Fig 2: IMD inhibited Notch1 signaling activation by ADAM10. (A) Quantitative real-time PCR analysis of mRNA levels of ADAM10 and ADAM17 in aortas of Control, AngII, AngII+IMD and AngII+DAPT in mice. (B) Western blot analysis of protein level of ADAM10 in aortas of Control, AngII, AngII+IMD and AngII+DAPT mice. Western blot analysis of protein levels of (C, D) NICD, the intracellular fragment of Notch1 and the full length Notch1 receptor and (E) ADAM10 and ADAM17 in AngII-induced macrophage cell line Raw264.7. n=3, Data are mean ± SD. *P<0.05, **P<0.01 vs. Control. #P<0.05, ##P<0.01 vs. AngII. (F) Western blot analysis of protein level of ADAM10 in AngII-induced macrophage cell line Raw264.7. Except for the first 3 groups, IMD receptor blocker IMD17-47 group was added. n=3, Data are mean ± SD. *P<0.05 vs. Control. #P<0.05 vs. AngII. & P<0.05 vs. AngII+IMD. (G) Western blot analysis of protein level of ADAM10 in AngII-induced macrophage cell line Raw264.7. Except for the first 3 groups, cAMP/PKA inhibitor H89, cGMP/PKG inhibitor Ly83583 and PI3K/Akt inhibitor LY294002 were added. n=3, Data are mean ± SD. *P<0.05 vs. Control. #P<0.05 vs. AngII. $ P<0.05 vs. AngII+IMD.

Fig 3: Physical exercise reverses the decreased Notch signaling pathway in the hippocampus induced by chronic restraint stress. (a) The experimental timeline. (b) Sucrose preference test. (c) Forced swim test. (d) Locomotor activity. Total distance traveled in 30 min. Control, n = 8; Restraint, n = 12 in b, c, and d. Mice of all groups were 2‐month‐old. ***p < 0.001 in b and c. (e) Representative immunoblots and quantification of hippocampal (including Notch1, NICD, ADAM10, ADAM17) and serum protein levels (adiponectin). (f) qRT‐PCR analysis of gene expression for components of a Notch signaling pathway in hippocampus after restraint, including Notch1‐4, ADAM10, ADM17, Hes1, Hes5, Hey1, and HeyL Control, n = 8; Restraint, n = 9 in e and f. Mice of all groups were 2‐month‐old. ***p < 0.001 in e and f. (g) The experimental timeline. (h) Sucrose preference test. (i) Forced swim test. (j) Locomotor activity. Total distance traveled in 30 min. Control, n = 10; Restraint, n = 12; Restraint + Running, n = 12 in b, c, and d. Mice of all groups were 2‐month‐old. *p < 0.05, ***p < 0.001 in h and i. (k) Representative immunoblots and quantification of hippocampal (including Notch1, NICD, ADAM10, ADAM17) and serum protein levels (adiponectin). (l) qRT‐PCR analysis of gene expression for components of a Notch signaling pathway in hippocampus, including Notch1‐4, ADAM10, ADM17, Hes1, Hes5, Hey1, and HeyL Control, n = 10; Restraint, n = 10; Restraint + Running, n = 10 in e and f. Mice of all groups were 2‐month‐old. *p < 0.05, **p < 0.01, ***p < 0.001 in k and l. Data are presented as means ± SEM

Fig 4: 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.

Fig 5: Summarizing cartoon of our findings combined with data published by other groups before. CD44 is cleaved by ADAM10 to generate a CD44 C-terminal fragment (CD44 CTF). This CTF is then prone to y-secretase (y-sec) cleavage within the membrane, which generates a CD44 intracellular domain (CD44 ICD). This ICD translocates to the nucleus and induces the expression of e.g. MMP14, MMP2 and MMP9. At the cell surface CD44, MMP14, MMP2, MMP9 and the tissue inhibitor of metalloproteinases 2 (TIMP2) form a multi-protein complex and attach to intracellular F-actin. This complex influences cell-cell and cell-matrix contacts and thereby changes cell migration, spheroid formation and attachment to hyaluronan.