MAZ mediates the cross-talk between CT-1 and NOTCH1 signaling during gliogenesis

Abstract

Neurons and glia cells are differentiated from neural stem/progenitor cells (NSCs/NPCs) during brain development. Concomitant activation of JAK/STAT and NOTCH1 signaling is required for gliogenesis, a process to generate glia cells to ensure proper brain functions. NOTCH1 signaling is down-regulated during neurogenesis and up-regulated during gliogenesis. However, the underlying mechanism remains elusive. We report here that cardiotrophin-1 (CT-1) activates NOTCH1 signaling through the up-regulation of ADAM10, a rate-limiting factor of NOTCH1 signaling activation. We found that a transcriptional factor, Myc-associated zinc finger protein (MAZ), plays an important role in ADAM10 transcription in response to CT-1 in NPCs. MAZ knockdown inhibits CT-1 stimulated gliogenesis and it can be rescued by over-expressing human NICD. Our results provide a link between NOTCH1 activation and neuronal secreted CT-1, suggesting that CT-1 plays an important role in ensuring the coordinated activation of NOTCH1 signaling during gliogenesis.

Figure 1. CT-1 up-regulates ADAM10 and NICD levels in NPCs and NIH3T3 cells.

NPCs isolated from E11.5–13.5 mouse embryonic cortex were stimulated with CT-1 (100 ng/ml) or buffer (control, Cont) for 72 hrs, and NIH3T3 cells were stimulated with CT-1 (100 ng/ml) or buffer (control, Cont) for 24 hrs. Cell lysates were subjected to Western Blot analysis for both NICD (A) and full length NOTCH1 receptor (B) and two S2 enzymes ADAM10 (C) and ADAM17 (D). GAPDH was used as loading control. Representative blots and statistical analysis are shown in the upper and lower panels respectively. Two downstream effectors of NOTCH1 pathway, Hes1 & Hes5 were examined by qRT-PCR after CT-1 stimulation for 24 hrs in NIH3T3 cells (E) or 48 hrs in NPCs (F). The blots were cropped to improve the clarity and conciseness and full-length blots are presented in Supplementary Fig. S3. All data represent means ± SEM (one-way ANOVA). N ≥ 5, *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2. ADAM10 is essential for CT-1 induced NICD increase.

(A) Confirmation of ADAM17 KO 293T cell lines (1–3 and 13–1) and ADAM10 KO 293T cell line (51) by Western blot analysis. (B,C) Different cell lines were stimulated with 100 ng/ml CT-1 for 24 hrs. Cell lysates were analyzed for NICD, ADAM10, or ADAM17 levels with GAPDH as loading control. Representative blots are shown in (B,C). (D,E) Statistical analysis for NICD and ADAM10 levels from (B,C). The blots were cropped to improve the clarity and conciseness and full-length blots are presented in Supplementary Fig. S3. All data represent means ± SEM (one-way ANOVA). N = 4, *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3. CT-1 induces ADAM10 transcription through MAZ.

(A) NIH3T3 cells were treated with CT-1 at different time points. Adam10 mRNA levels were determined by qRT-PCR. (B) Luciferase reporter constructs with different regions of Adam10 promoter were transfected into NIH3T3 cells. Relative luciferase activities were determined after stimulation with CT-1 or control buffer for 12 hrs. (C) After incubation with CT-1 for 24 hrs, nuclei of NIH3T3 cells were extracted and ChIP were performed with anti-MAZ antibody or IgG control followed by amplification with primers targeting the −526 to −410 bp of Adam10 promoter region. An adjacent fragment of Adam10 intron1 was included as a negative control. ‘Input’ indicates PCR amplification of total DNA. (D) The relative amount of Adam10 promoter fragments by CHIP assay in (D) were determined by qRT-PCR. All data represent means ± SEM (one-way ANOVA). N = 4, *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4. MAZ regulates CT-1 induced expression of NICD, ADAM10 and GFAP in NPCs.

NPCs isolated from E11.5–13.5 mouse embryonic cortex were transfected with MAZ, sh-MAZ, hNICD or control constructs as indicated before seeding onto culture dishes. Cell lysates were prepared 72 hrs after CT-1 stimulation and NICD, ADAM10, MAZ, GFAP, Nestin or Map2 levels were analyzed by Western blot analysis. The blots were cropped to improve the clarity and conciseness and full-length blots are presented in Supplementary Fig. S3. All data represent means ± SEM (one-way ANOVA). N ≥ 4, *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5. MAZ is required for CT-1 induced gliogenesis of NPCs.

NPCs were isolated from E13.5–14.5 mouse embryonic cortex and transfected with GFP, sh-MAZ, hNICD or control constructs as indicated. Cells were treated with or without CT-1 for 72 hrs and subjected to immunofluorescent analysis. Nestin, Tuj1, and GFAP positive (⁺) cells were counted in those GFP positive cells. (A) The representative images. (B–D) Statistical analyses of the percentage of GFAP+, Nestin+, and Tuj-1+ cells in GFP+ cells. All data represent means ± SEM (one-way ANOVA). N ≥ 4, *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 6. A schematic model for the crosstalk between CT-1 and NOTCH signalings during gliogenesis.

CT-1 plays an important role in ensuring co-activation of JAK/STAT and Notch1 signaling during gliogenesis. CT-1 regulates the rate-limiting S2 enzymes of NOTCH, ADAM10 via transcription factor, Maz. Maz is essential for CT-1 induced up-regulation of ADAM10, NICD and GFAP, as well as gliogenesis.