TGFß1 alters androgenic metabolites and hydroxysteroid dehydrogenase enzyme expression in human prostate reactive stromal primary cells: Is steroid metabolism altered by prostate reactive stromal microenvironment?

The inflammatory tissue microenvironment can be an active promoter in preneoplastic cancer lesions. Altered steroid hormone metabolism as induced by the inflammatory microenvironment may contribute to epithelial cancer progression. Dehydroepiandrosterone sulfate (DHEAS) is the most abundant endogenous steroid hormone present in human serum and can be metabolized to DHEA, androgens and/or estrogens in peripheral tissues. We have previously reported that TGFß1-induced reactive prostate stromal cells increase DHEA metabolism to active androgens and alter prostate cancer cell gene expression. While much of the focus on mechanisms of prostate cancer and steroid metabolism is in the epithelial cancer cells, this study focuses on TGFß1-induced effects on DHEA metabolic pathways and enzymes in human prostate stromal cells. In DHEA-treated primary prostate stromal cells, TGFß1 produced time- and dose-dependent increases in metabolism of DHEA to androstenedione and testosterone. Also TGFß1-treated prostate stromal cells exhibited changes in the gene expression of enzymes involved in steroid metabolism including up-regulation of 3ß hydroxysteroid dehydrogenase (HSD), and down-regulation of 17ßHSD5, and 17ßHSD2. These studies suggest that reactive prostate stroma and the inflammatory microenvironment may contribute to altered steroid metabolism and increased intratumoral androgens.

Transforming growth factor ß1 increase of hydroxysteroid dehydrogenase proteins is partly suppressed by red clover isoflavones in human primary prostate cancer-derived stromal cells.

Transforming growth factor ß1 (TGF-ß1) increases dehydro-epiandrosterone (DHEA) metabolism to androgens and prostate-specific antigen (PSA) in a prostate tissue model where stromal (6S) cells and epithelial (LAPC-4) cells are cocultured. Red clover (RC) isoflavones inhibits transforming growth factor (TGF)-ß-induced androgenicity. Mechanisms controlling those activities were explored. Three hydroxysteroid dehydrogenases (HSDs), 3ß-HSD, HSD-17ß1 and HSD-17ß5 involved in metabolizing DHEA to testosterone (TESTO) were investigated. Individual depletion of HSDs in 6S cells significantly reduced TGF-ß1/DHEA-induced PSA in LAPC-4 cells in cocultures. Monomer amounts of 3ß-HSD were similar without or with TGF-ß1 in both cell types but aggregates of 3ß-HSD in 6S cells were much higher than those in LAPC-4 cells and were upregulated by TGFß in 6S cells. Basal and TGF-ß1-treated levels of HSD-17ß1 and HSD-17ß5 in LAPC-4 cells were significantly lower than in 6S cells, whereas levels of HSD-17ß1 but not HSD-17ß5 were TGFß inducible. 6S cell HSD genes expression induced by TGFß or androgen signaling was insignificant to contribute TGF-ß1/DHEA-upregulated protein levels of HSDs. RC decreased TGF-ß1- upregulation of aggregates of 3ß-HSD but not HSD-17ß1. Depletion of TGFß receptors (TGFß Rs) reduced TGF-ß1/DHEA-upregulated HSDs and TESTO. Immunoprecipitation studies demonstrated that TGF-ß1 disrupted associations of TGFß Rs/HSDs aggregates, whereas RC suppressed the dissociations of aggregates of 3ß-HSD but not HSD-17ß1 from the receptors. Given that TGFß Rs are recycled with or without ligand, TGF-ß1-induced disassociation of the HSDs from TGFß Rs may increase stability and activity of the HSDs. These data suggest a pathway connecting overproduction of TGFß with increased PSA in prostate cancer.

Expression and localization of SWAP-70 in human fetomaternal interface and placenta during tubal pregnancy and normal placentation.

SWAP-70 has been demonstrated as a multiple functional signaling protein involved in formation of membrane ruffling induced by signal cascade of tyrosine kinase growth factor receptors. In the present study, the spatial and temporal expression pattern of SWAP-70 on human fetomaternal interface was investigated using specimens collected from tubal and normal pregnancies by in situ hybridization, immunohistochemistry, and Western blotting. Data showed an intense expression of SWAP-70 in trophoblasts at weeks 3-6 of fallopian implantation and at weeks 6-7 of normal pregnancy. The most intense expression was exhibited by those highly motile and invasive extravillous trophoblasts. From gestational week 8 on, the level of SWAP-70 in trophoblasts decreased significantly, and the signal was restricted in villous cytotrophoblast cells. In the in vitro cultured human trophoblast cell line, B6Tert-1, colocalization of SWAP-70 with F-actin was verified. Data in human placenta were similar to what we recently reported on rhesus monkey fetomaternal interface. Our results suggest that SWAP-70 may be involved in regulating migration and invasion of trophoblast cells during the processes of embryonic implantation and placentation in primates.

Gene expression pattern and hormonal regulation of small proline-rich protein 2 family members in the female mouse reproductive system during the estrous cycle and pregnancy.

Small proline-rich proteins (SPRR) are known to construct the cornified cell envelope (CE) in the stratified squamous epithelial cell. Their functions in the simple epithelium such as the uterine epithelium are not clear hitherto. In the present study, the mRNA expression patterns of sprr2 family members in the mouse uterus and vagina during the estrous cycle and pregnancy as well as their regulation by steroids were investigated. Using semi-quantitative RT-PCR, it was revealed that the transcripts of sprr2b, 2e and 2g genes were up-regulated in the proestrous and estrous uteri, and sprr2d was up-regulated only in the estrous uterus. In the vagina, transcription of sprr2a, 2b, 2d, 2e and 2k genes were up-regulated at the metestrous stage. Northern blot analysis demonstrated that the overall expression of sprr2 was highly up-regulated in the estrous uterus and the metestrous vagina. During pregnancy, the sprr2 mRNA in the uterus was sharply repressed from day 3 postcoitus on, and began to be induced around labor time. In situ hybridization showed that the sprr2 transcripts were localized in uterine luminal and glandular epithelial cells as well as vaginal stratified epithelial cells. In ovariectomized mice, the expression of sprr2a, 2d, 2e and 2f genes in the uterus were induced by estrogen, and the effect of estrogen on sprr2d and 2e expression could be partly abolished by progesterone. The data indicate that the sprr2 genes have unique regulation patterns in different reproductive tissues under different physiological conditions, and the encoded proteins might play diverse functions in the female reproductive system.

Molecular cloning and assessment of the immunocontraceptive potential of the zona pellucida subunit 3 from Brandt's vole (Microtus brandti).

A full-length cDNA encoding Brandt's vole (Microtus brandti) zona pellucida glycoprotein subunit 3 (vZP3) was isolated using rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR). The cDNA contains an open reading frame of 1254 nucleotides encoding a polypeptide of 418 amino acid residues. The deduced amino acid sequence of vZP3 revealed high overall homology with hamster (82.1%), mouse (81.3%) and rat (80.6%). A synthetic vZP3 peptide corresponding to amino acid residues 328-343 was conjugated to keyhole limpet hemocyanin (KLH-vZP3(328-343)) and used to immunise female Brandt's voles in order to test the efficacy of this peptide as a contraceptive antigen. High IgG antibody levels to the vZP3(328-343) peptide were present in the sera of female voles immunised with KLH-vZP3(328-343) and these also cross-reacted to the zona pellucida in ovaries of Brandt's vole. The fertility of the KLH-vZP3(328-343) -immunised voles was reduced by 50% compared with controls without evidence of significant ovarian pathology.

Differential regulation of estrogen receptor (ER)alpha and ERbeta in primate mammary gland.

Estrogen, mainly estradiol (E2), and progesterone (P) are essential for the growth and differentiation of the breast, but their roles in breast cancer are highly debated. To understand how E2 and P influence cell proliferation and differentiation, it is essential to know how their receptors are regulated. Because of limited tissue availability, little is known about regulation of the two estrogen receptors (ERalpha and ERbeta) and the two progesterone receptor isoforms (PR-A and PR-B) in the normal human breast. What we know comes from rodent studies, which are not always pertinent for the human breast. We report now on regulation of gonadal hormone receptors during the menstrual cycle, pregnancy, and lactation in rhesus monkey mammary gland and on the relationship of these receptors to proliferation. We found that ERalpha but not ERbeta is down-regulated when E2 levels increase and when cells enter the cell cycle. PR-B but not PR-A is expressed in proliferating cells. Thus under normal conditions, the ratio of ERalpha to ERbeta in the breast depends on plasma concentrations of E2. Elevated expression of ERalpha (as occurs in postmenopausal women) is a normal response to loss of E2 and indicates nonproliferating cells. As selective receptor ligands become available, they will be helpful in delineation of the functions of these receptors.

[Temporal and spatial expression of estrogen activity-related molecules in eutopic endometrium of adenomyosis].

OBJECTIVE: The aim of this study was to demonstrate the temporal and spatial expression of estrogen receptor (ER), 17beta-hydroxysteroid dehydrogenase (17beta-HSD) in uterine endometria and the endometrial-myometrial interface (EMI) of adenomyosis and the effects of estrogen activity-related molecules on the occurrence of adenomyosis. METHODS: Thirty-three cases of normal endometria (and myometrial) and eighteen cases of endometria (and myometrial) with adenomyosis were collected. Immunohistochemical assay was performed to locate the ERalpha, ERbeta, 17beta-HSDI and 17beta-HSDII in endometria and EMI. RESULTS: The ERalpha positive cell number in glandular epithelial cells at the early proliferative phase increased evidently in adenomyosis (90%), while it was 60% in normal endometria. We found ERalpha signal in cytoplasm in glandular epithelial cells of adenomyosis endometria as well as in nucleus. Compared with normal endometrium (early proliferative phase: +; late proliferative phase: ++; late secretory phase: +), eutopic endometrium with adenomyosis exhibited a higher level of 17beta-HSDI (early proliferative phase: ++; late proliferative phase: +++; late secretory phase: ++). The intensity of 17beta-HSDIIin glandular epithelial cells of eutopic endometrium with adenomyosis (early proliferative phase: +++; secretory phase: ++++) was also higher than that of normal endometrium (early proliferative phase: - approximately ++; secretory phase: +++). Higher intensities of ERalpha, ERbeta and 17beta-HSDIand lower intensities of 17beta-HSDII were observed in EMI than in the eutopic endometrium of adenomyosis. CONCLUSIONS: The elevation of ERalpha positive cell number, 17beta-HSDI level as well as the insufficient compensation of 17beta-HSDII in eutopic endometrium with adenomyosis and the change in expression pattern of ERalpha, ERbeta, 17beta-HSDI and 17beta-HSDII in EMI lead to the local enhancement of estrogen effect, which would promote cell proliferation.

Determination of genes involved in the early process of embryonic implantation in rhesus monkey (Macaca mulatta) by suppression subtractive hybridization.

Embryonic implantation is a temporally and spatially restricted process that involves a precise cross talk between the embryo and the receptive maternal endometrium. Underlying the complex changes in the uterus during implantation is the alteration in gene expression pattern, which is not fully understood for the primates. In the present study, suppression subtractive hybridization (SSH) was performed to screen genes that were differentially expressed in the implantation site of the pregnant rhesus monkey, and a subtractive cDNA library was constructed. Furthermore, with dot blot analysis, reverse Northern blot analysis, and semiquantitative reverse transcription-polymerase chain reaction, 76 of 376 clones randomly selected from the library were proven to be differentially expressed in the implantation site. With DNA sequencing and BLAST analysis against the GenBank/EMBL database, it was demonstrated that the cDNA fragments carried by 73 clones shared high homology with 31 human genes. Among them, 15 positive clones represented the S100A10 gene and 10 positive ones corresponded with the secreted frizzled-related protein 4 gene. The other two clones shared homology with one human EST. There was one clone homologous to a human DNA sequence, which indicated that it might be a novel gene. To our knowledge, this is the first report to determine genes involved in the early implantation stage in the rhesus monkey with high throughput technology.

Expression of P450 aromatase and 17beta-hydroxysteroid dehydrogenase type 1 at fetal-maternal interface during tubal pregnancy.

Steroidogenesis in the placenta has been studied widely, but little is known about steroid metabolism in ectopic pregnancy. Previous studies have indicated that trophoblast invasion and placentation in the uterus and the fallopian tube may be controlled by similar mechanisms. As far as 17beta-estradiol (E(2)) production is concerned, it has been well demonstrated that its biosynthesis in the placenta involves the action of P450 aromatase (P450arom) and 17beta-hydroxysteroid dehydrogenase type 1 (17HSD1). The purpose of this study was to characterize the expression pattern of P450arom and 17HSD1 at the fetal-maternal interface, particularly in various trophoblast cells, in tubal pregnancy. Using in situ hybridization, P450arom mRNA was localized in syncytiotrophoblast (ST) cells, which are mainly responsible for hormone production during pregnancy, whereas no signal was detected in villous cytotrophoblast (VCT), column CT and extravillous CT (EVCT) cells. Immunohistochemical assays revealed that 17HSD1 is present in ST cells, a large portion of EVCT cells and 20% of column CT cells. On the other hand, no expression of 17HSD1 was detected in VCT cells. In addition, 17HSD1 was found in epithelial cells of the fallopian tube. Interestingly, the expression level of 17HSD1 in fallopian tube epithelium during tubal pregnancy was significantly higher than that during normal cycle. Our data provide the first evidence that normal and tubal pregnancies possess identical expression of P450arom and 17HSD1 in ST cells and therefore, similar E(2) production in the placenta. Further, the association of 17HSD1 with EVCT cells indicates that 17HSD1 perhaps play a role in trophoblast invasion. Finally, increased expression of 17HSD1 in epithelial cells of fallopian tube may lead to a local E(2) supply sufficient for the maintenance of tubal pregnancy.

Regulation of postnatal lung development and homeostasis by estrogen receptor beta.

Estrogens have well-documented effects on lung development and physiology. However, the classical estrogen receptor alpha (ERalpha) is undetectable in the lung, and this has left many unanswered questions about the mechanism of estrogen action in this organ. Here we show, both in vivo and in vitro, that ERbeta is abundantly expressed and biologically active in the lung. Comparisons of lungs from wild-type mice and mice with an inactivated ERbeta gene (ERbeta(-/-)) revealed decreased numbers of alveoli in adult female ERbeta(-/-) mice and findings suggesting deficient alveolar formation as well as evidence of surfactant accumulation. Platelet-derived growth factor A (PDGF-A) and granulocyte-macrophage colony-stimulating factor (GM-CSF), key regulators of alveolar formation and surfactant homeostasis, respectively, were decreased in lungs of adult female ERbeta(-/-) mice, and direct transcriptional regulation of these genes by ERbeta was demonstrated. This suggests that estrogens act via ERbeta in the lung to modify PDGF-A and GM-CSF expression. These results provide a potential molecular mechanism for the gender differences in alveolar structure observed in the adult lung and establish ERbeta as a previously unknown regulator of postnatal lung development and homeostasis.

Temporal and spatial expression of integrins and their extracellular matrix ligands at the maternal-fetal interface in the rhesus monkey during pregnancy.

The integrin and extracellular matrix protein (ECM)-mediated adhesion and invasion of the receptive maternal uterine endometrium by trophoblasts is a critical event in the complex physiological process of pregnancy. Although the process has been largely characterized in mice, the relevant mechanism in primates remains unclear. We investigated the expression patterns and dynamic alterations of integrin subunits (alpha1, alpha5, alpha6, beta1, and beta4) and their ECM ligands, such as laminin (LN), type IV collagen (Col IV), and fibronectin (FN), at the maternal-fetal interface during Gestational Days 15, 25, 50, and 100 and at full term in 20 pregnant rhesus monkeys. Immunohistochemistry and in situ hybridization revealed that a relatively high expression of integrins occurred in trophoblast cells at Gestational Day 15, with the peak level occurring at Day 25. The expression level decreased from Day 50 to term. Along the invasive pathway, expression levels of integrin alpha1, alpha5, and beta1 subunits were gradually elevated from the proximal to distal column, reaching peak level in the trophoblast shell, but were reduced in those invasive extravillous cytotrophoblast (EVCT) cells in contact with the decidua. Integrin alpha1, alpha5, beta1, and beta4 subunits were also highly expressed in decidual stromal cells and moderately expressed in the maternal epithelium and endothelium. Immunoreactive FN, LN, and Col IV were distributed in EVCT and decidual stromal cells and part of the uterine epithelial and endothelial cells. These data suggest that the correlated expression of integrins and their ECM ligands at the maternal-fetal interface might be involved in regulation of cell proliferation and differentiation and the counterbalanced invasion-accelerating and invasion-restraining processes in trophoblast cells during the early stage of pregnancy.

Divergent effects of retinoic acids on the expression of ERalpha and 17beta-hydroxysteroid dehydrogenase type 2 in endometrial carcinoma cells (RL 95-2).

The effects of E2 are dependent on ERs and local E2 concentration in target cells. Modulation of intracellular E2 concentration involves the action of 17beta-hydroxysteroid dehydrogenase (17HSD) type 2, the enzyme converting E2 to estrone. In the present study, the influence of RAs on the growth of endometrial cancer cell line RL 95-2 as well as the expression of ERs and 17HSD type 2 have been investigated. It was found that RAs repress the growth of RL 95-2 cells, which express all subtypes of RXR and RAR, as examined by RT-PCR. Also, quantitative RT-PCR analysis showed that both ERalpha and ERbeta are present in RL 95-2 cells, and Western blot assay further revealed that ERalpha expression was decreased by all trans-RA treatment. In contrast, RAs induced 17HSD type 2 mRNA expression in a dose- and time-dependent fashion. This stimulatory effect was also detected at the level of in vivo oxidative 17HSD activity in cultured cells. On the other hand, the abundance of 17HSD type 2 mRNA was not altered by RAs in cultured normal epithelial cells isolated from human early- and late-secretory endometrium. The data indicate that RAs have an inhibitory effect on the growth of RL 95-2 cells and a cross-talk with the estrogen pathway in estrogen-responsive endometrial cancer cells.