A positive autoregulatory loop of Jak-STAT signaling controls the onset of astrogliogenesis

Abstract

During development of the CNS, neurons and glia are generated in a sequential manner. The mechanism underlying the later onset of gliogenesis is poorly understood, although the cytokine-induced Jak-STAT pathway has been postulated to regulate astrogliogenesis. Here, we report that the overall activity of Jak-STAT signaling is dynamically regulated in mouse cortical germinal zone during development. As such, activated STAT1/3 and STAT-mediated transcription are negligible at early, neurogenic stages, when neurogenic factors are highly expressed. At later, gliogenic periods, decreased expression of neurogenic factors causes robust elevation of STAT activity. Our data demonstrate a positive autoregulatory loop whereby STAT1/3 directly induces the expression of various components of the Jak-STAT pathway to strengthen STAT signaling and trigger astrogliogenesis. Forced activation of Jak-STAT signaling leads to precocious astrogliogenesis, and inhibition of this pathway blocks astrocyte differentiation. These observations suggest that autoregulation of the Jak-STAT pathway controls the onset of astrogliogenesis.

Figure 1

The late onset of astrocyte differentiation in vitro. (a) Western blot analysis of primary mouse E11 cortical cells cultured for 2, 5, or 8 DIV with or without LIF (100 ng ml⁻¹) continuous treatment. GAPDH signals were used as loading controls. (b) RT-PCR analysis of the expression of astrocytic markers GFAP and S100β, in 2-DIV or 5-DIV cultured E11 cortical cells. GAPDH RT-PCR signals were used as controls. (c,d) 1.9-kb GFAP and 2-kb S100β promoter luciferase reporter constructs were introduced into E11 cortical cells, either untreated or treated with LIF (100 ng ml⁻¹) for 1 d. Luciferase assays were performed between 1 to 2 DIV or at 5 DIV (with transfection and LIF treatment starting 1 d before harvesting) (*, P < 0.05 as compared to the non-LIF treated group, n ≥ 6). (e,f) Bacterially expressed phosphorylated STAT1 (pSTAT1) binds to the STAT binding site (DNA cis-element) within the GFAP and S100β promoters as determined by electrophoretic mobility shift assays (EMSA). Mutation studies of the STAT binding site indicated that STAT cis-elements are required for the activation of glial specific genes by LIF (*, P < 0.05 compared with non-LIF treated group, n ≥ 6). (g) ChIP assay showing that the association of the STAT3/CBP complex with the GFAP promoter is dynamically regulated, which correlates with the gliogenic potential of the cells. The STAT3/CBP complex associates with the GFAP promoter in 5-DIV, but not in 1-DIV cultured E11 cortical cells with 1 d of LIF treatment before cell harvesting. Ab, antibody. α, antibody against (in all figures).

Figure 2

Dynamic regulation of the Jak-STAT pathway during development. (a) Immunocytochemical studies of tyrosine-phosphorylated STAT3 (red) and nestin (green), a neural progenitor cell marker, in 2-, 5-, and 8-DIV cultured E11 cortical cells treated with LIF (100 ng ml⁻¹) for 20 min. Cell nuclei are indicated by DAPI (blue) staining. (b) Western blot analysis of the Jak-STAT pathway of E11 neural progenitor cells at different culturing time periods with 20-min LIF treatment before sample harvesting. (c) Long-term treatment with LIF seems to upregulate the expression of components in the Jak-STAT pathway (LIF was added on the first day of culturing). (d) Activation of a synthetic STAT reporter over time in culture. Luciferase analysis of a synthetic promoter that contains four tandem repeats of the STAT binding elements was used to evaluate the overall STAT-mediated transcription activity in E11 cortical progenitor cells cultured for 1–2 d or 5 d with or without 1-d treatment of LIF (*, P < 0.05 as compared with non–LIF treated group, n ≥ 6).

Figure 3

Sequential activation of the Jak-STAT pathway in vivo correlates with the timing of astrogliogenesis. (a) A fluorescent image of E15 cortical ventricular area from pNestin-GFP mice, demonstrating enriched GFP expression in the ventricular zone (VZ). (b,c) The ventricular zone (green) and non–ventricular zone (non-green) tissues were dissected from different developmental stages (E12, E16 or postnatal day (P) 0 from the pNestin-GFP transgenic mice under a fluorescent dissection microscope. Western blot analyses used a TuJ1 antibody that labels neuronal specific βIII tubulin, and a GFP antibody. β-actin blot indicates the loading control. (d–f) Western blot analyses of STAT activation and astrocyte differentiation in vivo at different developmental stages. After incubation with or without LIF (100 ng ml⁻¹) for 20 min, lysates of green VZ/SVZ tissues from various developmental stages (E12, E14, P0 and P4) were probed with antibodies against tyrosine-phosphorylated STAT1 or STAT3 (d,e), astrocyte marker GFAP (d), gp130, LIFRβ (f), GAPDH (e), and β-actin (d). Lysates from P0 or P4 without 20-min LIF treatment were also enriched for active forms of STAT1 and STAT3. (g) RT-PCR analysis of gp130, Jak1 and astrocytic markers GFAP and S100β at different developmental stages.

Figure 4

The positive autoregulatory loop of the Jak-STAT machinery. (a) STAT1/3 regulates the expression of STAT1. Two (−1741 bp, −1532 bp) of the three STAT binding elements within the 2-kb STAT1 promoter are capable of binding to recombinant pSTAT1 in EMSA assays. Luciferase analysis of the mutagenized STAT1 promoter indicates that both cis-elements are required for LIF induction of the promoter in 5-DIV cultured E11 cortical cells. Mut1, STAT element 1 mutant; Mut2, STAT element 2 mutant. (b) Luciferase analysis of the STAT1 promoter in E11 neural progenitor cell cultures treated with LIF for 1 d at different culturing time points. The STAT1 promoter becomes much more active in the 5-DIV long-term cultures than in 1- to 2-DIV cultures (*, P < 0.05 compared with the rest of the group, n ≥ 6). (c) STAT binding sites within the promoters of gp130 and STAT3 are conserved between human and mouse. (d) STAT1/3 can bind to the putative STAT-responsive element from −158 bp to −150 bp(5′-TTACGGGAA-3′) within the gp130 promoter, as shown by EMSA assay. (e) ChIP analyses showing the developmentally regulated association between STAT3 and CBP with STAT1 (left) and gp130 (right) promoters in E11 primary cortical cell culture at 2 and 5 DIV. (f) RT-PCR of the Jak-STAT signaling components in E11 primary cortical cells cultured for 2 and 5 DIV.

Figure 5

Inhibition of the Jak-STAT pathway suppresses astrogliogenesis. (a) STAT3F (STAT3 Y705F) suppresses LIF-triggered activation of both the synthetic STAT reporter (4STAT-Luc) and the GFAP promoter (GFAP-Luc) in 3-DIV E14.5 cortical cells that were treated with LIF for 1 d (*, P < 0.05 as compared with the rest of the groups, n ≥ 6). (b) Western blot analysis showing protein levels of the components of the Jak-STAT pathway after overexpression of STAT3F in E14.5 cortical cells either left untreated or treated with LIF for 2 d before harvesting at 3 DIV. (c) ChIP analyses demonstrate the association of the STAT/CBP complex with the GFAP (upper panel) and gp130 (lower panel) promoters with or without STAT3F expression in 4-DIV E11 primary neural progenitors treated with LIF for 2 d. (d) Inhibition of astroglial differentiation by various dominant interfering forms of STAT3. Cultures were either left untreated or treated with LIF (100 ng ml⁻¹) for 24 h before fixation at 4 DIV. Cells were stained with an antibody recognizing GFAP (red). Nuclei are shown by Hoechst staining (blue). (e) Quantification of GFAP-positive cells as a percentage of total cells in E14.5 cortical neural progenitor cells after 4 DIV in the presence of LIF, after transfection with control, STAT3F, STAT3D and STAT3 siRNA (*, P < 0.05 as compared with the rest of the groups using one-way ANOVA and Fisher’s post hoc test, n = 6).

Figure 6

Constitutively active STAT3C leads to precocious astrocyte differentiation in the presence of LIF. (a) Luciferase assay of the synthetic STAT reporter in the presence of STAT3C with or without LIF for 1 d in E11 cortical culture at 2 DIV (*, P < 0.05 as compared to the rest of the groups, n ≥ 6). (b,c) STAT3C leads to precocious activation of the Jak-STAT pathway. E11 cortical cell culture infected with STAT3C virus for 3 d were analyzed by western blot using antibodies against various components of the Jak-STAT pathway. (d) GFAP promoter activity is increased after overexpression of STAT3C. The combination of 2-d LIF and STAT3C treatment leads to a marked increase in GFAP promoter activity (*, P < 0.05 as compared with the rest of the groups, n ≥ 6). (e) Immunocytochemistry of GFAP expression of E11 cortical cell culture at 2 DIV in the absence or presence of STAT3C expression (adenoviral infection at 1 DIV) with or without 2-d LIF treatment. Glial cells were labeled with antibodies against GFAP (red) and nuclei were stained by Hoechst (blue). Lower panel shows quantification of GFAP-positive cells as a percentage of total cells in the immunostaining experiments (*, P < 0.05 as compared with the rest of the groups, one-way ANOVA, n = 6). (f) GFAP expression of E12 cortical cell culture at 4 DIV in the absence or presence of STAT3C (added at 1 DIV) with or without LIF longterm treatment were analyzed as in e. Quantification of the immunostaining experiments is shown in lower panel (*, P < 0.05 as compared with the rest of the groups, one-way ANOVA, n = 6). (g) ChIP analysis indicates association of STAT3/CBP, HDAC1 and N-CoR with the GFAP promoter in cultured 3-DIV E11 neural progenitor cells infected with the STAT3C virus (viral infection at day 1 and LIF treatment for 2 d).

Figure 7

De-repression of the Jak-STAT pathway owing to reduced neurogenin-1 and neurogenin-2 expression during the switch from neurogenesis to gliogenesis. (a) Reciprocal expression of proneural bHLH genes and astroglial differentiation factors in cortical germinal zones during development as shown by western blot analyses of the proneural bHLH gene, Ngn1, astrocyte differentiation–related factors such as tyrosine phosphorylated STAT1 (pSTAT1), and an astrocyte marker, GFAP, in cortical nestin-positive neural epithelial cells at different developmental stages. (b) Gain-of-function experiments demonstrate the suppression of the Jak-STAT signaling components by proneural bHLH genes Ngn1 and Ngn2, as shown by western blot. E11 mouse cortical neural stem cells cultures were infected with control, Ngn1, or Ngn2 adenoviruses, and 24–36 h after infection, the cells were either left untreated or treated with LIF for 20 min before harvesting. (c) Loss-of-function experiments using Ngn2 knockout mice indicate precocious activation of the Jak-STAT machinery and astroglial differentiation in the absence of Ngn2 expression. E14 mouse wild type, Ngn2^+/− and Ngn2^−/− cortices were dissected, followed by 20 min LIF treatment, and lysed for western blot analyses.