Exploring The Regulatory Mechanism Of Membrane-initiated Estrogen Signaling On Corticotropic-releasing Hormone Secretion

Corticotropin-releasing hormone(CRH)is the central driving force of the hypothalamopituitary-adrenal(HPA)axis.As an important stress molecule,CRH level changes play a crucial role in acute or chronic stress response,and in the pathogenesis of stress-related disorders such as depression.We have found that the classic nuclear-initiated estrogen signaling(NIES)stimulates CRH gene expression as a transcription factor.However,the possible effect and the mechanism of the membrane-initiated estrogen signaling(MIES)on the expression of CRH remains unclear.There have been indications that MIES may up-regulate nitric oxide synthase(NOS)expression via phosphatidylinositol 3-hydroxy kinase(PI3K)and mitogen-activated protein kinase(MAPK)pathways,which further increase the production of nitric oxide(NO).NO is also a key molecule in stress response and may stimulate CRH release.In the present study we,therefore,first investigated whether MIES may regulates CRH secretion by activating NO production through intracellular PI3 K and/or MAPK pathways.In the SK-N-SH cells which express estrogen receptor(ER)?,ER?,G protein coupled receptor 30(GPR30)that is a membrane estrogen receptor(m ER),NOS,and CRH,we used the specific m ER activator,the estradiol-bovine serum albumin(E2-BSA),to activate m ER.We observed that MIES can significantly increase NO(P < 0.001)and CRH(P =0.019)secretion,as well as the m RNA levels of NO synthase-1(NOS1)(P = 0.005)in 30 mins.A NO donor,sodium nitroprusside dehydrate(SNP),also significantly stimulated CRH secretion(P = 0.001),while a NOSinhibitor(7-nitroindazole,7-Ni)down-regulated CRH secretion(P = 0.008).In addition,giving the inhibitor of PI3K(LY294002,LY)alone,or giving LY plus the inhibitor of MAPK(PD98059,PD)to the cell culture medium appeared to block the stimulating effect of E2-BSA on NO synthesis(P < 0.001);while E2-BSA still significantly stimulated the CRH secretion(P < 0.001),indicating that there is(an)other MIES-mediated CRHstimulating pathway,independent of NO production.We next looked for other kinases that participate in the rapid MIES and were not reported to have a direct effect on NO secretion.This made protein kinase C(PKC)and PKA possible candidates,and PKA was found to be located in the PI3 K pathway.We further studied the effect of the MIES-PKCmediated pathway on CRH secretion,with or without NOS-inhibitor,taking the MIESPKA(-PI3K)pathway as a control.Using the inhibitor for PKA,H-89 dihydrochloride hydrate(H-89)and the inhibitor for PKC,bisindolylmaleimide IV(BIM),we found out that activating m ER with E2-BSA may upregulate CRH secretion via PKC pathway independent of NO synthesis.In addition,we confirmed that the MIES-PKA(-PI3K)indeed activates CRH release via increased NO synthesis.In summary,MIES can efficiently upregulate CRH secretion via various intracellular kinase pathways.Both MIES and NIES up-regulate the HPA axis activity by stimulating CRH,and thus get involved in the stress response and in the pathogenesis of stress-related disorders,such as depression.It deserves further study to reveal the possibility of MIES-,NIES-related molecules as the potential therapeutic targets for depression.