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. 2015 Sep;33(9):2762-72.
doi: 10.1002/stem.2055. Epub 2015 Jun 23.

Morphine Promotes Astrocyte-Preferential Differentiation of Mouse Hippocampal Progenitor Cells via PKCε-Dependent ERK Activation and TRBP Phosphorylation

Chi Xu  1 , Hui Zheng  2 , Horace H Loh  1 , Ping-Yee Law  1
Affiliations

Morphine Promotes Astrocyte-Preferential Differentiation of Mouse Hippocampal Progenitor Cells via PKCε-Dependent ERK Activation and TRBP Phosphorylation

Chi Xu et al. Stem Cells. 2015 Sep.

Abstract

Previously we have shown that morphine regulates adult neurogenesis by modulating miR-181a maturation and subsequent hippocampal neural progenitor cell (NPC) lineages. Using NPCs cultured from PKCε or β-arrestin2 knockout mice and the MAPK/ERK kinase inhibitor U0126, we demonstrate that regulation of NPC differentiation via the miR-181a/Prox1/Notch1 pathway exhibits ligand-dependent selectivity. In NPCs, morphine and fentanyl activate ERK via the PKCε- and β-arrestin-dependent pathways, respectively. After fentanyl exposure, the activated phospho-ERK translocates to the nucleus. Conversely, after morphine treatment, phospho-ERK remains in the cytosol and is capable of phosphorylating TAR RNA-binding protein (TRBP), a cofactor of Dicer. This augments Dicer activity and promotes the maturation of miR-181a. Furthermore, using NPCs transfected with wild-type TRBP, SΔA, and SΔD TRBP mutants, we confirmed the crucial role of TRBP phosphorylation in Dicer activity, miR-181a maturation, and finally the morphine-induced astrocyte-preferential differentiation of NPCs. Thus, morphine modulates the lineage-specific differentiation of NPCs by PKCε-dependent ERK activation with subsequent TRBP phosphorylation and miR-181a maturation.

Keywords: Adult stem cells; Cell signaling; MAPK; Neural differentiation; Neural stem cell; Progenitor cells; Signal transduction; miRNA.

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Figures

Figure 1
Figure 1. Morphine and fentanyl activate ERK via differential pathways
(A) ERK phosphorylation was tested in wild type, PKCε KO or β-arrestin2 KO NPCs cultured in differentiation media by immunoblotting after treatment of 1 μM morphine or 10 nM fentanyl for 10 min. PKC activities were determined with immunoblotting of PKC phosphorylated substrates. β-actin was used as the internal control. C: control; M: morphine; F: fentanyl. (B) Quantification of ERK phosphorylation as shown in (A), calculated as folds of control. *, p<0.05 compared to wild type cells treated with the same agonist. Data are the mean ± SEM of four independent experiments. (C) ERK phosphorylation was tested in NPCs transfected with control siRNA, PKCα siRNA and PKCε siRNA by immunoblotting after treatment of 1 μM morphine 10 min. (D) Quantification of ERK phosphorylation as shown in (C), calculated as folds of control. *, p<0.05 compared to cells transfected with control siRNA. Data are the mean ± SEM of four independent experiments. (E) ERK phosphorylation was tested in NPCs transfected with control siRNA, PKCα siRNA and PKCε siRNA by immunoblotting after treatment of 10 nM fentanyl for 10 min. (F) Quantification of ERK phosphorylation as shown in (E), calculated as folds of control. Data are the mean ± SEM of four independent experiments.
Figure 2
Figure 2. Morphine promotes astrocyte-preferential differentiation via PKCε-mediated ERK phosphorylation
(A) Hippocampal neural progenitor cells derived from wild type, PKCε knockout and β-arrestin2 knockout mice were cultured in complete differentiation medium for 4 d, with or without the treatment of 1 μM morphine. Cells were stained with markers for neurons (Tuj1), astrocytes (GFAP), oligodendrocytes (O4) and with DAPI. Images are representative four independent experiments with similar results. Scale bar, 25 μm. β-arr: β-arrestin. (B) Quantification of cells stained with each marker in (A), calculated as the percentage of the total number of cells stained with DAPI. Red: Tuj1; Green: GFAP; Purple: O4. *, p<0.05; **, p<0.01 compared to the same marker in control group of the same cell line. #, p<0.05 compared to the same marker in the wild type control group. Data are the mean ± SEM of four independent experiments. WT: wild type; KO: knockout. (C) Hippocampal neural progenitor cells were cultured in complete differentiation medium for 4 d, with or without the treatment of 1 μM morphine and pretreatment of 10 μM U0126 for 1 h. Cells were stained with Tuj1, GFAP and DAPI. Scale bar, 25 μm. Images are representative of four independent experiments with similar results. (D) Quantification of cells stained with each marker in (C), calculated as the percentage of the total number of cells stained with DAPI. Red: Tuj1; Green: GFAP. *, p<0.05 compared to the same marker in morphine-treated group without U0126 pretreatment. #, p<0.05 compared to the same marker in the control group. Data are the mean ± SEM of four independent experiments.
Figure 3
Figure 3. Morphine promotes TRBP phosphorylation and Dicer expression via OPRM1
(A) Adult hippocampus-derived neural progenitor cells were cultured in complete differentiation medium for 24 h, with or without the treatment of 1 μM morphine or 10 nM fentanyl, and pretreatment of 10 μM CTOP for 1 h. The protein levels of Drosha, Dicer, TRBP and ERK1/2 were determined by western blot. β-actin was used as the internal control. (B-D) Quantification of protein levels of Drosha (B), Dicer (C) and phospho-TRBP (D) as shown in (A), calculated as folds of control. *, p<0.05 compared to control. Data are the mean ± SEM of four independent experiments. (E) The expression of TRBP and Dicer mRNAs were determined by qRT-PCR after 24 h of differentiation with indicated treatments. The results were normalized against those of GAPDH. All data represent mean ± SEM of four independent experiments.
Figure 4
Figure 4. Morphine promotes TRBP phosphorylation and Dicer expression via PKCε and ERK activation
(A) NPCs derived from wild type, PKCε knockout and β-arrestin2 knockout mice were cultured in complete differentiation medium for 24 h, with or without the treatment of 1 μM morphine or 10 nM fentanyl. The protein levels of Dicer, TRBP, phosphorylated PKC substrates and β-arrestin1/2 were determined by western blot. β-actin was used as the internal control. c: control; m: morphine; f: fentanyl. (B) Quantification of protein levels of Dicer and phospho-TRBP as shown in (A), calculated as folds of control. *, p<0.05 compared to control group of the same cell line. #, p<0.05 compared to wild type cells with the same agonist treatment. Data are the mean ± SEM of four independent experiments. (C) Wild type, PKCε knockout and β-arrestin2 knockout mice were treated by subcutaneous implantation of one morphine pellet (75 mg free base per mouse) or placebo pellets for 4 days. The hippocampi of treated mice were collected and the protein levels of Dicer, TRBP, phosphorylated PKC substrates and β-arrestin1/2 were determined by western blot. β-actin was used as the internal control. p: placebo; m: morphine. (D) Quantification of protein levels of Dicer and phospho-TRBP as shown in (C), calculated as folds of control. *, p<0.05 compared to placebo of the same animal group. #, p<0.05 compared to wild type with the same treatment. Data are the mean ± SEM of eight independent experiments. (E) NPCs were cultured in complete differentiation medium for 24 h, with or without the treatment of 1 μM morphine or 10 nM fentanyl, and pretreatment of 10 μM U0126 for 1 h. The protein levels of Dicer, TRBP and ERK were determined by western blot. β-actin was used as the internal control. c: control; m: morphine; f: fentanyl. (F) Quantification of protein levels of Dicer and phospho-TRBP as shown in (E), calculated as folds of control. *, p<0.05 compared to control. Data are the mean ± SEM of four independent experiments.
Figure 5
Figure 5. TRBP phosphorylation mediates Dicer expression and activation of the miR-181a/Prox1/Notch1 cascade
(A) NPCs were transfected with wild type, SΔA or SΔD TRBP and cultured in complete differentiation medium for 24 h, and the expression of TRBP and Dicer mRNAs were determined by qRT-PCR. The results were normalized against those of GAPDH. All data represent mean ± SEM of four independent experiments. (B) NPCs transfected wild type, SΔA or SΔD TRBP were cultured in complete differentiation medium for 24 h, with or without the treatment of 1 μM morphine or 10 nM fentanyl. The protein levels of Dicer and TRBP were determined by western blot. β-actin was used as the internal control. con: control; mor: morphine; fen: fentanyl. (C) Quantification of protein levels of Dicer and phospho-TRBP as shown in (B), calculated as folds of wild type control. *, p<0.05; **, p<0.01 compared to wild type cells with the same agonist treatment. #, p<0.05 compared to wild type control. Data are the mean ± SEM of four independent experiments. (D) The expression of miR-181a-5p and mRNA levels of Prox1 and Notch1 were determined by qRT-PCR after 24 h of agonist treatment. The results were normalized against the level of GAPDH, and further normalized against the result obtained from wild type control group. *, p<0.05; **, p<0.01 compared to wild type cells with the same agonist treatment. #, p<0.05 compared to wild type control. All data represent the mean ± SEM of four independent experiments.
Figure 6
Figure 6. TRBP phosphorylation modulates morphine-induced astrocyte-preferential differentiation of NPCs
(A) NPCs transfected wild type, SΔA or SΔD TRBP were cultured in complete differentiation medium for 4 d, with or without the treatment of 1 μM morphine. Cells were stained with markers for neurons (Tuj1), astrocytes (GFAP) and with DAPI. Scale bar, 25 μm. Images are representative of four independent experiments with similar results. (B) Quantification of cells stained with each marker, calculated as the percentage of the total number of cells stained with DAPI. Red: Tuj1; Green: GFAP. *, p<0.05 compared to wild type groups with the same treatment. #, p<0.05 compared to the wild type control. (C) The mRNA levels of βIII-tubulin and GFAP were determined by qRT-PCR after 4 d of differentiation with indicated treatments. The results were normalized against GAPDH levels. *, p<0.05, **, p<0.01, compared to wild type groups with the same treatment. #, p<0.05 compared to the wild type control. All data represent mean ± SEM of four independent experiments. WT: wild type.
Figure 7
Figure 7. A schematic diagram summarizing the signaling pathways of morphine and fentanyl regulating adult neurogenesis
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