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  • We hypothesize that both IGF dependent kinase activation

    2020-03-18

    We hypothesize that both IGF-1-dependent kinase activation and locally synthesized neuroestrogens interactively regulate estrogen receptor activity in neuronal lenvatinib in the absence of exogenously applied estradiol. The Neuro-2A cell line was chosen as the model system for these studies because IGF-1-dependent regulation of nuclear estrogen receptors has already been established and has been shown to interact with the application of exogenous estradiol (Mendez and Garcia-Segura, 2006). The goal of the present work is to integrate the effects of MAPK activation, PI3K activation, exogenously applied estradiol, and endogenous neuroestrogen synthesis in determining the transcriptional output of estrogen receptors in Neuro-2A cells. In a series of experiments, we determined the sequence of kinase cascade activation events that occur following IGF-1 treatment. We then determined how activation of these two kinases contributes to regulation of estrogen receptor-dependent gene transcription. We finally determined the contribution of neuroestrogen synthesis to basal and IGF-1-dependent nuclear estrogen receptor activation.
    Methods
    Results
    Discussion Basal phosphorylation of both ERK and Akt was detectable by Western blot in the absence of any treatment, indicating that both MAPK and PI3K have some level of basal activity in Neuro-2A cells. Inhibition of basal MAPK activity with U0126 resulted in a significant increase in PI3K activity while inhibition of basal Akt activity with wortmannin had no effect on MAPK activity. This result suggests MAPK non-selectively suppresses PI3K while the observed PI3K-dependent suppression of MAPK is specific to IGF-1 treatment. Although only the main effects of MAPK and PI3K inhibition on ERE-dependent gene expression were statistically significant, a post-hoc test performed to directly compare ERE-dependent transgene expression between the DMSO pretreatment, vehicle treatment group and the U0126 pretreatment, vehicle treatment groups was also trending toward significance (p = 0.067). Furthermore, as wortmannin pretreatment alone resulted in a non-statistically significant increase in estrogen receptor activation, ERE-dependent gene expression was significantly higher in the wortmannin pretreatment, vehicle treatment group than the U0126 pretreatment, vehicle treatment group (p < 0.001). These results suggest that basal MAPK and PI3K activities might each perform a role in regulation of basal estrogen receptor activity in Neuro-2A cells, but this hypothesis would need to be confirmed with additional experimentation. A thorough identification of the sources of basal MAPK and PI3K activity in neuronal cells and how they affect estrogen receptor activity in the absence of applied estrogens is an interesting avenue for further investigation. IGF-1 treatment elicited a rapid and short burst of MAPK activation in Neuro-2A cells 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.