Militarinone A induces differentiation in PC12 cells via MAP and Akt kinase signal transduction pathways

Abstract

The fungal metabolite militarinone A (MILI A) promotes neurite outgrowth in PC12 cells. This study was conducted to investigate the signaling pathways involved in the cellular differentiation processes induced by the compound, with a focus on cascades implicated with nerve growth factor (NGF)-mediated neuritogenesis. MILI A possessed pronounced amphiphilic properties. The compound rapidly accumulated in the cell membrane and was slowly released into the cytoplasma. In primed PC12 cells, an early activation of protein kinase B (Akt), representing a downstream target of phosphoinositol 3 (PI 3 ) kinase, and a delayed phosphorylation of extracellular signalregulated kinases 1 and 2 (ERK1/2), and of transcription factor cAMP responsive element binding protein (CREB) was found. The NGF-dependent activation of c-Jun amino terminal kinase (SAPK/JNK1) was potentiated. Morphological differentiation of cells and the phosphorylation of specific signal molecules were blocked by the MAP kinase (MEK1) inhibitor PD098059, the PI 3 -kinase (PI 3 K) inhibitor wortmannin and the adenylyl cyclase inhibitor 9-cyclopentyladenine.

Promotion of cell death or neurite outgrowth in PC-12 and N2a cells by the fungal alkaloid militarinone A depends on basal expression of p53

“…When applying 40 lM MiliA to PC-12 cells stably over-expressing TrkA (TA) and naïve PC-12, we observed similar neuronal outgrowth in both cases ( Fig. 1 and [3]. In addition, phosphorylation of PKB and MAPKs in TA (not shown) and naïve PC-12 was found (Fig.…”

“…We previously reported [3] that addition of 40 lM MiliA to PC-12 cells for 24 h after pre-treatment with NGF [34] induced neurite outgrowth which was attributed to the NGFdependent up-regulation of its receptor TrkA. However, pretreatment with a commercially available TrkA-inhibitor that was able to prevent NGF-induced phosphorylation of PKB and MAPKs, did not prevent MiliA-dependent phosphorylation of these proteins (Fig.…”

“…Militarinone A has been reported to rapidly integrate into the membranous fraction [3] due to its amphiphilic properties [36], whereas release into the cytoplasm was markedly slower. As both PC-12 and N2a cells showed release of AIF, we isolated whole, viable mitochondria …”

“…Both cell types were grown in humified atmosphere containing 5% CO 2 . PC-12 cells stably overexpressing TrkA were a kind gift from Prof. Frank-D. Böhmer, (Institute of Molecular Cell Biology, Jena, Germany) and were treated as described [3]. The following chemicals have been used: LY294.002, zVAD, pifithrin a (PFTa) (all from Alexis Corporation, Lausen, Switzerland); N-Acetyl-L-cysteine (NAC) and nerve growth factor (NGF) (both from Sigma-Aldrich, Buchs, Switzerland).…”

“…Small non-peptidic molecules with neurotrophic properties, and capable of penetrating the blood-brain barrier, have been proposed as an alternative [2]. In the course of a screening for such molecules we discovered that the fungal alkaloid militarinone A (MiliA) induced neurite extension in PC-12 cells [3]. Due to its amphiphilic properties MiliA was rapidly integrated into the membranous fraction where it triggered PI3-K/ PKB-and ERK1/2-dependent differentiation [4,5].…”