Regulation of 11β-hydroxysteroid dehydrogenase type 1 gene expression in human ovarian surface epithelial cells by interleukin-1

Peter Y.K.Yong 1 Christopher Harlow 0 K.J.Thong 1 Stephen G.Hillier 0 0 Department of Reproductive and Developmental Sciences, University of Edinburgh Centre for Reproductive Biology , 49 Little France Crescent, Edinburgh EH16 4SB , UK 1 Assisted Conception Programme, Royal Infirmary of Edinburgh , Edinburgh EH16 4SA BACKGROUND: Local modulation of 11-hydroxysteroid dehydrogenase (11HSD) activity, to promote increased availability of anti-inflammatory glucocorticoids, is proposed as a compensatory response to inflammatory stimuli. Human 11HSD type 1 (11HSD1) is principally an 11-oxoreductase that reversibly reduces cortisone to cortisol. METHODS: Since ovulation is an acute inflammatory process, we examined the influence of proinflammatory cytokines on expression of 11HSD1 mRNA and metabolism of cortisone to cortisol by human ovarian surface epithelium (HOSE) in vitro. RESULTS: Northern analysis showed an 1.5 kb-sized 11HSD1 mRNA transcript in total RNA that was up-regulated 3-fold by interleukin (IL)-1 (0.5 ng/ml) at 24 h. By real-time RTPCR, induction of 11HSD1 mRNA by IL-1 was measurable at 6 h and maximal at 12 h. Primary HOSE cell cultures also showed low-level 11-oxoreductase activity that was stimulated time- and dose-dependently by IL-1 and IL-1. The 11HSD1 mRNA and 11-oxoreductase responses to 0.5 ng/IL were both suppressed by IL-1 receptor antagonist (25 ng/ml). CONCLUSIONS: Cultured HOSE cells express IL-1-responsive 11HSD1 and 11-oxoreductase activity mRNA in vitro. An 11HSD1-catalysed increase in anti-inflammatory glucocorticoid activity caused by pro-inflammatory cytokines could contribute to the local resolution of inflammation during ovulation. - Mammalian ovulation is a natural inflammatory reaction (Espey, 1994; Morris and Richards, 1995; Hellberg et al., 1996). The ovulation-inducing gonadotrophin surge triggers target ovarian cells to undergo profound structural and metabolic changes including mobilization of thecal fibroblasts, increased leukocyte migration, release of inflammatory media tors and loosening of connective tissue elements in the follicle wall and overlying tunica (Murdoch et al., 1999; Bukulmez and Arici, 2000). Within the ovulatory follicle, luteinising granulosa cells divert from estrogen biosynthesis to predominantly progesterone biosynthesis, anticipating formation of the corpus luteum. Simultaneously, granulosa cells increasingly express 11-oxoreductase (Tetsuka et al., 1997; Smith et al., 2000), the activity of which favours the accumulation of cortisol over cortisone within the follicular fluid (Andersen and Hornnes, 1994; Harlow et al., 1997; Andersen et al., 1999; Yong et al., 2000). Since cortisol is a potent anti-inflammatory agent (van der Burg and van der Saag, 1996; McKay and Cidlowski, 1999), locally activated cortisol may play a role in the resolution of this natural inflammatory event (Andersen and Hornnes, 1994; Hillier and Tetsuka, 1998; Irahara et al., 1999). The ovarian surface epithelium (OSE) is the outermost cellular layer breached during ovulation and is therefore intimately involved in the tissue remodelling that occurs (Auersperg et al., 2001; Murdoch et al., 2001). OSE cells near the site of stigma formation undergo apoptosis followed by inflammatory necrosis and slough off before the surface is ruptured (Murdoch et al., 1999). After which, adjacent OSE cells proliferate and recolonize the affected area (Osterholzer et al., 1985; Gillett et al., 1992). Repeated episodes of ovulation-associated injury and repair are presumed to underlie the high frequency of ovarian carcinoma arising from the OSE (Fathalla, 1971; Salazar et al., 1995), which account for 90% of all ovarian cancers (Ozols et al., 1991). Since factors related to inflammation of the OSE have been associated with increased risk of ovarian cancer (Ness and Cottreau, 1999; Ness et al., 2000), it is critically important to understand how inflammatory cell damage is normally resolved in the OSE. Exposure to inflammatory stimuli increases 11 -hydroxysteroid dehydrogenase type 1 (11HSD1) gene expression and enzymic activity in various epithelial cell types (Schleimer, 1991; Escher et al., 1997; Feinstein and Schleimer, 1999; Cai et al., 2001), including ovarian granulosa cells (Tetsuka et al., 1999). Since 11HSD1 is predominantly an 11-oxoreductase that reversibly metabolizes cortisone to cortisol (Tannin et al., 1991; Stewart and Mason, 1995), its increased expression at sites of inflammation has been proposed as part of a compensatory mechanism that promotes anti-inflammatory actions of glucocorticoids (Andersen and Hornnes, 1994; Escher et al., 1997; Hillier and Tetsuka, 1998). The ovulation-inducing LH surge triggers local production of inflammatory cytokines (Adashi, 1998), to which the OSE is inevitably exposed during ovulation. Thus, we sought to determine if cultured human OSE (HOSE) cells undertake cytokine-responsive 11HSD1 gene expression and 11-oxoreduction of cortisone to cortisol in vitro, focusing on founder members of the interleukin (IL)-1 gene family: IL-1, IL-1 and IL1-receptor antagonist (IL1-RA) (Sims et al., 2001). Subjects and methods Patients OSE cells were obtained from normal ovaries of premenopausal women (age range 3346 years) undergoing laparotomy for benign gynaecological conditions, and in whom there had not been exposure to exogenous hormones in the 2 months prior to surgery. Sampling was random with respect to stage of menstrual cycle (Table I). The specimens were obtained at the start of the operation before any surgical dissection so as to minimize blood contamination and devascularization. If oophorectomy was planned, the specimen was obtained before removal of the ovary. All specimens were obtained with informed consent, and local ethics committee approval was granted for the study. Experiments on HOSE cells collected and cultured from 15 patients are reported. Collection and culture of HOSE cells The exposed ovarian surface was gently scraped using a sterile Ayres spatula to remove a portion of the OSE layer, taking great care to avoid follicular rupture during handling. The specimen comprising flakes of OSE in peritoneal fluid, as a consequence of the sampling technique, was rinsed from the spatula into sterile culture medium, before being transported to the laboratory. The culture medium consisted of Medium 199:MCDB105 (1:1, v/v) supplemented with fetal calf serum (15% v/v), streptomycin (50 g/ml), penicillin (50 IU/ml), nystatin (500 IU/ml) and 1mmol/l L-glutamine (Kruk et al., 1990). All culture materials were from Gibco BRL (Life Technologies Ltd, Renfrewshire, UK) or Sigma Chemical Co. (Poole, Dorset, UK). The HOSE cells in culture medium were transferred to a serum-precoated 75 cm2 culture flask (Corning Inc. Glass Works, Corning, NY, USA) and incubated at 37C in a humidified tissue culture incubator gassed with 95% air5% CO2, for up to 28 days. The culture medium was renewed every 7 days. In this p (...truncated)