Motoneuron apoptosis is blocked by CEP-1347 (KT 7515), a novel inhibitor of the JNK signaling pathway - PubMed (original) (raw)

. 1998 Jan 1;18(1):104-11.

doi: 10.1523/JNEUROSCI.18-01-00104.1998.

M A Glicksman, A N Basma, K M Walton, E Knight Jr, C A Murphy, B A Bartlett, J P Finn, T Angeles, Y Matsuda, N T Neff, C A Dionne

Affiliations

Motoneuron apoptosis is blocked by CEP-1347 (KT 7515), a novel inhibitor of the JNK signaling pathway

A C Maroney et al. J Neurosci. 1998.

Abstract

Neurons undergoing apoptosis can be rescued by trophic factors that simultaneously increase the activity of extracellular signal-regulated kinase (ERK) and decrease c-Jun N-terminal kinase (JNK) and p38. We identified a molecule, CEP-1347 (KT7515), that rescues motoneurons undergoing apoptosis and investigated its effect on ERK1 and JNK1 activity. Cultured rat embryonic motoneurons, in the absence of trophic factor, began to die 24-48 hr after plating. During the first 24 hr ERK1 activity was unchanged, whereas JNK1 activity increased fourfold. CEP-1347 completely rescued motoneurons for at least 72 hr with an EC50 of 20 +/- 2 nM. CEP-1347 did not alter ERK1 activity but rapidly inhibited JNK1 activation. The IC50 of CEP-1347 for JNK1 activation was the same as the EC50 for motoneuron survival. Inhibition of JNK1 activation by CEP-1347 was not selective to motoneurons. CEP-1347 also inhibited JNK1 activity in Cos7 cells under conditions of ultraviolet irradiation, osmotic shock, and inhibition of glycosylation. Inhibition by CEP-1347 of the JNK1 signaling pathway appeared to be selective, because CEP-1347 did not inhibit p38-regulated mitogen-activated protein kinase-activated protein kinase-2 (MAPKAP2) activity in Cos7 cells subjected to osmotic shock. The direct molecular target of CEP-1347 was not JNK1, because CEP-1347 did not inhibit JNK1 activity in Cos7 cells cotransfected with MEKK1 and JNK1 cDNA constructs. This is the first demonstration of a small organic molecule that promotes motoneuron survival and that simultaneously inhibits the JNK1 signaling cascade.

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Figures

Fig. 1.

Fig. 1.

Structure of CEP-1347. CEP-1347 was synthesized by derivitization of K-252a, an indolocarbazole isolated from culture broths of Nocardiosis.

Fig. 2.

Fig. 2.

Time course of motoneuron death in the absence or presence of CEP-1347. Cells were plated at a density of 6 × 104 cells/cm2 in chemically defined N2 medium. After 2 hr to allow for attachment, cells were incubated with 0.006% DMSO (control), 250 n

m

CEP-1347, or 5–10 μ

m

SB203580 and monitored for cell viability over 3 d. Cell viability was measured by using the calcein AM assay as described in Materials and Methods. Experimental data represent the mean ± SD, _n_= 4; DMSO control data represent the mean ± SD,n = 12. Three independent experiments were performed; data presented are from one representative experiment. *p < 0.05, by Dunnett’s _t_statistics, significantly different from control cultures.

Fig. 3.

Fig. 3.

Apoptosis of enriched E14.5 motoneurons in the absence or presence of CEP-1347. Cells were plated at a density of 6 × 104 cells/cm2 in chemically defined N2 medium. After 2 hr to allow for attachment, control cells were incubated with 0.006% DMSO control (a, c) or 250 n

m

CEP-1347 (b, d) for 5 d, followed by fixation and photography with Hoffman modulating contrast optics (a, b), or for 2 d, followed by staining with Hoechst dye (c, d) to detect condensed chromatin.

Fig. 4.

Fig. 4.

ERK1 and JNK1 activity in the absence or presence of CEP-1347. Cultures of enriched E14.5 motoneurons were treated with 0.01% DMSO control or 500 n

m

CEP-1347 for various times, as indicated. Cells were lysed in 1% Triton buffer, and the lysate was immunoprecipitated with the ERK1 (A) or JNK1 (B) antibody. The immunoprecipitates were assayed for kinase activity by using myelin basic protein or c-Jun, respectively, as substrates. Experiments were performed at least two times, and results from representative experiments are shown.Points represent the average of duplicate samples; error bars indicate the SEM.

Fig. 5.

Fig. 5.

Dose–response of inhibition of JNK1 activity and cell survival by CEP-1347. Cultures of enriched E14.5 motoneurons were plated and allowed to adhere 2.5 hr before the addition of the indicated concentrations of CEP-1347. For JNK1 activity, cells were collected 22 hr after the addition of compound and assayed for kinase activity as described in Figure 4; cell viability was determined by calcein AM assay after 5 d in culture. The percentage of cell viability is relative to untreated controls, which is equivalent to 100%. Points represent the average of duplicate samples; the error bars indicate the SEM.

Fig. 6.

Fig. 6.

JNK1 activity in Cos7 cells overexpressing HA–JNK1 alone or with MEKK1. Cos7 cells were grown to 80% confluency and transfected with HA-tagged JNK1 alone or with MEKK1 at various amounts of cDNA, as indicated. After a 48 hr period the cells were incubated with 0.01% DMSO or 500 n

m

CEP-1347 for 2 hr, followed by lysis in 1% Triton buffer. Lysate was normalized to protein and immunoprecipitated with HA antibody. The immunoprecipitates were assayed for kinase activity with the _c-jun_substrate. Experiments were performed at least two times, and results from representative experiments are shown. Activity is expressed by fold increase relative to untreated HA–JNK-transfected cells.Columns represent the average of duplicate samples; the error bars indicate the SEM.

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