Induction of G protein-coupled estrogen receptor (GPER) and nuclear steroid hormone receptors by gonadotropins in human granulosa cells

Estradiol and progesterone mediate their actions by binding to classical nuclear receptors, estrogen receptor α (ERα) and estrogen receptor β (ERβ) and progesterone receptor A and B (PR-A and PR-B) and the non-classical G protein-coupled estrogen receptor (GPER). Several animal knock-out models have shown the importance of the receptors for growth of the oocyte and ovulation. The aim of our study was to identify GPER in human granulosa cells (GC) for the first time. Moreover, the effect of different doses of gonadotropins on estrogen and progesterone receptors in the human ovary should be investigated as follicle stimulating hormone (FSH) and luteinizing hormone (LH) are also responsible for numerous mechanisms in the ovary like induction of the steroid biosynthesis. Human GC were cultured in vitro and stimulated with different doses of recombinant human FSH or LH. Receptor expression was analyzed by immunocytochemistry and quantitative real-time RT-PCR. GPER could be identified for the first time in human GC. It could be shown that high concentrations of LH increase GPER protein expression. Furthermore FSH and LH increased ERβ, PR-A and PR-B significantly on protein level. These findings were verified for high doses of FSH and LH on mRNA level. ERα was not affected with FSH or LH. We assume that gonadotropins induce GPER, ERβ and PR in luteinized granulosa cells.     

Resveratrol enhances anti-proliferative effect of VACM-1/cul5 in T47D cancer cells

Vasopressin-activated calcium-mobilizing (VACM-1) protein is a cul-5 gene product that forms complexes with a subclass of ubiquitin E3 ligases involved in proteasomal protein degradation. The expression of VACM-1 cDNA in the T47D breast cancer cell line inhibits growth and decreases phosphorylation of mitogen activated protein kinase. Factors that regulate expression or stability of VACM-1 protein have not been identified, however. In our search to identify drugs/substances that may control VACM-1 protein expression, we examined the effects of resveratrol (trans-3,5,4′-trihydroxystilbene), a natural component in the human diet which inhibits tumor initiation and promotion. CMV vector and VACM-1 cDNA stably transfected T47D breast cancer-derived cells were treated with resveratrol and cell growth and VACM-1 protein concentrations were measured. Since the cellular mechanism of resveratrol-dependent inhibition of cell growth also involves the regulation of estrogen receptors, the effect of 17-β−estradiol and resveratrol on ERα levels and on cell growth was examined in control and in VACM-1 cDNA transfected cells. Our results demonstrate that antiproliferative effect of resveratrol observed in the control T47D cancer cells was significantly enhanced in VACM-1 cDNA transfected T47D cells. Western blot results indicated that resveratrol increased VACM-1 protein concentration. Finally, treatment with resveratrol for 24 and 48 h attenuated 17-β−estradiol induced increase in cell growth both in control and in VACM-1 cDNA transfected cells. The effect was significantly higher in the VACM-1 cDNA transfected cells when compared to controls. These results indicate that the antiproliferative effect of resveratrol may involve induction of VACM-1/cul5.     

Predominance of gonadotropin α-subunit resulting from preferential loss of hCGβ production in an established cell line

Summary The BeWo line of trophoblastic cells, maintained in continuous culture since 1966, was employed to investigate the phenomenon of gonadotropin α-subunit predominance that exists in several cell lines. The secretion of complete human chorionic gonadotropin (hCG) relative to α-subunit was compared in several different BeWo sublines, all of which were derived from BeWo stock roller tube colonies. In all of the BeWo sublines, secretion of hCG originally exceeded secretion of α-subunit. With time in culture, however, there was a marked decline in production of hCG/hCGβ, but not in α-subunit. Thus it appears that the production of hCGβ by BeWo choriocarcinoma cells is more labile than the production of the α-subunit.     

Progesterone receptor plays a major antiinflammatory role in human myometrial cells by antagonism of nuclear factor-kappaB activation of cyclooxygenase 2 expression

Spontaneous labor in women and in other mammals is likely mediated by a concerted series of biochemical events that negatively impact the ability of the progesterone receptor (PR) to regulate target genes that maintain myometrial quiescence. In the present study, we tested the hypothesis that progesterone/PR inhibits uterine contractility by blocking nuclear factor kappaB (NF-kappaB) activation and induction of cyclooxygenase-2 (COX-2), a contractile gene that is up-regulated in labor. To uncover mechanisms for regulation of uterine COX-2, immortalized human fundal myometrial cells were treated with IL-1beta +/- progesterone. IL-1beta alone caused a marked up-regulation of COX-2 mRNA, whereas treatment with progesterone suppressed this induction. This was also observed in human breast cancer (T47D) cells. In both cell lines, this inhibitory effect of progesterone was blocked by RU486. Using chromatin immunoprecipitation, we observed that IL-1beta stimulated recruitment of NF-kappaB p65 to both proximal and distal NF-kappaB elements of the COX-2 promoter; these effects were diminished by coincubation with progesterone. The ability of progesterone to inhibit COX-2 expression in myometrial cells was associated with rapid induction of mRNA and protein levels of inhibitor of kappaBalpha, a protein that blocks NF-kappaB transactivation. Furthermore, small interfering RNA-mediated ablation of both PR-A and PR-B isoforms in T47D cells greatly enhanced NF-kappaB activation and COX-2 expression. These effects were observed in the absence of exogenous progesterone, suggesting a ligand-independent action of PR. Based on these findings, we propose that PR may inhibit NF-kappaB activation of COX-2 gene expression and uterine contractility via ligand-dependent and ligand-independent mechanisms.     

Variable responsiveness of hormone-inducible hybrid genes in different cell lines

Altered steroid responsiveness leads to various pathological conditions and is a particular problem for the treatment of cancers arising in steroid-sensitive cells. To develop cellular model systems for the analysis of the molecular mechanisms mediating altered steroid responses, we have analyzed the inducibility of a steroid-responsive promoter in different cell lines. In vitro constructs containing the mouse mammary tumor virus promoter fused to the herpes simplex virus thymidine kinase gene or the bacterial neo gene were transfected into four different cell lines [Rat-2, CHO chinese hamster ovary cells, F9, and T47D). Thymidine kinase+ clones and neo-resistant clones were selected in the presence of dexamethasone (dex) and/or other steroid hormones. We find that the mouse mammary tumor virus promoter activity is completely dependent on the presence of dex in Rat-2 cells but is constitutively active in CHO cells and is inactive in F9 teratocarcinoma cells in the presence and absence of dex. In the human breast cancer cell line T47D, we observe no response to dex but do observe an inducibility by progesterone. Examination of glucocorticoid receptors in these cell lines showed that Rat-2, CHO, and F9 cells contain sufficient receptors to allow a hormonal response, whereas in T47D cells several glucocorticoid binding activities appear to be present. Our results indicate that the presence of receptor in cells is not always sufficient to allow hormonal activation and that, in some cell lines, like CHO, other factors are present that can substitute for an activated steroid hormone receptor complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of estrogen receptor-alpha in cells of the osteoclastic lineage

 Estrogen deficiency at the menopause is associated with an increased rate of bone loss and subsequent risk of skeletal fracture. Whilst cells of the osteoblastic lineage are known to express estrogen receptors, the presence of estrogen receptors in osteoclasts remains controversial. We have examined expression of the classic estrogen receptor, estrogen receptor-alpha (ERα), during osteoclast differentiation. In situ mRNA hybridisation with a digoxygenin-labelled riboprobe to ERα mRNA, together with immunocytochemical analysis using a human ERα-specific monoclonal antibody demonstrated similar findings and confirmed the expression of ERα in chondroblasts and osteoblasts from human fetal bone and mineralising human bone marrow cultures. ERα expression was detected in human bone marrow cultures treated with 1,25(OH)₂D₃ and macrophage colony-stimulating factor and in macrophage cultures treated with 1,25(OH)₂D₃. However, in an in vitro model of human osteoclast formation, no ERα expression was observed in the osteoclasts that developed. The human preosteoclast TCG 51 cell line showed strong expression of ERα in contrast to the low levels observed in the more mature bone resorptive TCG 23 cell line. No expression was detectable in osteoclasts cultured from giant cell tumour of bone (GCTB) tissue or in osteoclasts in Pagetic, GCTB, or hyperparathyroid bone tissues. In conclusion, preosteoclasts express detectable levels of ERα, but osteoclast maturation and bone resorption is associated with loss of ERα expression. This indicates that ERα expression and regulation may play a role in osteoclast formation. Accepted: 4 November 1998     

Regulation of ERα Signaling Pathway in Neuronal HN10 Cells: Role of Protein Acetylation and Hsp90

Estrogen has a variety of neuroprotective effects but the molecular basis of its function is still mainly unclear. Estrogen receptor (ER) signaling is highly dependent on posttranslational modifications and the assembly of coactivator and corepressor complexes. Several proteins involved in ERα signaling have recently been found to be acetylated, including ERα itself and Hsp90, a key chaperone in the functional regulation of ERα. ERα complexes also contain histone deacetylases (HDAC) which repress transactivation. Our purpose was to clarify the role of protein acetylation and Hsp90 function in the ERE-mediated ERα signaling in neuronal HN10 cells. We observed that increasing protein/histone acetylation status with trichostatin A, a potent HDAC inhibitor, increased the 17β-estradiol (E2)-induced transactivation of ERE-driven luciferase in non-transfected cells, and even more extensively in pERα-transfected cells. E2-induced ERE-driven transactivation was blocked by ICI 182.780. Several ER antagonists, such as raloxifene and tamoxifen, were unresponsive. Valproate, an antiepileptic drug which is recently characterized as a HDAC inhibitor, was also able to potentiate the E2-induced ERE-transactivation. Inhibition of the function of Hsp90 chaperone with geldanamycin strongly inhibited the E2-induced ERE-transactivation. Overexpression of SIRT2 protein deacetylase did not inhibit the acetylation-potentiated ERE-driven transactivation indicating that SIRT2 deacetylase is not involved in ERα signaling. Our results reveal that ERα signaling is dependent on protein acetylation and epigenetic regulation.     

Regulation of the human endogenous retroviral Syncytin-1 and cell-cell fusion by the nuclear hormone receptors PPARγ/RXRα in placentogenesis

Cytotrophoblast (CT) cell fusion into a syncytiotrophoblast is obligatory for placentation and mediated by the human endogenous retrovirus (HERV)-W envelope gene Syncytin-1. Abnormal placentation is associated with preeclampsia (PE), HELLP and intrauterine growth restriction (IUGR). In placentogenesis, the MAP-kinase p38α regulates PPARγ/RXRα signaling and target genes, like leptin, resistin, ABCG2, and hCG. The aim of this study was to analyze PPARγ/RXRα signaling and target gene regulation using primary CT cultures, the trophoblastic cell line BeWo and placental tissues from patients with normal and abnormal placentation. CT from four different human control placentae and BeWo cells demonstrated that Syncytin-1, other signaling members and CT cell fusions were regulated with PPARγ/RXRα activators troglitazone and 9-cis retinoic acid, via protein kinase A and p38α inhibition. Significant discordant regulations between CTs and BeWo were found. Two PPARγ/RXRα-response-elements from upstream regulatory elements and the 5'LTR of HERV-W were confirmed with DNA-protein binding assays using nuclear extracts and recombinant PPARγ/RXRα proteins. These promoter elements were validated with luciferase assays in the presence of PPARγ/RXRα modulators. Furthermore, troglitazone or 9-cis retinoic acid treatment of siRNA-PPARγ and siRNA-RXRα transfected BeWo cells proved the requirement of these proteins for Syncytin-1 regulation. Thirty primary abnormal placentae from PE, HELLP and IUGR patients compared to 10 controls showed significant deregulation of leptin RNA and protein, p38α, phospho-p38α, PPARγ, ABCG2, INSL4 and Syncytin-1. Our study characterized PPARγ/RXRα signaling in human CT and cell fusions identifying Syncytin-1 as a new target gene. Based on these results, a disturbed PPARγ/RXRα pathway could contribute to pathological human pregnancies.     

Pituitary estrogen receptor α and dopamine subtype 2 receptor gene expression in transgenic mice with overproduction of heterologous growth hormones

 Pituitary somatotrophs are suppressed in mice transgenic for human (h) or bovine (b) growth hormone (GH) genes fused with metallothionein (MT) or phosphoenolpyruvate carboxykinase (PEPCK) promoters. Previous morphologic studies revealed that lactotrophs are inhibited in hGH transgenic lines probably due to prolactin-like effects of hGH whereas in female bGH transgenics, the lactotrophs are stimulated. In the present study, estrogen receptor (ERα) mRNA was studied by autoradiographic in situ hybridization (ISH), ERα protein by immunocytochemistry, and dopamine subtype 2 receptor (D2R) mRNA by ISH. In MT/ and PEPCK/hGH transgenic mice, silver grains signaling ERα mRNA were significantly decreased compared to controls; the reduction was stronger in males (8.6 and 37%) than in females (4.6 and 11%). The decrease in the number of ERα-immunoreactive nuclei followed the same pattern (13.3 and 6% in males vs 3.2 and 5.2% in females). In MT/hGH mice the D2R mRNA signal was significantly increased in males (6 and 15.4%) and females (16%). In MT/bGH transgenics, ERα mRNA and ERα-immunoreactive nuclei were significantly increased (25 and 6%) only in males; D2R mRNA was more decreased in females (23%) than in males (15%). In conclusion, the opposite changes in ERα and D2R gene expressions are correlated with lactotroph inhibition in hGH transgenic mice and their stimulation in bGH transgenic mice. The changes in ERα expression were stronger in males, whereas those of D2R were more pronounced in females. Accepted: 16 November 1998     

Expression level of ABCG2 in the placenta decreases from the mid stage to the end of gestation

The aim of this study was to elucidate the expression pattern of ABCG2 in the placenta from the mid stage to the end of gestation. rABCG2 expression was investigated in rats on the 14th gestation day (gd) and the 20th gd. Expression of the rABCG2 gene and expression of rABCG2 protein in the placenta were detected on gd 14 by RT-PCR and Western blot analysis respectively. The expression level of rABCG2 on gd 20 was less than that on gd 14. We investigated whether progesterone, secreted from the placenta, regulates the expression of ABCG2 in BeWo cells. Expression levels of the ABCG2 gene and protein in BeWo cells were decreased by progesterone treatment. We conclude that progesterone plays a role in reduction in the expression level of ABCG2 in the placenta with the advance of gestation from the mid stage to the end of gestation.     

Cellular localization of estrogen receptor-alpha (ERα) and -beta (ERβ) mRNA in the boar testis

Boar testes synthesize high amounts of estrogens which are known to stimulate several male sexual functions in a variety of extragonadal target tissues. Possible effects within the testis depend on the existence of the estrogen receptor subtypes α and β (ERα, ERβ). The precise cellular localization of these subtypes within the testis was, so far, based mainly on protein expression studies using different antibodies in several species including boars shows contradictory results. Therefore, we investigated the ERα and ERβ gene expression using RT-PCR of testis homogenates and RT-PCR after UV-single cell microdissection combined with in-situ hybridization of four fertile boars with an average age of 32 weeks. Both ERα and ERβ mRNA were found in testis homogenates. Using in-situ hybridization and UV-single cell microdissection ERα mRNA was present in type A and type B spermatogonia up to mid-pachytene primary spermatocytes in stage V–VIII and stage I of the seminiferous epithelial cycle, but not in other cells. ERβ mRNA was found only in Sertoli cells. Interstitial Leydig cells revealed neither ERα nor ERβ mRNA. The data suggest a direct impact of estrogen in the boar on Sertoli cell function via ERβ and germ cell formation via ERα.     

Estrogen receptor beta yield from baculovirus lytic infection is higher than from stably transformed Sf21 cells

The production of estrogen receptors (ER) in cultured insect cells is advantageous because these cells are relatively easy to culture and they perform post-translation modifications necessary for protein stability and function. There are three options for protein expression in insect cells: transient transfection, lytic baculovirus infection, or transfection followed by selection to create stable cell lines. Stable transfection has been promoted to be advantageous for the production of recombinant proteins because no re-infection is required, which might provide better lot-to-lot reproducibility in protein production. In this paper, we demonstrate that lytic baculovirus infection of Sf21 cells yields approximately tenfold more bioactive ERβ than cells stably transformed with pIZ/V5-His plasmid under OpIE2 promoter. We provide the first evidence that stable expression of recombinant human ERβ decreases the proliferation of Sf21 cells by inhibition of cell replication in a ligand-independent manner. These results mirror findings in breast cancer cells showing that an increase in ERβ expression decreases cell proliferation. We conclude that baculovirus infection of Sf21 cells is better for human ERβ production than stable-transformation of Sf21 cells.     

Diethylstilbestrol increases the density of prolactin cells in male mouse pituitary by inducing proliferation of prolactin cells and transdifferentiation of gonadotropic cells

Diethylstilbestrol (DES) has been implicated in mammalian abnormalities. We examined the effects of DES on follicle-stimulating hormone (FSH), luteinizing hormone (LH), and prolactin (PRL) cells in the pituitaries of male mice treated with various doses of DES for 20 days. DES reduced the density of FSH and LH cells in a dose-dependent manner, but increased that of PRL cells. When the expression of estrogen receptor (ER) α and β was assessed, an induction of ERβ by DES was found predominantly in PRL cells. However, since these effects were abolished in ERα knockout mice, DES appears to act primarily through ERα. When the expression of Ki-67 and Pit-1 in PRL cells was examined at various time-points after DES treatment, some PRL cells became Ki-67 positive at 10–15 days, and Pit-1-positive cells were increased at 5–15 days. Furthermore, some FSH and LH cells became Pit-1 positive, and co-localized with PRL at 5–10 days. Our results indicate that DES increases PRL cells by inducing proliferation of PRL cells and transdifferentiation of FSH/LH cells to PRL cells.     

Cyclin b1 promoter activity and functional cdk1 complex formation in G1 phase of human breast cancer cells.

Overexpression of cyclin B has been detected in various human breast cancer cell lines, breast tumor tissues, and immortalized but nontransformed breast cells. The cause of this overexpression has not been thoroughly investigated, nor is it known if cyclin B protein forms a functional complex with its partner, cdk1, at inappropriate cell cycle periods. In this study we examined the pattern of cyclin B1 promoter activity in three breast cancer cell lines, BT-549, MDA-MB-157, T-47D, and the immortalized breast cell line MCF-10F. Using cells stably transfected with a cyclin B1 promoter-luciferase reporter, luciferase activity was measured throughout the cell cycle in lovastatin synchronized cells and in G1 and S/G2 phases of asynchronized cells by flow cytometry. Results demonstrate that the cyclin B1 promoter activity increases, as expected, during the S/G2 period in all the cell lines. However, some promoter activity can be detected in G1 phase of the different cell line with BT-549 displaying the more altered pattern. Functional cyclin B1-cdk 1 protein complex was detected in G1 phase of BT-549 and T-47D cell lines. These results suggest that in a subset of transformed breast cancer cells altered cyclin B1 promoter activity may contribute to the misexpression of cyclin B protein.