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K S Fegan, M T Rae, H O D Critchley and S G Hillier

Peritoneal surface epithelial (PSE) cells participate in adhesion formation following inflammatory injury yet adjacent ovarian SE (OSE) cells regenerate without scarification after ovulation. OSE cells show inflammation-associated expression of 11β hydroxysteroid dehydrogenase type 1 (11βHSD1) enzyme, enabling intracrine generation of anti-inflammatory cortisol to minimise tissue damage. We asked if human PSE cells show an 11βHSD1 response to pro-/anti-inflammatory stimulation and if so, how the 11-oxoreductase activity generated compares with OSE. PSE collected from premenopausal women undergoing surgery for benign gynaecological conditions were used to establish primary PSE cell cultures that were treated for 48 h with interleukin-1α (IL-1α) with/without anti-inflammatory steroid (cortisol or progesterone). mRNA levels corresponding to the genes of interest (11βHSD1, 11βHSD2, cyclooxygenase-2, COX-2) were measured by quantitative RT-PCR. IL-1α (0.5 ng/ml) stimulated 11βHSD1 and COX-2 mRNA levels in PSE cells but 11βHSD2 was unaffected. Cortisol (1 μM), not progesterone (1 μM), increased 11βHSD1 mRNA and synergistically enhanced IL-1α action. Cortisol suppressed IL-1α-stimulated COX-2 more effectively than progesterone. PSE cells had a significantly lower basal 11-oxoreductase enzyme activity than OSE cells; IL-1α did not significantly increase the 11-oxoreductase activity in PSE cells but did so in OSE cells. We conclude that PSE cells respond to IL-1α and anti-inflammatory steroids in qualitatively similar ways as OSE. However, the enzymatic activity of 11βHSD1 is lower in PSE and less responsive to IL-1α. This could help explain why peritoneal healing often leads to adhesion formation, whereas postovulatory ovarian healing is scar-free.

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M T Rae, D Niven, A Ross, T Forster, R Lathe, H O D Critchley, P Ghazal and S G Hillier

The human ovarian surface epithelium (HOSE) is a common site of gynaecological disease including endometriosis and ovarian cancer, probably due to serial injury-repair events associated with successive ovulations. To comprehend the importance of steroid signalling in the regulation of the HOSE, we used a custom microarray to catalogue the expression of over 250 genes involved in the synthesis and reception of steroid hormones, sterols and retinoids. The array included a subset of non-steroidogenic genes commonly involved in pro-/anti-inflammatory signalling. HOSE cells donated by five patients undergoing surgery for non-malignant gynaecological conditions were cultured for 48 h in the presence and absence of 500 pg/ ml interleukin-1α (IL-1α). Total RNA was reverse-transcribed into biotin-labelled cDNA, which was hybridised to the array and visualised by gold-particle resonance light scattering and charge-coupled device (CCD) camera detection. Results for selected genes were verified by quantitative reverse-transcription PCR. In five out of five cases, untreated HOSE cells expressed genes encoding enzymes required for de novo biosynthesis of cholesterol from acetate and subsequent formation of C21-pregnane and C19-androstane steroids. Consistent with the inability of HOSE cells to synthesise glucocorticoids, oestrogens or 5α-reduced androgens de novo, CYP21, CYP19 and 5α-reductase were not detected. The only steroidogenic gene significantly up-regulated by IL-1α was 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1). Other cytokine-induced genes were IL-6, IL-8, nuclear factor κB (NFκB) inhibitor α, metallothionein-IIA and lysyl oxidase: inflammation-associated genes that respond to glucocorticoids. The only steroidogenic gene significantly suppressed by IL-1α was 3βHSD1. Other genes suppressed by IL-1α were aldehyde dehydrogenase (ALDH) 1, ALDH 10, gonadotrophin hormone-releasing hormone receptor, peroxisome proliferation-activated receptor-binding protein (PPAR-bp) and nuclear receptor subfamily 2 group F member 2. These results define a steroidogenic phenotype of cultured HOSE cells and provide a limited expression profile for genes with associated signalling functions. IL-1α co-ordinately induces 11βHSD1 and a panel of glucocorticoid-regulated, inflammation-associated genes in HOSE cells, providing further evidence that cortisol generated by 11βHSD1 could participate in the local resolution of inflammation associated with ovulation.

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O Gubbay, M T Rae, A S McNeilly, F X Donadeu, A J Zeleznik and S G Hillier

cAMP response-element binding (CREB) transcription factors transduce cell survival responses to peptide hormones and growth factors in normal tissues and mutant CREB proteins are implicated in tumorigenesis. Ovarian cancer most frequently arises from the ovarian surface epithelium (OSE), possibly due to repeat inflammation-associated injury-repair episodes that promote neoplasia. We asked if post-receptor signalling involving the CREB family of proteins plays a role in OSE cell survival. In an ovine ovulation model, abundant expression of phospho-CREB/activating transcription factor (ATF) protein was detected immunohistochemically, strongly localised to OSE cells in the proximity of pre-ovulatory follicles. Treatment of primary sheep OSE cell cultures with LH stimulated cAMP accumulation and reduced apoptosis (caspase 3/7 activity) in response to serum withdrawal. When OSE cells were infected with an adenovirus containing a CRE-luciferase construct, exposure to LH and FSH induced CRE-directed transcription. Finally, when a non-phosphorylatable mutant of CREB (Ad CREBS133A) was adenovirally expressed, apoptosis measured by activation of caspases was increased several fold relative to that caused by transfection with wild-type CREB (Ad CREBWT) or lacZ (Ad lacZ). To test the potential clinical relevance of these findings, we expressed mutant CREB protein in normal human OSE cells from four women and a series of cell lines derived from human ovarian cancers. Infection with Ad CREBS133A markedly increased apoptosis in normal human OSE but had no detectable effect on apoptosis in any of the cancer cell lines. We conclude that CREB/ATF signalling is important for the maintenance of OSE cell survival in vitro and is altered in human cell lines derived from ovarian cancers.

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M A Hyatt, G S Gopalakrishnan, J Bispham, S Gentili, I C McMillen, S M Rhind, M T Rae, C E Kyle, A N Brooks, C Jones, H Budge, D Walker, T Stephenson and M E Symonds

The liver is a major metabolic and endocrine organ of critical importance in the regulation of growth and metabolism. Its function is determined by a complex interaction of nutritionally regulated counter-regulatory hormones. The extent to which hepatic endocrine sensitivity can be programed in utero and whether the resultant adaptations persist into adulthood is unknown and was therefore the subject of this study. Young adult male sheep born to mothers that were fed either a control diet (i.e.100% of total live weight-maintenance requirements) throughout gestation or 50% of that intake (i.e. nutrient restricted (NR)) from 0 to 95 days gestation and thereafter 100% of requirements (taking into account increasing fetal mass) were entered into the study. All mothers gave birth normally at term, the singleton offspring were weaned at 16 weeks, and then reared at pasture until 3 years of age when their livers were sampled. NR offspring were of similar birth and body weights at 3 years of age when they had disproportionately smaller livers than controls. The abundance of mRNA for GH, prolactin, and IGF-II receptors, plus hepatocyte growth factor and suppressor of cytokine signaling-3 were all lower in livers of NR offspring. In contrast, the abundance of the mitochondrial protein voltage-dependent anion channel and the pro-apoptotic factor Bax were up regulated relative to controls. In conclusion, maternal nutrient restriction in early gestation results in adult offspring with smaller livers. This may be mediated by alterations in both hepatic mitogenic and apoptotic factors.