Regulation Of Neurite Development-Associated Proteins By Corticotropin-Releasing Hormone Receptors In Cultured Hippocampal Neurons

The hippocampus is one of the basal structures of the limbic system. It plays a fundamental role in learning and memory process. Corticotropin-releasing hormone (CRH) is the adrenocorticotropin secretion from the anterior pituitary and capable of integrating the neuroendocrine, autonomic, behavioural, neurite growth and immune response to stress by CRH receptors (CRH-Rs). CRH receptors include two major characterized members: CRH-R1 and CRH-R2. At present, the study on the functions of CRH receptors (CRH-Rs) remains elusive.The research showed CRH promoted neurite growth in hippocampal neurons. Therefore, we first examined the regulation of neurite development by CRH-Rs in cultured rat hippocampal neurons.Then we studied the regulation of collapsin response mediator protein 3 (CRMP3) and mitotic kinesin like protein 1 (MKLP1) by CRH-Rs in cultured hippocampal neurons. And the mechanism of this effect was further explored.Main results:1. Regulation of neurite development by CRH-Rs in cultured hippocampal neurons.By transfection with enhanced green fluorescent protein (EGFP), CRH was found to increase the total dendritic branch length (TDBL) in cultured hippocampal neurons, which can be blocked by specific CRH-R1 antagonist antalarmin, not by specific CRH-R2 antagonist astressin 2B. And UrocortinⅡ, the specific CRH-R2 agonist, was found to decrease TDBL. The results demonstrated that activated CRH-R1 promoted neurite development but CRH-R2 inhibited it in cultured hippocampal neurons.2. Regulation of neurite development-associated proteins by CRH-Rs in cultured hippocampal neurons.1) Regulation of CRMP3 expression by CRH-Rs(1) Immunofluorescence analysis showed CRMP3 protein expression in cultured hippocampal neurons.(2) Real-time PCR and Western blot showed that CRH decreased CRMP3 mRNA and protein expression dose-dependently, which can be blocked by nonselective CRH receptor antagonist astressin or specific CRHR2 antagonist astressin 2B, not by specific CRHR1 antagonist antalarmin. UrocortinⅡ, the specific CRHR2 agonist had the greater effect on CRMP3 expression. The results demonstrated that activated CRH-R2 but not-R1 mediated the regulatory effect on CRMP3 expression.(3) UrocortinⅡincreased phosphorated PLC-β3 expression which can be blocked by astressin 2B at protein level.(4) Treatment of cells with PLC inhibitor U73122 exhibited a similar effect as PKCα/βinhibitor Go6976, completely blocking UrocortinⅡaction on CRMP3 expression. And PMA could significantly decrease CRMP3 expression. Treatment of cells with AC inhibitor SQ22536 or PKA inhibitor H89 did not block CRHR2-induced CRMP3 expression. And Forskolin could not decrease CRMP3 expression.2) Regulation of MKLP1 expression by CRH-Rs(1) Immunofluorescence analysis showed MKLP1 protein expression in cultured hippocampal neurons.(2) Western blot showed that CRH up-regulated MKLP1 protein expression dose-dependently, which can be blocked by antalarmin, not by astressin 2B. The results demonstrated that activated CRH-R1 but not-R2 mediated the regulatory effect on MKLP1 protein expression.(3) Treatment of cells with U73122 or Go6976 did not block CRHR1-induced MKLP1 expression at mRNA and protein level. SQ22536 or H89 also did not block CRHR1 action. And ERK inhibitor PD98059, JNK inhibitor SP600125 and p38 inhibitor SB203580 also did not block CRHR1-induced MKLP1 expression at mRNA level.Conclusions:In conclusion, these results suggest that in hippocampal neurons, CRH-R1 activates to accelerate neurite development contrary to CRH-R2. Moreover, activated CRH-R2 can decrease CRMP3 expression, which is dependent on PLC/PKC signaling pathways. In addition, activated CRH-R1 also can increase MKLP1 expression at protein level.