Role Of Human Chorionic Gonadotropin In Maintaining 11β-Hydroxysteroid Dehydrogenase Type 2 Expression In Human Placental Syncytiotrophoblasts

Proper glucocorticoid exposure in utero is vital for normal fetal organ maturation, but excess glucocorticoids are detrimental to fetal growth and can even predispose the individuals to the high risk of having certain diseases in adulthood, such as hypertension, diabetes and strokes. It has been proposed that the fetus is protected from 10 times higher maternal glucocorticoid levels by the placental enzyme 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2), which converts biologically active cortisol to inactive cortisone requiring NAD as its cofactor. Importantly,11β-HSD2, with a Michaelis constant (Km) value in the nanomolar range, is suited well to regulate the passage of maternal glucocorticoids into the fetal circulation. It was demonstrated that at term all the cortisone in the fetus was of maternal origin, suggesting that placental 11β-HSD2 was acting as an effective glucocorticoid barrier at term. Alterations in placental 11β-HSD2 expression and activity are known to be associated with reduced fetal growth in placentae of human pregnancies complicated by intrauterine growth restriction (IUGR) and adult hypertension. This placental glucocorticoid barrier was functional not only at term, but also at the early and mid gestational ages.The expression of 11β-HSD2 was localized to the syncytiotrophoblast, which is the component of the human placenta involved not only in fetal maternal exchanges, but also in secretion of pregnancy-specific hormones such as human chorionic gonadotropin (hCG). Human CG is a glycoprotein hormone composed of two nonidentical subunits,αandβ, that are associated nonconvalently. The role of hCG in the maintenance of pregnancy is well known for the most extensively studied function of stimulating progesterone synthesis as well as the tocolytic action of promoting uterine quiescence. Studies have also shown its involvement in the regulation of implantation, trophoblastic hormonal secretion and trophoblast differentiation. It is well established that the stimulation of hCG receptors activates adenylate cyclase, resulting in the production of cyclic adenosine monophosphate (cAMP) and subsequent protein kinase A (PKA) activation. Intact hCG has been detected in maternal blood as early as around the 8th day of gestation. After the 6th week of gestation, the major site of hCG synthesis is switched from cytotrophoblast to syncytiotrophoblast with the maximal concentrations of hCG in maternal plasma attained at 8 th to 10 th weeks gestation, a period when the glucocorticoid barrier is beginning to take shape. In addition, in parallel with a significant decrease in serum hCG level 2-3 weeks before the spontaneous start of labor, there was a decline in placental 11β-HSD2 activity as well. The coincidence of hCG secretion and 11β-HSD2 expression in the placenta strongly indicates a close relationship between these two events. Although accumulating evidence indicates that activation of cAMP/PKA pathway plays an important role in up-regulating 11β-HSD2 expression in placental syncytiotrophoblasts, the endogenous hormone produced by the placenta utilizing this pathway to maintain 11β-HSD2 expression remains largely unknown. Previous studies have found that injections of hCG could stimulate 11β-HSD2 expression in the fish testis and rat uterus, suggesting it is very likely that hCG could be one of the endogenous hormones utilizing cAMP/PKA pathway to maintain 11β-HSD2 expression in human placenta.As the substrate for 11β-HSD2, glucocorticoids have been shown to increase placental 11β-HSD2 expression in vitro, which would represent an important safeguard mechanism by which the fetus be protected from detrimental exposure to elevated levels of maternal glucocorticoids. Since glucocorticoids have been reported to stimulate hCG secretion from the placenta, we speculate that the effect of glucocorticoids on placental 11β-HSD2 expression could be mediated at least in part through the stimulation of hCG secretion.In this study, we examined the role of hCG in maintaining placental 11β-HSD2 expression and in mediating the induction of placental 11β-HSD2 expression by cortisol in cultured human placental syncytiotrophoblasts.Materials and Methods1,Placenta Trophoblast Cell Culture. Human placentae were obtained from uncomplicated normal term pregnancies after elective caesarean section. Placental trophoblast cells were prepared using a modification of the method of Kliman. After being cultured for 48h placental trophoblast cells fused to form syncytiotrophoblasts spontaneously which were used for study.2,Treatment of Syncytiotrophoblasts in Culture. The cells were treated with forskolin (the adenylate cyclase stimulator, 100uM), H89 (PKA inhibitor, 20uM), hCG, hCG antibody, cortisol respectively for 12h or 24h.3,The total RNA and protein were extracted from the cells treated as above for analysis with realtime-PCR and Western blotting for the purpose of quantitative analysis of the target genes.4,Using hematoxylin and eosin stain (HE stain) and microscope to determine the maximal formation of syncytiotrophoblasts and the purity of syncytiotrophoblasts.5,Using chemiluminescence immunoassay (CLIA) for the quantitative analysis of p-hCG in the culturing medium.6,Using cAMP-GloTM Assay kit to measure intracellular cAMP concentration.7,Transfection of Placental Trophoblasts with pGL3 Plasmid Carrying 11β-HSD2 Promoter-driven Reporter Gene followed by measurement of Promoter Activity.Results1,Microscopic morphological examination of the cells showed that maximal fusion of the cells was found to occur at 48 h after plating. Measurement of the hCG level in the culture medium showed that the secretion of hCG by the cells reached a plateau at 48 h after plating suggesting maximal formation of syncytiotrophoblasts occurred at this time point. Thus this incubation time was chosen to allow maximal formation of syncytiotrophoblasts before experimentation in this study.2,Human Chorionic Gonadotropin Up-regulates 11β-HSD2 Expression via cAMP/PKA Pathway in Human Placenta Syncytiotrophoblasts. Measurement with qRT-PCR revealed that treatment of the syncytiotrophoblasts with adenylate cyclase stimulator forskolin (100uM) for 24 h increased 11β-HSD2 mRNA level significantly, while inhibition of PKA with H89 (20uM) decreased 11β-HSD2 mRNA level significantly. Neutralization of hCG secreted by the syncytiotrophoblasts with different concentrations of hCG antibody (1:80,1:125,1:250) for 24 h reduced 11β-HSD2 mRNA level in a concentration-dependent manner. Blocking the endogenous hCG with its antibody (1:100) also decreased 11β-HSD2 protein level. In the mean time, treatment of the cells with hCG antibody (1:100) for 4 h decreased cAMP level in the syncytiotrophoblasts. In contrast to the results of hCG antibody treatment, treatment of the syncytiotrophoblasts with exogenous hCG (10 IU/ml) for 12 h increased both 11β-HSD2 and protein levels, which were blocked by H89 (20uM). Forskolin (100uM) treatment of the syncytiotrophoblasts transfected with pGL3 basic plasmid carrying 330bp 11β-HSD2 promoter for 24 h increased the promoter activity significantly, while both H89 (20uM,24h) and hCG antibody (1:100) treatment of the syncytiotrophoblasts for 24 h significantly decreased 11 p-HSD2 promoter activity.3,Involvement of hCG in the Up-regulation of 11β-HSD2 Expression by Cortisol in Human Placenta Syncytiotrophoblasts. Treatment of the syncytiotrophoblasts with cortisol (0.01-luM) for 24 h increased 11β-HSD2 mRNA level in a concentration dependent manner. Protein synthesis inhibitor CHX (10uM) completely abolished the induction of 11β-HSD2 mRNA expression by cortisol (1uM), suggesting the induction by cortisol requires de novo protein synthesis. Cortisol (1uM) treatment of the syncytiotrophoblasts for 24h increased both hCG and subunit mRNA levels and hCG subunit level in the culture medium. Furthermore, the induction of 11β-HSD2 mRNA and protein expression by cortisol (1uM,24h) could be blocked by either hCG antibody (1:100) or H89 (20uM).ConclusionWe demonstrated for the first time that hCG is an important paracrine or autocrine hormone maintaining 11β-HSD2 expression and the up-regulation of 11β-HSD2 expression by cortisol may be mediated in part by hCG in the syncytiotrophoblasts.