Pierre R. Provost

Sex hormone metabolism in lung development and maturation

Sex hormones are increasingly recognized as regulators of lung development. Respiratory distress syndrome (RDS) is the leading cause of morbidity in preterm neonates and occurs with a higher incidence in males. The mechanisms underlying the effects of androgens on lung development and the occurrence of RDS are only partially deciphered, and positive roles of estrogens on surfactant production and alveologenesis are relevant to our understanding of pulmonary diseases. This manuscript reviews current knowledge on androgen and estrogen metabolism and on relevant hormone targets in the fetal lung. Further investigations are needed to elucidate mechanisms orchestrating sex hormone effects on lung development. These studies aim to decrease mortality and morbidity associated with RDS and other pathologies related to lung immaturity at birth.

Androgen Formation and Metabolism in the Pulmonary Epithelial Cell Line A549: Expression of 17β-Hydroxysteroid Dehydrogenase Type 5 and 3α-Hydroxysteroid Dehydrogenase Type 31

Surfactant synthesis within developing fetal lung type II cells is affected by testosterone and 5alpha-dihydrotestosterone (5alpha-DHT). The pulmonary epithelial cell line A549, isolated from a human lung carcinoma, like normal lung type II cell, produces disaturated phosphatidylcholines and has been widely used for studying the regulation of surfactant production. Androgen receptor has been detected in A549 cells; however, the capacity of these cells for androgen synthesis and metabolism has not been investigated at molecular level. This study was undertaken to identify the steroidogenic enzymes involved in the formation and metabolism of androgens from adrenal C19 steroid precursors in A549 cells. When cultured in the presence of normal FCS, A549 intact cells converted DHEA to androstenediol, androstenedione principally to testosterone, and 5alpha-DHT to 5alpha-androstane 3alpha,17beta-diol. High levels of 17beta-hydroxysteroid dehydrogenase (HSD) and 3alpha-HSD activities were detected in both cytosol and microsomes isolated from homogenates. Analysis of A549 RNA indicated the presence of 17beta-HSD type 4 and type 5, and of 3alpha-HSD type 3 messenger RNAs. Very low levels of 3beta-HSD type 1 and 5alpha-reductase type 1 messenger RNAs and activities were detected. With regard to active androgen formation, there was little or no capacity for the conversion of DHEA to 5alpha-DHT. In contrast, androstenedione was rapidly transformed to testosterone. The pattern of steroid metabolism was not affected by the use of charcoal-stripped FCS or by the synthetic glucocorticoid dexamethasone. Together, our findings show that A549 cells express a pattern of steroid metabolism in which 17beta-HSD type 5 and 3alpha-HSD type 3 are the predominant enzymes. The level of androgens is regulated at the level of catalysis in intact cells such that the intracellular level of testosterone is stabilized, whereas 5alpha-DHT is rapidly inactivated by reduction to 3alpha,17beta-diol. This pattern of androgen metabolism has implications for the relative importance of testosterone and 5alpha-DHT in normal lung development and surfactant production.

Effect of insulin on serum levels of dehydroepiandrosterone metabolites in men.

OBJECTIVE Insulin was found to decrease the concentration of serum dehydroepiandrosterone (DHEA) and DHEA‐sulphate (DHEAS) and recent data suggest that an increase in the metabolic clearance rate of DHEA (MCRDHEA) may be involved. In this study, we have investigated the effects of insulin on DHEA metabolism in men.

Gene expression profile of androgen modulated genes in the murine fetal developing lung

BackgroundAccumulating evidences suggest that sex affects lung development. Indeed, a higher incidence of respiratory distress syndrome is observed in male compared to female preterm neonates at comparable developmental stage and experimental studies demonstrated an androgen-related delay in male lung maturation. However, the precise mechanisms underlying these deleterious effects of androgens in lung maturation are only partially understood.MethodsTo build up a better understanding of the effect of androgens on lung development, we analyzed by microarrays the expression of genes showing a sexual difference and those modulated by androgens. Lungs of murine fetuses resulting from a timely mating window of 1 hour were studied at gestational day 17 (GD17) and GD18, corresponding to the period of surge of surfactant production. Using injections of the antiandrogen flutamide to pregnant mice, we hunted for genes in fetal lungs which are transcriptionally modulated by androgens.ResultsResults revealed that 1844 genes were expressed with a sexual difference at GD17 and 833 at GD18. Many genes were significantly modulated by flutamide: 1597 at GD17 and 1775 at GD18. Datasets were analyzed by using in silico tools for reconstruction of cellular pathways. Between GD17 and GD18, male lungs showed an intensive transcriptional activity of proliferative pathways along with the onset of lung differentiation. Among the genes showing a sex difference or an antiandrogen modulation of their expression, we specifically identified androgen receptor interacting genes, surfactant related genes in particularly those involved in the pathway leading to phospholipid synthesis, and several genes of lung development regulator pathways. Among these latter, some genes related to Shh, FGF, TGF-beta, BMP, and Wnt signaling are modulated by sex and/or antiandrogen treatment.ConclusionOur results show clearly that there is a real delay in lung maturation between male and female in this period, the latter pursuing already lung maturation while the proper is not yet fully engaged in the differentiation processes at GD17. In addition, this study provides a list of genes which are under the control of androgens within the lung at the moment of surge of surfactant production in murine fetal lung.

Apolipoprotein A-I, A-II, C-II, and H expression in the developing lung and sex difference in surfactant lipids

A sex difference in surfactant lipids is associated with a higher incidence of respiratory distress syndrome for males in cases of preterm birth. In animal models, the sex difference in surfactant lipids was shown to be androgen receptor-dependent. This report examines expression of apolipoprotein (apo)A-I, apoA-II, apoC-II, apoE, apoH, and lipoprotein lipase (LPL) by quantitative real-time PCR in pools of male and female fetal lung tissues from various mouse litters from gestation day (GD) 15.5 to 18.5, and in various adult tissues. Although the expression profiles of ApoA-I, ApoA-II, ApoC-II, and ApoH are complex, these genes are co-regulated and they all present a sex difference (P=0.0896, 0.0896, 0.0195, and 0.0607 respectively) with higher expression for females for several litters. Pulmonary expression of apoA-I, apoA-II, and apoH were specific to the developing lung. ApoE and LPL mRNAs showed a significant increase from GD 17.5 to 18.5. An increase in apoA-I-, apoA-II-, apoC-II-, and apoH-mRNA accumulation was observed from GD 16.5 to 17.5 in correlation with the emergence of mature type II pneumonocytes. These four apolipoprotein genes are co-regulated with type 2 and 5 17beta-hydroxysteroid dehydrogenases, which are respectively involved in inactivation and synthesis of androgens. Finally, apoC-II was detected by immunohistochemistry in epithelial cells of the distal epithelium. Positive signals looking like secretory granules were located near the basal membrane. Our results are compatible with a role for apolipoproteins in lipid metabolism and transport in the developing lung in association with the sex difference in surfactant lipid synthesis.

Epithelial cells are the major site of hydroxysteroid (17β) dehydrogenase 2 and androgen receptor expression in fetal mouse lungs during the period overlapping the surge of surfactant

Many genes involved in the peripheral metabolism of androgens, including hydroxysteroid (17beta) dehydrogenases (HSD17B) 2 and 5, steroid 5alpha reductase 1, and 3alpha-HSD, are expressed in the developing lung. Because lung development is delayed by androgens and pathologies related to lung immaturity are major concerns for preterm neonates, we are interested in the elucidation of the androgen metabolism in developing lung. In the present report we have identified the cell types expressing HSD17B2 (testosterone into androstenedione) and androgen receptor in normal male and female mouse developing lung between the gestation days 15.5 and 17.5. In situ hybridization and immunohistochemistry revealed that HSD17B2 is expressed in epithelial cells of respiratory and conducting zones, and in mesenchymal cells. The androgen receptor protein was observed in the same cell types that HSD17B2, and in alpha-smooth muscle actin-positive cells surrounding arteries. No difference was observed for the location of HSD17B2 and androgen receptor expression at any time points studied, or according to sex. Taken together, our results are in concordance with the hypothesis that in mouse fetal lungs the level of androgen receptor occupancy is finely tuned by local HSD17B2 expression.

Type 2 and 5 17β-hydroxysteroid dehydrogenases and androgen receptor in human fetal lungs

Androgens delay fetal lung maturation through an androgen receptor (AR)-dependent mechanism. Type 2 and 5 17beta-hydroxysteroid dehydrogenases (17betaHSD) are involved in androgen inactivation and synthesis, respectively. We aimed to further characterize the human fetal lung potential for androgen metabolism and response. 17betaHSD2, 17betaHSD5, and AR mRNA levels were determined in lungs of mid-late gestation and in adult lungs, while protein detections were performed at mid-gestation. Relationships between levels of each mRNA and gestational age were observed. AR protein levels showed important differences among individuals of the same gestational window. 17betaHSD2 and AR were co-localized in epithelial and mesenchymal cells. AR was detected in both, cytoplasm and nucleus, which suggests fine-tuning of AR occupancy. In contrast, 17betaHSD5 was localized in a few epithelial cells of conducting zones. Our results support the existence of a local androgen metabolism in male and female human fetal lungs during the period of high-risk premature birth.

Androgen receptor and 17β-HSD type 2 regulation in neonatal mouse lung development

A QPCR analysis of androgen receptor and several androgen metabolizing genes was performed during the saccular and alveolar stages of mouse lung development. Androgen receptor expression showed a statistically significant increase during the alveolar stage while levels of 17beta-hydroxysteroid dehydrogenase type 2 (17beta-HSD 2) expression significantly decreased at the end of the saccular stage and remained low throughout the alveolar period. 17beta-HSD 1, 17beta-HSD 5, 5alpha-reductase type 1, and mouse 3alpha-HSD did not present such a regulation. The androgen receptor protein was primarily detected in the nucleus of airway epithelial cells and of a subset of respiratory epithelial cells. 17beta-HSD 2 mRNA co-localized with androgen receptor protein during saccularization, but was absent from airway epithelium during alveolarization. Taken together, our results demonstrate temporal and spatial regulation of androgen receptor and 17beta-HSD 2 during the sacculo-alveolar transition period of mouse lung development suggesting control of androgen action.

Sexually dimorphic gene expression that overlaps maturation of type II pneumonocytes in fetal mouse lungs

BackgroundIn human, respiratory distress of the neonates, which occurs in prematurity, is prevalent in male. Late in gestation, maturation of type II pneumonocytes, and consequently the surge of surfactant synthesis are delayed in male fetuses compared with female fetuses. Although the presence of higher levels of androgens in male fetuses is thought to explain this sex difference, the identity of genes involved in lung maturation that are differentially modulated according to fetal sex is unknown. We have studied the sex difference in developing mouse lung by gene profiling during a three-day gestational window preceding and including the emergence of mature PTII cells (the surge of surfactant synthesis in the mouse occurs on GD 17.5).MethodsTotal RNA was extracted from lungs of male and female fetal mice (gestation days 15.5, 16.5, and 17.5), converted to cRNA, labeled with biotin, and hybridized to oligonucleotide microarrays (Affymetrix MOE430A). Analysis of data was performed using MAS5.0, LFCM and Genesis softwares.ResultsMany genes involved in lung maturation were expressed with no sex difference. Of the approximative 14 000 transcripts covered by the arrays, only 83 genes presented a sex difference at one or more time points between GDs 15.5 and 17.5. They include genes involved in hormone metabolism and regulation (i.e. steroidogenesis pathways), apoptosis, signal transduction, transcriptional regulation, and lipid metabolism with four apolipoprotein genes. Genes involved in immune functions and other metabolisms also displayed a sex difference.ConclusionAmong these sexually dimorphic genes, some may be candidates for a role in lung maturation. Indeed, on GD 17.5, the sex difference in surfactant lipids correlates with the sex difference in pulmonary expression of apolipoprotein genes, which are involved in lipid transport. This suggests a role for these genes in the surge of surfactant synthesis. Our results would help to identify novel genes involved in the physiopathology of the respiratory distress of the neonates.

Expression of genes related to the hypothalamic-pituitary-adrenal axis in murine fetal lungs in late gestation

BackgroundLung maturation is modulated by several factors, including glucocorticoids. Expression of hypothalamic-pituitary-adrenal (HPA) axis-related components, with proposed or described local regulatory systems analogous to the HPA axis, was reported in peripheral tissues. Here, HPA axis-related genes were studied in the mouse developing lung during a period overlapping the surge of surfactant production.MethodsExpression of genes encoding for corticotropin-releasing hormone (CRH), CRH receptors (CRHR) 1 and 2beta, CRH-binding protein, proopiomelanocortin (POMC), melanocortin receptor 2 (MC2R), and glucocorticoid receptor was quantified by real-time PCR and localized by in situ hydridization in fetal lungs at gestational days (GD) 15.5, 16.5, and 17.5, and was also quantified in primary mesenchymal- and epithelial cell-enriched cultures. In addition, the capability of CRH and adrenocorticotropic hormone (ACTH) to stimulate pulmonary expression of enzymes involved in the adrenal pathway of glucocorticoid synthesis was addressed, as well as the glucocorticoid production by fetal lung explants.ResultsWe report that all the studied genes are expressed in fetal lungs according to different patterns. On GD 15.5, Mc2r showed peaks in expression in samples that have previously presented high mRNA levels for glucocorticoid synthesizing enzymes, including 11beta-hydroxylase (Cyp11b1). Crhr1 mRNA co-localized with Pomc mRNA in cells surrounding the proximal epithelium on GD 15.5 and 16.5. A transition in expression sites toward distal epithelial cells was observed between GD 15.5 and 17.5 for all the studied genes. CRH or ACTH stimulation of genes involved in the adrenal pathway of glucocorticoid synthesis was not observed in lung explants on GD 15.5, whereas CRH significantly increased expression of 21-hydroxylase (Cyp21a1) on GD 17.5. A deoxycorticosterone production by fetal lung explants was observed.ConclusionsTemporal and spatial modulations of expression of HPA axis-related genes in late gestation are consistent with roles for these genes in lung development. Our data are likely to lead to valuable insights in relation to lung diseases originating from lung immaturity.