IGF treatment elicited a rapid and short burst

IGF-1 treatment elicited a rapid and short burst of MAPK activation in Neuro-2A 487 that is terminated within 15 min of stimulation. The MAPK inhibitor, U0126, completely blocked IGF-1-dependent induction of ERE-dependent gene expression, indicating that it is the critical kinase that mediates IGF-1-dependent activation of estrogen receptors. We additionally identified a novel function of PI3K in rapid regulation of estrogen receptor activation that is distinct from the previously described long-term regulation of estrogen receptor stability (Mendez and Garcia-Segura, 2006). In the present work, application of IGF-1 in the presence of the PI3K inhibitor, wortmannin, enhanced both the magnitude and duration of the transient MAPK burst and also enhanced IGF-1-dependent induction of ERE-dependent gene expression. Furthermore, wortmannin-dependent enhancement of nuclear estrogen receptor activity was not observed in the presence of the MAPK inhibitor U0126. This indicates that PI3K-dependent regulation of nuclear estrogen receptors is achieved through regulation of MAPK activity and not through some unidentified, independent pathway. However, support for this interpretation is limited by the use of a single pharmacological agent for each kinase pathway. We cannot rule out the possibility that these inhibitors had unexpected off target effects similar to previous work with the PI3K inhibitor LY294002 (Pasapera Limón et al., 2003). The present results are also limited by the lack of replication of IGF-1-dependent rapid activation of MAPK 5 min after treatment. However, this effect has already been described previously in a number of cell types including the Neuro-2A cell line used in the present work (Munderloh et al., 2009). The use of additional pharmacological inhibitors or genetic techniques for manipulation of MAPK and PI3K activity would confirm and refine the mechanisms of IGF-1-dependent interaction between these two kinases. The previously described endogenous patterns of IGF-1-induced kinase signaling, and resulting interaction between MAPK and PI3K, differ across neuronal sub-types. In R28 retinal neuron-like cells, IGF-1 treatment elicits prolonged activation (at least 80 min) of both PI3K and MAPK (Kong et al., 2016). IGF-1 activates PI3K but not MAPK in PC12 neural crest cells but activates both PI3K and MAPK in primary culture of cortical neurons (Wang et al., 2012). IGF-1 activates PI3K and inhibits MAPK in substantia nigra pars compacta dopaminergic neurons in rat model of Parkinson\'s disease (Quesada et al., 2008). IGF-1R-dependent activation of PI3K activates ERK via c-raf but independent of Akt in trigeminal ganglion neurons (Wang et al., 2014). Chochlear hair cell survival is enhanced by IGF-1 through a mechanism that requires both PI3K and MAPK and results in transient (12 h) burst in survival-related gene expression (Hayashi et al. 2013, 2014). The pattern of activation in Neuro-2A cells described here is most similar to that of primary cultured hippocampal neurons. IGF-1 treatment results in sustained activation of PI3K but transient activation of MAPK while BDNF treatment results in sustained activation of both cascades in primary cultured hippocampal neurons (Zheng and Quirion, 2004).