Expression of steroid metabolizing enzymes by aggregating fetal brain cells in culture: a model for developmental regulation of the progesterone 5 alpha-reductase pathway.

In this study, we established an in vitro model system for the study of developmental regulation of steroid enzyme expression in the perinatal brain. Single cell suspensions were prepared from the hypothalamic-olfactory tubercle region of 18-day-old rat fetuses, and aggregates were formed by incubation under constant rotation. On day 0, 3, 6, or 12 of culture, aggregates were incubated for 4 or 20 h with 3H-progesterone (P4) and the profile of 3H-steroids in the medium analyzed. Five major metabolites were formed from 3H-P4: 5 alpha-pregnan-3, 20-dione (DHP), 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha-OH-DHP), 3 beta-hydroxy-5 alpha-pregnan-20-one (3 beta-OH-DHP), and two unidentified polar substances designated A and B. Progressively with aggregate-age in culture, there was a decrease in the relative amounts of 3H-P4 recovered in the medium and a sequential increase in DHP, 3 beta-OH-DHP, 3 alpha-OH-DHP, A and B. Aggregates maintained in a chemically defined, serum-free medium metabolized P4 at an accelerated rate compared to those maintained in serum. An inhibitor of the enzyme 5 alpha-reductase completely inhibited P4 metabolism, indicating that 5 alpha-reduction is the primary step in this pathway. Thus, the aggregates express three key enzymes in P4 metabolism: 5 alpha-reductase, 3 alpha- and 3 beta-hydroxysteroid oxidoreductases and the operation of this pathway is proposed: P4----DHP----3 alpha-OH-DHP + 3 beta-OH-DHP; A and B are derived from one or more of the latter three. Hence, this culture system can serve as a model to study regulatory processes in the developing steroidogenic brain.

Dexamethasone-induced accumulation of neuropeptide-Y by aggregating fetal brain cells in culture: a process dependent on the developmental age of the aggregates.

Neuropeptide-Y (NPY) and glucocorticoid receptors are coexpressed in many neurons in the brain. We addressed the question: Do glucocorticoids regulate the accumulation and/or secretion of immunoreactive (IR) NPY by fetal rat brain cells in culture, and if so, is the effect developmental stage dependent? Aggregates, formed from dissociated cells obtained from the hypothalamus-olfactory tubercle of 17-day-old fetuses, were cultured in serum-free medium for 23 days. On day 23, the aggregate NPY content was 6 ng/flask, and secretion (last 2 days) was approximately 12 ng/24 h. Exposure to dexamethasone (Dex; 20 nM) between days 0-23 led to a 1.9-fold increase in the aggregate content of NPY, whereas NPY secretion was not altered. When Dex exposure was limited to days 12-23, 16-23, 19-23, or 21-23, only a 12- to 23-day exposure induced NPY accumulation, and it was as effective as a 0- to 23-day exposure. The Dex-induced increase in NPY content was evident after a lag period of 4 days or more. When Dex exposure occurred on days 0-12, the aggregate NPY content on day 12 or 23 was not altered. None of these treatments altered the aggregate/medium content of immunoreactive somatostatin (SRIF) or the response to a 48-h exposure to forskolin (10 microM). Dex induction of NPY accumulation was a saturable function of the Dex concentration (maximal at 20 nM), and it was completely inhibited by RU486, a glucocorticoid/progesterone receptor antagonist; neither progesterone, 17 beta-estradiol, nor testosterone altered aggregate/medium NPY contents. Protein/DNA contents of the aggregates were either unaffected or slightly reduced by Dex. Thus, 1) Dex stimulates the accumulation of immunoreactive NPY, but not SRIF, by cultured fetal brain cells; 2) this effect requires a continuous 8-12 days of exposure to Dex during a late developmental stage in culture; 3) Dex does not potentiate or attenuate forskolin action on the NPY neuron; and 4) Dex action appears to be mediated by the glucocorticoid receptor. These results are consistent with glucocorticoid induction of production and/or decreased intracellular degradation of NPY, and with glucocorticoids regulating the NPY neuron in the perinatal brain in a developmental age-dependent manner.

Regulated production and secretion of immunoreactive neuropeptide Y by aggregating fetal brain cells in cultures.

The aim of this study was to establish a culture system of fetal brain cells that could serve as a model for the study of the developmental regulation of the neuropeptide Y (NPY) neuron. Single cell suspensions were prepared from the hypothalamic-olfactory tubercle region of 18-day-old rat fetuses, and aggregates were formed by incubation in serum-free medium under constant rotation. Aggregate formation was complete within 24-48 h, and cultures were maintained for up to 23 days. The content of immunoreactive (IR) NPY in the medium and in the aggregates increased progressively with time in culture and at each time point, the medium contained 5- to 10-fold more NPY-IR. A 48-hour exposure to forskolin resulted in a 2-fold increase in the accumulation of NPY-IR in the aggregates and in the medium, indicating that both production and secretion of NPY are regulated by the cAMP intracellular pathway. Sephadex gel filtration revealed the presence of proNPY- and NPY-size substances. The ratio of NPY- to proNPY-size substances increased progressively with age of the aggregates as well as in tissues obtained from perinatal rats of comparable age. Thus, production and secretion of NPY-IR in the cultured aggregates are regulated processes and hence, this culture system can serve as a model to study regulatory processes in the developing NPY neuron.

Characterization of the promoter of human prostaglandin H synthase-1 gene.

The gene of human prostaglandin H synthase-1 (PGHS-1,EC 1.14.99.1) was isolated from a human cosmid library and was contained in two overlapping clones. Multiple transcriptional start sites were identified. The major one located 136 bases upstream from the ATG initiation codon. The promoter region contains no canonical TATA box but possesses a high G+C content. These observations suggest that PGHS-1 gene has the characteristics of a housekeeping gene. Both 0.8 and 0.4 kb of the 5'-flanking sequence of the PGHS-1 gene can confer transcriptional activity.

Transcriptional regulation of endothelial constitutive PGHS-1 expression by phorbol ester.

Human endothelial cells contain two isoforms of prostaglandin H synthase (PGHS). PGHS-1 is constitutively expressed, whereas PGHS-2 is inducible. To determine whether expression of PGHS-1 is regulated, we treated cultured human umbilical vein endothelial cells (HUVEC) with phorbol 12-myristate 13-acetate (PMA) or its inactive analogue and measured PGHS-1 mRNA levels by Northern analysis and competitive polymerase chain reaction. PMA increased PGHS-1 mRNA levels determined by both techniques in a time- and concentration-dependent manner. The mRNA level was increased about twofold over the basal level after 4-6 h of PMA (10-50 nM) treatment. The level of PGHS-1 protein was similarly increased by PMA. Stimulation of PGHS-1 mRNA levels was abrogated by cycloheximide, actinomycin D, staurosporine, or calphostin C. The 5'-promoter activity of human PGHS-1 gene was increased twofold over the basal level by PMA in NS-20 cells. These results indicate that the constitutive PGHS-1 in HUVEC is transcriptionally stimulated by PMA in a protein kinase C-dependent manner.