Table 1.
Inhibitors or antagonists used in this study.
Table 2.
Primer sequences for real-time PCR used in this study.
Fig 1.
Induction of neurite outgrowth and the related genes by IR in C17.2 cells.
C17.2 cells were exposed to IR at 4, 6 and 8 Gy or treated with NGF and BDNF at 20 ng/ml, and incubated at 37°C for 72 hr. The morphological change for neurite outgrowth by IR was observed in microscopic images (×200 magnification) (A). To assess rate of neurite-bearing cells, each 200 cells in three randomly taken images were analyzed by Image J software (A, right-down panel). To confirm IR-induced mRNA expression of neurite outgrowth-related genes, GAP43 and Rab13, irradiated Neuro-2a cells were analyzed by real-time PCR (B). The results represent the mean ± SD from triplicate data. *p < 0.05, **p < 0.01 vs naive group.
Fig 2.
Induction of neuronal marker by IR in C17.2 cells.
C17.2 cells were exposed to IR at 6 Gy or treated with NGF and BDNF at 20 ng/ml, and incubated at 37°C for 72 hr. Effects of IR on expression of NSC marker, nestin, neuronal marker, β-III tubulin, and astrocyte marker, GFAP were analyzed by Western blot and quantified by Image J software. The results represent the mean ± SD from triplicate data. **p < 0.01 vs naive group.
Fig 3.
Induction of neuronal function-related genes by IR in C17.2 cells.
C17.2 cells were exposed to IR at 6 Gy or treated with NGF and BDNF at 20 ng/ml, and incubated at 37°C for 72 hr. To assess effect of IR on neuronal function-related genes, expression of synaptophysin and synaptotagmin1 (A), GABAA and GABAB receptors (B), ionotropic glutamate receptors (C), and metabotropic glutamate receptors (D) was analyzed by real-time PCR. The results represent the mean ± SD from triplicate data. *p < 0.05, **p < 0.01 vs naive group.
Table 3.
Summary of expression profile of neuronal function-related genes in IR-stimulated and neurotrophin-stimulated C17.2 cells.
Fig 4.
Suppression of IR-induced neuronal properties by mGluR1 antagonist, LY367385 in C17.2 cells.
C17.2 cells treated with the antagonists of mGluR1 (LY367385) at 25 μM, mGluR5 (MPEP) at 5 μM, Group II mGluRs (mGluR2 and mGluR3; LY341495) at 100 nM and Group III mGluRs (mGluR4, mGluR7 and mGluR8; MSOP) at 100 μM for 2 hr, and then exposed to IR at 6 Gy and incubated at 37°C for 72 hr. To assess rate of neurite bearing cells, each 200 cells in three randomly taken images were analyzed by Image J software. IR-induced neurite outgrowth is not affected by the antagonists of mGluR5, Group II mGluRs nad Group III mGluRs but was blocked by mGluR1 antagonist, LY367385 (A). Expression of NSC marker, nestin, and neuronal marker, β-III tubulin was analyzed by Western blot (B, upper panel), and then quantified by Image J software (B, down panel). To assess effect of mGluR1 inhibition on IR-induced neuronal function-related genes, mRNA expression of synaptophysin, synaptotagmin1 and GABAA receptors was analyzed by real-time PCR (C). *p < 0.05, **p < 0.01 vs naive group, †p < 0.05, ††p < 0.01 vs radiation only group.
Fig 5.
Effect of STAT3 and p53 inhibitors on IR-induced neuronal properties in C17.2 cells.
C17.2 cells were treated with the inhibitors of JNK (SP600125) at10 μM, STAT1 (Fludarabine) at 10 μM, STAT3 (S3I-201) at 10 μM, Rac (NSC23766) at 20 μM, B-Raf (GDC-0879) at 20 μM and p53 (Pft-α) at 20 μM for 2 hr, and then exposed to IR at 6 Gy and incubated at 37°C for 72 hr. To assess rate of neurite bearing cells, each 200 cells in three randomly taken images were analyzed by Image J software. IR-induced neurite outgrowth is not affected by the inhibitors of JNK, STAT1, Rac and B-Raf but was blocked by the STAT3 inhibitor, S3I-201 and the p53 inhibitor, Pft-α (A). Expression of NSC marker, nestin, and neuronal marker, β-III tubulin was analyzed by Western blot (B, upper panel), and then quantified by Image J software (B, down panel). **p < 0.01 vs naive group, †p < 0.05, ††p < 0.01 vs radiation only group.
Fig 6.
Suppression of IR-induced mGluR1 expression by STAT3 inhibitor in C17.2 cells.
C17.2 cells were treated with STAT3 inhibitor, S3I-201 at 10 μM and p53 inhibitor, Pft-α at 20 μM for 2 hr, and then exposed to IR at 6 Gy and incubated at 37°C for 72 hr. Level of mGluR1 mRNA was analyzed by real-time PCR (A) and expression of mGluR1 was analyzed by Western blot (B). The results represent the mean ± SD from triplicate data. **p < 0.01 vs naive group, ††p < 0.01 vs radiation only group.
Fig 7.
Suppression of IR-induced neuronal differentiation signaling by PI3K inhibitor in C17.2 cells.
C17.2 cells treated with the inhibitors of MEK (PD98059), p38 (SB203580), PKA (H-89) and PI3K (LY294002) at 10 μM for 2 hr, and then exposed to IR at 6 Gy and incubated at 37°C for 72 hr. To assess rate of neurite bearing cells, each 200 cells in three randomly taken images were analyzed by Image J software. IR-induced neurite outgrowth is not affected by inhibitors of MEK, p38 and PKA but was blocked by PI3K inhibitor, LY294002 (A). Level of β-III tubulin and mGluR1, and activation of AKT, p53 and STAT3 were analyzed by Western blot (B). The results represent the mean ± SD from triplicate data. **p < 0.01 vs naive group, ††p < 0.01 vs radiation only group.
Fig 8.
Effect of STAT3 inhibitor and mGluR1 antagonist on the IR-induced expression of neuronal markers in C17.2 cells.
C17.2 cells were treated with S3I-201 at 10 M and LY367385 25 M at for 2 hr, and then exposed to IR at 6 Gy and incubated at 37°C for 72 hr. NSC marker, nestin positive (red) and neuronal marker, β-III tubulin positive (green) cells were analyzed by immunocytochemistry (×100 magnification) (A). Nuclei were labeled with Hoechst (blue). The rate of antibody positive cells was quantified by Image J software (B). **p < 0.01 vs naive group, ††p < 0.01 vs radiation only group.
Fig 9.
Effect of STAT3 inhibitor and mGluR1 antagonist on the IR-induced neuronal markers in mouse primary neural stem cells.
Isolated NSCs were treated with S3I-201 at 5 M and LY367385 10 M at for 2 hr, and then exposed to IR at 1 Gy and incubated at 37°C for 72 hr. NSC marker, nestin positive (red) and neuronal marker, β-III tubulin positive (green) cells were analyzed by immunocytochemistry (×100 magnification) (A). Nuclei were labeled with Hoechst (blue). The rate of antibody positive cells was quantified by Image J software (B). **p < 0.01 vs naive group, ††p < 0.01 vs radiation only group.
Fig 10.
Schematic representation of signaling pathway in IR-induced neuronal differentiation.
Ionizing radiation promotes the neuronal differentiation of C17.2 cells via two independent signaling pathways, PI3K-p53 and PI3K-STAT3-mGluR1. p53 and STAT3 may play as the downstream target of PI3K signaling in IR-induced neuronal differentiation of C17.2 cells. The activation of p53 via PI3K may be involved in the IR-induced neurite outgrowth and neuronal marker expressions. mGluR1 signaling of PI3K-STAT3 downstream may also be involved in the IR-induced neuronal function-related protein expressions as well as neurite outgrowth and neuronal marker expressions.