Finally we have determined for the first time
Finally, we have determined for the first time that aromatase enzyme activity is necessary to observe IGF-1-dependent ddhUTP structure receptor activation. Neuroestrogen synthesis has been shown to be important for the maintenance of synaptic spine density in hippocampal slice cultures (Kretz et al., 2004) and has been shown to support cognitive function in postmenopausal women (Bender et al., 2015; Collins et al., 2009; Bayer et al., 2015) and ovariectomized rodents (Vierk et al., 2012; Martin et al., 2003). The present work shows a specific requirement for aromatase enzyme activity in the short-term IGF-1-dependent activation of ERE-dependent gene expression. Similar work has shown that aromatase enzyme activity was necessary for rapid activation of Arc and PSD-95 mRNA synthesis in the hippocampal H19-7 immortalized cell line (Chamniansawat and Chongthammakun, 2012). In vivo work in a mouse model of Alzheimer's disease pathology found that levels of brain-derived estradiol alter the efficacy of estrogen replacement therapy in a mouse model of Alzheimer's disease pathology (Li et al., 2013). Like the present work, these observations indicate that exogenously applied estradiol and endogenously synthesized neuroestrogens are not always interchangeable and result in distinct patterns of cell signaling and estrogen dependent gene regulation. Previous results suggest that relief of ligand-binding domain inhibition is necessary to unmask MAPK-dependent activation of ERα (Ali et al., 1993; Kato et al., 1995). We suggest that in Neuro-2A cells, endogenous estrogen synthesis provides a constant source of minimal ligand sufficient to relieve ligand-binding domain inhibition and permit responsiveness to the IGF-1 stimulatory signal. However, the present work does not rule out alternative interpretations of the data presented. Aromatase activity can also be rapidly activated through intracellular kinase signaling (Fester et al., 2015). It is possible that IGF-1 dependent activation of MAPK directly upregulates aromatase enzyme activity, activating nuclear estrogen receptor activity indirectly through an autocrine mechanism. Additionally, estrogen signaling has also been shown to activate a variety of kinase pathways in neuronal (Boulware et al., 2005) and neuroblastoma cells (Clark et al., 2014; Ni et al., 2015; Takahashi et al., 2011). It is therefore also possible that local aromatase enzyme-dependent estrogen synthesis initiates estrogen-dependent kinase signaling that interacts with IGF-1-dependent kinase signaling and modulates IGF-1-dependent effects on nuclear estrogen receptor activation. Further experimentation will be necessary to refine these observations and elucidate the full mechanism of interaction. Unexpectedly, co-application of exogenous estradiol along with IGF-1 after aromatase enzyme inhibition did not result in the same short-term level of estrogen receptor activation as application of exogenous estradiol alone after aromatase enzyme inhibition. Small differences in the sequence of stimulatory events experienced by each treatment group can account for this difference. IGF-1 dependent MAPK activation reaches its maximal level within 5 min and returns to near baseline levels after 15 min of treatment. It has been shown that estradiol-induced dimerization of ERα is negligible within the first 15 min of estradiol application and does not reach maximum levels until 2 h after treatment (Powell and Xu, 2008). Cultures pretreated with the aromatase enzyme inhibitor followed by treatment with the combination of estradiol and IGF-1 therefore first experienced rapid activation of MAPK and PI3K followed by slower ligand-induced dimerization, while cultures pretreated with aromatase enzyme inhibitor followed by treatment with exogenous estradiol alone experienced only slower ligand-induced dimerization with no intervening kinase activation. The high temporal resolution of the Firefly luciferase reporter systems may be able to detect these differences where other reporter systems with lower temporal resolution would not. Furthermore, aromatase enzyme inhibition had no effect on the response of nuclear estrogen receptors to co-application of estradiol and IGF-1 24 h after treatment; long after IGF-1-dependent regulation of nuclear estrogen receptors had ceased. Aromatase enzyme inhibition only interfered with the response to co-application of estradiol and IGF-1 within the first 6 h when IGF-1 treatment was still effective.