Complete inhibition of anisomycin and UV radiation but not cytokine induced JNK and p38 activation by an aryl-substituted dihydropyrrolopyrazole quinoline and mixed lineage kinase 7 small interfering RNA.

Mixed lineage kinase 7 (MLK7) is a mitogen-activated protein kinase kinase kinase (MAPKKK) that activates the pro-apoptotic signaling pathways p38 and JNK. A library of potential kinase inhibitors was screened, and a series of dihydropyrrolopyrazole quinolines was identified as highly potent inhibitors of MLK7 in vitro catalytic activity. Of this series, an aryl-substituted dihydropyrrolopyrazole quinoline (DHP-2) demonstrated an IC50 of 70 nM for inhibition of pJNK formation in COS-7 cell MLK7/JNK co-transfection assays. In stimulated cells, DHP-2 at 200 nM or MLK7 small interfering RNA completely blocked anisomycin and UV induced but had no effect on interleukin-1beta or tumor necrosis factor-alpha-induced p38 and JNK activation. Additionally, the compound blocked anisomycin and UV-induced apoptosis in COS-7 cells. Heart tissue homogenates from MLK7 transgenic mice treated with DHP-2 at 30 mg/kg had reduced JNK and p38 activation with no apparent effect on ERK activation, demonstrating that this compound can be used to block MLK7-driven MAPK pathway activation in vivo. Taken together, these data demonstrate that MLK7 is the MAPKKK required for modulation of the stress-activated MAPKs downstream of anisomycin and UV stimulation and that DHP-2 can be used to block MLK7 pathway activation in cells as well as in vivo.

Stress-inducible protein 1 is a cell surface ligand for cellular prion that triggers neuroprotection.

Prions are composed of an isoform of a normal sialoglycoprotein called PrP(c), whose physiological role has been under investigation, with focus on the screening for ligands. Our group described a membrane 66 kDa PrP(c)-binding protein with the aid of antibodies against a peptide deduced by complementary hydropathy. Using these antibodies in western blots from two-dimensional protein gels followed by sequencing the specific spot, we have now identified the molecule as stress-inducible protein 1 (STI1). We show that this protein is also found at the cell membrane besides the cytoplasm. Both proteins interact in a specific and high affinity manner with a K(d) of 10(-7) M. The interaction sites were mapped to amino acids 113-128 from PrP(c) and 230-245 from STI1. Cell surface binding and pull-down experiments showed that recombinant PrP(c) binds to cellular STI1, and co-immunoprecipitation assays strongly suggest that both proteins are associated in vivo. Moreover, PrP(c) interaction with either STI1 or with the peptide we found that represents the binding domain in STI1 induce neuroprotective signals that rescue cells from apoptosis.

Cellular prion protein transduces neuroprotective signals.

To test for a role for the cellular prion protein (PrP(c)) in cell death, we used a PrP(c)-binding peptide. Retinal explants from neonatal rats or mice were kept in vitro for 24 h, and anisomycin (ANI) was used to induce apoptosis. The peptide activated both cAMP/protein kinase A (PKA) and Erk pathways, and partially prevented cell death induced by ANI in explants from wild-type rodents, but not from PrP(c)-null mice. Neuroprotection was abolished by treatment with phosphatidylinositol-specific phospholipase C, with human peptide 106-126, with certain antibodies to PrP(c) or with a PKA inhibitor, but not with a MEK/Erk inhibitor. In contrast, antibodies to PrP(c) that increased cAMP also induced neuroprotection. Thus, engagement of PrP(c) transduces neuroprotective signals through a cAMP/PKA-dependent pathway. PrP(c) may function as a trophic receptor, the activation of which leads to a neuroprotective state.

Differential effects of stress stimuli on a JNK-inactivating phosphatase.

Stress signals elicit a wide variety of cellular responses, many of which converge on the phosphorylation of JNK and p38 kinases, the activation of which has been well-characterized. How these kinases are switched off by dephosphorylation is not well understood. Here we describe how diverse cellular stresses affect differently the stability and activity of a JNK-inactivating dual-specificity threonine-tyrosine phosphatase M3/6. Both anisomycin and arsenite activate the JNK pathway and, in addition, inactivate the M3/6 phosphatase. However, while anisomycin treatment of cells leads to M3/6 protein degradation, arsenite appears to inactivate M3/6 directly. These results might have implications for the mechanism of tumour promotion by arsenic.

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 approximately 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.

[Effect of DGAVP and Org2766 on intracellular free Ca2+ concentration in dissociated brain cells].

Intracellular free calcium concentration ([Ca2+]i) was measured with Fura-2 in freshly dissociated brain cells isolated from newborn (1-2 day) mouse pups using AR-CM-MIC cation measurement system, and the effects of DGAVP and Org2766 on the changes in [Ca2+]i induced by the protein synthesis inhibitor anisomycin (ANI) were studied. The results indicate that anisomycin caused dose-dependent increases in [Ca2+]i; and DGAVP itself showed no significant effect on [Ca2+]i, but an appropriate dose of DGAVP antagonized the increases induced by the selective dose-range of ANI, suggesting that the antagonism of ANI-induced inhibition of protein synthesis by DGAVP was likely achieved by preventing ANI from increasing [Ca2+]i, but this mechanism did not apply to the other neuropeptide Org2766. Therefore, we suppose that the mechanism of the two neuropeptides are different in terms of their effect on intracellular free calcium concentration.

The antagonistic effects of naloxone on cycloheximide and anisomycin-induced amnesia.

The amnesia induced by cycloheximide (CXM) injected SC and by CXM or anisomycin injected ICV immediately after the training test was antagonized in combination with an opiate antagonist, naloxone (NLX). This antagonism occurred on both the passive avoidance- and escape-learning responses in a dose-dependent manner in mice. NLX alone (0.3-10.0 mg/kg) did not alter the performances of these tasks. Furthermore, the decrease in retention performance on shuttle avoidance in rats induced by CXM was also antagonized by NLX. Treatment with CXM and/or NLX did not affect spontaneous locomotor activity. The interaction of these drugs on the performance of the passive avoidance- and escape-learning and the shuttle avoidance tasks may be related to neurochemical memory processes. These results suggest that an opioid system may participate in the amnesic actions induced by protein synthesis inhibitors in these models.

Alleviation of anisomycin-induced amnesia by pre-test treatment with lysine- vasopressin.

Amnesia in mice for a passive avoidance response induced by anisomycin injection immediately after training was reversed by 40 micrograms of lysine-vasopressin given one hour before testing. Control groups receiving non-contingent shock instead of training were used to demonstrate that the effects of vasopressin were due to memory of shock received in a particular place, rather than non-specific suppression of locomotion. The effects of vasopressin on retention were not mimicked by either pentylenetetrazol or epinephrine suggesting that the enhanced latencies were probably not the result of increases in fear or arousal. These data support the hypothesis that the retrieval of memory can be facilitated by vasopressin. The possibility of a relationship between the effects of vasopressin and those of catecholamine manipulations on memory is discussed.