Transcription factor klf12 negatively regulates estradiol synthesis in ovarian granulosa cells

To explore the mechanism of KLF12 (Krüppel-like factor 12) involved in regulation of estradiol(E2) synthesis in ovarian granulosa cells. Adenovirus-mediated overexpression of KLF12 in KGN cells to detection of CYP19A1 expression and E2 release. Ovarian granulosa cell line (KGN) were cultured in phenol red-free DMEM/F12 medium containing 10% charcoal/dextran-treated FBS, with added 2 μM androstendione. E2 release were measured using Access Immunoassay System. The chromatin immunoprecipitation technique/PCR (ChIP/PCR) and avidin-biotin conjugate DNA precipitation (ABCD) assay were used to identify the target gene for KLF12 in KGN cells. Otherwise, the effect of KLF12 in the regulation of CYP19A1 gene expression in KGN was determined by luciferase reporter assay and Q-PCR. The data are expressed as the means ± SEM from at least three independent experiments. Student's t-test and ANOVA were performed to detect differences between two groups and among more than two groups, respectively. The values were determined to be significant when P < 0.05. Immunocytochemistry for the zinc finger-containing transcription factor KLF12 protein was high level expression in the cytoplasm of granulosa cells and oocyte of maturing wild-type follicles. Adenovirus-mediated overexpression of KLF12 significantly suppressed E2 release in KGN cells culture media (3075.0 ± 430.1 vs 4817.5 ± 464.2, P < 0.05, compared to Ad-GFP group). In addition, Q-PCR demonstrated that KLF12 decreased CYP19A1 mRNA expression by 40% in KGN. Moreover, luciferase reporter, ChIP/PCR and ABCD assay further confirmed that KLF12 directly binds to the CAGTGGG sequence within the promoter region of CYP19A1 gene. Furthermore, KLF12 mRNA and protein expression in KGN cells were significantly decreased after treatment with FSH. Importantly, overexpression of KLF12 significantly attenuated FSH-induced CYP19A1 mRNA expression and E2 release in KGN (6071.8 ± 974.5 vs 12818.4 ±1116.6, P < 0.01, compared to FSH). Our study provides the first evidence that KLF12 is a novel transcription factor directly binds to CYP19A1 promoter and suppressed CYP19A1 expression, and then negatively regulates ovarian granulosa cell estradiol synthesis.