Quantitative appreciation of steroidogenic gene expression in mouse tissues: new roles for type 2 5alpha-reductase, 20alpha-hydroxysteroid dehydrogenase and estrogen sulfotransferase

We have recently developed an improved method for the RealTime PCR quantification of reversed transcribed mRNA (Q_RTPCR) that allows to obtain absolute mRNA expression levels with high sensitivity and accuracy. Using this Q_RTPCR method to assess the mRNA expression levels of genes encoding steroidogenic enzymes in male and female mouse tissues allows us to gain quantitative appreciation of the function of these genes. We could thus identify the existence of two types of steroidogenic tissues: those of classical endocrine glands such as the testis, ovary and adrenals which deliver steroids into the circulation, and in which millions of copies/mug total RNA are detected, and those of peripheral intracrine tissues where steroids are synthesized locally and exert their action at the site where they are produced (prostate, uterus, etc.), and in which the expression level of steroidogenic enzymes is much lower. We also observed an abnormally high expression levels of type 2 5alpha-reductase and 20alpha-HSD in the male and female adrenals, respectively, thus indirectly suggesting new roles for these sex-specific enzymes. On the other hand estrogen sulfotransferase, the enzyme that inactivates estrogen, has been found selectively expressed in male tissues, thus suggesting a role for this enzyme to protect male-specific tissues against estrogenic activity.     

Proinflammatory gene induction by platelet-activating factor mediated via its cognate nuclear receptor

It has been postulated that intracellular binding sites for platelet-activating factor (PAF) contribute to proinflammatory responses to PAF. Isolated nuclei from porcine cerebral microvascular endothelial cells (PCECs) produced PAF-molecular species in response to H(2)O(2). Using FACS analysis, we demonstrated the expression of PAF receptors on cell and nuclear surfaces of PCECs. Confocal microscopy studies performed on PCECs, Chinese hamster ovary cells stably overexpressing PAF receptors, and isolated nuclei from PCECs also showed a robust nuclear distribution of PAF receptors. Presence of PAF receptors at the cell nucleus was further revealed in brain endothelial cells by radioligand binding experiments, immunoblotting, and in situ in brain by immunoelectron microscopy. Stimulation of nuclei with methylcarbamate-PAF evoked a decrease in cAMP production and a pertussis toxin-sensitive rise in nuclear calcium, unlike observations in plasma membrane, which exhibited a pertussis toxin-insensitive elevation in inositol phosphates. Moreover, on isolated nuclei methylcarbamate-PAF evoked the expression of proinflammatory genes inducible nitric oxide synthase and cyclooxygenase-2 (COX-2) and was associated with augmented extracellular signal-regulated kinase 1/2 phosphorylation and NF-kappaB binding to the DNA consensus sequence. COX-2 expression was prevented by mitogen-activated protein kinase kinase/extracellular signal-regulated kinase 1/2 and NF-kappaB inhibitors. This study describes for the first time the nucleus as a putative organelle capable of generating PAF and expresses its receptor, which upon stimulation induces the expression of the proinflammatory gene COX-2.     

Rhein inhibits angiogenesis and the viability of hormone-dependent and -independent cancer cells under normoxic or hypoxic conditions in vitro

Hypoxia is a hallmark of solid tumors, including breast cancer, and the extent of tumor hypoxia is associated with treatment resistance and poor prognosis. Considering the limited treatment of hypoxic tumor cells and hence a poor prognosis of breast cancer, the investigation of natural products as potential chemopreventive anti-angiogenic agents is of paramount interest. Rhein (4,5-dihydroxyanthraquinone-2-carboxylic acid), the primary anthraquinone in the roots of Cassia alata L., is a naturally occurring quinone which exhibits a variety of biologic activities including anti-cancer activity. However, the effect of rhein on endothelial or cancer cells under hypoxic conditions has never been delineated. Therefore, the aim of this study was to investigate whether rhein inhibits angiogenesis and the viability of hormone-dependent (MCF-7) or -independent (MDA-MB-435s) breast cancer cells in vitro under normoxic or hypoxic conditions. Rhein inhibited vascular endothelial growth factor (VEGF₁₆₅)-stimulated human umbilical vein endothelial cell (HUVEC) tube formation, proliferation and migration under normoxic and hypoxic conditions. In addition, rhein inhibited in vitro angiogenesis by suppressing the activation of phosphatidylinositol 3-kinase (PI3K), phosphorylated-AKT (p-AKT) and phosphorylated extracellular signal-regulated kinase (p-ERK) but showed no inhibitory effects on total AKT or ERK. Rhein dose-dependently inhibited the viability of MCF-7 and MDA-MB-435s breast cancer cells under normoxic or hypoxic conditions, and inhibited cell cycle in both cell lines. Furthermore, Western blotting demonstrated that rhein inhibited heat shock protein 90alpha (Hsp90α) activity to induce degradation of Hsp90 client proteins including nuclear factor-kappa B (NF-κB), COX-2, and HER-2. Rhein also inhibited the expression of hypoxia-inducible factor-1 alpha (HIF-1α), vascular endothelial growth factor (VEGF₁₆₅), epidermal growth factor (EGF), and the phosphorylation of inhibitor of NF-κB (I-κB) under normoxic or hypoxic conditions. Taken together, these data indicate that rhein is a promising anti-angiogenic compound for breast cancer cell viability and growth. Therefore, further studies including in vivo and pre-clinical need to be performed.     

Specificity of the stimulatory effect of prostaglandins on hormone release in rat anterior pituitary cells in culture

Specificity of the effect of prostaglandins (PGs) on hormone release by the anterior pituitary gland was studied using cells in primary culture. Growth hormone (GH) release is stimulated by all eight PGs studied, PGE₁ and E₂ being 1000-fold more potent than the corresponding PGFs. The release of luteinizing hormone (LH), follicle-stimulating hormone (FSH), and prolactin (PRL) remains unchanged upon addition of PGEs. While the basal release of thyrotropin (TSH) is only slightly stimulated by concentrations of PGEs above 10⁻⁶M, an important potentiation of the stimulatory effect of thyrotropin-releasing hormone on TSH release is observed. The release of GH, TSH and LH is stimulated equally well by PGAs and PGBs at concentrations higher than 10⁻⁶M, 3 × 10⁻⁶M, and 10⁻⁵M, respectively. PGFs do not affect the release of any of the measured pituitary hormones at concentrations below 10⁻⁴M. The stimulation of GH release by PGE₂ can be inhibited by the PG antagonist 7-oxa-13-prostynoic acid, a half-maximal inhibition being found at a concentration of 4 × 10⁻⁵M of the antagonist in the presence of 10⁻⁶M PGE₂. In the presence of somatostatin (10⁻⁸M), the inhibition of GH release cannot be reversed by PGE₂ at concentrations up to 10⁻⁴M. 8-bromo-cyclic AMP-induced GH release is additive with that produced by PGE₂.The present data show that 1) of the five pituitary hormones measured, only GH release is stimulated by prostaglandins at relatively low concentrations, 2) the PGE-induced GH release can be competitively inhibited by 7-oxa-13-prostynoic acid, 3) the inhibition of GH release by somatostatin cannot be reversed by PGE₂ and 4) the PGEs increase the responsiveness of the thyrotrophs to TRH.     

The effects of O2 on net photosynthesis at low temperature (5°C)

Abstract. It has been shown that atmospheric O₂ can either depress or stimulate the rate of apparent photosynthesis of white mustard depending on the environmental conditions: CO₂ concentration, light intensity and temperature. Stimulation by O₂ was observed only under high photon fluence rate and at high CO₂ concentrations. The critical CO₂ concentration below which O₂ was inhibiting and above which it was stimulating was dependent on the temperature of the assay: for plants grown at 12°C the critical CO₂ concentration was 13.35 mmol at 5° C and 21.92 mmol at 10° C. Stimulation by O₂ depended also on the growth temperature: for measurements at 26.31 mmol m^?3 CO₂, O₂ was stimulating at temperatures less than 12°C for plants grown at 12°C and less than 19°C for plants grown at 27°C. The efficiency of the O₂-dependent stimulation of net photosynthesis was maximum at 9.21 mol m^?3 O₂ at 26.31 mmol m^?3 CO₂. Oxygen-stimulation of net photosynthesis was detected in Nicotiana tabacum L. var Samsun, Lycopersicum esculentum L. and Chenopodium album L. At 5°C and under high photon fluence rate, O₂ increased the carboxylation capacity of the photosynthetic apparatus of mustard and decreased its affinity for CO₂. The O₂ inhibition of the net CO₂ uptake observed at low CO₂ concentrations was the result of a decrease in the affinity for carbon dioxide. The nature of the mechanism which causes the stimulation of photosynthesis is discussed.     

Characterization of type 12 17beta-hydroxysteroid dehydrogenase, an isoform of type 3 17beta-hydroxysteroid dehydrogenase responsible for estradiol formation in women

A novel 17beta-hydroxysteroid dehydrogenase (17beta-HSD) chronologically named type 12 17beta-HSD (17beta-HSD12), that transforms estrone (E1) into estradiol (E2) was identified by sequence similarity with type 3 17beta-HSD (17beta-HSD3) that catalyzes the formation of testosterone from androstenedione in the testis. Both are encoded by large genes spanning 11 exons, most of them showing identical size. Using human embryonic kidney-293 cells stably expressing 17beta-HSD12, we have found that the enzyme catalyzes selectively and efficiently the transformation of E1 into E2, thus identifying its role in estrogen formation, in contrast with 17beta-HSD3, the enzyme involved in the biosynthesis of the androgen testosterone in the testis. Using real-time PCR to quantify mRNA in a series of human tissues, the expression levels of 17beta-HSD12 as well as two other enzymes that perform the same transformation of E1 into E2, namely type 1 17beta-HSD and type 7 17beta-HSD, it was found that 17beta-HSD12 mRNA is the most highly expressed in the ovary and mammary gland. To obtain a better understanding of the structural basis of the difference in substrate specificity between 17beta-HSD3 and 17beta-HSD12, we have performed tridimensional structure modelization using the coordinates of type 1 17beta-HSD and site-directed mutagenesis. The results show the potential role of bulky amino acid F234 in 17beta-HSD12 that blocks the entrance of androstenedione. Overall, our results strongly suggest that 17beta-HSD12 is the major estrogenic 17beta-HSD responsible for the conversion of E1 to E2 in women, especially in the ovary, the predominant source of estrogens before menopause.     

Global warming will affect the genetic diversity and uniqueness of Lycaena helle populations

The climate warming of the postglacial has strongly reduced the distribution of cold‐adapted species over most of Central Europe. Such taxa have therefore become extinct over most of the lowlands and shifted to higher altitudes where they have survived to the present day. The lycaenid butterfly Lycaena helle follows this pattern of former widespread distribution and later restriction to mountain areas such as the European middle mountains. We sampled 203 individuals from 10 populations representing six mountain ranges (Pyrenees, Jura, Massif Central, Morvan, Vosges and Ardennes) over the species' western distribution. Allozyme and microsatellite polymorphisms were analysed to study the genetic status of these highly fragmented populations. Both molecular marker systems revealed a strong genetic differentiation among the analysed populations, coinciding with the orographic structure and highly restricted gene flow among them. The large‐scale genetic differentiation is more pronounced in allozymes (F_CT: 0.326) than in microsatellites (R_CT: 0.113), but microsatellites show a higher resolution on the regional scale (R_SC: 0.082) compared with allozymes (F_SC: n.s.). For both analytical tools, we found private alleles occurring exclusively in a single mountain area. The highly fragmented and isolated occurrence of populations is supported by the distribution pattern of potentially suitable climate suggested by species distribution models. Model projections under two climate warming scenarios predict a decline of climatically suitable areas, which will result in the extinction of most of the populations showing unique genetic characteristics.     

Primary production in the deep chlorophyll maximum of the central North Sea

Deep chlorophyll maxima (CM) are commonly observed in the summerstratified North Sea. This feature was studied north of DoggerBank in August and showed high chlorophyll a (Chl a) concentration(     

Synthesis and structure-activity relationships of analogs of EM-652 (acolbifene), a pure selective estrogen receptor modulator. Study of nitrogen substitution

EM-652 (acolbifene) analogs have been synthesized as selective estrogen receptor modulators. Substitution on the nitrogen atom of these 2H-1-benzopyran derivatives has been studied for its influence on antiestrogenic activity. Binding to the rat estrogen receptor, inhibition of estradiol-stimulated proliferation of T-47D breast cancer cells, as well as antiuterotrophic and uterotrophic activities in ovariectomized mice have been evaluated. 2H-1-Benzopyran 1b (EM-343, racemic form of EM-652), which contains a piperidine ring, shows the best pharmacological profile; RBA = 380, IC50 value = 0.110 nM (in T-47D cells), as well as 63% and 84% antiuterotrophic inhibitions at the 7.5 and 75 nmol doses, respectively.