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Comparative Study
. 2005 May 18;25(20):5066-78.
doi: 10.1523/JNEUROSCI.1427-05.2005.

Estradiol activates group I and II metabotropic glutamate receptor signaling, leading to opposing influences on cAMP response element-binding protein

Marissa I Boulware  1 , Jason P Weick, Bryan R Becklund, Sidney P Kuo, Rachel D Groth, Paul G Mermelstein
Affiliations
Comparative Study

Estradiol activates group I and II metabotropic glutamate receptor signaling, leading to opposing influences on cAMP response element-binding protein

Marissa I Boulware et al. J Neurosci. .

Abstract

In addition to mediating sexual maturation and reproduction through stimulation of classical intracellular receptors that bind DNA and regulate gene expression, estradiol is also thought to influence various brain functions by acting on receptors localized to the neuronal membrane surface. Many intracellular signaling pathways and modulatory proteins are affected by estradiol via this unconventional route, including regulation of the transcription factor cAMP response element-binding protein (CREB). However, the mechanisms by which estradiol acts at the membrane surface are poorly understood. Because both estradiol and CREB have been implicated in regulating learning and memory, we characterized the effects of estradiol on this transcription factor in cultured rat hippocampal neurons. Within minutes of administration, estradiol triggered mitogen-activated protein kinase (MAPK)-dependent CREB phosphorylation in unstimulated neurons. Furthermore, after brief depolarization, estradiol attenuated L-type calcium channel-mediated CREB phosphorylation. Thus, estradiol exhibited both positive and negative influences on CREB activity. These effects of estradiol were sex specific and traced to membrane-localized estrogen receptors that stimulated group I and II metabotropic glutamate receptor (mGluR) signaling. Activation of estrogen receptor alpha (ERalpha) led to mGluR1a signaling, triggering CREB phosphorylation through phospholipase C regulation of MAPK. In addition, estradiol stimulation of ERalpha or ERbeta triggered mGluR2/3 signaling, decreasing L-type calcium channel-mediated CREB phosphorylation. These results not only characterize estradiol regulation of CREB but also provide two putative signaling mechanisms that may account for many of the unexplained observations regarding the influence of estradiol on nervous system function.

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Figures

Figure 1.
Figure 1.
Estradiol rapidly stimulates MAPK-dependent CREB phosphorylation. A, Example confocal images of cultured CA3-CA1 female hippocampal neurons immunolabeled with MAP2 (green) and pCREB (red). Pyramidal neurons exhibited heightened nuclear staining for pCREB after a 5 min application of estradiol (17βE; 1 nm). No Stim, No stimulation. Scale bar, 20 μm. B, Quantification of the immunolabeling demonstrated that estradiol significantly increased CREB phosphorylation (F = 183.79; **p < 0.01). Furthermore, the effect of estradiol was eliminated after inhibition of MEK with PD98059 (PD; 25 μm). C, Estradiol increased CREB phosphorylation in a concentration-dependent manner (F = 14.73; p < 0.05 at 1 pm; p < 0.01 at all other concentrations when compared with control). D, Time course of estradiol-induced CREB phosphorylation. E, Western blot verification of estradiol-induced CREB phosphorylation (∼46 kDa band). ERK was used as the loading control. F, Application of estradiol also increased CRE-dependent transcription (T = 4.42; **p < 0.01). Error bars indicate SEM.
Figure 2.
Figure 2.
Estradiol attenuates L-type calcium channel-mediated CREB phosphorylation. A, Estradiol alone resulted in an increase (F = 16.97; **p < 0.01) in CREB phosphorylation (left 2 bars). However, estradiol treatment in the presence of a 20 mm K+ stimulus led to a significant (**p < 0.01) attenuation of depolarization-induced CREB phosphorylation (middle 2 bars). Pretreatment with the L-type calcium channel blocker nifedipine (NIF; 5 μm) eliminated depolarization-dependent CREB phosphorylation and unmasked estradiol-induced CREB phosphorylation (right 2 bars; **p < 0.01). B, Estradiol produced a rightward shift in the cumulative percentile of CREB fluorescence under basal conditions. C, Conversely, estradiol generated a leftward population shift in CREB phosphorylation after a 20 mm K+ depolarization. Error bars indicate SEM.
Figure 3.
Figure 3.
The bidirectional effects of estradiol on CREB phosphorylation are sex specific. A, B, Estradiol, in the absence of depolarization, increased (F = 26.82; **p < 0.01) CREB phosphorylation and decreased (**p < 0.01) L-type calcium channel-dependent CREB phosphorylation in neurons derived from female but not male animals. Error bars indicate SEM.
Figure 4.
Figure 4.
The effects of estradiol occur via action at the plasma membrane, are mimicked by the mixed estrogen receptor agonist/antagonist 4-hydroxy-tamoxifen, and are blocked by the estrogen receptor antagonist ICI 182,780. A, B, The membrane-impermeable estrogen analog EBSA mimicked the bidirectional effects of estradiol on CREB phosphorylation [F = 42.02 and 79.60; **p < 0.01 vs basal (A) and 20 mm K+ (B)]. C, D, The effect of 4-hydroxy-tamoxifen (4-OH-TMX) also paralleled the effect of estradiol [F = 31.15 and 41.27; **p < 0.01 vs basal (C) and 20 mm K+ (D)]. E, ICI 182,780 (ICI; 100 nm) eliminated both the estradiol-induced increase in CREB phosphorylation (F = 81.28; **p < 0.01 vs 17βE) and the estradiol-induced attenuation of L-type calcium channel-mediated CREB phosphorylation (**p < 0.01 vs 20 mm K+ plus 17βE). Error bars indicate SEM.
Figure 5.
Figure 5.
Estradiol-induced CREB phosphorylation is dependent on activation of PLC, PKC, and IP3R signaling. A, The effect of estradiol was reduced (F = 16.76; **p < 0.01) by inhibition of PKC with bisindolylmaleimide (500 nm). B, Similar results (F = 11.37; **p < 0.01) were observed after inhibition of IP3R with XC (1 μm). C, Pretreatment with both bisindolylmaleimide (Bis) and XC eliminated estradiol-induced CREB phosphorylation (F = 25.38; **p < 0.01). D, Consistent with estradiol activation of PLC, pretreatment with the inhibitor U73122 (1 μm) significantly (F = 19.89; **p < 0.01) attenuated estradiol-induced CREB phosphorylation. Error bars indicate SEM.
Figure 6.
Figure 6.
Estradiol-mediated CREB phosphorylation occurs via activation of mGluR1a signaling, independent of glutamate release. A, The mGluR1a antagonist/inverse agonist LY367385 (100 μm) eliminated estradiol-mediated CREB phosphorylation, without affecting estradiol inhibition of depolarization-induced CREB phosphorylation (F = 45.18; **p < 0.01 vs 17βE). B, The mGluR1/5 agonist DHPG (50 μm) in the presence of the mGluR5 antagonist MPEP (5 μm) increased CREB phosphorylation in cultures derived from female animals similar to estradiol and occluded the steroid effect (F = 49.98; **p < 0.01). C, DHPG (in the presence of MPEP) also resulted in an increase in CREB phosphorylation in cultures derived from male animals (F = 67.87; **p < 0.01). D, E, Whole-cell recordings of female hippocampal neurons (n = 9) revealed that estradiol does not evoke glutamate release or alter the frequency of mEPSCs. Calibration: 50 pA, 25 ms. F, Estradiol increased CREB phosphorylation after the removal of extracellular calcium, further supporting the notion that estradiol activation of mGluR1a signaling is independent of presynaptic glutamate release. Error bars indicate SEM.
Figure 7.
Figure 7.
Estradiol attenuates L-type calcium channel-mediated CREB phosphorylation via activation of group II mGluR signaling. A, The group II mGluR antagonist LY341495 (100 nm) eliminated the effect of estradiol on L-type calcium channel-mediated CREB phosphorylation (F = 38.19; **p < 0.01 vs 20K+ plus LY341495 and 20K+ plus 17βE) but had no effect on estradiol-induced CREB phosphorylation. B, The group II mGluR agonist APDC (10 μm) significantly attenuated L-type calcium channel-mediated CREB phosphorylation (F = 126.56) similar to estradiol and also occluded the steroid effect (**p < 0.01). C, The effect of APDC was also observed in cultures derived from male animals (F = 57.74; **p < 0.01). Error bars indicate SEM.
Figure 8.
Figure 8.
Estradiol activation of mGluR signaling is mediated by classical estrogen receptors located at the membrane surface. A, The ERα-specific agonist PPT (1 nm) led to an increase (F = 22.51; **p < 0.01) in CREB phosphorylation, similar to the actions of estradiol. The ERβ-specific agonist DPN (10 nm) was without effect. B, Conversely, both PPT and DPN were equally effective to estradiol in the attenuation of L-type calcium channel-dependent CREB phosphorylation (F = 74.17; **p < 0.01). Error bars indicate SEM.
Figure 9.
Figure 9.
Activation of mGluR2/3 and ERβ reduce L-type calcium channel currents. Furthermore, estradiol attenuation of L-type calcium channel-dependent CREB phosphorylation is mediated through Gi/o and PKA. A, Example traces of the whole-cell calcium channel current under control conditions and in the presence of the mGluR2/3 agonist APDC (10 μm). Calibration: 400 pA, 20 ms. B, Plot of the peak current as a function of time. The application of APDC produced a reduction in the whole-cell calcium current. The effect was eliminated in the presence of the L-type calcium channel blocker nifedipine (5 μm), indicating that activation of mGluR2/3 results in a decrease in the L-type calcium channel current. C, Both APDC (n = 9) and the ERβ agonist DPN (10 nm, n = 7) resulted in a decrease in the L-type calcium channel current. D, Estradiol reduction of L-type calcium channel-mediated CREB phosphorylation was eliminated after a 24 h pretreatment with the Gi/o inhibitor PTX (500 ng/ml). E, Inhibition of PKA with Rp-cAMPs (50 μm) also occluded the effect of estradiol on L-type calcium channel-mediated CREB phosphorylation. Neither PTX nor Rp-cAMPs affected estradiol-induced CREB phosphorylation (F = 47.79 and 216.05; **p < 0.01 vs baseline). Error bars indicate SEM.
Figure 10.
Figure 10.
Proposed mechanism by which estradiol produces bidirectional effects on CREB phosphorylation. Via interactions with classical estrogen receptors localized at the neuronal membrane, estradiol activates both group I and group II mGluR signaling. In one case, estradiol stimulation of mGluR1a leads to PLC/MAPK-induced CREB phosphorylation. Concurrently, activation of group II mGluR signaling decreases L-type calcium channel-dependent CREB phosphorylation through Gi/o inhibition of AC and PKA. The shaded arrows indicate decreased activity. CaM, Calmodulin; RSK, p90 ribosomal protein S6 kinase; CaMKIV, calmodulin-dependent protein kinase IV.
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