Cross talk of CCK8 and EGF in mouse neurons |
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Authors: | EGF in mouse neurons LI Juan WANG Li CHEN Rong LIU Xiao-yan MEI Zhi-qiang HE Tao |
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Affiliation: | Molecular Biology Laboratory, Luzhou Medical College, Luzhou 646000, China |
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Abstract: | AIM: To explore the mechanism of signaling transduction and cross talk between cholecystokinin octapeptide (CCK8) and epidermal growth factor (EGF) in mouse neurons and to observe the effect of CCK8 in coordination with EGF on neuron growth and cell viability. METHODS: For determining which kind of CCK receptor mediated the phosphorylation of EGF receptor, the cultured neurons were randomly divided into control group, CCK8 stimulation group, CCKA receptor antagonist group, CCKB receptor antagonist group, and CCKA+CCKB receptor antagonist group. Control and stimulation groups were stimulated with DMEM and CCK8 (10-7 mol/L) for 5 min, respectively, while antagonist groups were pre-incubated with different types of receptor antagonists (10-8 mol/L) for 10 min and followed by stimulating the neurons with CCK8. For observing the effect of CCK8 and EGF on the phosphorylation of EGFR in neurons and on neuron growth and cell viability, the cultured neurons were randomly divided into control group, CCK8 stimulation group, EGF stimulation group and CCK8+EGF stimulation group, which were stimulated with DMEM, CCK8 (10-7 mol/L), EGF (40 μg/L) and CCK8+EGF for 5 min, respectively. Reactions were terminated by freezing the neurons in liquid nitrogen and the phosphorylated EGFR was detected by Western blotting. Meanwhile, the viability of the neurons was observed by MTT method after stimulated for 24 h, 48 h, 72 h and 96 h. RESULTS: The phosphorylation levels of EGFR were decreased in the neurons treated with either of the two CCK receptor antagonists, and more obvious decrease was observed when the two CCK receptor antagonists were used in combination. Compared with control group, the phosphorylation levels of EGFR in the neurons were significantly increased(P<0.05) after stimulated with CCK8 or EGF, and the increase was more remarkable in CCK8+EGF stimulation group. CCK8 or EGF improved the viability and prolonged the life span of the neuron, and synergism of these two reagents was observed. CONCLUSION: Both CCKA and CCKB receptors are involved in the phosphorylation of EGFR in the neurons stimulated by CCK8, and the type A receptor may play a more important role. There is cross-talk between CCK8 and EGF signaling pathways in neurons. The signaling cross-talk between CCK8 and EGF may be the underlying molecular mechanism responsible for the synergistic effect on the neuron growth and viability in vitro. |
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Keywords: | Cholecystokinin Neurons Receptors epidermal growth factor |
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