Stress-activated Protein Kinase Inhibitor Research Articles

Discovery of a Stress-Activated Protein Kinase Inhibitor for Lymphatic Filariasis.

Lymphatic filariasis infects over 120 million people worldwide and can lead to significant disfigurement and disease. Resistance is emerging with current treatments, and these therapies have dose limiting adverse events; consequently new targets are needed. One approach to achieve this goal is inhibition of parasitic protein kinases involved in circumventing host defense mechanisms. This report describes structure-activity relationships leading to the identification of a potent, orally bioavailable stress activated protein kinase inhibitor that may be used to investigate this hypothesis.

Multiple neurotrophic effects of VEGF on cultured neurons.

A large literature demonstrates the multifunctional nature of vascular endothelial growth factor (VEGF). Though initially characterized as an endothelial cell-specific factor, recent studies reveal that VEGF has numerous effects on diverse cell types in the brain including neurons. The objective of this study is to examine the effects of VEGF in cultured cortical neurons on survival, p38 mitogen-activated protein kinase (p38 MAP kinase) activity, pro- and anti-apoptotic protein expression and on release of neurotrophic and neurotoxic factors. The results show that VEGF dose-dependently enhances the survival of neurons in culture. VEGF decreases active caspase 3 levels and increases expression of the anti-apoptotic protein Bcl-2. VEGF decreases phosphorylated p38 MAP kinase level and activity in cortical neurons. In addition to modulating survival/death pathways in cortical neurons, VEGF also regulates release of proteins that affect neuronal viability. VEGF causes a dose-dependent release of the neurotrophic protein pigment epithelial-derived factor (PEDF), while significantly decreasing release of the neurotoxic protein amyloid beta. The VEGF-mediated decrease in amyloid beta is dependent on a functional Flt-1 receptor and is inhibited by dicoumarol, a multifunctional inhibitor of stress-activated protein kinase (SAPK)/JNK and NFkappaB pathways. Taken together, these data demonstrate that the neurotrophic effects of VEGF are likely mediated directly by increasing survival and decreasing apoptotic proteins and signals as well as indirectly by modulating release of proteins that affect neuronal viability.

Betacellulin Protects From Pancreatitis by Activating Stress-Activated Protein Kinase

Acute pancreatitis (AP) is a serious, unpredictable clinical problem, the pathophysiology of which is poorly understood. Here, we evaluate whether betacellulin (BTC), a ligand of the epidermal growth factor receptor also able to activate the proapoptotic ERBB4 receptor, can protect against experimental AP. AP was induced in transgenic mice overexpressing BTC (BTC-tg), control mice, or control mice after administration of recombinant BTC. The severity of pancreatitis was assessed by measurements of serum amylase and lipase and histologic grading. The involvement of the stress-activated protein kinase (SAPK) was evaluated by treating BTC-tg mice with an SAPK inhibitor before induction of AP. BTC-tg mice showed increased apoptosis and proliferation in the exocrine pancreas, indicating an increased cell turnover. There was a marked, epidermal growth factor receptor-independent decrease in pancreas weight. After induction of AP by cerulein injection, BTC-tg mice showed a significantly lower increase in serum amylase and lipase levels as well as less pronounced tissue necrosis, edema, and inflammation, as compared to nontransgenic littermates. This protective effect, also confirmed in the L-arginine AP model, was associated with increased phosphorylation of SAPK and abrogated after treatment of BTC-tg mice with a SAPK inhibitor. Finally, the protective effect of BTC against AP was confirmed by treating nontransgenic mice with recombinant BTC. These findings indicate a potential application of the BTC/ERBB4 pathway for modulating the course of AP.

Up-regulation of CAR expression through Elk-1 in HepG2 and SW480 cells by serum starvation stress

Constitutive androstane receptor (CAR) is a transcription factor regulating the expression of several genes related to drug metabolism. CAR expression was elevated in human HepG2 and SW480 cells by serum starvation. From reporter gene assays, mutagenesis, RNA interference, and chromatin immunoprecipitation assays, we identified the serum response element at −142/−139 in the CAR gene transactivated by Elk-1. Whereas treatment with U0126 (ERK inhibitor) enhanced CAR expression, SP600125 (stress-activated protein kinase inhibitor, SAPK) suppressed the phosphorylation of Elk-1 caused by serum-starvation stress and the elevation of CAR mRNA, suggesting that CAR expression may be mediated by phosphorylated Elk-1 via the SAPK signaling pathway.

(−)-Epigallocatechin gallate reduces transforming growth factor β-stimulated HSP27 induction through the suppression of stress-activated protein kinase/c-Jun N-terminal kinase in osteoblasts

We previously reported that transforming growth factor-β (TGF-β) stimulates heat shock protein 27 (HSP27) induction through p38 mitogen-activated protein (MAP) kinase and extracellular signal-regulated kinase 1/2 (ERK1/2) in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether (−)-epigallocatechin gallate (EGCG), the major polyphenol found in green tea, affects the TGF-β-stimulated induction of HSP27 in these cells, and its underlying mechanism. EGCG significantly suppressed the HSP27 induction stimulated by TGF-β in a dose-dependent manner between 10 and 30 µM without affecting the HSP70 levels. TGF-β with or without EGCG did not affect the advanced oxidation protein products. The TGF-β-induced phosphorylation of p38 MAP kinase and ERK1/2 was not affected by EGCG. SP600125, a specific inhibitor of stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK), markedly reduced the HSP27 expression induced by TGF-β. EGCG significantly suppressed the TGF-β-induced phosphorylation of SAPK/JNK without affecting the phosphorylation of Smad2. EGCG attenuated the phosphorylation of both MKK4 and TAK1 induced by TGF-β. These results strongly suggest that EGCG suppresses the TGF-β-stimulated induction of HSP27 via the attenuation of the SAPK/JNK pathway in osteoblasts, and that this effect is exerted at a point upstream from TAK1.

164 HEAT STRESS ENHANCES THE DEVELOPMENT OF PORCINE PARTHENOGENETIC EMBRYOS, BUT NOT IN VITRO-FERTILIZED EMBRYOS

Recent research in our laboratory has demonstrated that a borderline supra-physiological heat stress (42�C) of moderate duration (9 h) can have beneficial effects on the development of porcine parthenogenetic embryos depending on the timing of heat stress application relative to oocyte activation. A 9-h heat stress (HS) applied to presumptive parthenotes immediately after oocyte activation by electrical stimulus (0-9 h post-activation [hpa]; 09HS) significantly accelerated the timing of the first cleavage event: only 26% of the non-heat stressed (NHS) controls had cleaved by 24 hpa, but more than 60% of the 09HS embryos had cleaved after 24 h (P < 0.0001; ANOVA as determined using the PROC GLM commands in SAS; SAS Institute, Inc., Cary, NC, USA). This discrepancy had all but disappeared by 48 hpa (P > 0.1). After 7 days in culture, however, 38.0% of the 09HS embryos developed to blastocyst whereas 18.5% of the NHS embryos developed to that stage (P < 0.0001). Pharmaceutical and biomolecule inhibitors were used to block several candidate biochemical pathways and processes that were thought to be involved in bringing about the phenotype observed in the 09HS embryos: alpha amanitin (an RNA polymerase inhibitor), roscovitine (a cdc2/MPF inhibitor), cordycepin (an inhibitor of polyadenylation), SP600125 (a stress-activated protein kinase inhibitor), LY294002 (a phosphoinositide 3-kinase inhibitor), and MG132 (a 26S proteasome inhibitor). NHS and 09HS embryos were incubated with and without inhibitors. At least three replicates of each experiment were performed. Although some interesting data have been generated, none of these pathways individually appears to be responsible for the observed beneficial effect of the heat stress on early embryo development in parthenotes (P > 0.01). Efforts to replicate in in vitro-fertilized (IVF) embryos the beneficial effects of the heat stress observed in parthenogenetic embryos have been largely unsuccessful, even if the timing of stressor application was altered. IVF embryos were heat stressed at 42�C for 9 h at different time points after insemination: 4-13 h post-insemination (hpi; 413HS), 13-22 hpi (1322HS), or 22-31 hpi (2231HS). The 24- and 48-h cleavage rates, and Day 7 blastocyst rates were assessed. These observations were repeated at least five times. Although the heat stress was able to alter the 24-h cleavage rates (P < 0.0001), 48-hpi cleavage rates and Day 7 blastocyst rates were not significantly different between groups (P > 0.1). We have observed similar results in nuclear transfer (NT) embryos (Lai et al. 2005), leading us to postulate that the nuclear remodeling that is necessary in IVF and NT embryos prevents these embryos from responding positively to the HS in a manner similar to that in the parthenogenetic embryos. Experiments to test this hypothesis as well as further genomic and proteomic analyses of this phenomenon are ongoing in our laboratory. This work was funded in part by Food for the 21st Century.

Regulation of the Circadian Oscillator in Xenopus Retinal Photoreceptors by Protein Kinases Sensitive to the Stress-activated Protein Kinase Inhibitor, SB 203580

Circadian rhythms are generated by transcriptional and translational feedback loops. Stress-activated protein kinases (SAPKs) are known to regulate transcription factors in response to a variety of extracellular stimuli. In the present study, we examined whether the SAPKs play a role in the circadian system in cultured Xenopus retinal photoreceptor layers. A 6-h pulse of SB 203580, an inhibitor of SAPKs, reset the circadian rhythm of melatonin in a phase-dependent manner similar to dark pulses. This phase-shifting effect was dose-dependent over the range of 1-100 microm. Treatment with SB 203580 also affected light-induced phase shifts, and light had no effect on the circadian oscillator in the presence of 100 microm SB 203580. In-gel kinase assays showed that SB 203580 selectively inhibited a small group of protein kinases in the photoreceptor cells. These SB 203580-sensitive kinases correspond to two isoforms of phosphorylated p38 MAPK and three isoforms of c-Jun N-terminal kinase (JNK). Further in vitro study demonstrated that SB 203580 also inhibited casein kinase Iepsilon (CKIepsilon), which has been shown to regulate circadian rhythms in several organisms. However, a pharmacological inhibition of CKI reset the circadian oscillator in a phase-dependent manner distinct from that of SB 203580. This argues against a primary role of CKI in the phase-shifting effects of SB 203580. These results suggest that SB 203580 affects the circadian system by inhibiting p38 MAPKs or JNKs and that these protein kinases are candidate cellular signals in the regulation of the circadian oscillator in the Xenopus retinal photoreceptors.

P38 stress-activated protein kinase inhibitor reverses bradykinin B 1 receptor-mediated component of inflammatory hyperalgesia

The effects of a p38 stress-activated protein kinase inhibitor, 4-(4-fluorophenyl)-2-(-4-methylsulfonylphenyl)-5-(4-pyridynyl) imidazole (SB203580), were evaluated in a rat model of inflammatory hyperalgesia. Oral, but not intrathecal, administration of SB203580 significantly reversed inflammatory mechanical hyperalgesia induced by injection of complete Freund's adjuvant into the hindpaw. SB203580 did not, however, affect the increased levels of interleukin-1β and cyclo-oxygenase 2 protein observed in the hindpaw following complete Freund's adjuvant injection. Intraplantar injection of interleukin-1β into the hindpaw elicited mechanical hyperalgesia in the ipsilateral paw, as well as in the contralateral paw, following intraplantar injection of the bradykinin B 1 receptor agonist des-Arg 9-bradykinin. Oral administration of SB203580 1 h prior to interleukin-1β administration prevented the development of hyperalgesia in the ipslateral paw and the contralateral bradykinin B 1 receptor-mediated hyperalgesia. In addition, following interleukin-1β injection into the ipsilateral paw, co-administration of SB203580 with des-Arg 9-bradykinin into the contralateral paw inhibited the bradykinin B 1 receptor-mediated hyperalgesia. In human embryonic kidney 293 cells expressing the human bradykinin B 1 receptor, its agonist des-Arg 10-kallidin produced a rapid phosphorylation of endogenous p38 stress-activated protein kinase. Our data suggest that p38 stress-activated protein kinase is involved in the development of inflammatory hyperalgesia in the rat, and that its pro-inflammatory effects involve the induction of the bradykinin B 1 receptor as well as functioning as its downstream effector.

Stress-induced glucose uptake in bovine chromaffin cells: a comparison of the effect of arsenite and anisomycin

The effect of the toxic chemical Na-arsenite and the protein synthesis inhibitor anisomycin on glucose transport in primary cultures of bovine chromaffin cells was compared using the effect of insulin-like growth factor I (IGF-I) as a reference. The enhanced uptake of glucose obtained in response to arsenite and anisomycin reached maximum after 60 min, with the response to anisomycin being delayed in onset relative to that of arsenite. At maximal doses the arsenite effect was consistently higher than that of anisomycin and comparable to the ∼2-fold effect produced by IGF-I. The selective inhibitor of stress-activated protein kinase 2 (SAPK2), SB 203580, inhibited completely anisomycin-induced glucose uptake but only partly suppressed uptake stimulated by arsenite. Both substances, in concentrations producing maximal effects on glucose transport, led to a strong phosphorylation of SAPK2. In contrast to the effect on glucose transport, the arsenite-induced phosphorylation of SAPK2 was relatively slow compared to the anisomycin-induced activation. The results indicate that glucose uptake induced by the two types of cellular stress are mediated by at least two different signaling pathways, which also differ from that activated by IGF-I.

The p38/Reactivating Kinase Mitogen-activated Protein Kinase Cascade Mediates the Activation of the Transcription Factor Insulin Upstream Factor 1 and Insulin Gene Transcription by High Glucose in Pancreatic β-Cells

Insulin upstream factor 1 (IUF1), a transcription factor present in pancreatic beta-cells, binds to the sequence C(C/T)TAATG present at several sites within the human insulin promoter. Here we isolated and sequenced cDNA encoding human IUF1 and exploited it to identify the signal transduction pathway by which glucose triggers its activation. In human islets, or in the mouse beta-cell line MIN6, high glucose induced the binding of IUF1 to DNA, an effect mimicked by serine/threonine phosphatase inhibitors, indicating that DNA binding was induced by a phosphorylation mechanism. The glucose-stimulated binding of IUF1 to DNA and IUF1-dependent gene transcription were both prevented by SB 203580, a specific inhibitor of stress-activated protein kinase 2 (SAPK2, also termed p38 mitogen-activated protein kinase, reactivating kinase, CSBP, and Mxi2) but not by several other protein kinase inhibitors. Consistent with this finding, high glucose activated mitogen-activated protein kinase-activated protein kinase 2 (MAPKAP kinase-2) (a downstream target of SAPK2) in MIN6 cells, an effect that was also blocked by SB 203580. Cellular stresses that trigger the activation of SAPK2 and MAPKAP kinase-2 (arsenite, heat shock) also stimulated IUF1 binding to DNA and IUF1-dependent gene transcription, and these effects were also prevented by SB 203580. IUF1 expressed in Escherichia coli was unable to bind to DNA, but binding was induced by incubation with MgATP, SAPK2, and a MIN6 cell extract, which resulted in the conversion of IUF1 to a slower migrating form. SAPK2 could not be replaced by p42 MAP kinase, MAPKAP kinase-2, or MAPKAP kinase-3. The glucose-stimulated activation of IUF1 DNA binding and MAPKAP kinase-2 (but not the arsenite-induced activation of these proteins) was prevented by wortmannin and LY 294002 at concentrations similar to those that inhibit phosphatidylinositide 3-kinase. Our results indicate that high glucose (a cellular stress) activates SAPK2 by a novel mechanism in which a wortmannin/LY 294002-sensitive component plays an essential role. SAPK2 then activates IUF1 indirectly by activating a novel IUF1-activating enzyme.