limit activation of death protein kinases JNK and p38.MethodsWe treated hUCBC with H₂O₂ and measured growth factors and cytokines secreted by hUCBC. We then treated cardiomyocytes with H₂O₂ for 24 h and measured Akt, JNK and p38 activation by means of Western blots. We also measured myocyte viability and apoptosis with the use of fluorescence-activated cell-sorting cytometry. We then investigated myocytes treated for 24 h with H₂O₂ plus hUCBC and myocytes without hUCBC or H₂O₂. Four million hUCBC were placed in transwells permeable only to hUCBC paracrine factors, and the transwells were placed in flasks with H₂O₂+Dulbecco's modified Eagle's medium or in flasks with myocytes plus H₂O₂+Dulbecco's modified Eagle's medium.ResultshUCBC increased secretion during H₂O₂ of hepatocyte growth factor by 338%, insulin-like growth factor by 200%, interleukin-4 by 200%, vascular endothelial cell growth factor by 192%, placental growth factor by 150%, interleukin-10 by 150% and angiogenin by 121%. H₂O₂ increased myocyte JNK activation by 237% and p38 activation by 60%, decreased myocyte viability by 38% and increased necrosis by 34% (all P < 0.01). hUCBC paracrine factors increased in myocytes with H₂O₂ Akt activation by ≥25%, decreased JNK and p38 activation by >35%, increased viability by >22% and decreased apoptosis by >33% (all P < 0.05). Akt inhibitor API-1 prevented the effects of hUCBC and enhanced H₂O₂ decrease of myocyte viability. Addition of JNK inhibitor SP600125 or p38 inhibitor SB203580 to myocytes plus H₂O₂ prevented H₂O₂ decrease in viability and increased hUCBC beneficial effects.ConclusionsDuring free radical stress, hUCBC paracrine factors activate myocyte Akt, which increases myocyte viability by decreasing activation of death-promoting protein kinases JNK and p38.     

DNA Replication Arrest in Response to Genotoxic Stress Provokes Early Activation of Stress-Activated Protein Kinases (SAPK/JNK)

The impact of DNA damage-induced replication blockage for early activation of stress kinases [stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK)] is largely unknown. Here, we show that induction of dual phosphorylation of SAPK/JNK by the DNA polymerase inhibitor aphidicolin was not ameliorated by additional exposure to ultraviolet (UV) light, indicating that overlapping mechanisms participate in signaling to SAPK/JNK triggered by both agents. UV-induced DNA replication blockage, cyclobutane pyrimidine dimer formation and DNA strand break induction coincided with SAPK/JNK phosphorylation at early (≤ 30 min) but not late (≥ 2 h) time points after exposure. Genotoxin-stimulated SAPK/JNK activation was attenuated in nonproliferating cells, indicating that S phase-dependent mechanisms are involved in signaling to SAPK/JNK. Correspondingly, UV-induced phosphorylation of SAPK/JNK was higher in S-phase cells as compared with G₁-phase cells. Activation of SAPK/JNK by genotoxins was below detection limit in nonproliferating human peripheral blood lymphocytes, whereas peripheral blood lymphocytes stimulated to proliferation displayed clear SAPK/JNK activation. UV-induced phosphorylation of SAPK/JNK was attenuated in XPC-defective cells, ameliorated in BRCA2 mutated cells and not changed in cells lacking ATM, DNA-PK, CSB, XPA, p53, ERCC1 or PARP as compared with the corresponding wild types. Based on these data, we suggest that DNA replication blockage caused by genotoxin-induced DNA damage contributes to early activation of SAPK/JNK.