Activation Of Stress-activated Protein Kinase Research Articles

Vitamin C Derivative Ascorbyl Palmitate Promotes Ultraviolet-B-Induced Lipid Peroxidation and Cytotoxicity in Keratinocytes

Among the preventative and protective strategies against the harmful effects of ultraviolet radiation to the skin is the application of antioxidants. Ascorbic acid has been shown to protect against sunburn, delay the onset of skin tumors, and reduce ultraviolet-B-radiation-induced skin wrinkling. In this work, we sought to determine the antioxidative properties of a lipid-soluble derivative of ascorbic acid, ascorbic acid-6-palmitate. We found that ascorbic acid-6-palmitate reduced cellular levels of reactive oxygen species following ultraviolet B irradiation. Treatment of keratinocytes with ascorbic acid-6-palmitate inhibited ultraviolet-B-mediated activation of epidermal growth factor receptor, extracellular regulated kinases 1 and 2, and p38 kinase because of its ability to prevent reduced glutathione depletion and scavenge hydrogen peroxide. Ascorbic acid-6-palmitate strongly promoted ultraviolet-B-induced lipid peroxidation, c-Jun N-terminal kinase activation, and cytotoxicity, however. End products of lipid peroxidation, such as 4-hydroxy-2-nonenal, have been reported to mediate stress-activated protein kinase activation and cell toxicity in epithelial cells. The lipid component of ascorbic acid-6-palmitate probably contributes to the generation of oxidized lipid metabolites that are toxic to epidermal cells. Our data suggest that, despite its antioxidant properties, ascorbic acid-6-palmitate may intensify skin damage following physiologic doses of ultraviolet radiation.

Hepatic ischemic preconditioning in mice is associated with activation of NF-κB, p38 kinase, and cell cycle entry

A brief period of hepatic ischemia protects the liver against subsequent ischemia-reperfusion (IR) injury, but the mechanism of such preconditioning is poorly understood. We examined whether preconditioning activated nuclear factor kappa B (NF-κB), the stress-activated protein kinases (SAPK), c-Jun N-terminal kinase-1 (JNK-1) and p38, and entry into the cell cycle. We used a murine model of partial hepatic ischemia. Preconditioning was performed by clamping the vasculature for 2 to 20 minutes, and allowing reperfusion for 10 minutes before 90-minute ischemia or IR. As assessed by serum alanine aminotransferase (ALT) levels and liver histology, preconditioning periods of 5 and 10 minutes were highly protective against IR injury, whereas 2-, 15-, and 20-minute intervals were ineffective. Preconditioning was associated with entry of hepatocytes into the cell cycle within 2 hours of subsequent IR, as indicated by proliferating cell nuclear antigen (PCNA) nuclear staining, induction of cyclin D1 and numerous mitotic figures; in the absence of preconditioning, such changes were not seen until 24 hours. Preconditioning increased nuclear binding of NF-κB within 30 minutes of the subsequent ischemic interval, paralleled by degradation of inhibitory (binding) protein for NF-κB (IκBα). Ischemic preconditioning also activated p38 kinase and JNK-1, which are known to converge on cyclin D1 regulation. The protective effect of the preconditioning regimen was more closely associated with p38 kinase than JNK-1 activation. In conclusion, the hepatoprotective effects of ischemic preconditioning are associated with activation of NF-κB and SAPKs that are associated with entry of hepatocytes into the cell cycle, a critical biological effect that favors survival of the liver against ischemic and IR injury. (H EPATOLOGY 2002;36:94-102.)

Vitamin D inhibits the activation of stress-activated protein kinases by physiological and environmental stresses in keratinocytes.

In addition to its known effects on keratinocyte proliferation and differentiation, the hormonal form of vitamin D, 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), has been shown to protect keratinocytes from UV- and chemotherapy-induced damage. Epidermal keratinocytes contain both the machinery needed to produce 1,25(OH)(2)D(3) and vitamin D receptors. The activation of the stress-activated protein kinases (SAPKs), such as c-Jun N-terminal kinase (JNK) and p38, is an early cellular response to stress signals and an important determinant of cell fate. This study examines whether modulation of these SAPKs is associated with the effects of 1,25(OH)(2)D(3) on keratinocytes under stress. HaCaT keratinocytes were exposed to heat shock, hyperosmotic concentrations of sorbitol, the epidermal growth factor receptor tyrosine kinase inhibitor AG1487, the pro-inflammatory cytokine tumor necrosis factor alpha, and H(2)O(2). These stresses activated both SAPKs. Pretreatment with 1,25(OH)(2)D(3) inhibited the activation of JNK by all stresses and the activation of p38 by heat shock, AG1478 and tumor necrosis factor alpha. Under the same conditions, treatment with 1,25(OH)(2)D(3) protected HaCaT keratinocytes from cytotoxicity induced by exposure to H(2)O(2) and hyperosmotic shock. The effect of 1,25(OH)(2)D(3) was dose-dependent, already apparent at nanomolar concentrations, and time-dependent, maximal after a 24-h pre-incubation. We suggest that inhibition of SAPK activation may account for some of the well-documented protective effects of 1,25(OH)(2)D(3) on epidermal cells during exposure to UV or chemotherapy and may also be related to the anti-inflammatory actions of the hormone in skin.

Inhibition of c-Abl with STI571 attenuates stress-activated protein kinase activation and apoptosis in the cellular response to 1-beta-D-arabinofuranosylcytosine.

The response of myeloid leukemia cells to treatment with 1-beta-D-arabinofuranosylcytosine (ara-C) includes activation of the c-Abl protein tyrosine kinase and the stress-activated protein kinase (SAPK). The present studies demonstrate that treatment of human U-937 leukemia cells with ara-C is associated with translocation of SAPK to mitochondria. STI571 (imatinib mesylate), an inhibitor of c-Abl, blocked both activation and mitochondrial targeting of SAPK in the ara-C response. In concert with these effects of STI571, similar findings were obtained in c-Abl-deficient cells. The results further show that STI571 inhibits ara-C-induced loss of mitochondrial transmembrane potential, caspase-3 activation, and apoptosis. These findings demonstrate that STI571 down-regulates c-Abl-mediated signals that target the mitochondria in the apoptotic response to ara-C.

Hyposmotic Stress Induces Cell Growth Arrest via Proteasome Activation and Cyclin/Cyclin-dependent Kinase Degradation

Ordered cell cycle progression requires the expression and activation of several cyclins and cyclin-dependent kinases (Cdks). Hyperosmotic stress causes growth arrest possibly via proteasome-mediated degradation of cyclin D1. We studied the effect of hyposmotic conditions on three colonic (Caco2, HRT18, HT29) and two pancreatic (AsPC-1 and PaCa-2) cell lines. Hyposmosis caused reversible cell growth arrest of the five cell lines in a cell cycle-independent fashion, although some cell lines accumulated at the G(1)/S interface. Growth arrest was followed by apoptosis or by formation of multinucleated giant cells, which is consistent with cell cycle catastrophe. Hyposmosis dramatically decreased Cdc2, Cdk2, Cdk4, cyclin B1, and cyclin D3 expression in a time-dependent fashion, in association with an overall decrease in cellular protein synthesis. However, some protein levels remained unaltered, including cyclin E and keratin 8. Selective proteasome inhibition prevented Cdk and cyclin degradation and reversed hyposmotic stress-induced growth arrest, whereas calpain and lysosome enzyme inhibitors had no measurable effect on cell cycle protein degradation. Therefore, hyposmotic stress inhibits cell growth and, depending on the cell type, causes cell cycle catastrophe with or without apoptosis. The growth arrest is due to decreased protein synthesis and proteasome activation, with subsequent degradation of several cyclins and Cdks.

Toll-like Receptor 2 and 4 (TLR2 and TLR4) Agonists Differentially Regulate Secretory Interleukin-1 Receptor Antagonist Gene Expression in Macrophages

Treatment of macrophages with lipopolysaccharide (LPS) from Gram-negative bacteria or peptidoglycan (PGN) from Gram-positive bacteria activates multiple intracellular signaling pathways and a large, diverse group of nuclear transcription factors. The signaling receptors for PGN and LPS are now known to be the Toll-like receptors 2 and 4 (TLR2 and -4, respectively). While a large body of literature indicates that the members of the TLR family activate nearly identical cytoplasmic signaling programs, several recent reports have suggested that the functional outcomes of signaling via TLR2 or TLR4 are not equivalent. In the current studies, we compared the responses of the secretory IL-1 receptor antagonist (sIL-1Ra) gene to both LPS and PGN. Both LPS and PGN induced IL-1Ra gene expression; however, the combination of both stimuli synergistically increased sIL-1Ra mRNA expression and promoter activity, suggesting that the signals induced by PGN and LPS are not equivalent. While both LPS and PGN utilized the PU.1-binding sites in the proximal sIL-1Ra promoter region to generate a full response, additional distinct promoter elements were utilized by LPS or PGN. Activation of p38 stress-activated protein kinase was required for LPS- or PGN-induced IL-1Ra gene expression, but the p38-responsive promoter elements localized to distinct regions of the sIL-1Ra gene. Additionally, while the LPS-induced, p38-dependent response was dependent upon PU.1 binding, the PGN-induced, p38 response was not. Collectively, these data indicated that while some of the intracellular signaling events by TLR2 and TLR4 agonists are similar, there are clearly distinct differences in the responses elicited by these two bacterial products.

ROS, stress‐activated kinases and stress signaling in cancer

Anticancer therapy is frequently efficient in early stages of the disease, whereas advanced tumors are usually resistant to the same treatments. The molecular basis for this change is not entirely understood. Many anticancer agents are DNA- or cytoskeleton-damaging drugs that show some specificity towards dividing cells. However, recent studies show that these agents also activate stress-signaling cascades that may play a role in eliciting the observed therapeutic effects. We discuss recent findings that suggest that induction of stress signaling in oncogenically transformed cells is integrated into apoptotic pathways. Reactive oxygen species (ROS) and stress-activated protein kinases (SAPKs), which are potentiated in recently transformed cells, emerge as key effectors of cell death. In advanced tumors, however, these agents are downregulated and, consequently, death signaling is suppressed. Such changes in ROS and SAPK activity levels during the course of tumor development may underlie the changes in responsiveness to anticancer therapy.

Fas, ceramide and serum withdrawal induce apoptosis via a common pathway in a type II Jurkat cell line

Ceramide is a key mediator of apoptosis, yet its role in Fas-mediated apoptosis is controversial. Some reports have indicated that ceramide is either a primary signaling molecule in Fas-induced cell death, or that it functions upstream of Fas by increasing FasL expression. Other studies have suggested that ceramide is not relevant to Fas-induced cell death. We have approached this problem by studying ceramide-induced apoptosis in unique Jurkat cell clones selected for resistance to membrane-bound FasL-induced death. Resistance of the mutant Jurkat cells was specific for FasL killing, since the mutant clones were sensitive to other apoptotic stimuli such as cycloheximide and staurosporine. We tested the effects of serum withdrawal, one of the strongest inducers of ceramide, and of exogenous ceramide on apoptosis of both wild-type and FasL-resistant clones. Wild-type Jurkat cells were remarkably sensitive to serum withdrawal and to exogenous ceramide. In contrast all FasL-resistant mutant clones were resistant to these apoptosis-inducing conditions. In contrast to previous work, we did not detect an increase in FasL in either wild-type or mutant clones. Moreover activation of stress-activated protein kinases (JNK/SAPKs) after serum withdrawal and exogenous ceramide treatment was detected only in the wild-type and not in the resistant clones. Because of the parallel resistance of the mutant clones to Fas and to ceramide-induced apoptosis, our data support the notion that ceramide is a second messenger for the Fas/FasL pathway and that serum withdrawal, through production of ceramide, shares a common step with the Fas-mediated apoptotic pathway. Finally, our data suggest that activation of JNK/SAPKs is a common mediator of the three pathways tested.

Activation of stress-activated protein kinases (SAPK) in tendon cells following cyclic strain: the effects of strain frequency, strain magnitude, and cytosolic calcium

Cyclic strain has been shown to benefit tendon health. However, repetitive loading has also been implicated in the etiology of tendon overuse injuries. Recent studies demonstrated that in several cell lines cyclic strain was associated with an activation of stress-activated protein kinases (SAPKs). These SAPKs, in turn, were shown to be important upstream regulators of a variety of cell processes including apoptosis. To examine the effect of repetitive loading on SAPK activation in tendon cells in vitro, canine patellar tendon cells were cyclically strained, and the cellular stress response evaluated by measuring c-Jun N-terminal kinase (JNK) activation. The effects of strain frequency and strain magnitude as well as the role of calcium signaling in this mechanotransduction mechanism were also examined. Cyclic strain resulted in an immediate activation of JNK, which peaked at 30 min and returned to resting levels by 2 h. This activation was regulated by a magnitude-dependent but not frequency-dependent response and appeared to be mediated through a calcium-dependent mechanotransduction pathway. While transient JNK activation is associated with normal cell processes, persistent JNK activation has been linked to the initiation of the apoptotic cascade. A similar mechanism could be responsible for initiating the pathological events (localized cell death) seen in tendon overuse injury.

Regulation of COX-2 expression in human intestinal myofibroblasts: mechanisms of IL-1-mediated induction.

Elevated mucosal interleukin-1 (IL-1) levels are frequently seen during acute and chronic intestinal inflammation, and IL-1 neutralization lessens the severity of inflammation. One major effect of IL-1 is the increased release of eicosanoid mediators via induction of cyclooxygenase-2 (COX-2). One site of COX-2-derived prostaglandin synthesis during acute and chronic intestinal inflammation is the intestinal myofibroblast. COX-2 expression has also been documented in these cells in colonic neoplasms. Thus an understanding of the regulation of COX-2 expression in human intestinal myofibroblasts is important. As an initial step toward this goal we have characterized IL-1alpha signaling pathways that induce COX-2 expression in cultured human intestinal myofibroblasts. IL-1 treatment resulted in a dramatic transcriptional induction of COX-2 gene expression. Activation of nuclear factor-kappaB (NF-kappaB), extracellular signal-regulated protein kinase (ERK), p38, and protein kinase C (PKC) signaling pathways was each necessary for optimal COX-2 induction. In contrast to what occurs in other cell types, including other myofibroblasts such as renal mesangial cells, PKC inhibition did not prevent IL-1-induced NF-kappaB or mitogen activated protein kinase/ stress-activated protein kinase activation, suggesting a novel role for PKC isoforms during this process. The stimulatory effects of PKC, NF-kappaB, ERK-1/2, and presumably c-Jun NH(2)-terminal kinase activation were exerted at the transcriptional level, whereas p38 activation resulted in increased stability of the COX-2 message. We conclude that, in intestinal myofibroblasts, IL-1-mediated induction of COX-2 expression is a complex process that requires input from multiple signaling pathways. Each parallel pathway acts in relative autonomy, the sum of their actions culminating in a dramatic increase in COX-2 transcription and message stability.

Phosphorylation of microtubule-associated protein tau by stress-activated protein kinases in intact cells

Tau is a microtubule-associated protein that is abnormally hyperphosphorylated in the filamentous lesions that define a number of neurodegenerative diseases collectively referred to as tauopathies. We previously showed that stress-activated protein (SAP) kinases phosphorylate tau protein at many of the hyperphosphorylated sites in vitro. Here we have developed a system to study the effects of five SAP kinases (SAPK1c/JNK1, SAPK2a/p38α, SAPK2b/p38β, SAPK3/p38γ and SAPK4/p38δ) on tau phosphorylation in intact cells. All kinases phosphorylated tau, albeit at different efficiencies. Tau was a good substrate for SAPK3/p38γ and SAPK4/p38δ, a reasonable substrate for SAPK2b/p38β and a relatively poor substrate for SAPK2a/p38α and SAPK1c/JNK1. These findings indicate that the aberrant activation of SAP kinases, especially SAPK3/p38γ and SAPK4/p38δ, could play an important role in the abnormal hyperphosphorylation of tau that is an invariant feature of the tauopathies.

Neurofilaments in diabetic neuropathy

This review discusses the role of abnormal neurofilament (NF) expression, processing, and structure as an etiological factor in diabetic neuropathy. Diabetic sensory and autonomic neuropathy in humans is associated with a spectrum of structural changes in peripheral nerve that includes axonal degeneration, paranodal demyelination, and loss of myelinated fibers-- the latter is probably the result of a dying-back of distal axons. NF filaments are composed of three subunit proteins, NFL, NFM, and NFH, and are major constituents of the axonal cylinder. It is clear that any abnormality in synthesis, delivery, or processing of these critical proteins could lead to severe impairments in axon structure and function. This article describes mechanisms of synthesis, phosphorylation, and delivery of NF and discusses how these processes may be abnormal in diabetics. The pathological alterations in the ganglion and preipheral nerve that occur in sensory and autonomic neuropath will be outlined and related to possible abnormal processing of NF. A major focus is the role or aberrant NF phosphorylation and its possible involvement in the imparied delivery of NF to the distal axon. Identification of stress-activated protein kinases (SAPKs) as NF kinases is discussed in detail and it is proposed that hyperglycemia-induced activation of SAPKs may be a primary etiological event in diabetic neuropathy.

EDG-1 links the PDGF receptor to Src and focal adhesion kinase activation leading to lamellipodia formation and cell migration.

Sphingosine-1-phosphate (SPP), formed by sphingosine kinase, is the ligand for EDG-1, a GPCR important for cell migration and vascular maturation. Here we show that cytoskeletal rearrangements, lamellipodia extensions, and cell motility induced by platelet-derived growth factor (PDGF) are abrogated in EDG-1 null fibroblasts. However, EDG-1 appears to be dispensable for mitogenicity and survival effects, even those induced by its ligand SPP and by PDGF. Furthermore, PDGF induced focal adhesion formation and activation of FAK, Src, and stress-activated protein kinase 2, p38, were dysregulated in the absence of EDG-1. In contrast, tyrosine phosphorylation of the PDGFR and activation of extracellular signal regulated kinase (ERK1/2), important for growth and survival, were unaltered. Our results suggest that EDG-1 functions as an integrator linking the PDGFR to lamellipodia extension and cell migration. PDGF, which stimulates sphingosine kinase, leading to increased SPP levels in many cell types, also induces translocation of sphingosine kinase to membrane ruffles. Hence, recruitment of sphingosine kinase to the cell's leading edge and localized formation of SPP may spatially and temporally stimulate EDG-1, resulting in activation and integration of downstream signals important for directional movement toward chemoattractants, such as PDGF. These results may also shed light on the vital role of EDG-1 in vascular maturation.

Influence of cytokines tumor necrosis factor-α and interferon-γ on signaling cascades associated with apoptosis in rat PC12 cells

During cell-mediated immune response, increased amounts of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) are released. In the present study, we investigated the potential of these two cytokines to mediate apoptosis and to alter signal transduction pathways involved in undifferentiated PC12 cells. To induce apoptosis, the pteridine 7,8-dihydroneopterin (NH2) was used. TNF-α alone and TNF-α in combination with IFN-γ led to no alteration in cell viability during 48 h of incubation. TNF-α was able to slightly elevate apoptosis compared with cells stimulated with NH2 alone. The combination of TNF-α and IFN-γ almost completely abrogated the rate of apoptosis induced by NH2. Similar degrees of activation of extracellular protein kinase were found after the addition of cytokines or cytokines in combination with NH2. Stress-activated protein kinase (SAPK) was not activated by the cytokines alone, whereas adding the cytokine TNF-α to NH2-stimulated cells resulted in activation of SAPK after 15 min.

Mechanism of increased renal gene expression during metabolic acidosis.

Increased renal catabolism of plasma glutamine during metabolic acidosis generates two ammonium ions that are predominantly excreted in the urine. They function as expendable cations that facilitate the excretion of acids. Further catabolism of alpha-ketoglutarate yields two bicarbonate ions that are transported into the venous blood to partially compensate for the acidosis. In rat kidney, this adaptation is sustained, in part, by the induction of multiple enzymes and various transport systems. The pH-responsive increases in glutaminase (GA) and phosphoenolpyruvate carboxykinase (PEPCK) mRNAs are reproduced in LLC-PK(1)-fructose 1,6-bisphosphatase (FBPase) cells. The increase in GA activity results from stabilization of the GA mRNA. The 3'-untranslated region of the GA mRNA contains a direct repeat of an eight-base AU sequence that functions as a pH-response element. This sequence binds zeta-crystallin/NADPH:quinone reductase with high affinity and specificity. Increased binding of this protein during acidosis may initiate the pH-responsive stabilization of the GA mRNA. In contrast, induction of PEPCK occurs at the transcriptional level. In LLC-PK(1)-FBPase(+) kidney cells, a decrease in intracellular pH leads to activation of the p38 stress-activated protein kinase and subsequent phosphorylation of transcription factor ATF-2. This transcription factor binds to cAMP-response element 1 within the PEPCK promoter and may enhance its transcription during metabolic acidosis.

Impaired Synergistic Activation of Stress-activated Protein Kinase SAPK/JNK in Mouse Embryonic Stem Cells Lacking SEK1/MKK4: DIFFERENT CONTRIBUTION OF SEK2/MKK7 ISOFORMS TO THE SYNERGISTIC ACTIVATION

Stress-activated protein kinase/c-Jun NH(2)-terminal kinase (SAPK/JNK), which is a member of the mitogen-activated protein kinase (MAPK) family, plays an important role in a stress-induced signaling cascade. SAPK/JNK activation requires the phosphorylation of Thr and Tyr residues in its Thr-Pro-Tyr motif, and SEK1 (MKK4) and MKK7 (SEK2) have been identified as the upstream MAPK kinases. Here we examined the activation and phosphorylation sites of SAPK/JNK and differentiated the contribution of SEK1 and MKK7alpha1, -gamma1, and -gamma2 isoforms to the MAPK activation. In SEK1-deficient mouse embryonic stem cells, stress-induced SAPK/JNK activation was markedly impaired, and this defect was accompanied with a decreased level of the Tyr phosphorylation. Analysis in HeLa cells co-transfected with the two MAPK kinases revealed that the Thr and Tyr of SAPK/JNK were independently phosphorylated in response to heat shock by MKK7gamma1 and SEK1, respectively. However, MKK7alpha1 failed to phosphorylate the Thr of SAPK/JNK unless its Tyr residue was phosphorylated by SEK1. In contrast, MKK7gamma2 had the ability to phosphorylate both Thr and Tyr residues. In all cases, the dual phosphorylation of the Thr and Tyr residues was essentially required for the full activation of SAPK/JNK. These data provide the first evidence that synergistic activation of SAPK/JNK requires both phosphorylation at the Thr and Tyr residues in living cells and that the preference for the Thr and Tyr phosphorylation was different among the members of MAPK kinases.

Proteinase-activated Receptor-2-mediated Activation of Stress-activated Protein Kinases and Inhibitory κB Kinases in NCTC 2544 Keratinocytes

In this study we examined the regulation of the stress-activated protein (SAP) kinases and inhibitory kappa B kinases (IKKs) through stimulation of the novel G-protein-coupled receptor proteinase-activated receptor-2 in the human keratinocyte cell line NCTC2544. Trypsin and the peptide SLIGKV stimulated a time-dependent increase in both c-Jun N-terminal kinase and p38 mitogen-activated protein kinase activity. Trypsin also stimulated NF kappa B-DNA binding and the activation of the upstream kinases IKK alpha and -beta. Phorbol 12-myristate 13-acetate also strongly activated both SAP kinases and IKK isoforms, suggesting the potential for a protein kinase C-mediated regulatory mechanism underlying the effects of trypsin. Pre-incubation with selective protein kinase C (PKC) inhibitors GF109203X and Gö6983, or transfection of dominant negative (DN)-PKC alpha, abolished phorbol 12-myristate 13-acetate-mediated c-Jun N-terminal kinase activity, although it only partially inhibited the response to trypsin. In contrast, Gö6983 reduced trypsin-stimulated p38 mitogen-activated protein kinase activity to a greater extent than GF109203X, although DN-PKC alpha or PKC zeta had no substantial effect. Additionally, inhibitors of PKC partially reduced trypsin-stimulated IKK alpha activity but abolished that of IKK beta, whereas DN-PKC alpha but not DN-PKC zeta substantially reduced trypsin-stimulated Flag-IKK beta activity. This study shows for the first time proteinase-activated receptor-2-mediated stimulation of both SAP kinase and IKK signaling and differing roles for PKC isoforms in the regulation of each pathway.

The involvement of a stress-activated pathway in equine influenza virus-mediated apoptosis.

We have shown elsewhere that equine-2 influenza virus (EIV; subtype H3N8) induced pronounced cell death in infected cells through apoptosis as demonstrated by DNA fragmentation assay and a combined TUNEL and immunostaining scheme. In this study, we investigated the mechanism of EIV-mediated cytotoxicity on a permissive mammalian epithelial cell line, Madin-Darby canine kidney (MDCK) cells. EIV infection increased the cellular levels of oxidative stress and c-Jun/AP-1 protein (which is known to be affected by oxidative stress), as well as its DNA binding activity. Increased production of TGF-beta1, an inducer of c-Jun N-terminal kinase or stress-activated protein kinase (JNK/SAPK) activation, was also detected in EIV-infected MDCK cells. It has been reported that TGF-beta may initiate a signaling cascade leading to JNK/SAPK activation. Addition of c-Jun antisense oligodeoxynucleotide, antioxidant N-acetyl-cysteine (NAC), JNK/SAPK inhibitor carvedilol, or TGF-beta-neutralizing antibody effectively blocked c-Jun/AP-1 upregulation and TGF-beta1 production mediated by EIV infection. These treatments also attenuated EIV-induced cytopathogenic effects (CPE) and apoptosis. Our results suggest that a stress-activated pathway is involved in apoptosis mediated by EIV infection. It is likely that EIV infection turns on the JNK/SAPK cascade, which modulates the activity of apoptosis-promoting regulatory factor c-Jun/AP-1 and epithelial growth inhibitory cytokine TGF-beta.

Inhibition of stress-activated protein kinase in the ischemic/reperfused heart: role of magnesium tanshinoate B in preventing apoptosis

The activation of stress-activated protein (SAP) kinase may lead to an induction of apoptosis that is responsible for part of the cardiomyocyte death in reperfusion injury. The objective of the present study was to investigate the mechanism by which magnesium tanshinoate B (MTB), a bioactive compound isolated from Danshen, prevents apoptosis in cardiomyocytes in the ischemic/reperfused heart. Isolated adult rat hearts were perfused by the Langendorff mode with medium containing MTB prior to the induction of normothermic global ischemia. At the end of the 30-min ischemic period, the heart was reperfused with the same medium with or without MTB for an additional 20 min. In the MTB-treated ischemic/reperfused heart, the number of apoptotic nuclei was reduced by 2.5-fold in comparison to that in untreated ischemic/reperfused controls [23 ± 4 vs 57 ± 7 (mean ± SD) TUNEL-positive cells, respectively, N = 3–4, P < 0.001]. SAP kinase activity was elevated 1.7-fold in ischemic/reperfused rat hearts [35.6 ± 3.8 vs 21.2 ± 3.3 (control) (mean ± SEM) relative densitometric units, N = 4–6, P < 0.05]. Treatment with MTB abolished this elevation in SAP kinase activity (25.0 ± 5.2 relative densitometric units), which was also decreased by 40% in the nucleus. When the heart was subjected to ischemia alone, there was no significant change in SAP kinase activity in the presence or absence of MTB. MTB did not appear to affect the p38 mitogen-activated protein kinase activity in this model system. In conclusion, MTB was shown to have cardioprotective activity against apoptosis, probably through the inhibition of SAP kinase activity.

Presenilin Links Alzheimer's Disease and Stress

Presenilin 1 (PS1) is a transmembrane protein localized to internal cell membranes and is involved in processing amyloid β precursor protein to produce Aβ (a protein implicated in Alzheimer's disease) and processing of Notch (a signaling protein involved in development). Kim et al. show that overexpression of PS1 in embryonic kidney cells or neuroblastoma cells inhibits activation of the stress-activated protein kinase (SAPK) pathway and suppresses peroxide-induced apoptosis. Experiments in which PS1 and constitutively active mutants of the proteins in the SAPK pathway were coexpressed indicate that PS1 exerts its inhibitory effect upstream of the guanosine triphosphatase Rac1 or via a Rac1-independent pathway. PS1-deficient mouse embryo fibroblasts exhibited enhanced SAPK activity under resting conditions and increased apoptosis upon treatment with peroxide. The γ-secretase activity of PS1 was determined to be essential for the inhibition of SAPK signaling based on analysis of various PS1 mutants. The authors discuss how the γ-secretase activity may impact Notch signaling, which apparently affects SAPK signaling through some type of pathway cross talk. J. W. Kim, T.-S. Chang, J. E. Lee, S.-H. Huh, S. W. Yeon, W. S. Yang, C. O. Joe, I. Mook-Jung, R. E. Tanzi, T.-W. Kim, E.-J. Choi, Negative regulation of the SAPK/JNK signaling pathway by presenilin 1. J. Cell Biol. 153 , 457-464. [Abstract] [Full Text]