Molecular Mechanisms Underlying The Regulation Of Aromatase By Cortisol In Human Placental Syncytiotrophoblasts

Context:According to the data provided by the World Health Organization, there are about 15 million cases of preterm children worldwide, and about one million cases of preterm infants died from complications each year, and this figure is still rising. However, the lack of identification of the mechanisms underlying human parturition has limited the specific and effective diagnosis and treatment of preterm labor. Therefore elucidation of the molecular mechanisms underlying human parturition has become one of the intensive research areas in reproductive biology.In a number of mammalian species including humans, the onset of parturition is preceded by a rise in estrogen production, which is believed to be a key event leading to birth. This rise of estrogen plays a pivotal role in transforming the myometrium from a quiescent to a contractile state. Thus, understanding the regulation of estrogen synthesis in late gestation is crucial in elucidating the mechanism initiating parturition. In several animal species, glucocorticoids derived from the fetal adrenal glands are known to drive this rise of estrogen production before parturition. Extensive studies in sheep show that glucocorticoids stimulate the placental enzyme P450 c17 hydoxylase that converts progesterone to C19-steroids, which are substrates for the production of estrogen, leading to simultaneous drop in progesterone and rise in estrogen toward the end of pregnancy. However, the placenta of primates, including humans, lacks P450 c17 hydroxylase and cannot use progesterone as a precursor for the synthesis of estrogen. Instead, it relies on dehydroepiandrosterone sulfate (DHEAS) from the maternal and fetal adrenal glands as precursors for estrogen synthesis. Upon reaching the placenta, subsequent to desulfuration, aromatization of DHEA by aromatase in placenta is the crucial step in estrogen formation. However, it is not known whether glucocorticoids, which drive estrogen formation in other animal species, also affect aromatase expression and estrogen formation in the human placenta. Elucidation of this question may help us to understand better the molecular mechanisms underlying human parturition.In this study, we used human term placenta without labor and syncytiotrophoblasts prepared from the placental villi to examine the effect of cortisol on aromatase expression, and the signaling pathway and transcriptional factors involved were investigated by using immunohistochemical staining, quantitative real time PCR (qRT-PCR), Western blotting, luciferase reporter gene assays, enzyme-link immunoassay (EIA), chemiluminescent microparticle immunoassay (CMIA), siRNA interference, and chromatin immunoprecipitation assay (ChIP) etc.Methods and Results:1. Immunohistochemical staining of the paraffin section of human placental villi revealed strong aromatase staining in the syncytial layer but little staining in the villous core, which is consistent with the increased aromatase mRNA and protein levels along with the syncytialization as measured with qRT-PCR and Western blotting. The above results indicate that aromatase is highly expressed in human placental syncytotrophoblasts, while cytotrophoblasts express low level of aromatse.2. By using qRT-PCR, Western blotting, we demonstrated that cortisol (0.01-1μM, 24h) dose-dependently stimulated aromatase expression in primary human placental syncytiotrophoblasts. Furthermore, we also demonstrated that cortiosol (1 μM,24h) increased the conversion of DHEA to estradiol in primary human placental syncytiotrophoblasts with enzyme-link immunoassay.3. Regular PCR, Western blotting and immunohistochemistry revealed that GR mRNA and protein were detectable in human placental syncytiotrophoblasts. Further studies with qRT-PCR and Western blotting illustrated that cortisol-induced aromatase mRNA and protein expression could be blocked by GR antagonist RU486 (1 μM), GR siRNA (120 μM) and the cortisol-induced aromatase mRNA expression could be blocked by the protein synthesis inhibitor CHX (10 μM), suggesting that the increase of aromatase level by cortisol is mediated by GR and dependent on synthesis of at least one other protein.4. We further found that treatment of the human placental syncytiotrophoblasts with db-cAMP (100 μM) and forskolin (100 μM), stimulators for the cAMP pathway, for 24h increased the expression of aromtase mRNA and protein as measured with qRT-PCR and Western blotting. Furthermore, we demonstrated that cortisol (1 μM,12h) significantly increased the intracellular cAMP level in syncytiotrophoblasts with cAMP EIA assay. In addition, qRT-PCR and Western blotting illustrated that cortisol-induced aromatase expression could be blocked by H89 (10 μM), a PKA inhibitor and NF449 (20 μM), a Gas inhibitor. These data suggested that activation of cAMP/PKA pathway was involved in the induction of aromatase expression by cortisol in human placental syncytiotrophoblasts.5. It is well known that the receptor that mediating the effects of glucocorticoids is an intracellular receptor and glucocorticoids could not activate cAMP/PKA pathway directly. Thus we investigated the possible mechanism of activating cAMP/PKA pathway by cortisol. Of interest, it has been shown that glucocorticoids could induce CRH and hCG expression and production in placental syncytiotrophoblasts, and both CRH and hCG operate their effect via the cAMP/PKA signaling pathway. We thus tested if CRH and hCG mediate the regulation of aromatase expression by cortisol. qRT-PCR illustrated that cortisol (0.01-1 μM,12h) dose-dependently induced CRH, hCG a and β subunit mRNA expression in human placental syncytiotrophoblasts, and EIA and CMIA showed increased production of CRH and hCG β subunit in the culture medium upon treatment with cortisol (1 μM) for 12 and 24 h. Combination neutralization of hCG with its antibody (1:100) and blockade of CRH receptors with non-selective antagonist a-h-CRH (1 μM) reduced basal levels of intracellular cAMP (12h), aromatase mRNA and protein (24h) as measured with cAMP EIA assay, qRT-PCR and Western blotting, suggesting that maintenance of the basal expression of aromatase might be the joint effects of hCG and CRH. In addition, measurements with cAMP EIA assay, qRT-PCR and Western blotting also revealed that the induction of intracellular cAMP level and aromatase expression by cortisol treatment (1 μM) could be partially attenuated by either hCG antibody or a-h-CRH, and completely blocked by combined treatment with hCG antibody and a-h-CRH. These data suggested that both CRH and hCG were involved in the induction of aromatase expression by cortisol in human placental syncytiotrophoblasts.6. Because the transcription of aromatase gene can be initiated at differential sites in human placenta, we designed different forward primers aligning to exon Ⅰ.1,1.2, or 2a paired with a common reverse primer aligned to exon Ⅱ to examine which transcript was induced by cortisol with qRT-PCR. Our results showed that human placental syncytiotrophoblasts mainly used the promoter upstream of exon Ⅰ.Ⅰ rather than exon 2a and 1.2 for aromatase transcription, and cortisol (1 μM,24h) could induce the transcription of exon Ⅰ.1 and 2a but not 1.2. Furthermore, luciferase reporter assays demonstrated that cortisol (1 μM,24h) and forskolin (100 μM,24h) stimulated aromatase gene promoter Ⅰ.1 activity in human placental syncytiotrophoblasts.7. Bioinformatic analysis of the promoter sequence flanking the 5’end of aromatase gene exon 1.1 revealed several GC boxes that bind Sp1, which is believed to be responsible for the high transcriptional activity of aromatase in human placenta in previous study. In this study, we found that cortisol (0.01-1 μM,24h) increased Sp1 mRNA and protein level, which could be completely blocked by RU486 (1 μM) in human placental syncytiotrophoblasts with qRT-PCR and Western blotting. Treatment of the syncytiotrophoblasts with mithramycin A (50 μM), an inhibitor of Spl, significantly attenuated the increase of aromatase mRNA and protein level induced by cortisol and forskolin as measured with qRT-PCR and Western blotting. Consistently, Knock-down of Sp1 expression with siRNA (60 nM) significantly decreased Spl protein levels and dramatically attenuated the induction of aromatase protein level by cortisol and forskolin. Furthermore, ChIP assay demonstrated that cortisol, forskolin and db-cAMP increased the binding of Sp1 to aromatase gene promoter Ⅰ.1. We also demonstrated that hCG antibody (1:100) or a-h-CRH (1μM) alone partially attenuated the induced Spl expression and its enrichment at the aromatase gene promoter Ⅰ.1 by cortisol (1 μM,24h). Furthermore, cortisol-enhanced Sp1 expression and its enrichment at the aromatase gene promoter Ⅰ.1 was completely blocked by combined treatment with hCG antibody and a-h-CRH. These results suggest that Sp1 may play a pivotal role in cortisol-induced aromatase expression in human placental syncytiotrophoblasts. Besides Sp1, ChIP assay also revealed that RNA polymerase II as well as the acetylation of H3K9 associated with aromatase gene promoter Ⅰ.1 was increased, whereas the dimethylation of H3K9 was decreased by cortisol, forskolin, or db-cAMP treatment for 24h, which suggesting that epigenetic modification of histone 3 was also involved in the regulation ofaromatase expression by cortisol in human placental syncytiotrophoblasts.Conclusion:In this study, we demonstrated that the expression of aromatase was mainly in the syncytial layer of human placental villi, which was upregulated in the course of cytotrophoblast syncytiaization, and human placental syncytiotrophoblasts mainly used the promoter upstream of exon Ⅰ.1 for aromatase transcription, and cortisol could induce the transcription of exon Ⅰ.1 as well as stimulate aromatase gene promoter Ⅰ.1 activity in human placental syncytiotrophoblasts. We also found that cortisol stimulated the conversion of DHEA to estradiol via the induction of aromatase in human placental syncytiotrophoblasts. We further demonstrated that this induction was through activation of the cAMP/PKA pathway indirectly via CRH and hCQ which increased Spl expression and its enrichment at the aromatase promoter flanking the 5’end of exon 1.1 as well as changed the acetylation and dimethylation of H3K9.This study provides new evidences for the involvement of glucocorticoids in human parturition by up-regulation of aromatase expression thereby increasing estrogen production in human placenta syncytiotrophoblasts. These findings may provide novel ways for intervention in human preterm pregnancy.