Influence of progestagens on bone health. Bone changes related to ovulatory disturbances and low progesterone levels

Clinical symptoms of endometriosis, such as pain and infertility, can be described as persistent stressors. Such continuous exposure to stress may severely affect the equilibrium and bidirectional communication of the endocrine and immune system, hereby further aggravating the progression of endometriosis. In the present study, we aimed to tease apart mediators that are involved in the stress response as well as in the progression of endometriosis. Women undergoing diagnostic laparoscopy due to infertility were recruited (n = 69). Within this cohort, early stage of endometriosis were diagnosed in n = 30 and advanced stage of endometriosis in n = 8. Levels of progesterone in serum were determined. Frequency of progesterone receptor (PR) expression on CD56(+) and CD8(+) peritoneal lymphocytes was analysed by flow cytometry. The production of tumour necrosis factor (TNF) and interleukin (IL)-10 by peritoneal leukocytes upon stimulation with the potent stress mediator corticotropin-releasing hormone (CRH) and the progesterone derivative dydrogesterone, or both, were evaluated. Furthermore, the production of progesterone-induced blocking factor (PIBF) by peritoneal leukocytes and the expression of PR in endometriotic tissue were investigated. Levels of progesterone in serum were decreased in women with endometriosis and inversely correlated to pain scores. Furthermore, an increased frequency of CD56(+)PR(+) and CD8(+)PR(+) peritoneal lymphocytes was present in advanced endometriosis. The TNF/IL-10 ratio, reflecting cytokine secretion by peritoneal cells, was higher in cells derived from endometriosis patients and could be further heightened by CRH stimulation, whereas stimulation with dydrogesterone abrogated the CRH-mediated inflammation. Finally, the expression of PIBF by peritoneal leukocytes was increased in endometriosis. Low levels of progesterone in the follicular phase could be responsible for the progression of endometriosis and related pain. Peripheral CRH, increasing upon high psychological stress, might contribute to the peritoneal inflammation present in endometriosis. The therapeutic application of progesterone derivatives, CRH blocking agents as well as improvement of stress coping may disrupt the vicious circle between the chronic peritoneal inflammation and high perception of psychological stress in endometriosis.

In this study we evaluated the effect of manipulating the estrogen and androgen environment of the neonatal male rat on subsequent immunoexpression of sex steroid receptors in the seminal vesicles (SVs) at age 18 days. The aim was to establish to what extent such changes were associated with and predictive of changes in SV structure/composition. Treatments were either diethylstilbestrol (DES; 10, 1, or 0.1 microg/injection), ethinyl estradiol (EE; 10 microg/injection), tamoxifen (2 mg/kg/day), flutamide (50 mg/kg), a gonadotropin-releasing hormone antagonist (GnRHa; 10 mg/kg), genistein (4 mg/kg/day), octylphenol (2 mg/injection), or bisphenol A (0.5 mg/injection). Compared with controls, treatment with DES (10 microg) induced loss of epithelial and stromal androgen receptor (AR) immunoexpression coincident with induction of stromal progesterone receptor (PR) immunoexpression and upregulation of stromal immunoexpression of estrogen receptor-alpha (ERalpha). These changes were associated with gross distortion (increase) of the normal stromal:epithelial tissue proportions in the SVs. DES (1 microg) and EE induced similar but less pronounced changes, and DES (0.1 microg) had no noticeable effect. Tamoxifen and flutamide induced PR and slightly upregulated ERalpha immunoexpression but had only a minor or no effect on AR expression and the stromal:epithelial ratio, though flutamide retarded normal development of the SVs. The latter was also evident in GnRHa-treated males, but otherwise this treatment had no effect on AR and PR immunoexpression. None of the foregoing treatments had any detectable effect on the immunoexpression of ERss in stromal or epithelial cells. The major treatment-induced changes in immunoexpression of AR, PR, and ERalpha and lack of change in ERss were confirmed by Western blots of SV protein extracts. None of the three weak (environmental) estrogens tested caused any detectable change in sex steroid receptor immunoexpression or SV tissue composition. We conclude that treatment-induced loss of AR is a prerequisite for altered stromal:epithelial proportions in the SVs and that such loss is always associated with induction of PR and upregulation of ERalpha; the latter two changes are insufficient on their own to bring about such a change. Nevertheless, induction of PR expression was always associated with altered SV development and is a potentially useful marker because it is not normally expressed in male reproductive tissues.

The prevalence of endometrial cancer (EC) is increasing worldwide. Progestin therapy is effective for both early stage EC patients who require preserving fertility and advanced or recurrent patients. Progestin resistance resulting from downregulation of progesterone receptor (PR) remains a major problem, and its mechanism is currently unclear. It was demonstrated that Sirtuin 1 (SIRT1), forkhead transcription factor 1 (FoxO1) and sterol regulatory element binding protein-1 (SREBP-1) may act as a pathway and play crucial roles in the development of EC in our previous studies. In the present study, we investigated the effect on the development of progestin resistance and the relationship with PR of SIRT1/FoxO1/SREBP-1. A progestin-resistant Ishikawa cell line was established in the stimulation and selection of medroxyprogesterone acetate (MPA), and the resistance was analyzed by MTT assay, flow cytometry, and Transwell invasion assay. qRT-PCR and western blotting were conducted to detect the expression of SIRT1, FoxO1, SREBP-1 and PR. SIRT1 knockdown progestin-resistant cells were established by lentiviral transduction. The new progestin-resistant cell line presented sufficient resistance to MPA in aspects of proliferation, distribution of cell cycle and apoptosis compared with original Ishikawa cells. Besides, the invasion capability of progestin-resistant cells was observably increased. In both protein and mRNA levels, SIRT1 and SREBP-1 were upregulated in progestin-resistant cells, while PR and FoxO1 were downregulated. SIRT1 was knocked down by lentivirus transfection in progestin-resistant cells, resulting in upregulation of PR, FoxO1 and downregulation of SREBP-1, thereby SIRT1 knockdown cells were more sensitive to MPA compared with progestin-resistant cells. SIRT1/FoxO1/SREBP-1 act as a pathway targeting PR and involve in the development of progestin resistance in Ishikawa cells.

Estrogen antagonists are widely used in the treatment of breast cancer, and studies of their mechanism of action may provide clues to an understanding of tumor growth regulation and mechanisms of normal estrogen action. We have used human breast cancer cells in long term culture as an in vitro model to study the roles of estradiol and the antiestrogens, tamoxifen and nafoxidine, on cell growth and progesterone receptor (PgR) induction. Tamoxifen is found to have dual dose-dependent estrogenic/antiestrogenic properties. With 1 micrometer tamoxifen, cell growth and PgR induction are suppressed. These effects are reversed by estradiol. At lower doses (less than 0.1 micrometer), however, tamoxifen is a potent estrogen and rapidly induces (24--48 h) PgR, which increases 4- to 10-fold after 4--6 days and falls if tamoxifen is removed. Induction of PgR by estradiol is weaker but follows a similar time course. Tamoxifen-induced PgR is similar to that induced by estradiol; it sediments at 8S on sucrose density gradients, is a tight binder (R5020 Kd, 1.7 micrometer at 4 C and 0.87 nM at 15 C), and can be translocated to the nucleus by R5020. The dual properties of tamoxifen are not due to metabolic formation of an active antiestrogen from a prohormone precursor. In contrast, the action of the antiestrogen nafoxidine is not biphasic in MCF-7 cells; it does not induce PgR over a wide dose range and at high doses, the compound inhibits cell growth.

Progestin resistance is the main obstacle to successful conservative therapy in young endometrial cancer patients. To investigate the molecular events that lead to progestin resistance and to find a possible way to reverse progestin resistance in endometrial cancer, we established a progestin-resistant Ishikawa cell line by long-term progestin treatment to downregulate progesterone receptor (PR) expression. Both medoxyprogesterone acetate (MPA) and LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, were assayed for their effects on the proliferation of progestin-sensitive and progestin-resistant cancer cells, respectively. The MPA inhibited the PI3K/Akt pathway and suppressed cell proliferation in progestin-sensitive Ishikawa cells, but activated the PI3K/Akt pathway and had no effect on cell proliferation in progestin-resistant Ishikawa cells or HEC-1A cells. Inhibiting the PI3K/Akt pathway by LY294002 upregulated PR expression and diminished cell growth, especially in progestin-resistant endometrial cancer cells. In vivo endometrial cancer xenograft studies in nude mice also showed that inhibiting the PI3K/Akt pathway reversed progestin resistance in endometrial cancer. Our results indicate that activation of the PI3K/Akt pathway by progestin without PR mediation plays an important role in progestin resistance to endometrial cancer cells. In addition, inhibiting the PI3K/Akt pathway might reverse progestin resistance in endometrial cancer.

Although the in vivo effect of estrogen on myometrial differentiation is well documented, estrogen effects on primary myocytes in vitro have been difficult to demonstrate. To construct a stable uterine myocyte system, capable of direct estrogen responsiveness, we used a transformed hamster myometrial cell line. Since these cells expressed a low level of estrogen receptors (ERs), we have stably transfected them with a vector for the human ER. After transfection, ER concentration increased from less than 300 sites per cell to 17,000 +/- 2,000 sites per cell (mean +/- SEM). To test the functional integrity of the transfected receptors, a chloramphenicol acetyltransferase gene linked to an estrogen response element upstream of thymidine kinase promoter was transiently transfected, and the amount of chloramphenicol acetyltransferase activity, an indicator of estrogen responsiveness, was found to increase 20-fold in response to 17 beta-estradiol (1 nM for 48 h). Furthermore, we tested the ability of estrogen to activate endogenous genes by measuring progesterone receptor (PR) induction. PR concentration in the transfected cells was 3,700 +/- 800 and increased 9-fold to 33,000 +/- 6,000 with 17 beta-estradiol (2 nM). This receptor density increase was confirmed by immunoblotting. PR induction was maximal at 16 h, was concentration dependent, and was not elicited by tamoxifen or ICI 164,384. We conclude that transformed hamster myocytes transfected with an ER gene are capable of estrogen-dependent PR expression in vitro and may serve as a useful system to study estrogen effect on myocytes.

Although the in vivo effect of estrogen on myometrial differentiation is well documented, estrogen effects on primary myocytes in vitro have been difficult to demonstrate. To construct a stable uterine myocyte system, capable of direct estrogen responsiveness, we used a transformed hamster myometrial cell line. Since these cells expressed a low level of estrogen receptors (ERs), we have stably transfected them with a vector for the human ER. After transfection, ER concentration increased from less than 300 sites per cell to 17,000 +/- 2,000 sites per cell (mean +/- SEM). To test the functional integrity of the transfected receptors, a chloramphenicol acetyltransferase gene linked to an estrogen response element upstream of thymidine kinase promoter was transiently transfected, and the amount of chloramphenicol acetyltransferase activity, an indicator of estrogen responsiveness, was found to increase 20-fold in response to 17 beta-estradiol (1 nM for 48 h). Furthermore, we tested the ability of estrogen to activate endogenous genes by measuring progesterone receptor (PR) induction. PR concentration in the transfected cells was 3,700 +/- 800 and increased 9-fold to 33,000 +/- 6,000 with 17 beta-estradiol (2 nM). This receptor density increase was confirmed by immunoblotting. PR induction was maximal at 16 h, was concentration dependent, and was not elicited by tamoxifen or ICI 164,384. We conclude that transformed hamster myocytes transfected with an ER gene are capable of estrogen-dependent PR expression in vitro and may serve as a useful system to study estrogen effect on myocytes.

Progestin resistance limits the effectiveness of progestin therapy in endometrial carcinoma for patients who desire to preserve fertility. To investigate the molecular mechanism of progestin resistance in endometrial carcinoma, we performed microarray analysis among Ishikawa and progestin resistant cell IshikawaPR cells. We found that epithelial to mesenchymal transition (EMT) was involved in progestin resistance and dachshund family transcription factor 1 (DACH1) is positively correlated with progesterone receptor (PGR). Knockdown of DACH1 in Ishikawa cell promoted proliferation, metastasis ability, and resistance to progestin. Conversely, overexpression of DACH1 in IshikawaPR cell rendered more sensitive to progestin treatment. Xenograft model assay also had similar results. In addition, our data showed that DACH1 overexpression inhibited EMT and decreased c‐Jun, Notch1 and Hes1expression. Our study demonstrated for the first time that EMT is involved in progestin resistance of EC. The response to progestin could be reserved by DACH1 suppressed EMT through Notch1 pathway via c‐Jun.

Role of estrogen receptor α and β in the induction of progesterone receptors in hypothalamic ventromedial neurons

: Specific extracellular matrix (ECM) proteins and their cellular receptors (integrins) are required for normal mammary gland morphogenesis and differentiation, while their expression is dramatically altered during tumorigenesis. It is not clear how ECM proteins affect breast tumor growth, however. We have examined the effects of five ECM proteins on two estrogen (E)regulated events, proliferation and induction of progesterone receptor (PgR), using estrogen receptor positive (MCF-7, T47D) or negative (MDA MB23 1) breast cancer cells. In the presence of serum, E enhanced proliferation of MCF-7 or T47D cells on collagen I, collagen IV, fibronectin, and vitronectin, but was less effective on laminin. MDA MB23 1 cells were not E-responsive on any ECM protein in the presence or absence of serum. MCF-7 cell PgR induction was enhanced by E on all ECM proteins in the presence or absence of serum, though response on laminin was weaker. In the absence of serum, MCF-7 and T47D cells proliferated in response to E maximally on collagen I, followed by fibronectin or vitronectin. However, the growth factors, ICIF- and ECIF, were required for E-induced proliferation and PgR induction in the absence of serum. These results have demonstrated that laminin can inhibit, while collagen I, fibronectin or vitronectin may enhance E-mediated cell proliferation and PgR induction. Since specific ECM proteins may confer and others inhibit E-responsiveness in breast cancer cells, therapeutic strategies may be designed to alter the ECM or integrin- ECM adhesion in the tumor microenvironment to prevent tumor growth and spreading.

This study attempted to show directly how antiestrogens (tamoxifen and nafoxidine) affect the estrogen receptor (ER) in MCF-7 human breast cancer cells. The mechanisms of estrogen action through the unfilled ERs in both cytoplasm (Rc) and nuclei (Rn) of this cell line were contrasted with the effects of estradiol (E) and antiestrogens on receptor binding nuclear processing reactions and progesterone receptor (PgR) synthesis. Within 5 minutes E bound Rc and translocated it to the nucleus; E also bound Rn directly. Beginning 30 minutes after E and continuing for 3-5 hours a progressive depletion of about 70% of filled nuclear receptors (RnE) occurred without reappearance of unfilled receptor. From that point on the RnE levels stabilized a step coincident with PgR induction. The antiestrogens also bound Rc and Rn as demonstrated by in vitro competition studies and by exchange rates of tritiated E for ER complexed to antiestrogens in whole cells. However the nuclear antiestrogen receptor complexes were aberrantly processed; 3-5 hours after tamoxifen only 30% of nuclear-bound receptors were lost before nuclear receptor levels stabilized whereas nafoxidine-bound nuclear receptors were not processed at all. In spite of the only partial ER processing after tamoxifen PgR was induced as long as lower steady-state ER levels were maintained. When tamoxifen was removed total cell ERs returned to control levels as Rc were restored. Simultaneously PgR falls. Rc binding and translocation alone however are not sufficient for PgR induction since if nuclear processing fails PgR induction is prevented (as is the case with nafoxidine). These data suggest that nuclear processing steps may be integral to the function of estrogenic compounds as inducers of PgR.

MCF-7 cells of human breast cancer origin responded to estradiol (E) treatment with increased levels of progesterone receptor (PgR) showing that human breast cells which have undergone malignant changes can continue to synthesize a specific protein under hormone control. These results were obtained by studying the effects of addition and withdrawal of E on PgR synthesis and correlating this with estrogen receptor (ER) binding translocation nuclear processing reactions and restoration of unoccupied ER. In cells treated 1-5 days with E (.001-100 nM) basal PgR levels (.3-.7 pmol/mg of deoxyribonucleic acid) increased 3-6 fold. PgR response was dose-dependent with .1 nM E (a physiologic dose) the maximal effective dose. Growth and induction of PgR by .1 nM E was suppressed by an antiestrogen Tamoxifen at 10000-fold molar excess (1 mcgM) but was reversed by supraphysiological E (10 nM) which reduced the molar excess of Tamoxifen to only 100-fold. In this cell line ER unoccupied by hormone was not restricted to the cytoplasm (Rc) since a portion of the cellular receptor can also be found in the nucleus in unoccupied form (RN). The level of PgR induction correlated with the extent of Rc binding and translocation and also with the extent of Rn binding. At .1 nM E 85% of Rc and Rn were depleted and PgR was maximally stimulated. The levels of PgR also paralleled processing of bound ER (RnE) appearing in the nucleus. Processing during which a new steady-state level of RnE was achieved appeared to be a saturable event. At low E doses despite Rc and Rn binding of E RnE failed to accumulate and total ER decreased. The loss approached 70% at .1 nM E. But at higher E doses some nuclear receptor remained unprocessed and RnE levels increased. The processing effect was rapid beginning within 10 minutes of E addition and completed by 5 hours. Processed RnE levels were seen before PgR induction and during maintenance of induced PgR states. In estrogen withdrawn cells PgR fell in parallel with end of processing and restoration of Rc and Rn.

The purpose of the present studies was to investigate the role of epidermal growth factor (EGF) in the acquisition of estrogen (E) and progestin (P) responsiveness in the mouse mammary gland in vivo. Using the Elvax 40P implant technique to introduce bioactive molecules directly into the mammary gland to produce a localized effect, we have made the novel observation that EGF implanted into glands of pubertal mice followed by E treatment resulted in the precocious acquisition of E-inducible progesterone receptors (PR). In sexually mature mice, EGF implants alone were able to increase PR. A neutralizing antibody specific for EGF blocked E-dependent stimulation of end-bud development and PR induction. Furthermore, the antiestrogen ICI 182,780 blocked the EGF-induced stimulation end-buds and PR induction, indicating that these EGF effects are mediated via estrogen receptors (ER). Immunohistochemical analysis showed that the endogenous EGF content of mammary glands of mature mice was higher than pubertal mice, that E implants caused a localized increase in mammary gland EGF content in both pubertal and mature mice, and that in mature mice E caused an increase in stromal cell EGF content. We have previously shown that the acquisition of E-inducible PR can be modulated by mammary stroma, and the present results indicate that mammary stroma could modulate hormonal responsiveness through control of local growth factor concentration. Taken together, these results provide evidence that E-dependent responses of mouse mammary gland in vivo, such as end-bud proliferation and PR regulation, may be mediated by EGF through an ER-dependent mechanism.

Bone and soft tissue changes associated with a removable partial denture. A novel method with a fusion of CBCT and optical 3D images

An antibody (IgG-RB) against the chick oviduct progesterone receptor (PR) was used to study the regulation of PR by estrogen in chicken pituitary and hypothalamus. PR was revealed exclusively in cell nuclei, whatever the hormonal state, including the absence of progesterone. In immature untreated chickens, PR was not detected in the pars distalis, while the pars tuberalis, preoptic area, and infundibular region of hypothalamus showed IgG-RB-immunoreactive cells or neurons. Estrogenic stimulation induced the appearance of PR in cells of the pars distalis and increased the immunoreactivity in the hypothalamus of young chickens of both sexes. After hormonal withdrawal, PR immunostaining returned to the level in untreated immature animals. In young hens, before they laid the first egg, PR appears progressively in pars distalis cells during the pubertal period. Antibodies to pituitary hormones (LH, FSH, GH, PRL, and ACTH) were used to characterize the secretory properties of PR-containing cells. LH- and FSH-immunoreactive cells were localized throughout the pars distalis in untreated animals. After estradiol treatment of young sexually immature chickens, immunostaining of LH and FSH was strongly reduced, up to extinction, in many gonadotropic cells, and only few PR-containing cells demonstrated some immunoreactivity in the cytoplasm. In contrast, in juvenile hens, a majority of PR-containing cells were identified as LH immunoreactive, that is gonadotrophs. There are probably two reasons for the paucity of doubly reactive cells in estradiol-treated chickens. One is technical, the optimal fixation time for both LH (greater than 20 h) and PR (less than 7 h) makes it practically impossible to reveal the hormone and the receptor at the same time. The other is related to the physiology of the system, which involves the simultaneous decrease in LH immunoreactivity and the PR induction in gonadotrophs by estradiol. The presence of PR in gonadotrophs suggests a direct feedback mechanism of sex steroids on pituitary cells in addition to the indirect effect through GnRH modulation. The hormonal content of PRL-, GH-, and ACTH-producing cells was not modified by estradiol treatment, as judged by the appropriate immunoreactivity, and their nuclei did not display PR. However, PRL cells and PR-containing cells were frequently present near each other within the same cell cords.