Steroid signalling in the human ovarian surface epithelium wound healing

programme grant G0500047The human ovarian surface epithelium (hOSE) is a cell monolayer that covers the surface of the ovary. Natural events like incessant ovulation, associated reproductive hormone action prior to and post-ovulation, along with the ovulationassociated inflammation, that result in injury and repair of hOSE, are considered to have a role in the development of epithelial ovarian cancer (EOC). Progesterone is apoptotic and anti-inflammatory, whereas androgens appear cytoproliferative for hOSE. Local generation of these steroid hormones is subject to 3β-hydroxysteroid dehydrogenase (3β-HSD) activity. Moreover, action of these hormones is achieved through coupling to their cognate receptors, progesterone (PR) and androgen receptors (AR). The overall aim of this thesis is to elucidate in vitro the regulation of progesterone and androgen biosynthesis and downstream signalling during the injury and repair of primary hOSE cells that were collected from pre-menopausal women who underwent surgery for benign gynaecological disorders. Injury was mimicked by treatment of cells with several pro-inflammatory cytokines, whereas repair was mimicked with T-lymphocyte, ‘anti-inflammatory’ cytokines. Immunohistochemical studies showed immunodetectable 3β-HSD in the human ovarian cell surface of whole ovary and three-week cultured hOSE cells, establishing 3β-HSD expression in vivo and in vitro. Cross-reaction of the 3β-HSD antibody with both enzyme isoforms did not allow investigation of isoform expression pattern. However, mRNA transcriptional studies with isoform specific primers and probe sets for semi-quantitative (sq) and quantitative (q) PCR revealed expression of both isoforms in hOSE cells; 3β-HSD1 mRNA was expressed at higher levels relative to 3β-HSD2 mRNA in accordance with the preference of this isoform in peripheral non-steroidogenic tissues. Of the cytokines tested, only IL-1α and IL-4 affected 3β-HSD expression. IL- 1α suppressed 3β-HSD1 mRNA, whereas it up-regulated 3β-HSD2 mRNA as assessed with qPCR, without though affecting total 3β-HSD protein and activity levels as assessed with western immunoblotting and radiometric activity assays, respectively. IL-1α did not affect AR or PR mRNA levels, suggesting a balance in androgen and progesterone biosynthesis during post-ovulatory wounding. IL-4 massively induced 3β-HSD1 and 3β-HSD2 mRNA and total 3β-HSD protein and activity. It also attenuated AR mRNA and protein, without affecting PR mRNA. Collectively, these data demonstrate that IL-4 sustains progesterone rather than androgen signalling and this may be part of the anti-inflammatory steroid action that protects hOSE from genetic damage. IL-1α effects appear to be mediated by NF-κB signalling pathway. PI-3K and p38 MAPK appeared involved in IL-1α-induced 3β- HSD2. IL-4-induced 3β-HSDs required STAT-6 and PI-3K pathways and also p38 MAPK at the case of 3β-HSD2. IL-4-attenuated AR was reversed by a p38 MAPK inhibitor. These data suggest that steroid signalling by IL-1α and IL-4 involve multiple signalling pathways. In primary EOC, 3β-HSD1 and 3β-HSD2 transcripts were attenuated relative to hOSE cells, suggestive of an acquired feature of neoplastic transformation. However, both transcripts could be restored after IL-4 treatment, attesting a therapeutic advantage of this cytokine. In conclusion, we have shown that 3β-HSD is under inflammatory control during ovarian post-ovulatory wound healing of hOSE. IL-1α- and IL-4-mediated 3β-HSD1 and 3β-HSD2 are regulated by multiple signalling pathways. Also, IL-4 was identified as an anti-inflammatory agent in hOSE with putative therapeutic benefit in malignancy

Pancreatic cancer incidence and survival and the role of specialist centres in resection rates in England, 2000 to 2014: A population-based study.

BACKGROUND: The aim was to compare population-based survival for exocrine pancreatic cancer in England in the 23 regions covered by specialist centres. The centres were initiated in 2001, covering populations of 2-4 million. METHODS: We examined incidence for adults diagnosed with a pancreatic exocrine cancer during 1995-2014 and age-standardised net survival up to five years after diagnosis for patients diagnosed during 2000-2013. We examined variation in regional resection rates and survival for patients diagnosed during 2010-2013. The data were extracted from the National Cancer Registration and Analysis Service. RESULTS: Age-standardised annual incidence rates of exocrine pancreatic cancer increased from 17.1 per 100,000 during 1995-1999 to 18.7 during 2010-2014. Age-standardised one-year and five-year net survival increased from 17.9% and 3.6%, respectively, for 2000-2009, to 21.6% and 4.2% during 2010-2013. There were 2086 (8.9%) resections among 23,415 patients diagnosed with an exocrine tumour in 2010-2013. The proportion ranged from 5.1% to 19.6% between centres. Among resected patients, survival was 73.0% at one year and 20.2% at five years. Of the total 2118 resected patients, 18 (0.9%) were at stage 1; 34 (1.6%) at stage 2; 791 (37.3%) at stage 3 and 140 (6.6%) at stage 4, although 53.6% of stage information was missing. Five-year survival was 2.1% for those who were not resected. The number of resections performed in each centre was not correlated with one-year survival. CONCLUSIONS: Despite improvements in the management of pancreatic cancer in England with the introduction of specialist centres, resection rates remain relatively low, and survival remains lower than in comparably wealthy countries

Changes in LXR signaling influence early-pregnancy lipogenesis and protect against dysregulated fetoplacental lipid homeostasis

Human pregnancy is associated with enhanced de novo lipogenesis in the early stages followed by hyperlipidemia during advanced gestation. Liver X receptors (LXRs) are oxysterol-activated nuclear receptors that stimulate de novo lipogenesis and also promote the efflux of cholesterol from extrahepatic tissues followed by its transport back to the liver for biliary excretion. Although LXR is recognized as a master regulator of triglyceride and cholesterol homeostasis, it is unknown whether it facilitates the gestational adaptations in lipid metabolism. To address this question, biochemical profiling, protein quantification, and gene expression studies were used, and gestational metabolic changes in T0901317-treated wild-type mice and Lxrab-/- mutants were investigated. Here, we show that altered LXR signaling contributes to the enhanced lipogenesis in early pregnancy by increasing the expression of hepatic Fas and stearoyl-CoA desaturase 1 (Scd1). Both the pharmacological activation of LXR with T0901317 and the genetic ablation of its two isoforms disrupted the increase in hepatic fatty acid biosynthesis and the development of hypertriglyceridemia during early gestation. We also demonstrate that absence of LXR enhances maternal white adipose tissue lipolysis, causing abnormal accumulation of triglycerides, cholesterol, and free fatty acids in the fetal liver. Together, these data identify LXR as an important factor in early-pregnancy lipogenesis that is also necessary to protect against abnormalities in fetoplacental lipid homeostasis

Estrogen biosynthesis in human H295 adrenocortical carcinoma cells

Adrenocortical carcinoma is an uncommon malignancy and feminizing symptoms secondary to adrenal estrogen-secretion are extremely rare. The direct secretion of estradiol by adrenocortical tumors requires, in addition to the expression of aromatase (CYP19), the expression of one or more of the reductive 17β-hydroxysteroid dehydrogenases. The expression of CYP19 transcripts and protein were markedly induced in the H295 adrenocortical carcinoma cell line after treatment with either forskolin or vasoactive intestinal peptide (VIP). Western immunoblotting demonstrated a marked induction of the CYP19 protein of characteristic size after only a short (6 h) treatment period with VIP or forskolin. The CYP19 mRNA transcripts were derived from both promoters PII (Ic) and I.3 (Id) after treatment with both agents. The reductive type 5 17β-hydroxysteroid dehydrogenase (AKR1C3) was also constitutively expressed in the H295 cells but neither its mRNA transcript nor protein levels were altered after forskolin or VIP treatment. Western immunoblotting of an estrogen-secreting adrenal carcinoma revealed notable levels of both aromatase and AKR1C3 expression while an aldosterone-producing adrenal adenoma lacked aromatase expression and showed a reduced level of AKR1C3 expression. Immunohistochemistry of the carcinoma-bearing adrenal revealed localization of AKR1C3 not only in the tumor but also principally in the zona reticularis of the normal adrenal tissue. Adrenal aromatase and AKR1C3 expression therefore appear to be features of adrenocortical malignancies that are associated with biosynthesis of active estrogen

Nuclear receptor-driven alterations in bile acid and lipid metabolic pathways during gestation

AbstractNuclear receptor signalling is essential for physiological processes such as metabolism, development, and reproduction. Alterations in the endocrine state that naturally occur during pregnancy result in maternal adaptations to support the feto-placental unit. A series of studies have shown that nuclear receptor signalling is involved in maternal adaptations of bile acid, cholesterol, and lipid homeostasis pathways to ensure maintenance of the nutritional demands of the fetus. We discuss regulation of hepatic nuclear receptors and their target genes in pregnancy and their impact on the development of disorders such as intrahepatic cholestasis of pregnancy and oestrogen-induced hepatotoxicity. This article is part of a Special Issue entitled: Translating nuclear receptors from health to disease

Steroid signalling in the human ovarian surface epithelium wound healing

The human ovarian surface epithelium (hOSE) is a cell monolayer that covers the surface of the ovary. Natural events like incessant ovulation, associated reproductive hormone action prior to and post-ovulation, along with the ovulationassociated inflammation, that result in injury and repair of hOSE, are considered to have a role in the development of epithelial ovarian cancer (EOC). Progesterone is apoptotic and anti-inflammatory, whereas androgens appear cytoproliferative for hOSE. Local generation of these steroid hormones is subject to 3β-hydroxysteroid dehydrogenase (3β-HSD) activity. Moreover, action of these hormones is achieved through coupling to their cognate receptors, progesterone (PR) and androgen receptors (AR). The overall aim of this thesis is to elucidate in vitro the regulation of progesterone and androgen biosynthesis and downstream signalling during the injury and repair of primary hOSE cells that were collected from pre-menopausal women who underwent surgery for benign gynaecological disorders. Injury was mimicked by treatment of cells with several pro-inflammatory cytokines, whereas repair was mimicked with T-lymphocyte, ‘anti-inflammatory’ cytokines. Immunohistochemical studies showed immunodetectable 3β-HSD in the human ovarian cell surface of whole ovary and three-week cultured hOSE cells, establishing 3β-HSD expression in vivo and in vitro. Cross-reaction of the 3β-HSD antibody with both enzyme isoforms did not allow investigation of isoform expression pattern. However, mRNA transcriptional studies with isoform specific primers and probe sets for semi-quantitative (sq) and quantitative (q) PCR revealed expression of both isoforms in hOSE cells; 3β-HSD1 mRNA was expressed at higher levels relative to 3β-HSD2 mRNA in accordance with the preference of this isoform in peripheral non-steroidogenic tissues. Of the cytokines tested, only IL-1α and IL-4 affected 3β-HSD expression. IL- 1α suppressed 3β-HSD1 mRNA, whereas it up-regulated 3β-HSD2 mRNA as assessed with qPCR, without though affecting total 3β-HSD protein and activity levels as assessed with western immunoblotting and radiometric activity assays, respectively. IL-1α did not affect AR or PR mRNA levels, suggesting a balance in androgen and progesterone biosynthesis during post-ovulatory wounding. IL-4 massively induced 3β-HSD1 and 3β-HSD2 mRNA and total 3β-HSD protein and activity. It also attenuated AR mRNA and protein, without affecting PR mRNA. Collectively, these data demonstrate that IL-4 sustains progesterone rather than androgen signalling and this may be part of the anti-inflammatory steroid action that protects hOSE from genetic damage. IL-1α effects appear to be mediated by NF-κB signalling pathway. PI-3K and p38 MAPK appeared involved in IL-1α-induced 3β- HSD2. IL-4-induced 3β-HSDs required STAT-6 and PI-3K pathways and also p38 MAPK at the case of 3β-HSD2. IL-4-attenuated AR was reversed by a p38 MAPK inhibitor. These data suggest that steroid signalling by IL-1α and IL-4 involve multiple signalling pathways. In primary EOC, 3β-HSD1 and 3β-HSD2 transcripts were attenuated relative to hOSE cells, suggestive of an acquired feature of neoplastic transformation. However, both transcripts could be restored after IL-4 treatment, attesting a therapeutic advantage of this cytokine. In conclusion, we have shown that 3β-HSD is under inflammatory control during ovarian post-ovulatory wound healing of hOSE. IL-1α- and IL-4-mediated 3β-HSD1 and 3β-HSD2 are regulated by multiple signalling pathways. Also, IL-4 was identified as an anti-inflammatory agent in hOSE with putative therapeutic benefit in malignancy.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Steroid signalling in the human ovarian surface epithelium wound healing

The human ovarian surface epithelium (hOSE) is a cell monolayer that covers the surface of the ovary. Natural events like incessant ovulation, associated reproductive hormone action prior to and post-ovulation, along with the ovulationassociated inflammation, that result in injury and repair of hOSE, are considered to have a role in the development of epithelial ovarian cancer (EOC). Progesterone is apoptotic and anti-inflammatory, whereas androgens appear cytoproliferative for hOSE. Local generation of these steroid hormones is subject to 3β-hydroxysteroid dehydrogenase (3β-HSD) activity. Moreover, action of these hormones is achieved through coupling to their cognate receptors, progesterone (PR) and androgen receptors (AR). The overall aim of this thesis is to elucidate in vitro the regulation of progesterone and androgen biosynthesis and downstream signalling during the injury and repair of primary hOSE cells that were collected from pre-menopausal women who underwent surgery for benign gynaecological disorders. Injury was mimicked by treatment of cells with several pro-inflammatory cytokines, whereas repair was mimicked with T-lymphocyte, ‘anti-inflammatory’ cytokines. Immunohistochemical studies showed immunodetectable 3β-HSD in the human ovarian cell surface of whole ovary and three-week cultured hOSE cells, establishing 3β-HSD expression in vivo and in vitro. Cross-reaction of the 3β-HSD antibody with both enzyme isoforms did not allow investigation of isoform expression pattern. However, mRNA transcriptional studies with isoform specific primers and probe sets for semi-quantitative (sq) and quantitative (q) PCR revealed expression of both isoforms in hOSE cells; 3β-HSD1 mRNA was expressed at higher levels relative to 3β-HSD2 mRNA in accordance with the preference of this isoform in peripheral non-steroidogenic tissues. Of the cytokines tested, only IL-1α and IL-4 affected 3β-HSD expression. IL- 1α suppressed 3β-HSD1 mRNA, whereas it up-regulated 3β-HSD2 mRNA as assessed with qPCR, without though affecting total 3β-HSD protein and activity levels as assessed with western immunoblotting and radiometric activity assays, respectively. IL-1α did not affect AR or PR mRNA levels, suggesting a balance in androgen and progesterone biosynthesis during post-ovulatory wounding. IL-4 massively induced 3β-HSD1 and 3β-HSD2 mRNA and total 3β-HSD protein and activity. It also attenuated AR mRNA and protein, without affecting PR mRNA. Collectively, these data demonstrate that IL-4 sustains progesterone rather than androgen signalling and this may be part of the anti-inflammatory steroid action that protects hOSE from genetic damage. IL-1α effects appear to be mediated by NF-κB signalling pathway. PI-3K and p38 MAPK appeared involved in IL-1α-induced 3β- HSD2. IL-4-induced 3β-HSDs required STAT-6 and PI-3K pathways and also p38 MAPK at the case of 3β-HSD2. IL-4-attenuated AR was reversed by a p38 MAPK inhibitor. These data suggest that steroid signalling by IL-1α and IL-4 involve multiple signalling pathways. In primary EOC, 3β-HSD1 and 3β-HSD2 transcripts were attenuated relative to hOSE cells, suggestive of an acquired feature of neoplastic transformation. However, both transcripts could be restored after IL-4 treatment, attesting a therapeutic advantage of this cytokine. In conclusion, we have shown that 3β-HSD is under inflammatory control during ovarian post-ovulatory wound healing of hOSE. IL-1α- and IL-4-mediated 3β-HSD1 and 3β-HSD2 are regulated by multiple signalling pathways. Also, IL-4 was identified as an anti-inflammatory agent in hOSE with putative therapeutic benefit in malignancy.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

A progesterone-brown fat axis is involved in regulating fetal growth

Pregnancy is associated with profound maternal metabolic changes, necessary for the growth and development of the fetus, mediated by reproductive signals acting on metabolic organs. However, the role of brown adipose tissue (BAT) in regulating gestational metabolism is unknown. We show that BAT phenotype is lost in murine pregnancy, while there is a gain of white adipose tissue (WAT)-like features. This is characterised by reduced thermogenic capacity and mitochondrial content, accompanied by increased levels of markers of WAT and lipid accumulation. Surgical ablation of BAT prior to conception caused maternal and fetal hyperlipidemia, and consequently larger fetuses. We show that BAT phenotype is altered from day 5 of gestation, implicating early pregnancy factors, which was confirmed by reduced expression of BAT markers in progesterone challenged oophorectomised mice. Moreover, in vitro data using primary BAT cultures show a direct impact of progesterone on expression of Ucp1. These data demonstrate that progesterone mediates a phenotypic change in BAT, which contributes to the gestational metabolic environment, and thus overall fetal size