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Abstract: Heavy metals are important environmental pollutants that affect many cellular functions. The signaling pathways that lead to apoptosis of adrenocortical cells following exposure to cadmium chloride (CdCl2) have not yet been fully clarified. We developed a primary culture of guinea pig adrenocortical cells and treated it with various concentrations of CdCl2 for different time periods. The apoptosis induced by CdCl2 was detected by fluorescein isothiocyanate-labeled annexin V and propidium iodide staining using a flow cytometer. Stress-activated protein kinase (SAPK) activities were measured by immunoprecipitation and chemiluminescence assay. After 2h of treatment, the apoptotic cell rate increased in a dose-dependent manner. The difference between the high-dose group and the control group was significant (P<0.01). The apoptosis was also found to increase in a time-dependent manner in cells treated with 50μmol/L CdCl2. Among the various treatment period groups, the difference between more than 1h-treated groups and the control group was significant (P<0.01). The SAPK activity increased with an increase in CdCl2 dose after 5min of exposure. However, after 15min of exposure to CdCl2, the SAPK activity decreased with an increase in exposure time. Our findings suggest that the SAPK signaling pathway might play an important role in CdCl2-induced apoptosis of adrenocortical cells. [Copyright &y& Elsevier]

Abstract: MAPK Pmk1p is the central element of a cascade involved in the maintenance of cell integrity and other functions in Schizosaccharomyces pombe. Pmk1p becomes activated by multiple stressing situations and also during cell separation. GTPase Rho2p acts upstream of the protein kinase C homolog Pck2p to activate the Pmk1 signalling pathway through direct interaction with MAPKKK Mkh1p. In this work we analyzed the functional significance of both Rho2p and Pck2p in the transduction of various stress signals by the cell integrity pathway. The results indicate that basal Pmk1p activity can be positively regulated by alternative mechanisms which are independent on the control by Rho2p and/or Pck2p. Unexpectedly, Pck1p, another protein kinase C homolog, negatively modulates Pmk1p basal activity by an unknown mechanism. Moreover, different elements appear to regulate the stress-induced activation of Pmk1p depending on the nature of the triggering stimuli. Whereas Pmk1p activation induced by hyper- or hypotonic stresses is channeled through Rho2p–Pck2p, other stressors, like glucose deprivation or cell wall disturbance, are transduced via other pathways in addition to that of Rho2p–Pck2p. On the contrary, Pmk1p activation observed during cell separation or after treatment with hydrogen peroxide does not involve Rho2p–Pck2p. Finally, Pck2p function is critical to maintain a Pmk1p basal activity that allows Pmk1p activation induced by heat stress. These data demonstrate the existence of a complex signalling network modulating Pmk1p activation in response to a variety of stresses in fission yeast. [Copyright &y& Elsevier]

Abstract: The cellular response to insult occurs by signaling via the stress-activated protein kinases, p38, and c-Jun NH2-terminal kinase (JNK). In the present study, we determined if hyperosmotic stress to rat hippocampal slices activates p38 and JNK and whether hyperosmolarity is a potential apoptotic stimulus in this experimental paradigm. Hyperosmotic stress, produced by addition of sorbitol to the incubation buffer, increased p38 phosphorylation; in contrast, JNK phosphorylation was not increased above control levels. Activation of p38 by phosphorylation was detected within 15 min of osmotic stress and was maintained through 360 min of hyperosmolarity. Concurrently, levels of cytochrome c were increased in the cytosolic fraction, indicating a decline in mitochondrial integrity. To a greater extent than the apoptotic stimulus, staurosporine, hyperosmotic stress activated caspase-3. Exposure to sorbitol also resulted in cleavage of the nuclear repair enzyme, poly(ADP-ribose) polymerase (PARP) and induced DNA fragmentation in slices. Concomitant treatment with sorbitol and SB202190, a selective p38 inhibitor, prevented the increase in cytosolic cytochrome c, decreased caspase-3 activation, and partially reduced PARP cleavage in a concentration-dependent manner. Inhibition of JNK with SP600125 did not affect caspase-3 activation in hippocampal slices. These results reveal hyperosmotic stress to be a potent activator of caspase-3 in hippocampus and suggest that downstream correlates of p38 signaling through a mitochondrial apoptotic pathway contribute significantly in the response to this insult. [Copyright &y& Elsevier]

Abstract: The environmental neurotoxin arsenic has recently been associated with altered neurofilament (NF) content in sciatic nerve. We examined herein the impact of sodium arsenite (the inorganic form of arsenic) on NF dynamics. Treatment of differentiated NB2/d1 cells and cultured dorsal root ganglion neurons decreased NF transport into axonal neurites and increased perikaryal phospho-NF immunoreactivity. Both of these effects were prevented by a pharmacological inhibitor (SP600125) of c-jun terminal kinase and by expression of a dominant-negative form of this kinase. Arsenic-induced inhibition of NF transport was prevented by treatment with lithium, a selective inhibitor of glycogen synthase kinase-3beta. Pharmacological inhibitors of cyclin-dependent kinase 5 and p38 mitogen-activated protein kinase did not attenuate the effects of arsenic on NF dynamics. These latter findings suggest that this environmental neurotoxin could contribute to peripheral neuropathy by perturbing NF dynamics. [Copyright &y& Elsevier]

Abstract: c-Jun N-terminal kinase (JNK), along with its upstream activator MEKK-1, is typically thought of as a stress-activated kinase that mediates apoptosis. However, additional studies indicate that the MEKK-1/JNK pathway mediates critical aspects of neuronal survival and differentiation. Herein, we demonstrate that transfection of differentiated NB2a/d1 cells with a construct expression constitutively activated (ca) MEKK-1 increases levels of phospho-dependent neurofilament (NF) immunoreactivity within perikarya, while expression of a dominant-negative (dn) form of MEKK-1 decreases it. Steady-state levels of perikaryal phospho-NF immunoreactivity are reduced and the increase resulting from expression of caMEKK-1 is prevented, by the JNK inhibitor SP600125, suggesting that JNK is a major downstream effector of MEKK-1 on NF phosphorylation. Unexpectedly, both caMEKK-1 and dnMEKK-1 inhibited neuritogenesis as well as translocation of NFs into newly elaborated neurites. The JNK inhibitor SP600125 also inhibited NF transport in a dose-dependent manner. caMEKK-1 also prevented the increase in NF transport otherwise mediated by MAP kinase. Finally, both caMEKK-1 and dnMEKK-1 prevented initial neuritogenesis. These findings indicate that the MEKK-1/JNK pathway regulates critical aspects of initial outgrowth, and subsequent stabilization of axonal neurites. [Copyright &y& Elsevier]

Previously, we have shown that ceramide is able to directly bind to and activate c-Raf and to trigger the downstream classical mitogen-activated protein kinase (MAPK/ERK) cascade in glomerular mesangial cells [Proc. Natl. Acad. Sci. USA 93 (1996) 6959]. In this study, we show that ceramide acts differently in glomerular endothelial cells in that treatment of endothelial cells with exogenous ceramide leads to a potent activation of the stress-activated protein kinase (SAPK/JNK) cascade but not to an activation of the classical ERK cascade. A similar effect was observed with the inflammatory cytokines TNFαand IL-1β,which activate a sphingomyelinase and thereby increase intracellular ceramide levels. The activation of JNKs as shown by c-Jun phosphorylation assays was paralleled by increased phosphorylation of the two JNK isoforms, p45 and p54. In addition, also the activator of JNKs, SEK1, was found to be increasingly phosphorylated by exogenous ceramide as well as by TNFα. In contrast, dihydroceramide had no effect on JNK or SEK1 phosphorylation. To see whether ceramide directly binds to MEKK1, which is the c-Raf analog in the SAPK cascade, a radioiodinated photoaffinity labeling analogue of ceramide, (N-[3-[[[2-(125I)iodo-4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzyl]oxy]-carbonyl] propanoyl]-d-erythro-sphingosine) ([125I]TID-ceramide) was used. Stimulation of endothelial cells with this [125I]TID-ceramide for 5 min followed by a short photolysis defined MEKK1 as a direct target of ceramide. With the same method, protein kinase C-α (PKC-α) was identified as a ceramide target. In contrast, no binding to c-Raf or the MEKK1 activator p65-PAK could be detected. A direct binding of ceramide to MEKK1 was also confirmed by affinity chromatography using a ceramide-coupled sepharose column. Furthermore, the ceramide-activated SAPK/JNK cascade is clearly involved in the mechanism of apoptosis, since in the presence of a JNK inhibitor, ceramide-induced DNA fragmentation is significantly reduced.In summary, we have shown that ceramide potently activates the SAPK cascade but not the ERK cascade in endothelial cells, which contrasts to mesangial cells where ceramide activates the ERK pathway and has only a minor effect on the SAPK cascade. Regarding the direct target of ceramide binding and action in endothelial cells, we identified MEKK1 as a further member of the growing family of ceramide-activated protein kinases. [Copyright &y& Elsevier]

Multiple lines of evidence demonstrate that oxidative stress is an early event in Alzheimer''s disease (AD), occurring prior to cytopathology, and therefore may play a key pathogenic role in the disease. Indeed, that oxidative mechanisms are involved in the cell loss and other neuropathology associated with AD is evidenced by the large number of metabolic signs of oxidative stress as well as by markers of oxidative damage. However, what is intriguing is that oxidative damage decreases with disease progression, such that levels of markers of rapidly formed oxidative damage, which are initially elevated, decrease as the disease progresses to advanced AD. This finding, along with the compensatory upregulation of antioxidant enzymes found in vulnerable neurons in AD, indicates that reactive oxygen species (ROS) not only cause damage to cellular structures but also provoke cellular responses. Mammalian cells respond to extracellular stimuli by transmitting intracellular instructions by signal transduction cascades to coordinate appropriate responses. Therefore, not surprisingly stress-activated protein kinase (SAPK) pathways, pathways that are activated by oxidative stress, are extensively activated during AD. In this paper, we review the evidence of oxidative stress and compensatory responses that occur in AD with a particular focus on the roles and mechanism of activation of SAPK pathways. [Copyright &y& Elsevier]

The adenosine A3 receptor generally couples to the Gi class of heterotrimeric G proteins, thereby decreasing cAMP levels and also mediating signaling via release of βγ subunits. Here we describe the central role of phosphatidylinositol-3′-kinase (PI3K) for adenosine A3 receptor-induced intracellular signaling to the stress-activated protein kinase p38 and the extracellular signal-regulated protein kinases ERK1/2. We used Chinese hamster ovary cells expressing the human adenosine A3 receptor, phospho-specific antibodies and different pharmacological tools to dissect the signaling pathways involving PI3K. The adenosine receptor agonist 5′N-ethylcarboxamidoadenosine induced a time- and dose-dependent increase in p38 and ERK1/2 phosphorylation, two signaling pathways that appeared also to be activated in the immortalized microglia cell line N13, which expressed endogenous adenosine A3 receptors. The 5′N-ethylcarboxamidoadenosine-induced effects on p38 and ERK1/2 in CHO cells were blocked by pertussis toxin pretreatment and were sensitive to pharmacological inhibition of PI3K. In addition, inhibition of Rac/Cdc42, small GTPases of the Rho family, by clostridium toxin B, diminished p38 phosphorylation but did not affect ERK1/2. Furthermore, we identified the serine 727 site of signal transducer and activator of transcription STAT3 as a probable downstream target of ERK1/2, and thereby provide evidence that adenosine A3 receptor mediated ERK1/2 activation has functional consequences. [Copyright &y& Elsevier]

: BackgroundCholecystokinin-A (CCK-A) and cholinergic receptor pathways, capable of activating stress kinases p38 mitogen-activated protein kinase (p38MAPK) and cJUN N-terminal kinase (JNK), are implicated in the pathogenesis of ligation-induced acute pancreatitis in rats. As ligation-induced acute pancreatitis in rats is associated with CCK-A receptor induction and p38MAPK activation, and as receptor induction could amplify acinar hyperstimulation and exacerbate cell stress, we tested the hypothesis that the cholinergic M3 receptor is induced and JNK is activated in this model.: MethodsCholinergic M3 receptor expression and JNK activation was compared in rats 1, 3, or 24 hours after sham operation or duct ligation.: ResultsImmunoblot analysis of pancreatic homogenates showed a time-dependent increase in cholinergic M3 receptor protein, total JNK, and phospho-JNK after duct ligation.: ConclusionsThere is a rapid and progressive cholinergic M3 receptor induction and JNK activation in ligation-induced acute pancreatitis in rats. These findings may have significance in the mechanism of disease pathogenesis. [Copyright &y& Elsevier]

Multiple kinase pathways determine serotonin transporter (SERT) regulation. We hypothesized a decrease in kinase expression with chronic selective serotonin reuptake inhibitor (SSRI) administration necessary to regulate extracellular serotonin. We studied whole brain kinase mRNA expression on Affymetrix gene chips in rats treated with placebo 3 and 21 days, fluoxetine 3 and 21 days, and citalopram 21 days. Protein kinase C (PKC)-delta, PKC-gamma, stress-activated protein kinase, cAMP-dependent protein kinase beta isoform, Janus protein kinase, and phosphofructokinase M were all down regulated chronically with citalopram and fluoxetine, but not with acute fluoxetine. The results are consistent with homeostasis of SERT function through a decrease in PK expression. [Copyright &y& Elsevier]

A stress-activated protein kinase pathway comprising mitogen-activated protein kinase kinases (MKKs), c-Jun N-terminal kinase (JNK) and the transcription factor c-Jun is implicated in neuronal apoptosis. Using an immune-complex kinase assay, we measured the activation of MKK4 and MKK7 in low potassium (LK)-induced apoptosis of rat cerebellar granule neurons (CGN). MKK7, but not MKK4, was activated within the first 4–6 h in four independent sets of 14-h CGN apoptosis time-courses. CEP-1347 (500 nM), an mixed-lineage kinase 3 inhibitor, prevented MKK7 activation and cell death following exposure of CGN cultures to LK-induced apoptosis. Western blot analysis revealed that levels of phosphorylated c-Jun were elevated between 30 min and 10 h of CGN apoptosis, temporally consistent with MKK7 activation. These data suggest that MKK7 is responsible for activating the JNK pathway during LK-induced CGN apoptosis. [Copyright &y& Elsevier]

Nitric oxide modulates stretch activation of mitogen-activated protein kinases in mesangial cells. Background. In vivo, intraglomerular hypertension results in resident cell hypertrophy, proliferation and matrix protein production, leading to glomerulosclerosis. Mesangial cells (MCs) exposed to in vitro stretch also proliferate and produce matrix. We have shown activation of Jun N-terminal kinase/stress-activated protein kinase (SAPK) and p42/44 mitogen-activated protein kinase (MAPK) in stretched MCs and have also demonstrated that l-arginine decreases resident cell proliferation and protects against glomerulosclerosis in remnant kidney glomeruli, presumably by increasing nitric oxide (NO) production. Consequently, we studied whether NO could affect SAPK and p42/44 MAPK activation in stretched MCs. Methods. MCs (passages 5 to 10) cultured on type 1 collagen-coated, flexible-bottom plates were exposed to 0 to 30 minutes of cyclic strain (60 cycles per minute) by computer-driven generation of vacuum of -27 kPa, inducing 28% elongation in the diameter of the surface. Control MCs were grown on coated, flexible-bottom plates. Protein levels (by Western blot) and activity assays for SAPK/JNK and p42/44 MAPK were performed under these conditions. As maximal activation was at 10 minutes, with decay by 30 minutes, the effect of NO on kinase activation was studied at 0, 2, 5, and 10 minutes by preincubation with 70 μmol/L s-nitroso-n-acetylpenicillamine (SNAP; an NO donor) or 1 mmol/L 8-bromo cyclic guanosine monophosphate (8-bromo-cGMP). Downstream events in response to stretch and NO were studied at the time of maximal response (10 minutes) by examining nuclear translocation of SAPK with immunofluorescence microscopy and transcription factor activator protein-1 nuclear protein binding by gel mobility shift assay. The effect of kinase inhibition by NO donors on MC proliferation was studied by Western blotting for proliferating cell nuclear antigen (PCNA). Results.... [ABSTRACT FROM AUTHOR]

The present study examined the stressor-like effects of repeated (4 days) administration of cocaine hydrochloride(COC) (35 mg/kg, i.p.) on the expression of heat shock proteins (HSPs) (HSP27, HSP60, HSP70, HSC70) and stress-activated protein kinases (SAPKs) (SAPKα, SAPKβ, SAPKγ) in the rat hippocampus. The interactions with intraperitoneal ethanol and drugs known as antidotes against COC toxicity were also examined. Similar to the effects of a 10 min immobilization stress (IM) over 4 days, an early increase (5 h time point) in nerve cells immunoreactive for HSPs (HSP27, HSP60, HSP70, HSC70) and SAPKs (SAPKβ, SAPKγ) was observed in the COC group. At the 24 h time point, a recovery was observed only for SAPKs, which have been suggested to control the HSP levels. Before the 48 h time point, alterations in the number of HSP+cells as compared to the control group (increase for HSP27 and HSP70+cells, and attenuation for HSP60 and HSC70+cells) could still be observed. Stress-related, attenuated swimming behaviors in the forced swimming test were also the most severe at the 5 h time point. Ethanol (1.5 g/kg) cotreatment on each administration day, even at non-toxic and/or euphoric doses, enhanced these stressor-like alterations. On the other hand, the protective effects of daily coadministered drugs related to benzodiazepine (5 mg/kg Ro 15-4513), dopamine (0.5 mg/kg SCH 23390), muscarinic (0.25 mg/kg pirenzepine) and serotonin (5 mg/kg ketanserin) receptors could be observed on the number of HSP-immunoreactive (24 h) and SAPK-immunoreactive cells (5 h). Against the stressor-altered swimming behaviors, Ro 15-4513 and SCH 23390 were more effective as compared to pirenzepine and ketanserin. [Copyright &y& Elsevier]

Shear stress and the endothelium. Vascular endothelial cells (ECs) in vivo are influenced by two distinct hemodynamic forces: cyclical strain due to vessel wall distention by transmural pressure, and shear stress, the frictional force generated by blood flow. Shear stress acts at the apical cell surface to deform cells in the direction of blood flow; wall distention tends to deform cells in all directions. The shear stress response differs, at least partly, from the cyclical strain response, suggesting that cytoskeletal strain alone cannot explain it. Acute shear stress in vitro elicits rapid cytoskeletal remodeling and activates signaling cascades in ECs, with the consequent acute release of nitric oxide and prostacyclin; activation of transcription factors nuclear factor (NF)κB, c- fos , c- jun and SP-1; and transcriptional activation of genes, including ICAM-1, MCP-1, tissue factor, platelet-derived growth factor-B (PDGF-B), transforming growth factor (TGF)-β1, cyclooxygenase-II, and endothelial nitric oxide synthase (eNOS). This response thus shares similarities with EC responses to inflammatory cytokines. In contrast, ECs adapt to chronic shear stress by structural remodeling and flattening to minimize shear stress. Such cells become very adherent to their substratum and show evidence of differentiation. Increased adhesion following chronic shear stress has been exploited to generate vascular grafts with confluent EC monolayers, retained after implantation in vivo , thus overcoming a major obstacle to endothelialization of vascular prostheses. [ABSTRACT FROM AUTHOR]

Different activation of mitogen-activated protein kinases in experimental proliferative glomerulonephritis. Mitogen-activated protein (MAP) kinases are critical for cell signaling goals such as cellular proliferation and induction of apoptosis. We examined whether MAP kinases, as a point of convergence for multiple extracellular stimuli, are activated in proliferative glomerulonephritis (GN) in vivo . Accelerated crescentic anti-glomerular basement membrane (GBM) GN was induced in rats preimmunized with rabbit IgG by administration of rabbit anti-rat GBM serum. Whole cortical tissue and isolated glomeruli were then subjected to kinase activity assays and Western blot analysis. Cortical activity of the archetypal MAP kinase, extracellular signal-regulated kinase (ERK), was increased significantly one, three, and seven days after induction of GN. In contrast, activation of MAP kinases with antiproliferative actions, stress-activated protein kinase, and p38 MAP kinase was detectable only in the early stages of proliferative GN (days one and three), implying that different MAP kinases serve distinct roles in the pathogenesis of GN. [ABSTRACT FROM AUTHOR]