Mechanism Of Inhibition Of Nicotine On StAR Expression In Fetal Adrenal Gland Mediated By YY1

Objective:Prenatal nicotine exposure can induce intrauterine growth retardation(IUGR),Alteration of the hypothalamic-pituitary-adrenal(HPA)axis during fetal development resulted in IUGR.Adrenal gland is the terminal effector organ of HPA axis,which is responsible for the synthesis of a variety of steroid hormones.And steroidogenic acute regulatory protein(StAR)plays a key role in steroid hormone synthesis.On the basis of our early study that the nicotine-mediated reduction of steroidogenic factor-1 expression resulted in inhibition of fetal adrenal steroid hormone synthesisvia the action of histone deacetylases(HDACs),the purpose of this subject is to further confirm the inhibitory effect of nicotine on the expression of StAR in the fetal adrenal gland and to elucidate the potential mechanism of transcriptional factor yin yang 1(YY1)in this process.Methods:Pregnant Wistar rats were randomly divided into control group and nicotine group.Rats in nicotine group were given subcutaneous administration twice a day at a dose of 1 mg/kg during gestation day 11 to 20 and those in control group were injected with the same volume of saline.On pregnant day 20,the pregnant rats were killed,and the fetal adrenal glands were collected.The gene and protein levels of StAR and YY1 in fetal adrenal gland were detected by real-time quantitative PCR(qRT-PCR)and immunohistochemical staining.Chromatin immunoprecipitation(ChIP)was used to detect the histone modification and interaction between YY1 and StAR promoter.In cell culture experiments,NCI-H295A cells were treated with nicotine(25 and 50 μM)for different days,qRT-PCR and western blotting were used to detect the expression of StAR and YY1.ChIP was used to detect the histone modification and interaction between YY1 and StAR.Results:①Compared with the control group,StAR protein levels were decreased in fetal adrenal gland of the prenatal nicotine-exposed group(P<0.01).Furthermore,increase in YY1 mRNA(about 1.2 times of the control)and protein levels(about 5.2 times of the control)were observed in the prenatal nicotine-exposed group(P<0.01).②Compared with the control group,the acetylation of H3 acetyl-K9 and H3 acetyl-K14 at the StAR promoter region presented a decreasing trend in nicotine exposure group,however,there was an increasing trend for H3 acetyl-K14 in YY1 promoter region(-474～-350,-373～-215)whereas there was a decreasing trend for H3 acetyl-K9 in YY1 promoter region(-373～-215).③In nicotine-treated NCI-H295A cells,a nicotine treatment of 50 μM for 5 days also inhibited StAR mRNA and protein expression when compared with the control(P<0.01,P<0.05).Compared with the controls,a significant increase of YY1 mRNA expression was found at respective concentrations of 25 and 50 μM for 5 days(P<0.01).Furthermore,nicotine enhanced YY1 mRNA expression by 168.9%(P<0.01)and 169.5%(P<0.01)at a concentration of 50μM for 3 and 5 days,respectively.Treatment with 50 μM nicotine for 5 days increased YY1 protein expression when compared with the control(P<0.01).④ChIP assay results showed that nicotine treatment decreased the levels of H3 acetyl-K9(P<0.05)and H3 acetyl-K14(P<0.01)in the StAR promoter region in NCI-H295A cells.⑤ChIP assay showed,when compared with the controls,that the interaction between YY1 and StAR promoter in the nicotine treatment group was significantly increased(P<0.01).Conclusion:Prenatal nicotine exposure induced YY1 expression and suppressed StAR expression in fetal rat adrenal glands in vivo and in NCI-H295A cells in vitro.YY1/HDAC-synergistical medicated histone deacetylation modification in the StAR promoter might play an important role in the suppression of fetal adrenal StAR expression after nicotine treatment.