The long-range goal of this project is to define the molecular mechanisms that regulate the expression of the enzymes required for the synthesis of progesterone and estradiol-17beta (E20 in the human and sheep placenta and fetal adrenal. These steroidal secretions are important in effecting the maintenance of uterine quiescence; but the rates of secretion or bioaction must be altered to initiate the preparatory and active phases of labor. The molecular basis of regulation of 3beta-hydroxysteroid dehydrogenase/delta 5->4-isomerase (3beta-HSD) type I in human trophoblast will be determined: (i) The levels of 3beta-HSD MRNA and protein in cultured trophoblast, choriocarcinoma, chorion laeve, and amnion cells will be evaluated after treatments with agonists of protein kinases A and C, and Ca2+-dependent signal transduction; (ii) with selected placental-derived growth factors [insulin-like growth factors (IGFs) I and II, and transforming growth of factor-betas(TGF-betas)]. The nature of the cis- regulatory and trans-acting elements in the 5'-untranscribed flanking sequence, which account for the high expression of this gene in trophoblast, will be established using transfection assays of chimeric gene constructs and gel-shift assays. The level of 3 beta-HSD expression in human fetal adrenal (HFA) is low compared with that in placenta throughout most of pregnancy; but increased of the adrenal enzyme must occur near the time of parturition. The HFA 3beta-HSD is postulated to be the product of a gene distinct from that which encodes the placental (type I) isoform and regulated separately. The action of ACTH, TGF-betas, parathyroid hormone- related protein (PTH-rP), IGFs, and E2 on the level of MRNA encoding HFA 3beta-HSD and the cellular content and activity of this enzyme will be defined. By immunohistochemistry and in situ hybridization, the cell types that express 3beta-HSD and 17alpha-hydroxylase cytochrome P450 (P45017alpha) in the ovine placentome during dexamethasone-induced parturition will be determined. The in vitro action of potential regulators (e.g., IGF-1/II, TGF-betas, glucocorticosteroids) of 3beta-HSD and P450alpha expression in ovine trophoblast cells will be evaluated. Glucocorticosteroid-induced expression of P45017alpha in ovine placenta near the time of parturition may involve alternative, tissue-specific promoters. Unique, untranslated exonic sequences, obtained from amplification of full-length placental and adrenal CDNAS, will be sought to identify placental- and adrenal-specific P45017alpha transcripts and promoters in the CYP17 gene; by means of """"""""foot-printing"""""""" studies, the role of putative steroid enhancer and TGF-beta-inhibitory sequences in the 5'- flanking sequence will be investigated. We will determine whether the enhanced placental expression of this gene involves a trans-acting factor that interacts with a steroid enhancer-like consensus sequence. We will ascertain if differences in 5'-regulatory elements in the human an sheep CYP17 genes account for differences in placental P45017alpha expression.
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