The previously described endogenous patterns of IGF induced
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 86 5 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). Additionally, we show here that treatment with IGF-1 and 17-β-estradiol elicit distinct temporal transcriptional responses from estrogen receptors in the Neuro-2A cell line. Treatment with estradiol resulted in a progressively increasing rate of ERE-dependent gene expression over a 24-h time-course. In contrast, IGF-1 treatment elicited only a transient response, peaking at 6 h and terminating 12 h after treatment. It is not clear from the data presented here why the response to IGF-1 was transient. IGF-1 dependent activation of MAPK ceased within 15 min of treatment while IGF-1-dependent activation of PI3K lasted for at least 2 h but had terminated 24 h after treatment (see Supplemental Figure 1). The response may be actively terminated or the cells may gradually stop responding as the IGF-1 treatment is naturally degraded. In either case, the termination of response to IGF-1 mirrors the termination of the transient MAPK burst, which is accelerated by IGF-1-induced activation of PI3K. This potentially creates circumstances in which MAPK, the critical kinase that promotes estrogen receptor activation, is actively inhibited while PI3K, a kinase that opposes MAPK-dependent activation of estrogen receptors, remains highly active for at least 2 h. We hypothesize that this ultimately results in a prolonged refractory period during which estrogen receptors will be unable to respond to subsequent MAPK stimulatory events. Further studies would be necessary to confirm this hypothesis. The interaction of IGF-1 and estrogen signaling has been described in various models of neuroprotection (Quesada et al., 2008; Kong et al., 2016; Hayashi et al. 2013, 2014), but the direct involvement of ERE-dependent gene expression in these various models was not directly tested. MAPK has previously been shown to activate ERE-dependent gene expression by exogenously expressed ERα in the non-neuronal COS-1 cell line (Kato et al., 1995) and the SK-N-BE neuroblastoma cells (Patrone et al., 1998). Previous wok investigating activation of endogenous estrogen receptors in the Neuro-2A cell line found that IGF-1 and estradiol treatment both enhanced ERE-dependent expression of the SEAP reporter protein 24 h after treatment (Mendez and Garcia-Segura, 2006). However, co-application of IGF-1 and estradiol reduced the estrogen receptor response relative to treatment with estradiol alone. Application of wortmannin not only blocked the IGF-1-dependent reduction in estrogen receptor activity but enhanced activity to levels far above that of treatment with estradiol alone. The results presented here describe a dynamic time-course of IGF-1-dependent regulation of estrogen receptor activity. IGF-1 treatment rapidly activates both MAPK and PI3K, but MAPK activity is rapidly terminated while PI3K activity remains elevated for at least 2 h but not as long as 24 h after treatment (see Supplemental Figure 1). The SEAP reporter protein has a half-life of about 22 h in culture (Salucci et al., 2002), therefore this reporter system gives an indication about how active estrogen receptors have been over the entire previous day. In contrast, the Firefly luciferase reporter protein has a half-life of only about 2 h (Ignowski and Schaffer, 2004), meaning this reporter system gives an indication about how active estrogen receptors have been over the previous few hours. The high temporal resolution of this reporter system allowed us to separate the short-term MAPK-dependent estrogen receptor activation from the longer term PI3K-dependent inhibition of ERE-dependent gene expression described previously.