Bisphenol A promotes dendritic morphogenesis of hippocampal neurons through estrogen receptor-mediated ERK1/2 signal pathway

Abstract

Bisphenol A (BPA), an environmental endocrine disruptor, has attracted increasing attention to its adverse effects on brain developmental process. The previous study indicated that BPA rapidly increased motility and density of dendritic filopodia and enhanced the phosphorylation of N-methyl-d-aspartate (NMDA) receptor subunit NR2B in cultured hippocampal neurons within 30min. The purpose of the present study was further to investigate the effects of BPA for 24h on dendritic morphogenesis and the underlying mechanisms. After cultured for 5d in vitro, the hippocampal neurons from 24h-old rat were infected by AdV-EGFP to indicate time-lapse imaging of living neurons. The results demonstrated that the exposure of the cultured hippocampal neurons to BPA (10, 100nM) or 17β-estradiol (17β-E2, 10nM) for 24h significantly promoted dendritic development, as evidenced by the increased total length of dendrite and the enhanced motility and density of dendritic filopodia. However, these changes were suppressed by an ERs antagonist, ICI182,780, a non-competitive NMDA receptor antagonist, MK-801, and a mitogen-activated ERK1/2-activating kinase (MEK1/2) inhibitor, U0126. Meanwhile, the increased F-actin (filamentous actin) induced by BPA (100nM) was also completely eliminated by these blockers. Furthermore, the result of western blot analyses showed that, the exposure of the cultures to BPA or 17β-E2 for 24h promoted the expression of Rac1/Cdc42 but inhibited that of RhoA, suggesting Rac1 (Ras related C3 botulinum toxinsubstrate 1)/Cdc42 (cell divisioncycle 42) and RhoA (Ras homologous A), the Rho family of small GTPases, were involved in BPA- or 17β-E2-induced changes in the dendritic morphogenesis of neurons. These BPA- or 17β-E2-induced effects were completely blocked by ICI182,780, and were partially suppressed by U0126. These results reveal that, similar to 17β-E2, BPA exerts its effects on dendritic morphogenesis by eliciting both nuclear actions and extranuclear-initiated actions that are integrated to influence the development of dendrite in hippocampal neurons.

Keywords: 17beta-estradiol; 17β-E(2); AdV-EGFP; BPA; BSA; Bisphenol A; CNS; Cdc42; DAB; DMEM; DMSO; Dulbecco’s Modified Eagle Medium; ECL; ER; ERK1/2; ERKs; Estrogen receptor; F-actin; FBS; GPR30; Gprotein-coupled receptor 30; Hippocampal neurons; MAPK; MEK; Morphogenesis; N-methyl-d-aspartate; N-methyl-d-aspartate receptor subunit 2B; NMDA; NR2B; PBS; PBS/Tween-20; PBST; PMSF; Rac1; Ras homologous A; Ras related C3 botulinum toxinsubstrate 1; Rho A; RhoA and Rac1/Cdc42; SDS–PAGE; SEM; bisphenol A; bovine serum albumin; cell divisioncycle 42; central nervous system; diaminobenzidine; dimethyl sulfoxide; enhanced chemiluminescence; estrogen receptor; extracellular signal-regulated kinase 1/2; fetal bovine serum; filamentous actin; mitogen-activated ERK-activating kinase; mitogen-activated protein kinase; pNR2B; phenylmethanesulfonyl fluoride; phosphate buffered saline; phosphorylated N-methyl-d-aspartate receptor subunit 2B; sodium lauryl sulfate–polyacrylamide gel electrophoresis; standard error of mean; the recombinant adenovirus encoding enhanced green fluorescent protein.