Estradiol-Mediated Axogenesis of Hypothalamic Neurons Requires ERK1/2 and Ryanodine Receptors-Dependent Intracellular Ca2+ Rise in Male Rats

Abstract

17β-estradiol (E2) induces axonal growth through extracellular signal-regulated kinase 1 and 2 (ERK1/2)-MAPK cascade in hypothalamic neurons of male rat embryos in vitro, but the mechanism that initiates these events is poorly understood. This study reports the intracellular Ca²⁺ increase that participates in the activation of ERK1/2 and axogenesis induced by E2. Hypothalamic neuron cultures were established from 16-day-old male rat embryos and fed with astroglia-conditioned media for 48 h. E2-induced ERK phosphorylation was completely abolished by a ryanodine receptor (RyR) inhibitor (ryanodine) and partially attenuated by an L-type voltage-gated Ca²⁺ channel (L-VGCC) blocker (nifedipine), an inositol-1,4,5-trisphosphate receptor (IP₃R) inhibitor (2-APB), and a phospholipase C (PLC) inhibitor (U-73122). We also conducted Ca²⁺ imaging recording using primary cultured neurons. The results show that E2 rapidly induces an increase in cytosolic Ca²⁺, which often occurs in repetitive Ca²⁺ oscillations. This response was not observed in the absence of extracellular Ca²⁺ or with inhibitory ryanodine and was markedly reduced by nifedipine. E2-induced axonal growth was completely inhibited by ryanodine. In summary, the results suggest that Ca²⁺ mobilization from extracellular space as well as from the endoplasmic reticulum is necessary for E2-induced ERK1/2 activation and axogenesis. Understanding the mechanisms of brain estrogenic actions might contribute to develop novel estrogen-based therapies for neurodegenerative diseases.

Keywords: Ca2+ signaling; ERK1/2; axogenesis; estradiol; hypothalamic neurons; ryanodine receptors.

Figure 1

E2-induced extracellular signal-regulated kinase 1 and 2 (ERK1/2) activation depends on cytosolic Ca²⁺ increase mainly mediated by ryanodine receptors (RyRs). Effects of (A) 2 μM nifedipine, (B) 50 μM ryanodine, (C) 100 μM 2-APB or (D) 10 μM U-73122 on E2-induced ERK phosphorylation. After washing for 2 h, the cultures were treated with the inhibitors for 1 h and were then pulsed for 15 min with 17β-estradiol (E2) and harvested for Western blotting. Top: ratio of readings for pERK/ERK bands in arbitrary densitometric units. Bottom: examples of immunoblots showing a decrease of hormone-induced ERK phosphorylation in cultures pretreated with the inhibitors. Molecular masses (kDa) for ERK1 and ERK2 are 44 and 42, respectively. Blots shown are representative of the mean ± SEM of 3–4 different cultures. (A) Nifedipine: ANOVA F_(2,10) = 52.78; p ≤ 0.001. Least significant differences (LSDs) test indicated ***p < 0.001 vs. control and ^⧫⧫p < 0.01 vs. E2. (B) Ryanodine: ANOVA F_(2,6) = 5.856; p = 0.04. LSDs test indicated *p = 0.05 vs. control and ^⧫p = 0.05 vs. E2. (C) 2-APB: ANOVA F_(2,6) = 27.203; p ≤ 0.001. LSDs test indicated ***p < 0.001 and *p = 0.05 vs. control and ^⧫⧫p = 0.01 vs. E2. (D) U-73122: ANOVA F_(2,6) = 34.891; p ≤ 0.001. LSDs test indicated ***p < 0.001 and **p = 0.01 vs. control and ^⧫p ≤ 0.05 vs. E2.

Figure 2

17β-estradiol (E2) induces Ca²⁺ oscillations. (A) Hypothalamic neurons were loaded with Cal-520 AM for 30 min at 37°C, maintained in a Ca²⁺-containing buffer (Ca²⁺-HBSS) and changes in cytosolic Ca²⁺ concentration were measured using confocal microscopy [Olympus IX81 inverted microscope equipped with a Disk Spinning Unit (DSU)]. Time series of Cal-520 pseudocolor images is shown, before and after the addition of E2 and thapsigargin (tg). Pseudocolor scale bar: 0.3–0.001 arbitrary units. Length scale bar: 20 μm. (B) Representative Ca²⁺ traces [regions of interest labeled 1–4 in (A)] plotted as changes over time in fluorescence intensity of the indicator (ΔF) respect to resting values (Fo). Arrows indicate the addition time of E2 (30 s) and tg (3 min). Data are from one representative experiment out of six independent experiments.

Figure 3

Ryanodine, external Ca²⁺ and nifedipine modulate E2-induced Ca²⁺ increase. (A) Mean values of maximal ΔF/Fo for Ca²⁺ mobilized by 17β-estradiol (E2) in hypothalamic neurons recorded either in the presence or absence of extracellular Ca²⁺ (Ca²⁺-HBSS/EGTA-containing buffer, named High Ca²⁺/Low Ca²⁺), or after a pre-incubation period (1 h) with ryanodine or nifedipine. ANOVA F_(3,41) = 22.94; p ≤ 0.001. LSDs test indicated ***p < 0.001 vs. Ca²⁺-HBSS. (B) Mean values of integrated area under ΔF/Fo curve (AUC) after thapsigargin (tg) addition during Ca²⁺ imaging [same conditions as (A)]. ANOVA F_(3,26) = 9.18; p ≤ 0.001. LSDs test indicated ***p < 0.001 and **p = 0.01 vs. ryanodine. Bars represent mean ± SEM; n = 4–6 different cultures.

Figure 4

RyR activity is required to the E2-induced axogenesis. (A) Representative images of male hypothalamic neurons cultured for 48 h with (E2) or without (control) 10 nM 17β-estradiol in combination or not with 50 μM ryanodine (Ry) for the last 24 h of incubation (arrows indicate the axons of some neurons). (B) Mean of axonal length for each condition in (A). ANOVA F_(3,12) = 4.51; p = 0.02. LSDs test indicated *p = 0.05 vs. control and ^⧫⧫p = 0.01 vs. E2. Data represent the mean ± SEM; n = 4 independent cultures. Scale bar: 100 μm.