Retinoic acid induces neurogenesis by activating both retinoic acid receptors (RARs) and peroxisome proliferator-activated receptor β/δ (PPARβ/δ)

Abstract

Retinoic acid (RA) regulates gene transcription by activating the nuclear receptors retinoic acid receptor (RAR) and peroxisome proliferator-activated receptor (PPAR) β/δ and their respective cognate lipid-binding proteins CRABP-II and FABP5. RA induces neuronal differentiation, but the contributions of the two transcriptional pathways of the hormone to the process are unknown. Here, we show that the RA-induced commitment of P19 stem cells to neuronal progenitors is mediated by the CRABP-II/RAR path and that the FABP5/PPARβ/δ path can inhibit the process through induction of the RAR repressors SIRT1 and Ajuba. In contrast with its inhibitory activity in the early steps of neurogenesis, the FABP5/PPARβ/δ path promotes differentiation of neuronal progenitors to mature neurons, an activity mediated in part by the PPARβ/δ target gene PDK1. Hence, RA-induced neuronal differentiation is mediated through RAR in the early stages and through PPARβ/δ in the late stages of the process. The switch in RA signaling is accomplished by a transient up-regulation of RARβ concomitantly with a transient increase in the CRABP-II/FABP5 ratio at early stages of differentiation. In accordance with these conclusions, hippocampi of FABP5-null mice display excess accumulation of neuronal progenitor cells and a deficit in mature neurons versus wild-type animals.

FIGURE 1.

CRABP-II/FABP5 ratio shifts during RA-induced neuronal differentiation. P19 cells were induced to differentiate as described under “Experimental Procedures.” RA was added on day 0. a, immunoblots demonstrating expression of Oct3/4, β3-tubulin, NeuN, and MAP2 during differentiation. b, levels of nestin mRNA during differentiation were assessed by Q-PCR. c, levels of mRNAs for denoted genes in predifferentiated P19 cells were measured by Q-PCR and normalized to 18 S rRNA. Data are mean ± S.D. of three biological replicates. d, left panel, immunoblots showing levels of CRABP-II and FABP5 during differentiation. Right panel, quantitation of immunoblots showing mean band intensities from two independent experiments. e and f, levels of mRNA for CRABP-II (d) and FABP5 (e) during differentiation were assessed by Q-PCR and normalized to respective levels in pre-differentiated cells. Data are means ± S.D. of three independent experiments. g, ratio of CRABP-II/FABP5 mRNAs during differentiation, measured by Q-PCR. h, levels of RARβ mRNA during differentiation were assessed by Q-PCR and normalized to level in pre-differentiated cells. Data are means ± S.D. of three independent experiments.

FIGURE 2.

RA triggers neuronal differentiation by activating RAR. P19 cells were treated with RA, the PPARβ/δ agonist GW0742, or the RAR agonist TTNPB (1 μm), and differentiation was followed as described under “Experimental Procedures.” a, expression levels of CRABP-II and FABP5 mRNA were assessed by Q-PCR 2 days following induction of differentiation. Data are means ± S.D. of three independent experiments. *, p < 0.01; **, p < 0.001 versus nontreated controls. b, expression levels of CRABP-II and FABP5 were assessed by immunoblots at day 4 following treatment with RA, GW0742, or TTNPB. c, levels of nestin mRNA were measured by Q-PCR. Data are means ± S.D., n = 3. **, p < 0.001 versus GW0742-treated controls. d, cells were fixed at day 10 and stained using DAPI to visualize nuclei (blue) and with antibodies against Oct3/4 (green) and β3-tubulin (red). Images were obtained using confocal fluorescence microscopy. Bar, 20 μm.

FIGURE 3.

PPARβ/δ/FABP5 pathway inhibits RA-induced differentiation of P19 cells to neuronal progenitors. a and b, P19 cell lines with reduced expression of PPARβ/δ or FABP5 were established by stable transfection of corresponding shRNA. Scrambled shRNA was used as control. Down-regulation of PPARβ/δ and FABP5 expression was confirmed by Q-PCR (a, mean ± S.D. of three plates; *, p < 0.05; **, p < 0.01) and immunoblots (b). c and d, cells expressing scrambled shRNA (Ctrlsh) or denoted shRNAs were induced to differentiate by addition of RA and levels of nestin mRNA measured by Q-PCR. Data are mean ± S.D. of three independent experiments. ***, p < 0.001 versus control shRNA. e, P19 cells were transfected with vectors harboring GFP, PPARβ/δ, or FABP5 cDNAs. The empty vector pLVX-IRES-GFP was used as control. Top panel, overexpression was assessed 48 h post-transfection by immunoblots. Bottom panel, quantitation of band intensities from three plates. Data are means ± S.D.; **, p < 0.01; ***, p < 0.001 versus GFP-expressing cells. f, cells ectopically overexpressing GFP, PPARβ/δ, or FABP5 were induced to differentiate by addition of RA, and nestin expression was measured by Q-PCR at day 4. Data are mean ± S.D. of three independent experiments. ***, p < 0.001 versus GFP control.

FIGURE 4.

RA signaling through the PPARβ/δ/FABP5 pathway promotes differentiation of neuronal progenitor cells to mature neurons. P19 cells and corresponding cells with stable reduced levels of PPARβ/δ or FABP5 were induced to differentiate. a, cells were fixed at day 6, immunostained using antibodies against nestin (green) and β3-tubulin (red), and imaged by confocal fluorescence microscopy. A representative field for each cell line is shown. Bar, 20 μm. The experiment was independently repeated three times with similar results. b and c, top panel, expression of the neuronal markers β3-tubulin and NeuN in the cell lines was assessed by immunoblots at day 4 (b) and day 10 (c). Bottom panel, quantification of immunoblots from four independent experiments. Data are mean ± S.D. *, p < 0.01 versus Ctrlsh (Student's two-tail t test). d, P19 cells were induced to differentiate by addition of RA. On day 4, cells were transferred to poly-l-lysine-coated tissue plates and cultured in media containing charcoal-treated serum (CT-FBS) and supplemented with neurobasal medium containing B27 supplement devoid of retinoids (B27-retinoids), or supplemented with retinol (ROH, 0.35 μm) and retinyl ester retinyl acetate (RE, 0.3 μm), or RA (0.5 μm), or GW0742 (1 μm). The appearance of the mature neuronal markers MAP2 was assessed by immunoblots on day 12. e, quantitation of data as in d from three independent experiments. Data are means ± S.D. *, p < 0.05 versus FBS + B27 containing retinoids. #, p < 0.05 versus CT-FBS + B27-retinoids.

FIGURE 5.

SIRT1 and Ajuba mediate inhibition of P19 cell differentiation to neuronal progenitors by the PPARβ/δ/FABP5 pathway. a, immunoblots demonstrating overexpression of SIRT1 or Ajuba in P19 cells. b, cells ectopically expressing GFP, Ajuba, or SIRT1 were induced to differentiate, and nestin expression was accessed by Q-PCR at day 4. Data are means ± S.D. (four independent experiments). ***, p < 0.001 versus GFP-transfected cells. c, P19 cells were treated with RA (1 μm). Left panel, immunoblots of denoted proteins 24 h post-treatment. Right panel, quantification of immunoblots from three independent experiments. Data are means ± S.D. *, p < 0.05; **, p < 0.01 versus untreated cells. d, cells were pretreated with vehicle or cycloheximide (CHX) (10 mm, 30 min) and then treated with RA (1 μm, 4 h). Levels of mRNAs were measured by Q-PCR. Data are mean ± S.D. (three independent experiments). *, p < 0.05; **, p < 0.01 versus nontreated controls; #, p < 0.05; ##, p < 0.01 versus cycloheximide alone. Inset, cells were pretreated with cycloheximide (10 mm, 30 min) and then treated with GW0742 (1 μm, 4 h). Levels of mRNAs were measured by Q-PCR. Data from a representative experiment are shown. The experiment was carried out twice with similar results. e, P19 cells were treated with vehicle or GW0742 (1 μm, 1 h). ChIP assays were performed as described under “Experimental Procedures.” Rabbit IgG was used as control. f, quantitation of band intensities in e. g, cells stably overexpressing GFP, PPARβ/δ, or FABP5 were cultured in charcoal-treated media overnight and then treated with GW0742 (20 nm, 4 h). Levels of mRNAs were measured by Q-PCR. Data are means ± S.D. (three independent experiments). *, p < 0.001 versus GFP-expressing cells.

FIGURE 6.

PDK1 mediates promotion of progenitor cells to mature neurons by the PPARβ/δ/FABP5 pathway. a, P19 cells were treated with GW0742 (20 nm, 4 h) and PDK1 mRNA assessed by Q-PCR. **, p < 0.01 versus untreated control. b, levels of PDK1 mRNA in cells stably expressing control shRNA or PPARβ/δ shRNA. ***, p < 0.001. c, P19 cells were transfected with expression constructs for PPARβ/δ or FABP5. Empty pLVX-IRES-GFP vector was used as control. 24 h post-transfection, cells were treated with GW0742 (20 nm, 4 h) and levels of PDK1 mRNA measured by Q-PCR. **, p < 0.01 versus GFP-expressing cells. d and e, P19 cells were induced to differentiate by addition of RA. On day 4, cells were transferred to poly-l-lysine-coated tissue plates and cultured in media containing charcoal-treated serum (CT-FBS) and supplemented with neurobasal medium containing B27 supplement devoid of retinoids (B27-retinoids) or supplemented with retinol (ROH, 0.35 μm) and retinyl acetate (RE, 0.3 μm), or RA (0.5 μm), or GW0742 (1 μm). d, levels of PDK1 mRNA assessed by Q-PCR on day 11. **, p = 0.003; #, p < 0.002 versus FBS + B27. e, top panel, levels of PDK1 and actin proteins were assessed immunoblots on day 12. Bottom panel, quantitation of immunoblots from three independent experiments. **, p < 0.002 versus FBS+B27. f, left, top panel, immunoblots demonstrating that the appearance of β3-tubulin is suppressed in cells with reduced expression of PPARβ/δ and is rescued upon ectopic expression of PDK1. Bottom panel, quantitation of blots from three independent experiments. *, p < 0.05; **, p < 0.01. Right panel, overexpression of PDK1 in cells expressing PPARβ/δ shRNA. All data are means ± S.D. (three independent experiments). p values were determined by a two-tail Student's t test.

FIGURE 7.

Localization of FABP5 in mouse brain and effects of its ablation on neuronal markers in hippocampus. a and b, location of FABP5 in mouse brain, analyzed by confocal fluorescence microscopy (a, bar, 50 μm) and by immunoblots (b). OB, olfactory bulb; CB, cerebellum; BS, brain stem; HT, hypothalamus; TH, thalamus; HC, hippocampus; CX, cerebral cortex; MB, midbrain. c and d, expression of nestin (c) and SOX2 (d) in hippocampus of WT and FABP5^−/− mice assayed by Q-PCR. *, p < 0.04 versus WT mice. n = 4 mice/group. e, immunofluorescence microscopy showing expression of NeuN (red) and glial fibrillary acidic protein (GFAP) (green) in hippocampus of WT and FABP5^−/− mice. gcl, granule cell layer; h, hippocampus hilus. Bar, 50 μm.

FIGURE 8.

Hippocampi of FABP5^−/− display lower expression of mature neuronal markers. a, immunoblots of denoted proteins in lysates of hippocampus of three WT and three FABP5^−/− mice. b–e, quantitation of immunoblots in a. *, p < 0.01. Level of PDK1 protein (e) and mRNA (f) in hippocampus of WT and FABP5-null mice. *, p < 0.004. 3 mice/group. All data are mean ± S.E. GFAP, glial fibrillary acidic protein.

FIGURE 9.

Model for the involvement of the RA in neuronal differentiation. RA promotes differentiation of stem cells into neural progenitor cells by activating the CRABP-II/RAR pathway. The alternative RA path, mediated by FABP5 and PPARβ/δ, can interfere with this step by inducing the expression of the RAR inhibitors Ajuba and Sirt1. This inhibition is avoided in early neurogenesis by an increase in the CRABP-II/FABP5 ratio and in the expression level of RARβ, ensuring that RA signaling is directed toward RAR. In later stages of differentiation, The CRABP-II/FABP5 ratio decreases, enabling RA to activate PPARβ/δ. In turn, the FABP5/PPARβ/δ pathway induces the expression of PDK1, thereby promoting differentiation of progenitor cells into mature neurons.