Targeting lysyl oxidase reduces peritoneal fibrosis.

BACKGROUND: Abdominal surgery and disease cause persistent abdominal adhesions, pelvic pain, infertility and occasionally, bowel obstruction. Current treatments are ineffective and the aetiology is unclear, although excessive collagen deposition is a consistent feature. Lysyl oxidase (Lox) is a key enzyme required for crosslinking and deposition of insoluble collagen, so we investigated whether targeting Lox might be an approach to reduce abdominal adhesions. METHODS: Female C57Bl/6 mice were treated intraperitoneally with multiwalled carbon nanotubes (NT) to induce fibrosis, together with chemical (ß-aminoproprionitrile-BAPN) or miRNA Lox inhibitors, progesterone or dexamethasone. Fibrotic lesions on the diaphragm, and expression of fibrosis-related genes in abdominal wall peritoneal mesothelial cells (PMC) were measured. Effects of BAPN and dexamethasone on collagen fibre alignment were observed by TEM. Isolated PMC were cultured with interleukin-1 alpha (IL-1α) and progesterone to determine effects on Lox mRNA in vitro. RESULTS: NT-induced fibrosis and collagen deposition on the diaphragm was ameliorated by BAPN, Lox miRNA, or steroids. BAPN and dexamethasone disrupted collagen fibres. NT increased PMC Lox, Col1a1, Col3a1 and Bmp1 mRNA, which was inhibited by steroids. Progesterone significantly inhibited IL-1α induced Lox expression by PMC in vitro. CONCLUSION: Our results provide proof-of-concept that targeting peritoneal Lox could be an effective approach in ameliorating fibrosis and adhesion development.

Pregnancy-associated plasma protein A in human ovarian follicles and its association with intrafollicular hormone levels.

OBJECTIVE: To evaluate follicular fluid (FF) levels of pregnancy-associated plasma protein A (PAPP-A) in relation to levels of intrafollicular hormones. Furthermore, immunostaining of human follicles of varying diameters was studied for PAPP-A, antimüllerian hormone (AMH), and aromatase, and the biological activity of PAPP-A in FF was evaluated. DESIGN: Laboratory investigation. SETTING: University hospital. PATIENT(S): A total of 43 women with a total of 80 samples were obtained from three different size-groups of antral follicles collected before and after the LH surge. INTERVENTION(S): ELISA measurement of steroids, PAPP-A, and AMH, immunohistochemistry of PAPP-A, AMH, and aromatase on follicles of different diameter, and proteolytic activity of PAPP-A toward insulin-like growth factor (IGF)-binding protein 4 (IGFBP-4). MAIN OUTCOME MEASURE(S): Association between FF levels of PAPP-A and measured ovarian hormones, PAPP-A activity in FF, localization of PAPP-A, AMH, and aromatase in antral follicles. RESULT(S): A highly significant association between FF levels of PAPP-A and all measured hormones were obtained with positive associations toward E2 and P, whereas AMH, T, and A showed strong negative associations. PAPP-A proteolytic activity toward IGFBP-4 was detected in human FF. PAPP-A immunostaining shifted from being primarily present in theca cells of small antral follicles to being expressed in granulosa cells (GCs) of preovulatory follicles. In contrast, AMH expression became reduced with increasing follicular diameter. Aromatase expression was highly specifically localized to GCs of preovulatory follicles. CONCLUSION(S): The results suggest that PAPP-A is specifically involved in the regulation of steroidogenesis in human antral follicles. Local regulation of IGF-II activity may represent a mechanism by which PAPP-A exerts this function and highlights the importance of IGF signaling during follicular development.

Local estrogen metabolism in epithelial ovarian cancer suggests novel targets for therapy.

Epithelial ovarian cancer (EOC) accounts for about 90% of malignant ovarian tumors, and estrogen is often implicated in disease progression. We therefore compared the potential for gating of estrogen action via pre-receptor metabolism in normal human ovarian surface epithelium (OSE), EOC and selected EOC cell lines (SKOV3 and PEO1). Steroid sulphatase (STS), estrogen sulfotransferase (EST), 17ß-hydroxysteroid dehydrogenases 2 (17BHSD2) and 5 (17BHSD5) mRNAs, proteins and enzymatic activities were all detectable in primary cell cultures of OSE and EOC, whereas aromatase and 17BHSD1 expression was negligible. qRT-PCR assay on total mRNA revealed significantly higher EST mRNA expression in OSE compared to EOC (P<0.05). Radioenzymatic measurements confirmed reduced sulfoconjugation (neutralization) of free estrogen in EOC relative to OSE. OSE cells were more effective at converting free [(3)H]-E1 to [(3)H]-E1S or [(3)H]-E2S, while EOC cell lines mainly converted [(3)H]-E1 to [(3)H]-E2 with minimal formation of [(3)H]-E1S or [(3)H]-E2S. IL1α treatment suppressed EST (P<0.01) and 17BHSD2 (P<0.001) mRNA levels in OSE and stimulated STS mRNA levels (P<0.001) in cancer (SKOV3) cells. These results show that estrogen is differentially metabolized in OSE and EOC cells, with E2 'activation' from conjugated estrogen predominating in EOC. Inflammatory cytokines may further augment the local production of E2 by stimulating STS and suppressing EST. We conclude that local estrogen metabolism may be a target for EOC treatment.

Low 17beta-estradiol levels in CNR1 knock-out mice affect spermatid chromatin remodeling by interfering with chromatin reorganization.

The type 1-cannabinoid receptor, CNR1, regulates differentiation of spermatids. Indeed, we have recently reported that the genetic inactivation of Cnr1 in mice influenced chromatin remodeling of spermatids, by reducing histone displacement and then sperm chromatin quality indices (chromatin condensation and DNA integrity). Herein, we have studied, at both central and testicular levels, the molecular signals potentially involved in histone displacement. In particular, investigation of the neuroendocrine axis involved in estrogen production demonstrated down-regulation of the axis supporting FSH/estrogen secretion in Cnr1-knockout male mice. Conversely, Cnr1-knockout male mice treated with 17beta-estradiol showed a weak increase of pituitary Fsh-beta subunit mRNA levels and a rescue of sperm chromatin quality indices demonstrating that estrogens, possibly in combination with FSH secretion, play an important role in regulating chromatin remodeling of spermatids.

IL1α and IL4 signalling in human ovarian surface epithelial cells.

The human ovarian surface epithelium (hOSE) is a mesothelial layer that surrounds the ovary and undergoes injury and repair cycles after ovulation-associated inflammation. We previously showed that IL4 is a key regulator of progesterone bioavailability during post-ovulatory hOSE repair as it differentially up-regulated 3ß-HSD1 and 3ß-HSD2 mRNA transcripts and total 3ß-hydroxysteroid dehydrogenase activity whereas it inhibited androgen receptor (AR) expression. We now show that the pro-inflammatory effect of IL1α on 3ß-HSD1 expression is mediated by nuclear factor-κB (NF-κB), whereas its anti-inflammatory action on 3ß-HSD2 expression is exerted via p38 mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K) and NF-κB signalling pathways. The anti-inflammatory IL4 effects on 3ß-HSD1 and 3ß-HSD2 mRNA expression are mediated through STAT6 and PI3K signalling networks. IL4 effects on AR and 3ß-HSD2 expression involve the p38 MAPK pathway. We also document that IL4 up-regulates lysyl oxidase (LOX) mRNA transcripts, a key gene for extracellular matrix (ECM) deposition and inhibits IL1α-induced expression of cyclooxygenase-2 (COX-2) mRNA, a gene involved in breakdown of ECM, showing a further role in post-ovulatory wound healing. We conclude that IL1α and IL4 actions in the post-ovulatory wound healing of hOSE cells are mediated by different signalling transduction pathways. The p38 MAPK signalling pathway may have possible therapeutic benefit in inflammation-associated disorders of the ovary, including cancer.

Development of steroid signaling pathways during primordial follicle formation in the human fetal ovary.

CONTEXT: Ovarian primordial follicle formation is critical for subsequent human female fertility. It is likely that steroid, and especially estrogen, signaling is required for this process, but details of the pathways involved are currently lacking. OBJECTIVE: The aim was to identify and characterize key members of the steroid-signaling pathway expressed in the second trimester human fetal ovary. DESIGN: We conducted an observational study of the female fetus, quantifying and localizing steroid-signaling pathway members. SETTING: The study was conducted at the Universities of Aberdeen, Edinburgh, and Glasgow. PATIENTS/PARTICIPANTS: Ovaries were collected from 43 morphologically normal human female fetuses from women undergoing elective termination of second trimester pregnancies. MAIN OUTCOME MEASURES: We measured mRNA transcript levels and immunolocalized key steroidogenic enzymes and steroid receptors, including those encoded by ESR2, AR, and CYP19A1. RESULTS: Levels of mRNA encoding the steroidogenic apparatus and steroid receptors increased across the second trimester. CYP19A1 transcript increased 4.7-fold during this period with intense immunostaining for CYP19A detected in pregranulosa cells around primordial follicles and somatic cells around oocyte nests. ESR2 was localized primarily to germ cells, but androgen receptor was exclusively expressed in somatic cells. CYP17A1 and HSD3B2 were also localized to oocytes, whereas CYP11A1 was detected in oocytes and some pregranulosa cells. CONCLUSIONS: The human fetal ovary expresses the machinery to produce and detect multiple steroid signaling pathways, including estrogenic signaling, with the oocyte acting as a key component. This study provides a step-change in our understanding of local dynamics of steroid hormone signaling during the key period of human primordial follicle formation.

Functional characterisation of the H365Y mutation of the 21-hydroxylase gene in congenital adrenal hyperplasia.

The study subject was a 13 day-old boy admitted to hospital, with weight loss since birth. He presented with the vomiting and hypotension that are classical features of congenital adrenal hyperplasia (CAH). The most common type of CAH is an autosomal recessive disorder caused by mutations in the 21-hydroxylase (CYP21A2) gene. To examine the CYP21A2 gene, gene-specific PCR was carried out, followed by sequencing. The baby was shown to be a compound heterozygote H365Y/R356W for two CYP21A2 gene mutations each inherited from a different parent. One of the mutations has not previously been functionally characterised. The mutations were reconstructed in an expression plasmid and characterised in vitro after transient transfection into human embryonic kidney (HEK293T) and hepatoblastoma (C3A) cell lines followed by measurement of enzyme activity. The CYP21A2 H365Y mutant exhibited minimal 21-hydroxylase activity to convert 17-hydroxyprogesterone to 11-deoxycortisol or progesterone to 11-deoxycorticosterone. Western immunoblotting indicated that the H365Y enzyme was produced in more variable amounts than wild type; in particular, the H365Y mutant protein may be unstable and/or subject to a more rapid degradation by the human proteosome as well as catalytically inefficient. The double mutant genotype with a severe mutation on each allele is compatible with the clinical presentation.

Adult-onset hypogonadotropic hypogonadism caused by aberrant expression of aromatase in an adrenocortical adenocarcinoma.

Estrogen-secreting adrenal cancers are extremely rare, with feminizing symptoms attributed to aromatase expression in the adrenal tumor. We describe a case of hypogonadotropic hypogonadism as a consequence of aberrant aromatase expression in a patient with adrenocortical adenocarcinoma. A 54 year-old man presented with a two month history of gynecomastia and reduced libido. Endocrine biochemistry at presentation showed hypogonadotropic hypogonadism (LH 2.4 U/L, FSH <1.0 IU/L, testosterone 2.8 nmol/L) with increased serum estrone (E(1), 821 pmol/L) and estradiol (E(2), 797 pmol/L) and subclinical ACTH-independent hypercortisolism (serum cortisol post 1mg overnight dexamethasone suppression test, 291 nmol/L). A right adrenal mass was identified on CT scanning and the patient underwent an open adrenalectomy. Post-operative evaluation showed normalization of serum levels of E(1) (95 pmol/L), E(2 )(109 pmol/L), testosterone (11.4 nmol/L), LH (4.1 U/L) and FSH (5.9 IU/L), and of cortisol dynamics. Immunohistochemistry of the adrenal cancer confirmed aberrant expression of aromatase in most, although not all, carcinoma cells. Transcripts associated with utilization of promoters II, I.1 and I.3 were prominently represented in the tumor aromatase mRNA. This case highlights that clinical features of feminizing adrenocortical carcinomas can be secondary to estrogen production by aberrantly transcribed and translated aromatase within the tumor. Even in males, gonadotropin secretion is subject to predominantly estrogen-mediated feedback-inhibition. The diagnosis of adrenocortical adenocarcinoma should be considered in men presenting with low testosterone and gonadotropins, particularly in the presence of feminizing features.

Cyclin D1 regulates hepatic estrogen and androgen metabolism.

Cyclin D1 is a cell cycle control protein that plays an important role in regenerating liver and many types of cancer. Previous reports have shown that cyclin D1 can directly enhance estrogen receptor activity and inhibit androgen receptor activity in a ligand-independent manner and thus may play an important role in hormone-responsive malignancies. In this study, we examine a distinct mechanism by which cyclin D1 regulates sex steroid signaling, via altered metabolism of these hormones at the tissue and cellular level. In male mouse liver, ectopic expression of cyclin D1 regulated genes involved in the synthesis and degradation of sex steroid hormones in a pattern that would predict increased estrogen and decreased androgen levels. Indeed, hepatic expression of cyclin D1 led to increased serum estradiol levels, increased estrogen-responsive gene expression, and decreased androgen-responsive gene expression. Cyclin D1 also regulated the activity of several key enzymatic reactions in the liver, including increased oxidation of testosterone to androstenedione and decreased conversion of estradiol to estrone. Similar findings were seen in the setting of physiological cyclin D1 expression in regenerating liver. Knockdown of cyclin D1 in HuH7 cells produced reciprocal changes in steroid metabolism genes compared with cyclin D1 overexpression in mouse liver. In conclusion, these studies establish a novel link between the cell cycle machinery and sex steroid metabolism and provide a distinct mechanism by which cyclin D1 may regulate hormone signaling. Furthermore, these results suggest that increased cyclin D1 expression, which occurs in liver regeneration and liver diseases, may contribute to the feminization seen in these settings.

Selenium supplementation attenuates procollagen-1 and interleukin-8 production in fat-loaded human C3A hepatoblastoma cells treated with TGFbeta1.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is associated with obesity, insulin resistance and hepatic steatosis. Non-alcoholic steatohepatitis (NASH) is a serious consequence of NAFLD where chronic tissue damage and inflammation result in fibrosis which may progress to cirrhosis. Transforming growth factor beta1 (TGFbeta1), proinflammatory cytokines and oxidative stress are thought to play crucial roles in the pathogenesis of these conditions. The contributions of individual liver cell types to fibrogenesis remain controversial and the influence of selenium status has not been investigated. METHODS: In this study we have used a cell culture model of fat-loading using oleate-treated human hepatoblastoma (C3A) cells to investigate how fat-loading and selenium status might influence the production of collagen in response to TGFbeta1. The secretion of inflammatory cytokines was also investigated, together with the epithelial character of the treated cells. RESULTS: We found that in response to treatment with TGFbeta1, C3A cells produced mRNA encoding the pro-alphaI chain of procollagen I, secreted procollagen I peptide, up-regulated production of the proinflammatory cytokine interleukin-8 (IL-8) and the mesenchymal marker vimentin, and down-regulated albumin production. Most of these responses were considerably enhanced when cells were fat-loaded with oleate and were attenuated by selenium addition at a dose that optimised the expression of thioredoxin reductase and glutathione peroxidase. CONCLUSIONS: Our data establish that both fat-loading and suboptimal selenium status enhance collagen and IL-8 production by C3A hepatocytes in response to TGFbeta1, possibly as part of an epithelial to mesenchymal transition. GENERAL SIGNIFICANCE: These findings suggest that the hepatocyte may be an important contributor to the pathogenesis of fibrosis associated with NAFLD.

Steroidogenesis in the fetal testis and its susceptibility to disruption by exogenous compounds.

Masculinization depends on adequate production of testosterone by the fetal testis within a specific "masculinization programming window." Disorders resulting from subtle deficiencies in this process are common in humans, and environmental exposures/lifestyle could contribute causally because common therapeutic and environmental compounds can affect steroidogenesis. This evidence derives mainly from rodent studies, but because there are major species differences in regulation of steroidogenesis in the fetal testis, this may not always be a guide to potential effects in the human. In addition to direct study of the effects of compounds on steroidogenesis, information also derives from study of masculinization disorders that result from mutations in genes in pathways regulating steroidogenesis. This review addresses this issue by critically reviewing the comparative timing of production and regulation of steroidogenesis in the fetal testis of humans and of rodents and its susceptibility to disruption; where there is limited information for the fetus, evidence from effects on steroidogenesis in the adult testis is considered. There are a number of fundamental regulatory differences between the human and rodent fetal testis, most notably in the importance of paracrine vs. endocrine drives during masculinization such that inactivating LH receptor mutations block masculinization in humans but not in rodents. Other large differences involve the steroidogenic response to estrogens and GnRH analogs and possibly phthalates, whereas for other compounds there may be differences in sensitivity to disruption (ketoconazole). This comparison identifies steroidogenic targets that are either vulnerable (mitochondrial cholesterol transport, CYP11A, CYP17) or not (cholesterol uptake) to chemical interference.

Disruption in the expression and immunolocalisation of steroid receptors and steroidogenic enzymes in letrozole-induced polycystic ovaries in rat.

The objective of the present study was to characterise the expression and tissue distribution of steroid receptors (oestrogen receptor-alpha and -beta (ERalpha, ERbeta), androgen receptor (AR) and progesterone receptor (PR)) and steroidogenic enzymes (P450 aromatase (P450arom), 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and steroidogenic acute regulatory protein (StAR)) in letrozole-induced polycystic ovaries of rats. Changes in serum hormone levels, protein expression in whole ovaries by western blot analysis and protein localisation by immunohistochemistry were determined in female rats treated with the aromatase inhibitor letrozole and compared with controls in proestrous and diestrous rats. Increases in the serum LH, FSH and testosterone concentrations were observed in letrozole-treated rats whereas serum oestradiol and progesterone levels were reduced. Protein expression as analysed by western immunoblot was consistent with the immunohistochemical data. Letrozole treatment induced an increase in the expression of AR, StAR and 3beta-HSD and a decrease in ERbeta. ERalpha, PR and P450arom showed partial changes in relation to some cycle stages. These results indicate that cystogenesis in this experimental model is characterised by changes in steroid receptors and steroidogenic enzyme expression that may be essential to proper ovarian functioning and are in agreement with similar changes observed in women with PCOS.

Regulation of 3beta-hydroxysteroid dehydrogenase type 1 and type 2 gene expression and function in the human ovarian surface epithelium by cytokines.

The human ovarian surface epithelium (hOSE) is a squamous-to-cuboidal layer that surrounds the ovary. hOSE undergoes injury and repair cycles as a result of ovulation-induced inflammation, an event relevant to the development of epithelial ovarian cancer (EOC). Locally produced steroids mediate the response to inflammation. 3beta-Hydroxysteroid dehydrogenase (3beta-HSD) drives the intracrine generation of progestogens and androgens that potentially affect cell survival and proliferation. We therefore investigated the regulation of 3beta-HSD along with downstream steroid signalling in hOSE. Double immunofluorescence of cultured primary hOSE cells confirmed the expression of 3beta-HSD protein Interleukin (IL). IL-1alpha treatment of primary cells to mimic ovulation-associated inflammation suppressed 3beta-HSD1 expression and stimulated 3beta-HSD2 mRNA (P < 0.001), without affecting total 3beta-HSD protein and activity or androgen or progesterone receptor (PR) mRNA levels. Conversely, IL-4 as a proxy for a post-ovulatory healing cytokine increased both 3beta-HSD transcripts, total protein and activity (P < 0.01). IL-4 also suppressed androgen receptor expression (P < 0.01) without affecting that of the PR, thereby potentially sustaining both progesterone biosynthesis and its underlying signalling in the ovarian surface. 3beta-HSD protein was immunodetectable in primary ascites of women who were diagnosed with EOC but both mRNA transcripts were diminished relative to normal cells (P < 0.05). Notably, this difference was countered by IL-4 treatment (P < 0.01). We conclude that stimulation by IL-4 could be physiologically relevant to post-ovulatory ovarian healing and suggest a novel therapeutic strategy for the activation of progesterone-associated apoptosis in ovarian cancer. Also, our results suggest an attenuation of 3beta-HSD expression in EOC although further studies are required for confirmation.

Differentiation-dependent progesterone synthesis and metabolism in NT2-N human neurons.

Human embryonic teratocarcinoma-derived Ntera2/cl.D1 (NT2) cells recapitulate many features of embryonic neuronal progenitor cells. Upon retinoic acid (RA) treatment they terminally differentiate into post-mitotic neuron-like cells (NT2-N), akin to human fetal neurons, thus representing an in vitro model of human neuron terminal differentiation. Experimental evidence also indicate NT2-N cultures as a potential source for cell transplantation therapy. The neurosteroids progesterone and its metabolite 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-THP) promote neurogenesis and show anti-neurodegenerative properties. This study's aim was to assess the neurosteroidogenic competence of NT2 cells during RA-induced neuronal differentiation. Radioimmunoassay measurements revealed progesterone only in NT2-N cultures (4 week RA). Accordingly, progesterone synthesis from (3)H-pregnenolone was absent in NT2 cells and increased during RA exposure, being highest in NT2-N. [(3)H]-pregnenolone metabolism, yielding [(3)H]-progesterone and [(3)H]-5alpha-dihydroprogesterone ([(3)H]-5alpha-DHP), was time-dependent and inhibited by trilostane, a 3beta-hydroxysteroid-dehydrogenase (3beta-HSD) inhibitor. Conversely, (3)H-progesterone metabolism, which yielded [(3)H]-5alpha-DHP > [(3)H]-3beta,5alpha-THP > [(3)H]-3alpha,5alpha-THP, occurred at all time points examined, though showing a nadir in cultures treated with RA for 1 and 2 weeks. The differentiation-dependent increase of progesterone accumulation matched 3beta-HSD type I mRNA expression and 3beta-HSD immunoreactivity, that co-localized with Map2a/b- and GAD67 in NT2-N. Hence, in vitro differentiated human neurons, while retaining progesterone metabolic activity, also become competent in progesterone synthesis. These findings suggest an autocrine/paracrine role of neuronal progesterone, either on its own or through its 5alpha-reduced metabolites, in fetal brain development and allow speculation that NT2-N-produced neurosteroids may contribute to the encouraging results of NT2-N transplants in animal models of neurodegenerative diseases.

Cortisol inactivation by 11beta-hydroxysteroid dehydrogenase-2 may enhance endometrial angiogenesis via reduced thrombospondin-1 in heavy menstruation.

CONTEXT: Heavy menstrual bleeding (HMB; menorrhagia) impairs quality of life for women and requires medication or surgery. Because glucocorticoids inhibit angiogenesis in other organs, we hypothesized that endometrium of women with HMB is subject to decreased local glucocorticoid exposure and enhanced angiogenesis, thereby increasing menstrual bleeding. DESIGN: Endometrium was collected from 29 women with menstrual complaints. Menstrual blood loss was measured by alkaline-hematin assay (n = 12, > 80 ml (HMB); n = 17, < 80 ml). Quantitative RT-PCR for thrombospondin-1 (TSP-1) and glucocorticoid-metabolizing enzymes, 11beta-hydroxysteroid dehydrogenases-1 and -2 (11betaHSD1,2) was performed. Glucocorticoid effects on endometrial stromal cells and uterine endothelial cells (UECs) were determined. RNA interference studies in UECs examined the effect of TSP-1 ablation on cortisol action. RESULTS: Secretory phase endometrium mRNA levels for the cortisol inactivating enzyme 11betaHSD2 were higher [3.78 +/- 1.29 vs. 1.40 +/- 0.6 (arbitrary units), P < 0.05], whereas TSP-1 mRNA was lower [0.40 +/- 0.13 vs. 1.66 +/- 1.02 (arbitrary units), P < 0.05] in women with HMB. In cultured endometrial stromal cells and UECs, cortisol increased TSP-1 expression. Both cortisol and TSP-1 inhibited new vessel formation in endometrial explants embedded in Matrigel. In UECs cortisol inhibition of tube-like structure formation was blocked by small interfering RNA (siRNA) against TSP-1 (25 +/- 2.5% cortisol inhibition with scrambled siRNA vs. 0% cortisol inhibition with TSP-1 siRNA inactivation, P<0.01). CONCLUSIONS: Enhanced inactivation of cortisol by 11betaHSD2 in endometrium from women with HMB may explain reduced TSP-1 levels and hence endothelial cell dysfunction and abnormal angiogenesis. Inhibition of 11betaHSD2 may be a rational novel therapy for heavy menstrual bleeding.

3beta-Hydroxysteroid dehydrogenases and pre-receptor steroid metabolism in the human ovarian surface epithelium.

Ovulation-associated inflammation with accompanied cytokines and reproductive hormones impact upon the human ovarian surface epithelium (hOSE) and probably have a role in the aetiology of ovarian cancer. Progesterone and progestin-related events, i.e. pregnancy and oral contraception, protect from the disease. We have investigated the pre-receptor metabolism of progesterone in primary hOSE cells and an immortalised hOSE cell line, OSE-C2, focusing on transcriptional regulation of 3beta-hydroxysteroid dehydrogenase (3beta-HSD) by inflammatory, anti-inflammatory and apoptotic factors. In hOSE cells, we show that anti-inflammatory effects of IL-1alpha and IL-4 on 3beta-HSD2 mRNA involve a p38 MAPK signalling pathway, whereas pro-inflammatory response of IL-1alpha to 3beta-HSD1 mRNA involves a NF-kappaB inflammatory pathway. In OSE-C2 cells, retinoic acid and transforming growth factor-beta1 massively induce 3beta-HSD1 mRNA levels. In conclusion, we elaborate several mechanisms for intracrine formation of progesterone in hOSE that could contribute in the development of novel strategies to prevent, diagnose and/or treat ovarian cancer.

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 17beta-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 (6h) 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 17beta-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.

GnRH-mediated DAN production regulates the transcription of the GnRH receptor in gonadotrope cells.

The primary function of gonadotropin-releasing hormone (GnRH) is the regulation of pituitary gonadotropin hormone gene transcription, biosynthesis and release. These effects are mediated through intracellular mobilization of Ca2+ and activation of PKC isoforms and MAP kinases. We show here that DAN (differential screening-selected gene aberrative in neuroblastoma) which is a secreted bone morphogenic protein (BMP) antagonist belonging to the TGFbeta protein superfamily, is controlled by GnRH in murine gonadotrope cells. Acute GnRH stimulation induced a rapid, 27-fold, elevation of DAN mRNA, accompanied by an approximate 3-fold increase in the amount of mature DAN glycoprotein in the cell cytoplasm and in DAN secretion into the culture medium. Incubation of L beta T2 cells in DAN-containing medium altered the levels of a number of cellular proteins. Two of these were identified as the steroidogenic acute regulatory protein (StAR) and the actin-related protein 2/3 complex subunits 2 (p34-ARC) which are primarily involved in steroidogenesis and cytoskeleton remodelling, respectively. DAN caused an approximate 2-fold specific elevation in the cytoplasmic levels of both these proteins in L beta T2 cells. We further tested the effects of DAN on classical GnRH effects viz. gonadotropin and GnRH receptor gene expression. Co-transfection of L beta T2 cells with DAN and gonadotropin subunit promoter luciferase reporter genes had no effect on GnRH stimulation of alpha GSU and LH beta or on the additive GnRH and activin induction of FSH beta subunit transcription. However, co-transfection of DAN markedly inhibited the synergistic activation of GnRH and activin on GnRH receptor gene expression thus implicating DAN as a novel autocrine/paracrine factor that modulates GnRH function in pituitary gonadotropes.

Mid-luteal endometrial intracrinology following controlled ovarian hyperstimulation involving use of a gonadotrophin releasing hormone antagonist.

BACKGROUND: There are concerns of reduced pregnancy rates with the use of gonadotrophin-releasing hormone antagonists (GnRH antagonists) in IVF/ICSI cycles. Sex steroids and their metabolizing enzymes in the endometrium may play a vital role in embryo implantation. This study has evaluated the levels and localization of sex-steroid receptors and metabolizing enzymes, 3beta-hydroxysteroid dehydrogenases (3betaHSD) and selected 17beta-HSD (17betaHSD), in mid-luteal endometrium of women treated with GnRH antagonist (Cetrorelix) and recombinant FSH (rFSH; Gonal-F) with luteal phase progesterone supplementation. METHODS: Mid-luteal phase endometrial biopsies were obtained from oocyte donors undergoing ovarian stimulation and from control women with regular periods. Immunohistochemistry and real-time quantitative-polymerase chain reaction (QRT-PCR) were used to compare protein and mRNA expression of progesterone receptor (PR), estrogen receptor alpha (ERalpha), estrogen receptor beta (ERbeta), androgen receptor (AR), 3betaHSD1, 3betaHSD2, 17betaHSD2 and 17betaHSD5. RESULTS: Cetrorelix-rFSH treatment caused a mid-luteal suppression of PR protein expression in the endometrial stroma, surface epithelium and glands, although expression in the glands of control samples was variable. In contrast, the treatment caused an increase in PR staining in perivascular cells. No other significant differences in protein expression were observed between the two groups. mRNA levels of AR, ERalpha, 3betaHSD1 and 17betaHSD2 were significantly reduced in the treatment group. PR mRNA levels were also reduced by GnRH antagonist-rFSH treatment, but the difference was not significant. CONCLUSIONS: Changes in the expression of sex-steroid receptors and metabolizing enzymes may lead to alterations in the activity and intracellular availability of estrogens, progestogens and androgens in endometrium of women treated with Cetrorelix and rFSH. Their impact on embryo implantation merits further evaluation.

Functional expression and characterisation of human cytochrome P45017alpha in Pichia pastoris.

Human cytochrome P45017alpha (CYP17), present in mammalian adrenal and gonadal tissues, catalyses both steroid 17-hydroxylation and C17,20 lyase reactions, producing intermediates for the glucocorticoid and androgenic pathways, respectively. The characterisation of this complex enzyme was initially hampered due to low level in vivo expression of CYP17. Heterologous expression systems have contributed greatly to our current knowledge of CYP17's dual catalytic activity. However, due to the hydrophobic nature of this membrane-bound protein, primarily truncated and modified forms of CYP17 are currently being expressed heterologously. Although the N-terminally modified enzyme has been well characterised, protein structure and function studies still necessitate the expression of unmodified, wild-type CYP17. We report here the expression of a catalytically active, unmodified human CYP17 in the industrial methylotrophic yeast, Pichia pastoris. A typical P450 carbon monoxide difference spectrum, with an absorption maximum at 448nm and a substrate-induced type I spectrum were recorded using a detergent-solubilised cellular fraction containing CYP17. The expressed enzyme catalysed the conversion of progesterone to 17-hydroxyprogesterone as well as 16-hydroxyprogesterone, a product unique to human and chimpanzee CYP17. This is the first report showing the heterologous expression of a fully functional human steroidogenic cytochrome P450 enzyme in P. pastoris.