Nongenomic glucocorticoid receptor action regulates gap junction intercellular communication and neural progenitor cell proliferation

Abstract

Glucocorticoids (GCs) are used to treat pregnant women at risk for preterm delivery; however, prenatal exposure to GCs may trigger adverse neurological side effects due to reduced neural progenitor cell (NPC) proliferation. Whereas many established cell-cycle regulators impact NPC proliferation, other signaling molecules, such as the gap junction protein connexin-43 (Cx43), also influence proliferation. Gap junction intercellular communication (GJIC) is influenced by GCs in some cells, but such hormone effects have not been examined in coupled stem cells. We found that both continuous and transient exposure of embryonic day 14.5 mouse neurosphere cultures to dexamethasone (DEX) limits proliferation of coupled NPCs, which is manifested by both a reduction in S-phase progression and enhanced cell-cycle exit. A short (i.e., 1-h) DEX treatment also reduced GJIC as measured by live-cell fluorescence recovery after photobleaching, and altered the synchrony of spontaneous calcium transients in coupled NPCs. GC effects on GJIC in NPCs are transcription-independent and mediated through plasma membrane glucocorticoid receptors (GRs). This nongenomic pathway operates through lipid raft-associated GRs via a site-specific, MAPK-dependent phosphorylation of Cx43, which is linked to GR via caveolin-1 (Cav-1) and c-src. Cav-1 is essential for this nongenomic action of GR, as DEX effects on GJIC, Cx43 phosphorylation, and MAPK activation are not observed in Cav-1 knockout NPCs. As transient pharmacologic inhibition of GJIC triggers reduced S-phase progression but not enhanced cell-cycle exit, the nongenomic GR signaling pathway may operate via distinct downstream effectors to alter the proliferative capacity of NPCs.

Fig. 1.

Rapid inhibition of GJIC in NPCs by GCs is associated with Cx43 phosphorylation. (A and B) NPCs preloaded with Calcein AM were subjected to 1-h treatments with ethanol vehicle (Veh), 100 nM DEX (±1 μM RU-486), RU-486 alone, DEX–BSA (±RU-486), and 100 μM carbenoxolone (Cbx). Fluorescence recovery within individual bleached cells in a representative experiment is shown (A) along mean values for t_1/2 ± SEM of recovery (B; n = 4 independent experiments; one-way ANOVA, P < 0.0001; post hoc Tukey's multiple comparison test, P < 0.05). Representative Western blots are shown that measure Cx26 and Cx43 levels under analogous treatment conditions (C and D) along with mean ± SEM of densitometric scans of multiple blots (E and F) (n = 4; F: P = 0.0042; post hoc test, *P < 0.05). Stripped blots were probed with an anti-actin antibody. Positions of unphosphorylated (P0) and phosphorylated forms (P1, P2) of Cx43 are indicated (D).

Fig. 2.

Rapid activation of ERK-1/2 and site-specific phosphorylation of Cx43 by GCs in NPCs. Western blot analysis was used to measure phosphorylated ERK-1/2 (pERK-1/2) and total ERK-1/2 (tERK-1/2) (A and B) and phosphorylated Cx43 at serines 279 and 282 (pCx43s279s282) (C and D) following a 1-h treatment of NPCs with 100 nM DEX and/or 1 μM RU-486. Representative Western blots are shown (A and C) along with densitometric scans ±SEM of multiple blots (B and D). DEX treatment significantly enhanced pERK-1/2 (B) (n = 4; P = 0.0085; post hoc test, *P < 0.05) and pCx43s279s282 levels (D) (n = 3; P = 0.0023; post hoc test, *P < 0.05).

Fig. 3.

GC site-specific phosphorylation of Cx43 and inhibition of GJIC is ERK-1/2-dependent and occurs by a nongenomic GR signaling pathway. Western blot analysis was used to measure pCx43s279s282 following a 1-h treatment with 100 nM DEX and/or 40 μM PD98059 (A) or a 1-h pretreatment with 100 ng/mL ActD before a 1-h DEX treatment (C). Results of mean ± SEM of densitometric scans of multiple blots are shown (A: n = 4; P = 0.0012; post hoc test, *P < 0.05; C: n = 4; P = 0.0009; post hoc test, *P < 0.05). Representative blots are in Figs. S3 A and B and S4B. U0126 pretreatment (10 μM) had a similar effect as PD treatment (Fig. S3 C–E). NPCs preloaded with Calcein AM were subjected to analogous treatments [i.e., DEX ± PD (B) or ActD (D)] and mean values were measured for t_1/2 ± SEM of recovery (B: n = 5 independent experiments, P = 0.0002; post hoc test, *P < 0.05; D: n = 4 independent experiments, P < 0.0001; post hoc test, *P < 0.05). (E–I) Untransfected WT NPCs or CHO cells transiently transfected with an alanine-to-cysteine mutant (Mut) or WT human GR were stained as live cells with the fluorescent-tagged lectin membrane marker concavalin-A, followed by fixation and IIF staining for GR. In the merged images (G–I), an arrow indicates plasma membrane GR localization. Ninety-seven percent (34/35 from four separate fields) of WT GR and 96% (48/50 from five separate fields) of mutant GR-transfected cells displayed plasma membrane costaining of GR and concavalin-A.

Fig. 4.

GR is associated with Cav-1 in lipid rafts of NPCs, and Cav-1 along with c-src are necessary for rapid GC signaling. (A) NPC extracts were subjected to sucrose gradient fractionation to enrich for lipid rafts (i.e., fractions 4–6) and analyzed for GR and Cav-1 expression using Western blots (n = 2). The Golgi-associated protein ARF-1 was used to assess effective partitioning of the fractions. (B and C) Triton-soluble extracts were subjected to a coimmunoprecipitation (co-IP) assay with subsequent Western blots to reveal an association between GR and Cav-1. A nonimmune IgG was used in control co-IPs (n = 3). In both sucrose gradient fractionation and co-IP, NPCs were subjected to 1-h vehicle or 1-h 100 nM DEX. Western blot analysis was used to measure pERK-1/2 and tERK-1/2 (D) following pretreatment with PP2 for 30 min and a 1-h exposure to 100 nM DEX. Significant effects of DEX were revealed in results of the mean ± SEM ratio of pERK:tERK from densitometric scans (n = 6; P = 0.0001; post hoc test, Bonferroni, *P < 0.05). A representative blot is in Fig. S6A. Analogous results were seen for pCx43s279s282 following PP2 pretreatment (Fig. S6 B and C). (E) Western blot shows lack of Cav-1 in NPCs prepared from Cav-1 KO mice. Western blot analysis was used to measure pERK-1/2 and tERK-1/2 (F: n = 3; a representative blot is in Fig. S6D) and pCx43s279s282 (G: n = 3; a representative blot is in Fig. S6E) following 1-h treatments of Cav-1 KO NPCs with vehicle, 100 nM DEX (±1 μM RU-486), or RU-486 alone. No significant effects of DEX exposure were observed. (H) Cav-1 KO and WT and Cav-1 KO NPCs preloaded with Calcein AM were subjected to treatments with vehicle or 100 nM DEX. Mean values for t_1/2 ± SEM of recovery were obtained by fitting a decaying exponential to individual fluorescence recovery curves. No effects of DEX exposure were observed on GJIC in Cav-1 KO NPCs (n = 4 independent experiments; P = 0.0027; post hoc test, P < 0.05).

Fig. 5.

Transient GC exposure is sufficient to reduce NPC proliferation and enhance cell-cycle exit, whereas transient inhibition of GJIC limits S-phase progression but does not affect cell-cycle exit. (A and B) NPCs subjected to 24-h treatments with vehicle, 100 nM DEX (±1 μM RU-486), RU-486 alone, or a 1-h DEX pretreatment followed by a 23-h RU-486 exposure (PreDEX+RU) were treated with a 10 μM BrdU pulse during the final hour of treatment. Mean values for BrdU⁺/Ki67⁺ cells ±SEM obtained following IIF show a significant reduction in NPCs actively in S phase of the cell cycle (A: n = 3; four random fields per image; P = 0.0024; post hoc test, *P < 0.05), whereas analysis of Ki67-immunostained cells alone indicated a significant reduction in NPCs actively engaged in the cell cycle (i.e., G1-S-G2/M) (B: n = 4; four random fields per image; P < 0.0001; post hoc test, *P < 0.05). A representative image is in Fig. S7A. (C) Proliferation assays performed as described above reveal a significant effect of a limited (i.e., 1-h) 3 mM 1-heptanol exposure on NPCs actively progressing through S phase of the cell cycle measured 23 h following 1-heptanol removal and wash (1hr Hept/23hrWash) (mean number ± SEM of BrdU⁺/Ki67⁺ cells, n = 4; four random fields per image; P < 0.0001; post hoc test, *P < 0.05). (D) Analysis of mean ± SEM of Ki67-only labeled cells reveals no significant effect of 1-heptanol exposure on NPCs exiting the cell cycle (n = 4). FRAP results indicated rapid and reversible inhibition of NPC GJIC by 1-heptanol (Fig. S7B). (E) NPCs treated with vehicle, 1-h 100 nM DEX, or 1-h 3 mM 1-heptanol were preloaded with the ratiometric calcium indicator Fura-2 AM and then subjected to live-cell calcium imaging. Although vehicle-treated NPCs show highly synchronized Ca²⁺ bursts in coupled NPCs as determined by the ratio of Fura-2 excitation at 340:380 nm, both the synchronicity and bursting behavior is reduced in DEX- and 1-heptanol-exposed NPC pairs. (F) Analysis of Ca²⁺ bursts in 10 pairs of coupled NPCs indicated a significant reduction in the correlation of Ca²⁺ transients as measured by the Pearson correlation coefficient (r²) (n = 10 pairs; P = 0.0056; post hoc test, *P < 0.05).