Investigation of signalling cascades induced by neurotrophic synaptolepis factor K7 reveals a critical role for novel PKCε

Abstract

This study elucidates signalling cascades involved in the neurotrophic effects induced by an active compound of Synaptolepis kirkii, a plant that is used against snakebites and for treatment of epilepsy. The active compound of this plant, synaptolepis factor K7 (K7), is suggested to exert anti-tumoral and neurotrophic actions via modulation of PKC. In SH-SY5Y cells synthesis of the neuronal marker growth-associated protein 43 was increased upon 48 h treatment with K7. Immunofluorescent staining of neurites revealed an increased neurite formation by synaptolepis factor K7. Short-term signal transduction events were followed at the level of extracellular-regulated kinase phosphorylation. Extracellular-regulated kinase (ERK) phosphorylation was transiently increased upon stimulation with synaptolepis factor K7 (300 nM) with a maximal effect at 30 min. Use of the general PKC inhibitor bisindolylmaleimide I blocked the K7-induced ERK phosphorylation suggesting involvement of PKC. Conversely, inhibition of conventional PKCs, α, β and γ by treatment with Go6976 did not inhibit ERK phosphorylation up to 1 μM. Use of a specific-PKCε translocation inhibitor peptide or RNAi-mediated knockdown of PKC-epsilon (ε) abolished the K7-induced ERK phosphorylation implicating PKCε in K7 function. This was confirmed by the observed increase in PKCε translocation and autophosphorylation induced by the compound. These data show that synaptolepis factor K7 induces neuronal differentiation of SH-SY5Y cells concomitant with a transient increase in ERK phosphorylation that is mediated by activation of PKCε.

Introduction

Daphnane diterpene esters, a class of phytochemicals isolated from medicinal plants, have been shown to have neurotrophic and anti-tumoral properties (He et al., 2002). Although, other mechanisms of action have been reported (Zhang et al., 2006), some of these compounds were shown to modulate protein kinase C (PKC) activity (Yoshida et al., 1996). Given the fact that PKC isoforms are promising targets in the field of cancer therapy and treatment of neurological disorders, the mechanism of action via PKCs remains an interesting area of study (Teng and Tang 2006; Podar et al., 2007, Griner and Kazanietz, 2007, Battaini, 2001). PKCs can be divided in ‘classical’ (α, βI, βII, γ); ‘novel’ (δ, ε, η, θ); and ‘atypical’ (ζ, λ(mouse)/ι(human)) isoforms (Nishizuka, 1995, Hernandez et al., 2003). A crucial step in PKC activation is the translocation to distinct subcellular domains, mediated by C1 and C2 domains. While novel PKCs have an intrinsic high affinity for DAG, conventional PKCs need intracellular Ca²⁺ to bind DAG (Giorgione et al., 2006). DAG binding can be mimicked by phorbol esters like phorbol myristate acetate (PMA), a potent PKC activator. Interestingly, mezerein, a daphnane diterpene ester-related compound, has been shown to compete with phorbol ester binding, suggesting that this class of molecules have a similar mode of action (Driedger et al., 1994).

Synaptolepis kirkii Oliv. is a tropical plant from Central-East Africa of which the roots are used against snakebites and epilepsy. A dichloromethane extract from this plant resulted in different diterpene esters, including synaptolepis factor K7 (K7) (He et al., 2002). Besides inhibition of tumoral growth, this molecule has been reported to mediate neuronal survival in primary chicken dorsal root ganglion neurons (He et al., 2002). A model system to study actions of K7 is the human neuroblastoma SH-SY5Y cell line. This is a cell line of neuronal origin with many interesting features, particularly the ability to differentiate and form neurite processes. Expression of the neuronal marker growth-associated protein 43 (GAP43) is used as a biochemical marker of these neurite extensions (Monaghan et al., 2008, Olsson and Nanberg, 2001). Together with other functional neuronal cell markers, synaptophysin and synaptotagmin, its expression indicates the presence of functional synapses in vitro and in vivo (Korshunova and Mosevitsky, 2010, Masliah et al., 2001).

Different PKC isoforms have been reported to modulate neuroprotection and differentiation in SH-SY5Y cells (Levites et al., 2002, Troller et al., 2001). Overexpression of either PKCδ or PKCε has been shown to induce neurite outgrowth. This can be mimicked by transfection with PKCδ or ε regulatory domain, suggesting that kinase activity is not required for this effect. In addition, PKCε-induced neurite outgrowth has been shown to involve phosphatidylinositol-4,5-diphosphate binding (Shirai et al., 2007).

In this manuscript we show that K7 induces neuronal differentiation of SH-SY5Y cells and focus on the elucidation of the signalling cascades induced by this molecule. In this respect, a clear role for PKCε as one of the key mediators of K7-induced signal transduction pathways could be demonstrated.