br Effects of estradiol outside the
Effects of estradiol outside the nucleus are often mediated by classical estrogen receptors Today, it is widely accepted that steroid hormones can signal in ways that do not rely on receptors acting in the nucleus, including activation of receptors localized to the cellular membrane. Rapid effects of estradiol were first observed in uterine tissue over 50 years ago (Szego and Davis, 1967). Increased concentrations of cAMP induced by estradiol administration occurred within seconds, outside the timeframe that was traditionally attributed to actions of steroid hormones. These rapid effects indicated that alternate signaling mechanisms must exist, and it was hypothesized even at that time that these actions of estradiol were mediated by membrane-localized receptors. Subsequent support for this theory came from radioactive ligand binding assays that showed that synaptic plasma membranes have SB 239063 for estradiol (Towle and Sze, 1983), and from observations of estrogen receptors on the plasma membrane in Xenopus oocytes (Sadler et al., 1985; Sadler and Maller, 1982). The idea that estrogen receptors were physiologically active at the surface membrane was highly contentious, with these early findings often dismissed as technical artifacts. Nevertheless, the field persisted, accumulating more and more evidence of membrane-localized estrogen receptors (Chaban et al., 2003; Kelly et al., 1999; Kelly and Levin, 2001; Levin, 2001; Razandi et al., 1999, Razandi et al., 2003). Although several different membrane estrogen receptors have been reported (Qiu et al., 2008; Revankar et al., 2005), a large percentage of membrane-initiated steroid hormone signaling appears to be performed by a subpopulation of the same receptors that act in the nucleus. Specifically, estrogen receptors (ERs) α and β are found at the plasma membrane and are synthesized from the same transcript as their nuclear counterparts (Razandi et al., 1999). We have known that ERα and ERβ are critical for membrane signaling for at least a decade, as their genetic knockout interferes with rapid estrogen-mediated activation of the MAPK/ERK pathway (Ábrahám et al., 2004). While Ábrahám and colleagues did not examine the subcellular location where ERα and ERβ triggered MAPK signaling, the existence of these estrogen receptors at the plasma membrane has since been observed in many brain regions using a variety of techniques (Micevych and Mermelstein, 2008; Pedram et al., 2009; Razandi et al., 2003). Though the evidence for rapid, non-nuclear initiated action of ERα and ERβ had been compelling for many years, the mechanism(s) by which these receptors signaled outside the nucleus had remained frustratingly unclear. When our lab set about to investigate the signaling pathways responsible for the effects of membrane-initiated estradiol signaling in the mid-2000s, the fundamental issue was that previous reports had typically examined second messenger signals far downstream of the membrane-initiated event. As such, the literature contained a plethora of descriptive studies examining the impact of estrogens on a wide array of cellular processes. For example, estradiol was reported to attenuate L-type calcium currents (Chaban et al., 2003; Mermelstein et al., 1996) as well as the aforementioned activation of MAPK (Gu and Moss, 1996; Lee et al., 2004; Wade and Dorsa, 2003; Zhou et al., 1996). Hence, we wanted to understand the full signaling pathways that were responsible for multiple effects of estradiol. To do so, we utilized an in vitro assay monitoring phosphorylation of the transcription factor CREB as a measure of cellular activation. We first replicated what others had reported, finding that a brief application of physiological estradiol concentrations increased CREB phosphorylation in CA3-CA1 hippocampal neurons from 1 to 2 day-old female rat pups (Boulware et al., 2005). This effect was dose dependent, rapid, and blocked by MEK inhibitors. We then went on to examine the interaction of estradiol with L-type calcium channel signaling. Increased synaptic activity leads to increased CREB phosphorylation via activation of L-type voltage-gated calcium channels, and this activation can be reproduced in cell culture by K+-mediated depolarization. In this assay, a brief stimulation of cells with 20 mM K+ robustly increases CREB phosphorylation via CaMKIV signaling (Deisseroth et al., 1996; Mermelstein et al., 2000). Pretreatment with estradiol attenuates depolarization-induced CREB phosphorylation, revealing the bidirectional effects of estradiol in its modulation of these two discrete pathways. Our experiments additionally showed that these effects of estradiol are postsynaptic, occur via membrane receptors, and, importantly, do not occur in cultures from male animals (Boulware et al., 2005).