Intracrine induction of 11beta-hydroxysteroid dehydrogenase type 1 expression by glucocorticoid potentiates prostaglandin production in the human chorionic trophoblast

Glucocorticoids are involved in the modulation of the release of parturition hormones from the fetal membranes and placenta, where their actions are determined by the prereceptor glucocorticoid metabolizing enzyme 11beta-hydroxysteroid dehydrogenase (11beta-HSD). Two distinct isozymes of 11beta-HSD have been characterized. In the fetal membranes, 11beta-HSD1 is the predominate isozyme; it converts biologically inert 11-ketone glucocorticoid metabolites into active glucocorticoids. Sequence analysis of the cloned 11beta-HSD1 gene revealed a putative glucocorticoid response element in the promoter region. However, whether glucocorticoids modulate 11beta-HSD1 expression in the fetal membranes is unknown. In this study, 11beta-HSD1 and glucocorticoid receptor (GR) were coexpressed in the chorionic trophoblast. Radiometric conversion assay and Northern blot analysis revealed that both 11beta-HSD1 reductase activity and mRNA levels were increased by dexamethasone (1 microM, 0.1 microM) in the cultured chorionic trophoblast, and the effects were blocked by GR antagonist RU486 (1 microM). Prior induction of 11beta-HSD1 by dexamethasone potentiated the subsequent stimulation of prostaglandin H synthetase 2 expression and secretion of prostaglandin E(2) by cortisone in the chorionic trophoblast. There is colocalization of 11beta-HSD1 and GR in the chorionic trophoblast. By binding to GR, glucocorticoids induce the expression of 11beta-HSD1 by a possible intracrine mechanism, thereby amplifying the actions of glucocorticoids on prostaglandin production in the fetal membranes. This cascade of events initiated by glucocorticoids may play an important role in the positive feed-forward mechanisms of labor.     

Characterization and modulation of sex steroid metabolizing activity in normal human keratinocytes in primary culture and HaCaT cells

Skin, the largest organ of the human body, synthesizes active sex steroids from adrenal C19 precursor steroids. Normal human breast epidermal keratinocytes in primary culture were used to evaluate the enzymatic activities responsible for the formation and degradation of active androgens and estrogens during keratinocyte differentiation. Enzymatic activities, including 3beta-hydroxysteroid dehydrogenase/Delta5-Delta4 isomerase (3beta-HSD), 17beta-hydroxysteroid dehydrogenase (17beta-HSD), 5alpha-reductase and 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) were measured using [3H] steroids as substrates. After 10-60 days in culture, no 3beta-HSD activity was detected, but all other activities were measured, demonstrating the ability of keratinocytes to convert androstenedione (4-DIONE) into the potent androgen dihydrotestosterone (DHT). Furthermore, marked changes in enzymatic activity were observed during cell differentiation: 17beta-HSD was first detected during the third week of culture, the level of activity reaching a peak during the fourth week. This peak was followed by a progressive decrease during keratinization. On the other hand, 5alpha-reductase and 3alpha-HSD activities were first detected during the fourth week of culture. The enzymatic activities involved in the formation and degradation of sex steroids were also characterized in the immortalized human keratinocyte cell line HaCaT. It was then found that HaCaT cells possess a pattern of steroid metabolizing enzymes similar to that of human epidermal keratinocytes in culture. Since glucocorticoids are known to exert potent pharmacological effects on the skin, the effect of dexamethasone (DEX) on cell proliferation and enzymatic activities was determined using HaCaT cells. DEX causes a 55% decrease in HaCaT cell proliferation (IC50: 10nM) whereas DEX caused a three- to five-fold stimulation of oxidative 17beta-HSD activity in intact cells in culture (ED50: 30 nM) and this stimulatory effect was competitively blocked by the glucocorticoid antagonist RU486. A four-fold increase in type 2 17beta-HSD mRNA levels was also observed as measured by real-time PCR, correlating with the increase in oxidative activity. No effect of DEX on the other enzymatic activities (3beta-HSD, 5alpha-reductase, and 3alpha-HSD) was observed. Since increased levels of inflammatory cytokines have been detected in some skin diseases then these cytokines might play a role in the differentiation of keratinocytes. In this regard, we found that interleukin-4 (IL-4) induced the expression of 3beta-HSD in HaCaT cells, thus allowing the cells to produce a different set of sex steroids from adrenal C19 precursors. The present data thus indicate that HaCaT cells are a useful model to further study the regulation of the enzymes involved in the metabolism of sex steroids in keratinocytes.     

大鼠海马神经元内11β-HSD1和GR的共存及其意义

本研究旨在探讨糖皮质激素代谢酶-11β-羟基类固醇脱氢酶Ⅰ型（11β-HSD1）和糖皮质激素受体（GR）在大鼠海马神经元内的共同分布及其意义。用免疫细胞化学方法研究显示,海马神经元内不仅存在11β-HSD1免疫反应物质,还存在GR免疫反应物质,而且11β-HSD1与GR共存于同一个海马神经元内,用Western印迹杂交和薄层层析（TLC）方法研究表明,地塞米松（DEX）可以促进11β-HSD1与GR共存于同一个海马神经元内,用Western印迹杂交和薄层层析（TLG）方法研究表明,地塞米松（DEX）可以促进11β-HSD1蛋白表达及其酶的活性,利用11β-HSD1基因启动子区序列构建的以CAT酶为报告基因的pBLCAT6质粒转染PC12细胞,证实DEX能够促进CAT酶的表达。以上糖皮质激素的作用均可为GR受体阻断剂RU38486所阻断,结果提示;糖皮质激素（GC）与GR结合后,可以作用于与其共存的11β-HSD1基因启动子区,使11β-HSD1表达增加,从而使更多的GC代谢产物转化为有活性的GC,此机制可能与保证GC在海马神经元内与亲和力较低的GR结合有关。     

Developmental and steroid hormonal regulation of insulin-like growth factor II expression

We have investigated the influence of steroid hormones on insulin-like growth factor II (IGF-II) expression. Hepatic IGF-II mRNA decreased gradually during postnatal development, reaching adult levels at 3 weeks of age. Treatment of 1-day-old rats for 4 days with 10 micrograms/day of the glucocorticoid dexamethasone (DEX) reduced IGF-II mRNA levels 10-fold in liver and inhibited body weight gain. Estradiol and testosterone did not affect IGF-II expression. A dose-response relationship between IGF-II mRNA levels and the different amounts of DEX injected was seen. IGF-II levels remained low after withdrawal of DEX, indicating an irreversible effect. Albumin expression was increased in newborn rat livers after DEX treatment. Our results suggest that glucocorticoids play an important role in the regulation of IGF-II expression. The mechanism for glucocorticoid-induced reduction of IGF-II mRNA is still unclear; however, our findings indicate that DEX inhibits IGF-II by causing premature differentiation of the liver.     

Glucocorticoid regulation of phenylethanolamine N-methyltransferase in vivo.

In vivo, supraphysiological doses of glucocorticoids are required to restore adrenal medullary phenylethanolamine N-methyltransferase (PNMT, E.C. 2.1.1.28) activity after hypophysectomy. However, in vitro, phenylethanolamine N-methyltransferase gene expression appears normally glucocorticoid-responsive. To explore this paradox, rats were given dexamethasone or the type II-specific glucocorticoid RU28362 (1-1000 micrograms/day), and adrenal phenylethanolamine N-methyltransferase activity and mRNA levels were determined. At low doses (1-30 micrograms/day), neither steroid altered mRNA whereas at higher doses (100-1000 micrograms/day), mRNA rose 10- to 20-fold, with dexamethasone approximately 3 times as potent as RU28362. In contrast, enzyme activity fell with low doses of either steroid, consistent with suppression of ACTH and endogenous steroidogenesis. At higher doses of RU28362, enzyme activity remained low and unchanged despite increased mRNA expression, whereas higher doses of dexamethasone progressively restored the enzyme to normal. These findings suggest 1) that glucocorticoid regulation of phenylethanolamine N-methyltransferase activity occurs largely independent of gene expression; 2) that glucocorticoid effects on enzyme activity are primarily indirect, probably through cosubstrate regulation and/or enzyme stabilization; and 3) that these effects are not mediated via a classical (type II) glucocorticoid receptor mechanism, given the high doses of dexamethasone and corticosterone required and the inability of RU28362 to mimic the effects of these less selective steroids.     

Deleterious Effects of Supplementation with Dehydroepiandrosterone Sulphate or Dexamethasone on Rat Insulin-Secreting Cells Under In Vitro Culture Condition

Dehydroepiandrosterone (DHEA) and glucocorticoids are steroid hormones synthesised in the adrenal cortex. Administration of DHEA, its sulphate derivative, DHEAS, and more controversially dexamethasone (DEX), a synthetic glucocorticoid, have beneficial effects in diabetic animals. Cultivating BRIN-BD11 cells for 3 days with either DHEAS (30 μM) or DEX (100 nM), reduced total cell number and reduced cell viability and cellular insulin content. DHEAS-treated cells had poor glucose responsiveness and regulated insulin release, coupled with reduced basal insulin release. In contrast, DEX-treated cells lacked responsiveness to glucose and membrane depolarisation, and both protein kinase A (PKA) and protein kinase C (PKC) secretory pathways were desensitised. Therefore, we conclude that this steroid hormone and synthetic glucocorticoid are not beneficial to pancreatic β-cells in vitro.     

Effect of glucocorticoids pretreatment on steroidogenic capacity of adrenocortical cells isolated from Meishan piglets

The present in vitro experiment was designed to test whether 48 h of pretreatment with glucocorticoids, cortisol, or dexamethasone (DEX), would affect basal and corticotrophin (ACTH) stimulated (24 h) cortisol secretion from primary cultures of pig adrenocortical cells. Cells were divided into six groups: control pretreatment with or without ACTH challenge, cortisol pretreatment with or without ACTH challenge, and DEX pretreatment with or without ACTH. The culture medium and cells were collected at the end of treatment. Cortisol concentration in medium was measured by radioimmunoassay, and protein content of glucocorticoid receptor (GR) and key regulatory factors for steroidogenesis, including melanocortin type 2 receptor (MC2R), steroidogenic acute regulatory protein (StAR) and cholesterol side-chain cleavage cytochrome P450 (P450scc), were detected by Western blot analysis. The results showed that glucocorticoid pretreatment did not affect cortisol secretion under basal condition without ACTH challenge, but significantly enhanced ACTH-stimulated cortisol secretion. Furthermore, the protein content of GR, MC2R, StAR, and P450scc was all increased in groups pretreated with glucocorticoids. These results indicate that adrenocortical cells pretreated with glucocorticoids display higher steroidogenic capacity under ACTH challenge, through the upregulation of GR and other steroidogenic regulatory factors.     

Steroid control of steroidogenesis in isolated adrenocortical cells: molecular and species specificity

The molecular and species specificity of glucocorticoid suppression of corticosteroidogenesis was investigated in isolated adrenocortical cells. Trypsin-isolated cells from male rat, domestic fowl and bovine adrenal glands were incubated with or without steroidogenic agents and with or without steroids. Glucocorticoids were measured by radioimmunoassay or fluorometric assay after 1-2 h incubation. Glucocorticoids suppressed ACTH-induced steroidogenesis of isolated rat cells with the following relative potencies: corticosterone greater than cortisol = cortisone greater than dexamethasone. The mineralocorticoid, aldosterone did not affect steroidogenesis. Suppression by glucocorticoids was acute (within 1-2 h), and varied directly with the glucocorticoid concentration. Testosterone also suppressed ACTH-induced steroidogenesis. Glucocorticoid-type steroids have equivalent suppressive potencies, thus suggesting that these steroids may induce suppression at least partly by a common mechanism. Although corticosterone caused the greatest suppression, testosterone was more potent. The steroid specificity of suppression of cyclic AMP (cAMP)-induced and ACTH-induced steroidogenesis were similar, suggesting that suppression is not solely the result of interference with ACTH receptor function or the induction of adenylate cyclase activity. Exogenous glucocorticoids also suppressed ACTH-induced steroidogenesis of cells isolated from domestic fowl and beef adrenal glands, thus suggesting that this observed suppression may be a general mechanism of adrenocortical cell autoregulation.