Abstract
Small-molecule mixed-lineage kinase (MLK) inhibitors, such as CEP-1347 [3,9-bis[(ethylthio)methyl]-(8R*,9S*,11S*)-(–)-9-hydroxy-9-methoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H, 11H-2,7b,11a-triazadibenzo(a,g)cycloocta(cde)trinden-1-one] and CEP-11004 [3,9-bis-[(isopropylthio)methyl]-(8R*,9S*,11S*)-(–)-9-hydroxy-9-methoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b,11a-triazadibenzo(a,g)cycloocta(cde)trinden-1-one], prevent c-Jun NH2-terminal kinase (JNK) pathway activation as well as the consequent neuronal cell death in many cell culture and animal models. In the cell culture model of nerve growth factor (NGF)-deprived sympathetic neurons, we find that CEP-11004 induced a ∼3-fold increase in the mRNA and protein levels of TrkA, the NGF receptor. This resulted in ligand-independent activation of the TrkA receptor and the downstream phosphatidylinositol 3-kinase (PI3-kinase) pathway. Addition of the Trk inhibitor K252a [(8R*,9S*,11S*)-(–)-9-hydroxy-9-methoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b,11a-triazadibenzo(a,g)cycloocta(cde)-trinden-1-one] or the PI3-kinase inhibitor LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one] significantly decreased the protein synthesis rates, mitochondrial function, and neuronal survival maintained by CEP-11004. In contrast to sympathetic neurons, MLK inhibitors maintain only short-term survival of potassium- and serum-deprived rat cerebellar granule neurons (CGNs), despite continuous inhibition of the JNK pathway. We found that similar to sympathetic neurons, CEP-11004 increased the levels of the Trk receptor expressed in CGNs, TrkB. However, CGNs required the addition of the exogenous ligand brain-derived neurotrophic factor (BDNF) to activate the PI3-kinase pathway and to maintain long-term survival. BDNF activated TrkB, but caused rapid down-regulation of activated receptors and maintained only minimal survival. Therefore, increase in TrkB levels by CEP-11004 mediated a synergism with BDNF resulting in long-term survival in response to the combined treatment of CEP-11004 and BDNF. Taken together, our studies suggest that in addition to the direct inhibition of the JNK pathway, the indirect activation of the PI3-kinase pathway via Trk activation is important for MLK inhibitor-mediated neuronal survival and trophism.
Footnotes
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This work was supported by National Institutes of Health Grants R37AG12947 and R01NS38651 (E.M.J.).
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doi:10.1124/jpet.104.077800.
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ABBREVIATIONS: JNK, c-Jun NH2-terminal kinase; K252a, (8R*,9S*,11S*)-(–)-9-hydroxy-9-methoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b,11a-triazadibenzo(a,g)cycloocta(cde)trinden-1-one; CEP-1347, 3,9-bis[(ethylthio)methyl]-K252a; CEP-11004, 3,9-bis-[(isopropylthio)methyl]-K252a; MLK, mixed-lineage kinase; NGF, nerve growth factor; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PI3-kinase, phosphatidylinositol 3-kinase; CGNs, cerebellar granule neurons; BDNF, brain-derived neurotrophic factor; LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; BAF, boc-aspartyl fluoromethyl ketone; DMEM, Dulbecco's modified Eagle's medium; SCG, superior cervical ganglia; MEM, minimum essential medium; DIV, days in vitro; AM, acetoxymethyl ester; TBST, Tris-buffered saline/Tween 20; MAP, mitogen-activated protein; PCR, polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ANOVA, analysis of variance; ERK, extracellular signal-regulated kinase; ZM 241385, 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5] triazin-5-yl-amino]ethyl)phenol; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]-pyrimidine; SU6656, 2-oxo-3-(4,5,6,7-tetrahydro-1H-indol-2-ylmethylene)-2,3-dihydro-1H-indole-5-sulfonic acid dimethylamide; K25+S, high potassium and 10% serum; K5-S, low potassium and no serum; PACAP, pituitary adenylate cyclase-activating peptide.
- Received September 13, 2004.
- Accepted November 1, 2004.
- The American Society for Pharmacology and Experimental Therapeutics
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