Figure 1.
Long-term Continuous CORT treatment decreases Flk1 protein levels in vitro and in vivo.
(A) CORT (CORT; I µM) was applied to mouse primary cortical neurons at DIV 5. Flk1 protein levels were determined by western blotting analysis at 48 hand 72 h following CORT treatment. CON means DMSO treatment. Data represent mean±SE. (n = 6) expressed as fold change in Flk1 protein levels as compared to CON. β-actin is the loading control. *P<0.05 (Bonferroni's test). (B) Flk1 protein levels in frontal cortex of mice treated with CORT or vehicle control (CON; 0.45% hydroxypropyl-β-cyclodextrin) for 7 weeks. Data represent mean±SE (n = 6–8) expressed as fold change in Flk1 protein levels as compared to CON. *P<0.01 (t test).
Figure 2.
Long-term Continuous CORT treatment alters phospho PTEN, phospho Akt and phospho mTOR protein levels in cortical neurons.
CORT (CORT; I µM) was applied to mouse primary cortical neurons at DIV 5. Cell lysates collected at 48 h or 72 h following CORT treatment were used for western blot analysis. CON means DMSO treatment. Data represent mean±SE (n = 6) expressed as fold change in (A) phospho PTEN to total PTEN ratio, (B) phospho Akt to total Akt ratio and (C) phospho mTOR to total mTOR ratio as compared to CON. *P<0.01 versus CON; #P<0.01 versus values at 48 h (Bonferroni's test).
Figure 3.
Long-term Continuous CORT treatment increases VEGF protein levels in vitro and in vivo.
(A) CORT (CORT; 1 µM) was applied to mouse primary cortical neurons at DIV 5. VEGF protein levels were determined by western blotting analysis at 48 hand 72 h following CORT treatment. CON means DMSO treatment. Data represent mean±SE (n = 6) expressed as fold change in VEGF protein levels as compared to CON. *P<0.05 (Bonferroni's test). (B) VEGF protein levels in frontal cortex of mice treated with CORT (5 mg/kg) or vehicle control (CON; 0.45% hydroxypropyl-β-cyclodextrin) for 7 weeks were determined by western blot analysis. Data represent mean±SE (n = 5) expressed as fold change in VEGF protein levels as compared to CON. *P<0.01 (t test). (C) VEGF protein levels in serum samples collected from mice treated with CORT (CORT; 5 mg/kg) or vehicle control (CON; 0.45% hydroxypropyl-β-cyclodextrin) for 7 weeks were analysed by ELISA. Data represent mean±SE (n = 5–6) expressed as fold change in VEGF protein levels as compared to CON. *P<0.01 (t test).
Figure 4.
PI3K signaling is not involved in long-term continuous CORT-induced increases in VEGF protein levels.
(A) VEGF protein levels were determined by western blotting analysis in neuronal cell lysates treated with LY294002 (LY; 20 µM) for 48 h. CON means DMSO treatment. Data represent mean±SE (n = 6) expressed as fold change in VEGF protein levels as compared to CON. *P<0.05 (Bonferroni's test). (B) Pretreatment with LY did not prevent CORT-induced induction in VEGF protein levels in neurons. Cortical neurons at DIV 5 were treated with LY (20 µM) for 30 min followed by CORT (1 µM) exposure for 48 h. VEGF protein levels were determined in cell lysates by western blot analysis. CON means DMSO treatment. Data represent mean±SE (n = 6) expressed as fold change in VEGF protein levels as compared to CON. *P<0.05 (Bonferroni's test).
Figure 5.
Chronic CORT-induced Flk1 regulation is mediated through calcium.
(A) Calcium chelator BAPTA-AM blocked CORT (CORT)-induced reduction in Flk1 protein levels. BAPTA-AM (50 µM) was applied 30 min before CORT (1 µM) treatment to cultured neurons at DIV 5. Cell lysates were collected at 48 h after CORT treatment and Flk1 protein levels were determined by western blot analysis. CON means DMSO treatment. Data represent mean±SE (n = 5) expressed as fold change in Flk1 protein levels as compared to CON. *P<0.01 versus CON; #P<0.01 versus CORT (Bonferroni's test). (B) Chronic CORT treatment increases NCS-1 protein levels in neurons. CORT (CORT; 1 µM) was applied to mouse primary cortical neurons at DIV 5. NCS-1 protein levels were determined by western blotting analysis at 48 h following CORT treatment. CON means DMSO treatment. Data represent mean±SE (n = 5) expressed as fold change in NCS-1 protein levels as compared to CON. *P<0.01 (Bonferroni's test). (C) Chronic CORT treatment increases NCS-1 protein levels in mouse frontal cortex. NCS-1 protein levels in frontal cortex of mice treated with CORT (5 mg/kg) or vehicle control (CON; 0.45% hydroxypropyl-β-cyclodextrin) for 7 weeks were determined by western blot analysis. Data represent mean±SE (n = 6) expressed as fold change in NCS-1 protein levels as compared to CON. β-actin is the loading control.*P<0.05 (Bonferroni's test).
Figure 6.
GR downregulation is involved in chronic CORT-induced downregulation of Flk1.
(A) GR downregulation following chronic CORT exposure in neurons. CORT (CORT; 1 µM) was applied to mouse primary cortical neurons at DIV 5. GR protein levels were determined by western blotting analysis at 48 h following CORT treatment. CON means DMSO treatment. Data represent mean±SE (n = 5) expressed as fold change in GR protein levels as compared to CON. *P<0.05 (t test). (B) Chronic CORT treatment increases GR protein levels in mouse frontal cortex. GR protein levels in frontal cortex of mice treated with CORT (5 mg/kg) or vehicle control (CON; 0.45% hydroxypropyl-β-cyclodextrin) for 7 weeks were determined by western blot analysis. Data represent mean±SE (n = 5) expressed as fold change in GR protein levels as compared to CON. β-actin is the loading control.*P<0.05 (t test). (C) RU486 (RU, a GR antagonist) blocked CORT-induced reduction in GR protein levels. RU (1 µM) was applied 30 min before CORT (1 µM) treatment to cultured neurons at DIV5. Cell lysates were collected at 48 h after CORT treatment and GR protein levels were determined by western blot analysis. CON means DMSO treatment. Data represent mean±SE (n = 5) expressed as fold change in GR protein levels as compared to CON. *P<0.01 versus CON; #P<0.01 versus CORT (Bonferroni's test). (D) RU486 (RU, a GR antagonist) blocked CORT-induced reduction in Flk1 protein levels. Data represent mean±SE (n = 5) expressed as fold change in Flk1 protein levels as compared to CON. *P<0.01 versus CON; #P<0.01 versus CORT (Bonferroni's test). (E) Western blot analysis of Flk1 protein expression after immunoprecipitation with GR antibody in lysates collected from DIV6 neurons. NoAb: no anti-GR antibody; total: 10% input from total cell lysates. (F) Western blot analysis of GR protein expression after immunoprecipitation with Flk1 antibody in lysates collected from DIV6 neurons. NoAb: no anti-Flk1 antibody. (G) Immunoprecipitation of Flk1 in cell lysates from corticosterone (CORT) or vehicle control (CON; DMSO) treated for 48 h. Western blotting was performed with anti-GR and anti-Flk1 antibodies. Data represent mean±SE (n = 4) expressed as fold change in GR protein levels (normalized to Flk1 protein levels) as compared to CON. *P<0.05 versus CON (t test).
Figure 7.
Reduced Flk1 and GR protein levels in prefrontal cortex of schizophrenia subjects.
(A) Reduced Flk1 protein levels in prefrontal cortex samples from schizophrenia subjects. Flk1 protein levels in the prefrontal cortex of schizophrenia (SZ; n = 10) and control (CON; n = 8) subjects were determined by western blot analysis. Data represent mean±SE expressed as fold change in Flk1 protein levels as compared to CON. *P<0.05 versus CON (t test). (B) Reduced GR protein levels in prefrontal cortex samples from schizophrenia subjects (SZ; n = 10) as compared to control subjects (CON; n = 8). Data represent mean±SE expressed as fold change in GR protein levels as compared to CON. *P<0.05 versus CON (t test).
Figure 8.
Proposed model showing the effects of corticosterone on VEGF/Flk1 signaling pathway in mouse frontal cortex.
The signaling events induced by corticosterone (CORT) are mediated through the Glucocorticoid receptor (GR). The reduction in Flk1 levels following long-term continuous CORT exposure results in the activation of PTEN, but inhibition of Akt and mTOR phosphorylation. The effects of CORT on Flk1 are mediated through calcium (Ca2+). CORT exposure results in increased levels of VEGF in cortex. The role of VEGF in Flk1 regulation (or vice versa) under CORT exposure remains unknown. Solid arrows represent activation, whereas dashed arrows represent inhibition of the pathways.
Table 1.
Demographic data for postmortem samples.