Mitogen-activated Protein Kinase Members Research Articles

Effects of the brominated flame retardant tetrabromobisphenol-A (TBBPA) on cell signaling and function of Mytilus hemocytes: Involvement of MAP kinases and protein kinase C

Brominated flame retardants (BFRs) are a large group of compounds added to or applied as a treatment to polymeric materials to prevent fires. Tetrabisphenol A (TBBPA) is the most important individual BFR used in industry. Although TBBPA and its derivatives can be found in environmental samples, data are very limited on the presence of this compound in biota. Research on mammals indicates that TBBPA has low toxicity in vivo; however, in vitro TBBPA can act as a cytotoxicant, neurotoxicant, immunotoxicant, thyroid hormone agonist and has a weak estrogenic activity; in particular, the effects of TBBPA have been recently ascribed to its interactions with cellular signaling pathways, in particular with mitogen activated protein kinases (MAPKs). TBBPA has high acute toxicity to aquatic organisms, such as algae, molluscs, crustaceans and fish; however, little is known on the mechanisms of action of this compound in the cells of aquatic species. In this work, we investigated the possible effects and mechanisms of action of TBBPA on the immune cells, the hemocytes, of the marine mussel Mytilus galloprovincialis. The results demonstrate that TBBPA in the low micromolar range induces hemocyte lysosomal membrane destabilization. The effect was reduced or prevented by hemocyte pre-treatment by specific inhibitors of MAPKs and of protein kinase C (PKC). TBBPA stimulated phosphorylation of MAPK members and PKC, as evaluated by electrophoresis and Western blotting with anti-phospho-antibodies, although to a different extent and with distinct time-courses. A rapid (from 5 min) and transient increase in phosphoryation of the stress-activated JNK MAPKs and of PKC was observed, followed by a later increase (at 30–60 min) in phosphorylation of extracellularly regulated MAPKs (ERK2 MAPK) and of the stress-activated p38 MAPK. TBBPA significantly stimulated the hemocyte microbicidal activity towards E. coli, lysosomal enzyme release, phagocytic activity and extracellular superoxide (O 2 −) production. The results demonstrate that TBBPA in vitro activates the immune function of mussel hemocytes through kinase-mediated cell signaling and that common transduction pathways are involved in mediating the effects of this BFR in mammalian and aquatic invertebrate cells.

Co-activation of ERK, NF-κB, and GADD45β in Response to Ionizing Radiation

NF-kappaB has been well documented to play a critical role in signaling cell stress reactions. The extracellular signal-regulated kinase (ERK) regulates cell proliferation and survival. GADD45beta is a primary cell cycle element responsive to NF-kappaB activation in anti-apoptotic responses. The present study provides evidence demonstrating that NK-kappaB, ERK and GADD45beta are co-activated by ionizing radiation (IR) in a pattern of mutually dependence to increase cell survival. Stress conditions generated in human breast cancer MCF-7 cells by the administration of a single exposure of 5 Gy IR resulted in the activation of ERK but not p38 or JNK, along with an enhancement of the NF-kappaB transactivation and GADD45beta expression. Overexpression of dominant negative Erk (DN-Erk) or pre-exposure to ERK inhibitor PD98059 inhibited NF-kappaB. Transfection of dominant negative mutant IkappaB that blocks NF-kappaB nuclear translocation, inhibited ERK activity and GADD45beta expression and increased cell radiosensitivity. Interaction of p65 and ERK was visualized in living MCF-7 cells by bimolecular fluorescence complementation analysis. Antisense inhibition of GADD45beta strikingly blocked IR-induced NF-kappaB and ERK but not p38 and JNK. Overall, these results demonstrate a possibility that NF-kappaB, ERK, and GADD45beta are able to coordinate in a loop-like signaling network to defend cells against the cytotoxicity induced by ionizing radiation.

STAT3 and ras-MAPK signal transduction pathway in non-small cell lung cancer

Biochemical and genetic researches suggest that ras protein plays an important role in transduction process of cell proliferation differentiation signals from activated transmembrane receptors to substream protein kinases. This study is to explore the expression of ras, p38, both of which are members of MAPK (mitogen activated protein kinases) signal transduction pathway, and STAT3 (signal transducer and activator of transcription 3) in non-small cell lung cancer, and the association among them. Forty-two resected lung cancer and paracancerous lung tissue samples were used to determine the protein expression of ras, p38 and STAT3 with Western blot, the mRNA expression of p38 and STAT3 in lung cancer tissues of various ras protein expression with RT-PCR. The location of p38 and STAT3 in lung cancer tissues was revealed with immunofluorescent staining. The relative protein expressions of ras, p38 and STAT3 were 0.6012, 0.6724, 0.5119 in cancer tissues, and 0.2793, 0.3071, 0.1917 in paracancerous lung tissues, respectively (P < 0.01). The protein and mRNA expressions of p38 and STAT3 ( 0.7624 and 0.6262; 1.0309 and 1.0538) in cancer tissues with higher ras protein expression were remarkably higher than those with lower ras protein expression (0.4715 and 0.2569; 0. 6569 and 0.3437, P < 0.01). There was a significant correlation between the expression of ras, p38 and STAT3 (correlation coefficient: 0.809 and 0.842, P < 0.01), and so was the p38 and STAT3 (correlation coefficient: 0.829, P < 0.01). Abnormal expressions of STAT3 and some factors of ras-MAPK signal transduction pathway exist in the oncogenesis and development of non-small cell lung cancer, and many of them may have crosstalk.

Reversible Oxidation of ERK-directed Protein Phosphatases Drives Oxidative Toxicity in Neurons

Oxidative stress links diverse neuropathological conditions that include stroke, Parkinson's disease, and Alzheimer's disease and has been modeled in vitro with various paradigms that lead to neuronal cell death following the increased accumulation of reactive oxygen species. For example, immortalized neurons and immature primary cortical neurons undergo cell death in response to depletion of the antioxidant glutathione, which can be elicited by administration of glutamate at high concentrations. We have demonstrated previously that this glutamate-induced oxidative toxicity requires activation of the mitogen-activated protein kinase member ERK1/2, but the mechanisms by which this activation takes place in oxidatively stressed neurons are still not fully known. In this study, we demonstrate that during oxidative stress, ERK-directed phosphatases of both the serine/threonine- and tyrosine-directed classes are selectively and reversibly inhibited via a mechanism that is dependent upon the oxidation of cysteine thiols. Furthermore, the impact of ERK-directed phosphatases on ERK1/2 activation and oxidative toxicity in neurons was tested in a neuronal cell line and in primary cortical cultures. Overexpression of the highly ERK-specific phosphatase MKP3 and its catalytic mutant, MKP3 C293S, were neuroprotective in transiently transfected HT22 cells and primary neurons. The neuroprotective effect of the MKP3 C293S mutant, which enhances ERK1/2 phosphorylation but blocks its nuclear translocation, demonstrates the necessity for active ERK1/2 nuclear localization for oxidative toxicity in neurons. Together, these data implicate the inhibition of endogenous ERK-directed phosphatases as a mechanism that leads to aberrant ERK1/2 activation and nuclear accumulation during oxidative toxicity in neurons.

Developmental regulation of activated ERK expression in the spinal cord and dorsal root ganglion of the chick embryo

Mitogen-activated protein kinases (MAPKs) are involved in the intracellular pathways that respond to various extracellular signals. Extracellular signal-regulated kinase (ERK) is a member of MAPKs and has various functions in neural development. However, the in vivo distribution of the activated form of ERK (p-ERK) in the developing nervous system is not well understood. Here, we investigated the expression of p-ERK in the spinal cord and dorsal root ganglion (DRG) of chick embryos. In the spinal cord, p-ERK-positive cells appeared in the ventral ventricular zone on embryonic day 4 (E4). From E6 onward, they appeared in the gray matter and in the white matter, suggesting migration from the ventricular zone. A double labeling method revealed that these p-ERK-positive cells included oligodendrocyte precursors. In the dorsal horn, p-ERK-positive small cells appeared on E6. Subsequently, the positive cells in the dorsal horn increased transiently in number and then decreased markedly by E10. Motoneurons also expressed p-ERK transiently on E7. In the DRG, weak p-ERK immunoreaction appeared in the ventrolateral region on E5. From E6, the immunoreactivity became stronger and by E9 intense p-ERK-positive cells were observed throughout the DRG. These data provide a neuroanatomical framework to begin to examine the in vivo role of ERK in neural development.

‘In vivo’ effects of Bisphenol A in Mytilus hemocytes: modulation of kinase-mediated signalling pathways

Endocrine disrupting chemicals (EDCs) include a variety of natural and synthetic estrogens, as well as estrogen-mimicking chemicals. We have previously shown that in the hemocytes of the mussel Mytilus galloprovincialis Lam. both natural and environmental estrogens in vitro can rapidly affect the phosphorylation state of components of tyrosine kinase-mediated cell signalling, in particular of mitogen activated protein kinases (MAPKs) and signal transducers and activators of transcription (STAT), that are involved in mediating the hemocyte immune response. These effects were consistent with the hypothesis that ‘alternative’ modes of estrogen action involving kinase-mediated pathways similar to those described in mammalian systems are also present in invertebrate cells. This possibility was investigated in vivo with Bisphenol A (BPA): mussels were injected with BPA and hemocytes sampled at 6, 12, and 24 h post-injection. The results show that BPA (25 nM nominal concentration in the hemolymph) lead to a significant lysosomal membrane destabilisation at all times post-injection, indicating BPA-induced stress conditions in the hemocytes, whereas lower concentrations were ineffective. BPA induced significant changes in the phosphorylation state of MAPK and STAT members, as evaluated by SDS-PAGE and WB of hemocyte protein extracts with specific antibodies, although to a different degree at different exposure times. In particular, BPA induced a dramatic decrease in phosphorylation of the stress-activated p38 MAPK, whose activation is crucial in mediating the bactericidal activity. Moreover, BPA decreased the phosphorylation of a CREB-like transcription factor (cAMP-responsive element binding protein). The results demonstrate that BPA can affect kinase-mediated cell signalling in mussel hemocytes also in vivo, and suggest that EDCs may affect gene expression in mussel cells through modulation of the activity of transcription factors secondary to cytosolic kinase cascades. Overall, these data address the importance of investigating full range responses to EDCs in ecologically relevant marine invertebrate species.

Low-Density Lipoprotein Inhibits Secretion of Phospholipid Transfer Protein in Human Trophoblastic BeWo Cells

Human plasma phospholipid transfer protein (PLTP) plays an important role in lipoprotein metabolism. In this study, we investigated the effects of lipoproteins on the secretion of PLTP in cultured BeWo choriocarcinoma cells. Low-density lipoproteins (LDLs) decreased PLTP secretion in a dose- and time-dependent manner, whereas very low density lipoproteins and high-density lipoproteins (HDLs) had little effect. LDL suppression of PLTP secretion was not altered by the inhibition of both LDL receptor and LDL receptor-related protein with receptor-associated protein. Mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor, U0126, could abolish the LDL-mediated inhibition of PLTP secretion. Furthermore, LDL, but not HDL, could stimulate the expression of MAPK phosphatase-1 (MKP-1) in BeWo cells that resulted in the inactivation of p44/p42 extracellular signal-regulated kinase (ERK) 1 and 2, the family members of MAPKs. These results support the conclusion that LDL-mediated suppression of PLTP secretion in BeWo cells is through a LDL receptor-independent MAPK signaling pathway.

Iron-induced interleukin-6 gene expression: possible mediation through the extracellular signal-regulated kinase and p38 mitogen-activated protein kinase pathways.

Increased iron store in the body may increase the risk of many diseases such as cancer and inflammation. However, the precise pathogenic mechanism of iron has not yet been elucidated. In the present study, the early biological responses of cells to iron treatment were investigated in AP-1 luciferase reporter stably transfected mouse epidermal JB6 cells and primary rat hepatocytes. It was shown that water-soluble iron compounds, such as FeSO4 and Fe2(SO4)3, were more active in inducing AP-1 in JB6 cells than water-insoluble iron compounds, such as Fe2O3 and FeS. Iron stimulated mitogen-activated protein kinase (MAPK) family members of extracellular signal-regulated kinases (ERKs) and p38 MAPK but not c-jun NH2 terminal kinases (JNKs), both in JB6 cells and in primary rat hepatocytes, as determined by the phosphorylation assay. Interestingly, the increase in AP-1 luciferase activity by iron was inhibited by the pretreatment of the cells with PD98059, a specific MEK1 inhibitor, and SB202190, a p38 kinase inhibitor. Levels of interleukin-6 (IL-6), a pro-inflammatory cytokine, were increased in JB6 cells by iron in a dose-dependent manner. The increase in IL-6 and its mRNA by iron was also eliminated by the pretreatment of the cells with PD98059 and SB202190. Since the IL-6 promoter contains an AP-1 binding site, our studies indicate that the iron-induced IL-6 gene expression may be mediated through ERKs and p38 MAPK pathways, possibly one of the important mechanisms for the pathogenesis of iron overload.

Environmental estrogens can affect the function of mussel hemocytes through rapid modulation of kinase pathways

Estrogens and estrogenic chemicals can affect several vertebrate non-reproductive functions, the immune response in particular. We have previously shown that in the hemocytes of the marine mollusc Mytilus the natural estrogen 17β-estradiol (E 2) can affect the immune function through rapid tyrosine kinase-mediated signalling pathways converging on phosphorylation of both mitogen activated protein kinases (MAPKs) and signal transducers and activators of transcription (STATs), whose activation plays a key role in the immune response. In this work the effects of synthetic estrogens (such as DES), estrogenic chemicals (such as Bisphenol A, Nonylphenol), and plant estrogens (genistein) on mussel hemocytes were evaluated. The results demonstrate that all the EDCs tested exert in vitro effects similar to those of E 2 on lysosomal membrane stability, although at concentrations 1000 times higher than those of the natural estrogen. When the effects of DES, BPA, and NP on tyrosine kinase-mediated cell signalling were investigated, estrogenic compounds showed distinct effects on the phosphorylation state of MAPK and STAT members. In particular, only DES, like E 2, induced p38 MAPK phosphorylation, whereas BPA and NP seem to have opposite effects. Moreover, different EDCs significantly decreased the tyrosine phosphorylation state of STAT3 and STAT5, showing a distinct effect with respect to E 2. Experiments with specific kinase inhibitors showed that activation of p38 MAPK, but also of ERK MAPK and PI3-kinase, plays a key role in mediating the effect of DES. On the other hand, the effects of NP were partly mediated by ERK MAPK activation. BPA-induced lysosomal membrane destabilisation was unaffected by either MAPK or PI3-K inhibitors. However, hemocyte pre-treatment with the PKC inhibitor GF109203X prevented the effects of both BPA and NP, this indicating that kinase pathways other than those involving MAPKs are also responsible for mediating the effects of certain EDCs. Overall, the results support the hypothesis that EDCs may rapidly modulate the function of mussel hemocytes through activation of transduction pathways involving different kinase-mediated cascades. Moreover, the effects of EDCs on the phosphorylation state of transcription factor STATs suggest that these compounds may lead to changes in gene expression secondary to modulation of kinase/phosphatases. Our data address to the importance of investigating full range responses to estrogenic chemicals and may help understanding their basic mechanisms of action in ecologically relevant invertebrate species.

Nitric oxide prodrugs and metallochemotherapeutics: JS-K and CB-3-100 enhance arsenic and cisplatin cytolethality by increasing cellular accumulation

Development of chemotherapeutic resistance is a major cause of pharmacologic failure in cancer treatment. One mechanism of resistance in tumor cells is the overexpression of glutathione S-transferases (GSTs) that serve two distinct roles in the development of drug resistance via the formation of glutathione conjugates with drugs for their cellular efflux, and the inhibition of the mitogen-activated protein kinase pathway. To target GST-based resistance to chemotherapeutics, a series of nitric oxide (NO)-releasing diazeniumdiolates was synthesized and shown to release NO on reaction with GST and/or glutathione. Two diazeniumdiolates, JS-K [O(2)-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate] and CB-3-100 [O(2)-(2,4-dinitrophenyl) 1-[4-(N,N-diethylcarboxamido)piperazin-1-yl]diazen-1-ium-1,2-diolate], were studied on their ability in reversing arsenic and cisplatin resistance in a rat liver cell line that is tumorigenic and shows acquired tolerance to arsenic and cisplatin, with overexpression of GSTs. The enhanced cytolethality produced by the NO donors was accompanied by increased accumulation of arsenic and platinum within cells and by enhanced activation of mitogen-activated protein kinase members c-jun-NH-kinase and extracellular signal-regulated kinase. Our data indicate that JS-K and CB-3-100 are promising lead compounds for the possible development of a novel class of adjuvant chemotherapeutic agents potentially capable of reversing arsenic and cisplatin resistance in certain tumor cells.

From signal to cell polarity: mitogen-activated protein kinases as sensors and effectors of cytoskeleton dynamicity.

Mitogen-activated protein kinases (MAPKs) are ubiquitous phosphorylation enzymes involved in signal transduction, gene expression and activation of diverse cytoskeletal proteins. MAPKs participate in the regulation of a broad range of crucial cellular processes including cell survival, division, polarization, stress responses, and metabolism. Phosphorylation of cytoskeletal proteins usually results in the rearrangement of cytoskeletal arrays leading to morphological changes and cell polarization. On the other hand, some cytoskeletal motor proteins, such as kinesins, could activate MAPK members and participate in signal delivery to the proper cellular destination (e.g. during cell division). Moreover, changes in the integrity of cytoskeletal elements have direct impacts on MAPK activity. Recent evidence suggests that there is bi-directional signalling between MAPK cascades and cytoskeleton. The focus here is on this cross-talk between MAPK signalling and the cytoskeleton in various eukaryotic systems including yeast, plants, and mammals and a role is proposed for MAPKs as sensors monitoring the cytoskeleton-dependent balance of forces within the cell.

Interaction Codes within the Family of Mammalian Phox and Bem1p Domain-containing Proteins

The Phox and Bem1p (PB1) domain constitutes a recently recognized protein-protein interaction domain found in the atypical protein kinase C (aPKC) isoenzymes, lambda/iota- and zeta PKC; members of mitogen-activated protein kinase (MAPK) modules like MEK5, MEKK2, and MEKK3; and in several scaffold proteins involved in cellular signaling. Among the last group, p62 and Par6 (partitioning-defective 6) are involved in coupling the aPKCs to signaling pathways involved in cell survival, growth control, and cell polarity. By mutation analyses and molecular modeling, we have identified critical residues at the interaction surfaces of the PB1 domains of aPKCs and p62. A basic charge cluster interacts with an acidic loop and helix both in p62 oligomerization and in the aPKC-p62 interaction. Subsequently, we determined the abilities of mammalian PB1 domain proteins to form heteromeric and homomeric complexes mediated by this domain. We report several novel interactions within this family. An interaction between the cell polarity scaffold protein Par6 and MEK5 was found. Furthermore, p62 interacts both with MEK5 and NBR1 in addition to the aPKCs. Evidence for involvement of p62 in MEK5-ERK5 signaling is presented.

Angiopoietin-1 activates both anti- and proapoptotic mitogen-activated protein kinases.

In this study, we identified whether mitogen-activated protein kinases (MAPKs) mediate the effects of angiopoietin-1 (Ang-1) on endothelial cell apoptosis. Exposure of human umbilical vein endothelial cells to Ang-1 (300 ng/ml) evoked within 15-30 min a 15-fold and a 5-fold increase in phosphorylation of ERK1/2 and p38 MAPKs, respectively. Inhibitors of the PI-3 kinase pathway attenuated Ang-1-induced ERK1/2 phosphorylation at a level up-stream from Raf and MEK1/2, but these inhibitors augmented Ang-1-induced p38 phosphorylation. When serum and growth supplements were withdrawn, the percentage of endothelial apoptosis tripled over 24 h compared with control cells. The presence of Ang-1 (300 ng/ml) significantly attenuated endothelial cell apoptosis and inhibited caspase-9, -7, and -3 activation. These antiapoptotic effects were augmented when a p38 inhibitor was combined with Ang-1, whereas inhibition of ERK1/2 eliminated the antiapoptotic properties of Ang-1. We conclude that both anti- (ERK1/2) and pro- (p38) apoptotic members of MAPKs are simultaneously activated by Ang-1 in endothelial cells and that activation of ERK1/2 by Ang-1 is mediated through the PI-3 kinase pathway. The strong antiapoptotic effects of the ERK and the PI-3 kinase pathways mask the proapoptotic function of p38 MAPKs resulting in net attenuation of apoptosis by Ang-1.

Arsenic trioxide-induced apoptosis is independent of stress-responsive signaling pathways but sensitive to inhibition of inducible nitric oxide synthase in HepG2 cells.

Arsenic trioxide (As(2)O(3)) has been found to be remarkably effective in the treatment of patients with acute promyelocytic leukemia (APL). Although evidences for the proapoptotic activity of As(2)O(3) have been suggested in leukemic and other solid cancer cells, the nature of intracellular mechanisms is far from clear. In the present study, we investigated As(2)O(3) affect on the stress-responsive signaling pathways and pretreatment with antioxidants using HepG2 cells. When treated with micromolar concentrations of As(2)O(3), HepG2 cells became highly apoptotic paralleled with activation of caspase-3 and members of mitogen-activated protein kinases (MAPKs) including extracellular signal-regulated kinase (ERK) and c-jun NH(2)-terminal kinase (JNK) but not p38 MAP kinase. However, inhibition of each kinase activity failed to inhibit apoptosis by As(2)O(3). Addition of n-acetyl cysteine (NAC) or diphenyleneiodonium (DPI) effectively protected cells from apoptosis and significantly lowered As(2)O(3)-induced activation of caspase-3. However, neither NAC nor DPI was able to effect ERK or JNK activation induced by As(2)O(3). Guanidinoethyldisulfide dihydrochloride (GED) and 2-ethyl-2-thiopseudourea (ETU), known inhibitors of the inducible nitric oxide synthase (iNOS), also suppressed the apoptotic activity of As(2)O(3). These results suggest that As2O3 induces caspase-mediated apoptosis involving a mechanism generating oxidative stress. However, activation of some stress-responsive signaling pathways by As(2)O(3) may not be the major determinant in the course of apoptotic processes.

Pathogenic T cells in murine lupus exhibit spontaneous signaling activity through phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathways.

To determine the activation status of two cytoplasmic signaling pathways, phosphatidylinositol 3-kinase (PI 3-kinase) and the mitogen-activated protein kinase (MAPK) family. We studied the pathogenic CD4+ T cells that drive disease in the parent-into-F(1) mouse model of lupus-like chronic graft-versus-host disease (GVHD). We determined immunoprecipitated kinase activity for PI 3-kinase and MAPK members (Raf-1, extracellular signal-regulated kinase 1 [ERK-1], c-Jun N-terminal kinase 1 [JNK-1], and p38 MAPK) from either unfractionated splenocytes or purified donor CD4+ T cells. Uninjected normal mice served as negative controls, and acute GVHD mice served as positive controls. Compared with negative controls, unfractionated splenocyte kinase activity from chronic GVHD mice was significantly increased for PI 3-kinase and JNK-1, but not for Raf-1, p38 MAPK, or ERK-1. Increased PI 3-kinase and JNK-1 activity was also seen in acute GVHD splenocytes, as was increased Raf-1 and p38 MAPK activity. The pattern of increased PI 3-kinase and JNK-1 activity seen in unfractionated chronic GVHD splenocytes was also seen in isolated donor, but not host, CD4+ T cells from chronic GVHD mice, indicating that donor CD4+ T cell signaling activity accounted for at least a portion of the activity observed in unfractionated splenocytes. Increased ERK-1 activity was not seen in either donor or host CD4+ T cells. This pattern of cytoplasmic signaling pathway in donor CD4+ T cells was associated with increased T cell receptor membrane signaling activation (Lck and Fyn phosphorylation) and increased transcription activation (phosphorylation of inhibitor of nuclear factor kappaB), confirming the biologic significance of these observations. The pathogenic T cells driving disease in this murine model exhibit activation in the form of spontaneous cytoplasmic signaling pathway activity that can be detected without in vitro restimulation and involves a T cell-specific (PI 3-kinase) and a nonspecific stress/cytokine pathway (JNK-1). These results raise the possibility that a full characterization of the signaling pathways active in pathogenic lupus T cells might lead to new therapeutic targets.

Involvement of mitogen-activated protein kinases in Group B Streptococcus-induced macrophage apoptosis

We previously demonstrated that Group B Streptococcus (GBS), a pathogen that causes serious neonatal infections, induces macrophage apoptosis by β-hemolysin to avoid the host immune response. GBS-induced macrophage apoptosis is characterized by a calcium increase and is caspase-independent. This study reports the involvement of c-Jun NH 2-terminal kinase (JNK), p38 and extracellular signal-regulated kinase (ERK), three members of mitogen-activated protein kinases (MAPKs) family, in GBS-induced macrophage apoptosis. Our data indicate that during induction of apoptosis live GBS stimulates a strong persistent activation of JNK and p38 with concomitant inhibition of ERK. The time courses of MAPKs activation strongly correlate with GBS-induced macrophage apoptosis and are macrophage:GBS ratio-dependent. In fact, when GBS does not cause macrophage apoptosis, e.g. low macrophage:GBS ratio or non hemolytic GBS (gGBS), it induces a transient activation of JNK, p38, and ERK MAPKs. These latter results indicate that sustained and persistent activation of JNK and p38 and inhibition of ERK are involved in the GBS-induced macrophage apoptotic process and suggest that the time course and balance of MAPKs activation are critical for different macrophage responses to GBS (apoptosis versus antimicrobicidal activity). This study indicates a correlation between MAPKs activation and GBS-induced macrophage apoptosis. However, since neither ERK nor p38 inhibitors had an effect on GBS-induced apoptosis, their role in the complex signal network leading to GBS-induced macrophage apoptosis remains to be defined.

High glucose activates the p38 MAPK pathway in cultured human peritoneal mesothelial cells

Peritoneal fibrosis is a serious complication in long-term continuous ambulatory peritoneal dialysis (CAPD) patients, but the underlying mechanism is not well understood. Since high glucose activates the p38 mitogen-activated protein kinase (MAPK) pathway in various kinds of cells, and because mesothelial cells are always exposed to high glucose dialysate, we examined the activity and expression of p38 MAPK members in cultured human peritoneal mesothelial cells (HPMCs) under high glucose conditions. HPMCs were isolated from omentum and subcultured. After serum restriction, HPMCs were exposed to 5.6 mmol/L glucose (low glucose), 5.6 mmol/L glucose + 34.5 mmol/L mannitol (low glucose + mannitol), or 40 mmol/L glucose (high glucose) for 3 minutes to 48 hours with or without SB203580. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were performed to determine mRNA and protein expression, respectively. p38 MAPK and cyclic adenosine monophosphate (cAMP)-responsive element binding protein (CREB) activities and mRNA expressions were significantly increased in HPMCs exposed to high glucose compared to low glucose or low glucose + mannitol after 10 minutes and remain at higher levels to 48 hours (P < 0.05), but total p38 MAPK and CREB protein expressions did not differ. MAPK kinase 3/6 (MKK3/6) activity and mRNA expression were also higher in high glucose cells after 3 minutes (P < 0.05), and fibronectin mRNA expression was significantly increased in HPMCs exposed to high glucose after 2 hours (P < 0.05). In contrast, high glucose significantly inhibited MAPK phosphatase-1 (MKP-1) protein and mRNA expression after 10 minutes (P < 0.05). SB203580 (1 micromol/L) pretreatment for 1 hour significantly reduced high glucose-induced CREB activity and fibronectin mRNA expression by 89% and 75%, respectively (P < 0.05). p38 MAPK activity was increased in HPMCs exposed to high glucose, in parallel with increased MKK3/6 activity and decreased MKP-1 expression, resulting in CREB activation. This activated p38 MAPK pathway may play a role in the pathogenesis of peritoneal fibrosis.

Role of bioavailable iron in coal dust-induced activation of activator protein-1 and nuclear factor of activated T cells: difference between Pennsylvania and Utah coal dusts.

Activator protein-1 (AP-1) and nuclear factor of activated T cells (NFAT) are two important transcription factors responsible for the regulation of cytokines, which are involved in cell proliferation and inflammation. Coal workers' pneumoconiosis (CWP) is an occupational lung disease that may be related to chronic inflammation caused by coal dust exposure. In the present study, we demonstrate that coal from the Pennsylvania (PA) coalmine region, which has a high prevalence of CWP, can activate both AP-1 and NFAT in JB6 mouse epidermal cells. In contrast, coal from the Utah (UT) coalmine region, which has a low prevalence of CWP, has no such effects. The PA coal stimulates mitogen-activated protein kinase (MAPK) family members of extracellular signal-regulated kinases (ERKs) and p38 MAPK but not c-Jun-NH(2)-terminal kinases, as determined by the phosphorylation assay. The increase in AP-1 by the PA coal was completely eliminated by the pretreatment of cells with PD98059, a specific MAPK kinase inhibitor, and SB202190, a p38 kinase inhibitor, further confirming that the PA coal-induced AP-1 activation is mediated through ERKs and p38 MAPK pathways. Deferoxamine (DFO), an iron chelator, synergistically enhanced the PA coal-induced AP-1 activity, but inhibited NFAT activity. For comparison, cells were treated with ferrous sulfate and/or DFO. We have found that iron transactivated both AP-1 and NFAT, and DFO further enhanced iron-induced AP-1 activation but inhibited NFAT. These results indicate that activation of AP-1 and NFAT by the PA coal is through bioavailable iron present in the coal. These data are in agreement with our previous findings that the prevalence of CWP correlates well with levels of bioavailable iron in coals from various mining regions.

MAPKs mediate S phase arrest induced by vanadate through a p53-dependent pathway in mouse epidermal C141 cells.

Mitogen-activated protein (MAP) kinases play an important role in mediation of the signal transduction pathway in cellular response to genotoxic stress. Cell growth arrest is considered as an early stage in response to the genotoxic stress. p53 is well-known as a tumor suppression gene involved in both cell growth arrest and apoptosis. The present study investigated the involvement of MAP kinases in vanadate-induced cell growth arrest and the relationship of p53. DNA content analysis showed that vanadate-induced S phase arrest is time- and dose-dependent in p53 wild-type C141 cells but not in p53-deficient C141 cells. Western blotting results indicated that vanadate caused an inactivation of p-cdk2 at Thr160, which is an important kinase for the progression of S phase, and an increase in expression of p21, which is a key for S phase arrest. In p53-deficient cells, vanadate did not induce any observable change in p21 or p-cdk2 level. In addition, vanadate up-regulated phospho-p38 and ERK, two members of MAP kinases. At the same time, vanadate increased the p53 activity as measured by luciferase assay. Addition of PD98059 and SB202190, inhibitors of ERK and p38, respectively, decreased vanadate-induced S phase arrest, reduced p21 levels, restored activation of p-cdk2, and decreased p53 activity. The study demonstrated that vanadate-induced S phase arrest is mediated by both ERK and p38 in a p53-dependent pathway.

Cardiac Expression and Subcellular Localization of the p38 Mitogen-activated Protein Kinase Member, Stress-activated Protein Kinase-3 (SAPK3)

Despite the interest in the roles that mitogen-activated protein kinases (MAPKs) play in the heart, the role of the different MAPK isoforms has been relatively poorly defined. A third isoform of p38 MAPK, known variously as stress-activated protein kinase-3 (SAPK3), p38- γ or ERK6, has been previously shown to differ from p38-α / β both in its molecular weight and its lack of inhibition by the compound SB203580. We have generated monoclonal antibodies with specificity for SAPK3 demonstrated by immunoblot analysis, immunofluorescence studies, and cloning of SAPK3 from a rat heart cDNA expression library. By immunoblotting, we confirmed high expression of SAPK3 in fast, slow and mixed fibre types of murine skeletal muscle and observed significant expression restricted to heart, lung, thymus and testes. In addition to expression in normal heart (human, mouse, rat, dog and pig), we observed constant expression in diseased human heart, as well as control and hypertrophic cultured neonatal rat cardiac myocytes. Immunolocalization in cultured cardiac myocytes followed by confocal microscopy showed punctate, non-nuclear SAPK3 staining. In contrast, p38- α/ β staining was non-punctate and distributed throughout the cytosol and nucleus. Whereas treatment with Leptomycin B to prevent nuclear export processes promoted higher levels of p38- α/ β staining in cardiac myocyte nuclei, there was no apparent change in SAPK3 localization under these conditions. These differences between p38- α/β and SAPK3 probably reflect the specialized functions of SAPK3 and emphasize the need to evaluate SAPK3 upstream activators and downstream targets in the heart.