Cellular mechanisms for the control of glucocorticoid metabolism by 11-beta hydroxysteroid dehydrogenase in the human ovary
The aims of this thesis were to: (i) determine the regulatory properties of human follicular fluid (FF) on the activities and expression of 11(3HSD, (ii) investigate the effects of progesterone on llpHSD activity and expression (iii) investigate the effects of prostaglandins on basal and endocrine-stimulated llpHSD activity and expression (iv) to predict the topology and tertiary structure of 11 pHSDl. Human FF contains hydrophilic and hydrophobic compounds that stimulate and inhibit, respectively, the NADP(H)-dependent activities of 11 pHSDl. The predominant lipid inhibitors of 11(3HSD1 in FF are unlikely to be steroids or prostaglandins, and the effects of the FF components on enzyme activities occur without changes in 11 PHSDl protein expression. Inhibition of progesterone production by aminoglutethimide increased 1 lpHSD activities, without affecting 1 lpHSDl protein expression in human granulosa-lutein cells. Pharmacological inhibitors of prostaglandin-H synthase (PGHS)-2 (meclofenamic acid (MA) and NS-398) decreased cortisol-cortisone inter-conversion by up to 50%, suggesting that prostaglandins may stimulate ovarian llpHSD activities in a paracrine/autocrine manner. Furthermore, PGHS-2 inhibitors prevented human chorionic gonadotrophin (hCG) and IL-1(3 from stimulating 11(3HSD activities in human granulosa-lutein cells, implicating eicosanoids in the stimulation of ovarian llpHSD by gonadotrophins and cytokines. Since MA and NS-398 had no effect on 1 IpHSDl protein expression, the stimulation of 11 pHSD activities by eicosanoids occurs at the post-translational level. Finally, the structure of 1 lpHSDl protein was modelled to develop molecular mechanisms by which prostaglandins, gonadotrophins and cytokines might regulate enzyme activities. Although the primary sequence of 1 lpHSDl, which is highly conserved across 10 mammalian species, contains several consensus phosphorylation sites for serine/threonine and tyrosine kinases, models of 11 pHSDl topology predict that most phosphorylation sites would lie in the lumen of the endoplasmic reticulum. 1 lpHSDl includes hydrophobic a-helices neighbouring the active site, which may provide regions for the allosteric control of glucocorticoid metabolism by progesterone and eicosanoids in ovarian cells.