NF-κB-vimentin is involved in steroidogenesis stimulated by mono-butyl phthalate in primary cultured ovarian granulosa cells

Di-n-butyl phthalate (DBP) and its active metabolite, monobutyl phthalate (MBP) are the most common endocrine disrupting chemicals. Many studies indicated the effects of MBP on male steroidogenesis, however, little attention have been paid on the effects of low levels of MBP on female steroidogenesis. This study was aimed to assess steroidogenesis stimulated by low-dose MBP on primary cultured ovarian granulosa cells (mGCs). Ovarian granulosa cells were isolated from ICR female mice. Hormone levels in medium were detected by ELISA, mRNA and protein expressions of vimentin, NF-κB p65 and phosphorylation of NF-κB p65 (p-p65) were assayed by qRT-PCR, western blot and immunohistochemistry, respectively. Besides, confocal immunofluorescence and electrophoretic mobility shift assay (EMSA) were used for detecting vimentin expression and activity of NF-κB p65 binding to the promoter of vimentin, respectively. Progesterone levels, mRNA and protein levels of vimentin and p-p65 in cells were increased significantly in mGCs treated by MBP at 10− 10 M. Additionally, MBP-induced steroidogenesis was blocked when vimentin protein was knocked down or activity of NF-κB was inhibited. EMSA assay showed that binding activity of NF-κB to the promoter regions of vimentin was boosted after MBP exposure. Accordingly, the results suggested that MBP could up-regulated steroidogenesis through NF-κB-vimentin signal in mGCs.