Form Of Progesterone Receptor Research Articles

THE PROGINS VARIANT OF THE PGR GENE AND PLACENTAL ENDOCRINE FUNCTION AT RISK OF PRETERM LABOR: A PILOT STUDY

Progesterone receptors, which are encoded by the PGR gene, mediate the main physiological effects of progesterone – the hormone that is required for a successful pregnancy. The PROGINS variant is one of the most common variants of the PGR gene. Due to genetic heterogeneity, the presence of inferior forms of progesterone receptors in women can be a clinical problem. In addition to fertility problems, such women may theoretically respond differently to progesterone replacement therapy.Aim of the study to determine the effect of the PROGINS variant of the PGR gene on the hormonal status and results of progesterone deficiency treatment in pregnant women at risk of preterm labor.Material and methods. The main group included 30 pregnant women with progesterone deficiency and the threat of preterm labor, and the comparison group included 30 pregnant women with a normal course of pregnancy. The concentrations of progesterone, estradiol, and placental lactogen were measured in the pregnant women of both groups. Genotyping was assayed by PCR.Results. The genotypic frequencies of the PROGINS variant were not significantlydifferent between the main and comparison groups. Pregnant patients in the main group with the T2T2 genotype after treatment had significantly lower progesterone levels compared to patients with the T1T1 and T1T2 genotypes. Pregnant women in the main group with the T2T2 genotype delivered at a shorter gestational age.Conclusion. The results of our study showed the associations between the PROGINS variant of the PGR gene and the inefficacy of progesterone deficiency treatment in pregnant women at risk of preterm birth and shortened gestation.

How nuclear receptors transition between active and inactive forms: An energetic perspective.

Nuclear receptors regulate transcriptional programs in response to the binding of natural and synthetic ligands. These ligands modulate the receptor by inducing dynamic changes in the ligand binding domain that shift the C-terminal helix (H12) between active and inactive conformations. Despite decades of study, many questions persist regarding the nature of the inactive state and how ligands shift receptors between different states. Here, we use molecular dynamics (MD) simulations to investigate the timescale and energetic landscape of the conformational transition between inactive and active forms of progesterone receptor (PR) bound to a partial agonist. We observe that the microsecond timescale is insufficient to observe any transitions; only at millisecond timescales achieved via accelerated MD simulations do we find the inactive PR switches to the active state. Energetic analysis reveals that both active and inactive PR states represent energy minima separated by a barrier that can be traversed. In contrast, little or no transition is observed between active and inactive states when an agonist or antagonist is bound, confirming that ligand identity plays a key role in defining the energy landscape of nuclear receptor conformations.

Reasons and Mechanisms of Development of Mastopathy (Review of Literature)

This review article examines the relationship between various etiological factors in the development of mastopathy. Multiple exogenous and endogenous risk factors disrupt the normal ratio of sex hormones, causing hyperestrogenism and hyperproliferation of the epithelium in breast tissue, which leads to the development of mastopathy. In the presence of polymorphism of coding genes (ESR1 and PRG), mastopathy also increases the risk of developing breast cancer. Disturbances in the ESR1 receptor gene (Estrogen Receptor 1) lead to changes in the sensitivity of receptors to hormones and even to its loss. According to the literature, polymorphism in the intronic region of the ESR1 gene is associated with the risk of fibrocystic mastopathy. The presence of polymorphism of the PRG (Progesterone Receptor Genes) gene may cause the occurrence of defective forms of PR (Progesterone Receptor) with disruption of the ligand- and hormone-binding zones, which is confirmed by the effects of progesterone in tissues. Thus, progesterone deficiency makes possible excessive proliferation of not only epithelial cells, but also the connective tissue component of the mammary gland. In this case, the expression of the progesterone receptor gene is regulated by the level of estrogen. The association of various combinations of predisposition genes indicates the presence of a specific combination of genotypes for each clinical and pathogenetic variant. In proliferative forms of mastopathy, foci with an excess content of estrogen receptors in the epithelium and high activity of cell proliferation and apoptosis are formed in the breast tissue; there is an increase in the content of alpha-estrogen receptors, a change in the relative content of alpha- and beta-estrogen receptors, A- and B-progesterone receptors; the sensitivity of the epithelium to estradiol increases; a complex of cyclin-dependent kinases, alpha-transforming and epidermal growth factors is activated; the proliferative activity of the epithelium increases; apoptosis processes are disrupted; vascularization of the parenchyma increases. All these pathological mechanisms that increase the risk of breast cancer have not yet been sufficiently studied both at the experimental and clinical level. In the treatment of mastopathy, diet therapy, psychological correction, hormonal drugs, non-hormonal drugs, such as vitamins, minerals, hepatoprotectors, diuretics, enzyme drugs, immunocorrectors, adaptogens, non-steroidal anti-inflammatory drugs, herbal remedies are used. The general disadvantages of most of the remedies used today are attempts to eliminate the causes of mastopathy rather than conducting pathogenetic treatment.

Tumour and cellular distribution of activated forms of PR in breast cancers: a novel immunohistochemical analysis of a large clinical cohort

BackgroundThe progesterone receptor (PR) is expressed by ∼70% of early breast tumours and is implicated in the progression of breast cancer. In cancerous tissues PR may be activated in the absence of a ligand, or when ligand concentrations are very low, resulting in aberrantly activated PR (APR). The presence of APR may indicate that patients with breast cancer are more likely to respond to antiprogestins. The aims of this study were to describe and classify the histological subnuclear morphology of active and inactive PR in archival breast cancer samples.MethodsArchived tumour specimens from 801 women with invasive breast cancer were collected. Tissue samples (n=789) were analysed for PR isoforms A and B (PRA and PRB), Ki67 and estrogen receptors (ERα) status, using immunohistochemistry. Medical records were used to determine human epidermal growth factor 2 (HER2) status, tumour stage and grade.ResultsA total of 79% of tumours stained positive for either PRA or PRB, and of these 25% of PRA-positive and 23% of PRB-positive tumours had PR present in the activated form. APRA was associated with higher tumour grade (p=0.001). APRB was associated with a higher tumour grade (p=0.046) and a trend for a more advanced stage. Patients with PR-positive tumours treated with antiestrogens had better disease-free survival (DFS) than those with PR-negative tumours (p<0.0001). Cumulative progression rate and DFS were similar irrespective of APR status. Both APRA and APRB were independent of HER2, ERα and Ki67 expression.ConclusionsAPR had a binary mode of expression in the breast cancer specimens tested, allowing separation into two tumour subsets. APR is an independent target at the cellular and tumour level and may therefore be a suitable predictive marker for antiprogestins, such as onapristone. Using the described technique, a companion diagnostic is under development to identify APR in solid tumours.

SMC1 promotes epithelial-mesenchymal transition in triple-negative breast cancer through upregulating Brachyury.

Triple-negative breast cancer (TNBC) is a special subtype of breast cancer, which is characterized by the negative form of estrogen receptor (ER), progesterone receptor (PR) and human epithelial growth factor receptor 2 (HER2). TNBC accounts for ~15% of all breast cancer forms, and often leads to high mortality and poor prognosis. Structural maintenance of chromosome 1 (SMC1) is a subunit of the cohesion protein complex. Brachyury is a protein that is encoded by the T gene in humans, which is a transcription factor within the T-box complex of genes. Epithelial-mesenchymal transition (EMT) is a ubiquitous process in the body, and in particular, induces metastasis and the proliferation of cancer cells. In the present study, we found that SMC1 expression in TNBC tissues exceeded its expression in adjacent non-tumor tissues. Similarly, the expression of SMC1 in TNBC cell lines (hs578T and HCC1937) was found to be higher than in MCF10a and MCF7 cells. Subsequently, SMC1 was overexpressed and silenced in hs578T and HCC1937 cells through plasmid and siRNA transfection, respectively. The results showed that the high expression of SMC1 often promoted EMT, accompanied by the enhanced expression of Brachyury. Besides, upregulated expression of Brachyury through plasmid transfection also significantly improved the level of EMT, which further indicated that SMC1 increased EMT in TNBC through the induction of Brachyury expression. Taken together, these results contributed to a better understanding of the pathogenesis of TNBC, which also provided an experimental basis for the prevention, diagnosis and treatment of TNBC.

Abstract 2240: Progesterone receptor isoform ratio regulates the stem cell population in the mouse mammary gland

Abstract The ovarian steroid hormones progesterone and estrogen play vital roles in the development of normal mammary gland and are likewise linked to mammary carcinogenesis via their receptors. Progesterone receptor (PR) is expressed as two isoforms, PR-A and PR-B, both in human and mouse. It has been reported that in human breast tumors the PR-A/PR-B expression ratio is altered. To further study the role of PR isoforms on mammary gland biology, transgenic mice carrying either an additional A form of PR (PR-A) or the B form of PR (PR-B) were generated. Both mice strains present abnormal development in their mammary glands. Overexpression of the A isoform of PR leads to increased side branching and multilayered ducts, while overexpressing the B isoform leads to limited ductal growth. The characteristics of mammary glands from these mice provide direct evidence that an aberration in the mechanisms regulating the differential expression of the two isoforms of PR can have major implications to mammary carcinogenesis and might affect the stem cell population of the gland. Recent studies have shown that a hierarchy of mammary stem/progenitor cells exists among the mammary epithelium in both human and mouse and PR signaling has been shown to play a major role in the maintenance of the stem cell population. So far, however, the differential role of the PR isoforms in the regulation of the stem cell population in the breast has not been established. We propose that the altered ratio of PR A/B might affect the stem cell population expansion and self renewal in the mammary gland. We found that mammary glands of mice overexpressing the B isoform presented a higher percentage of cells positive for stem cell markers (CD24+/CD29+) than those derived from PR-A and WT mice. Furthermore cells derived from PR-B mammary glands had increased mammosphere forming capacity compared to both PR-A and WT. Moreover, when mammosphere cultures were carried out in the presence of 4-OH-tamoxifen 10-8M, the number of mammospheres formed from mammary glands of WT and PR-A mice was reduced compared to control cultures treated with vehicle, whereas there were no differences for cultures derived from mammary glands of PR-B mice. Additionally, upon ovariectomy (OVX), mammary glands from PR-A and WT mice showed a higher percentage of cells positive for stem cell markers by flow cytometry, and increase mammosphere forming capacity than control sham mice, while in mammary glands derived from PR-B OVX mice neither parameters changed. Our results suggest that the B isoform of PR, may play a predominant role in the maintenance of stem cell population of the mammary gland and in the development of hormone resistance given that ovarian regulation affected mammary glands of WT and PR-A mice, but not those derived from PR-B mice. Citation Format: Maria S. Recouvreux, Rocio Sampayo, Marina Simian. Progesterone receptor isoform ratio regulates the stem cell population in the mouse mammary gland. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2240. doi:10.1158/1538-7445.AM2015-2240

Abstract P6-05-13: Tumor and cellular distribution of activated forms of ER and PR in breast cancers

Abstract Background: Hormonal therapy is considered one of the most effective treatment options for patients with estrogen receptor (ER) and/or progesterone receptor (PR) positive breast cancers (BC); however only a 50% objective response rate is observed as 1st line therapy of ER positive (ERpos) tumors (Bonneterre, 2001). An IHC diagnostic test to identify the activated form of the ER and PR has previous been described (ASCO 2013 abst # 592, 593 &amp; 5602). The nuclear morphological of distribution of the ER and PR can be categorized in two patterns, a presumably not-activated D (diffuse pattern) or activated A (aggregated pattern). This classification is correlated with antiestrogen treatment outcome (ASCO 2013, abst # 592), and correlated with antiprogestin activity in vitro (AACR 2013, abst # 1317). Clinical activity of antiprogestins has been previously described (Robertson 1999, Jonat 2002). It is generally accepted that expression of PR is under the transcriptional control of ER, thus targeting ER can potentially suppresses PR expression. However, in breast cancer tissues although both ER and PR are present in the tumor specimen, there is low level of co-expression of ER/PR at the cellular level (ASCO 2013, abstract # 596). The goal of the study is to elucidate whether or not the activated form of ER and PR were coexpressed. Method: 293 ERpos or PRpos BC were obtained from the Oscar-Lambret Cancer Center, Lille, France. Each sample was analyzed with specific antibodies for ERα, PR A or PR B; a subset (53 for PR A and 49 for PR B cases) was analyzed by immunochemical duel staining on the same paraffin tumor section for ERα and PR A or PR B. Tumors were deemed receptor positive if &amp;gt;10% of the tumors cells (tc) were stained. Samples processed for duel staining came from the same dataset and were selected if they had both ERα and PR A and/or PR B present in more than 10% and less than 80% of stained tc. The distribution of ERα, PR A, PR B tc was described. Determination of the activated form of ERα (AERpos) and PR A (APRpos A), PR B (APRpos B) was performed using the previously described IHC technique at 100X magnification. Results: Standard receptor positivity (% of tumors tested) was as follows; ERα 85%, PR A 61% and PR B 65%, either PR A or B 68%. Duel staining receptor positivity (% of tumors) showed for the PR A subset: 72% ER pos, 74% PR A pos, and 9% ER/PR (% of tumors co-expressing); for the PR B subset: ER 60%, PR B 100%, and 13% ER/PR (% of co-expressing); Subnuclear Morphology: 216 cases were ER and PR positive, and of these, 23% were AERpos, 23% were APRpos A, 24%, APRpos B% and 31% APRpos for either A or B. In the 216 case dataset, 18% of the tumors had both AER and APR A or APR B; However, using the duel staining method no cases had more than 10% of the tc co-expressing i.e. AERpos and APRpos, indicating that the co-expression was principally at the tumor level and not at the cellular level. Conclusion: As known, BC commonly express both ER and PR; however the co-expression of ER and PR in malignant cells is uncommon, and this co-expression of ER and PR could be explained by chance. Although AER and APR are expressed within the same tumor, the activated forms of the ER and PR are not co-expressed at the cellular level. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-05-13.

Therapeutic Effects of Progesterone in Animal Models of Neurological Disorders

Substantial evidence supports that progesterone exerts many functions in the central and peripheral nervous system unrelated to its classical role in reproduction. In this review we first discussed progesterone effects following binding to the classical intracellular progesterone receptors A and B and several forms of membrane progesterone receptors, the modulation of intracellular signalling cascades and the interaction of progesterone reduced metabolites with neurotransmitter receptors. We next described our results involving animal models of human neuropathologies to elucidate the protective roles of progesterone. We described: (a) the protective and promyelinating effects of progesterone in experimental spinal cord injury; (b) the progesterone protective effects exerted upon motoneurons in the degenerating spinal cord of Wobbler mouse model of amyotropic lateral sclerosis; (c) the protective and anti-inflammatory effects of progesterone in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis and after lysolecithin demyelination; (d) the progesterone prevention of nociception and neuropathic pain which follow spinal cord injury; and (e) the protective effect of progesterone in experimental ischemic stroke. Whenever available, the molecular mechanisms involved in these progesterone effects were examined. The multiplicity of progesterone beneficial effects has opened new venues of research for neurological disorders. In this way, results obtained in animal models could provide the basis for novel therapeutic strategies and pre-clinical studies.

The role of Estrogen, Progesterone and Aromatase in Human Non-Small-Cell Lung Cancer

Lung cancer is the leading cause of cancer-related deaths in both men and women worldwide. Despite advances in treatment, patients have few effective therapeutic options and survival rates remain low. Emerging evidence suggests that the hormones estrogen and progesterone play a key role in the progression of non-small-cell lung cancer (NSCLC). The aromatase enzyme, which is responsible for a key step in estrogen biosynthesis, elicits higher levels of estrogen in lung tumors as well as in metastases compared with nonmalignant tissues. Thus, aromatase may prove to be a key predictive biomarker for treatment of NSCLC. Epidemiologic and preclinical data show estrogens play a critical role in lung tumor development and progression. Two estrogen receptors, α and β, are expressed in normal and in cancerous lung epithelium, and estrogen promotes gene transcription that stimulates cell proliferation and inhibits cell death. Furthermore, expression of both forms of estrogen receptor, progesterone receptor and aromatase in NSCLC specimens has been correlated with worse clinical outcomes. Combination therapies that include estrogen receptor downregulators and aromatase inhibitors are currently being assessed in Phase I-II clinical trials among patients with advanced NSCLC. Results will help guide future lung cancer management decisions, with a goal of achieving more effective and less toxic treatments for patients.

Estrogen and progesterone receptors have distinct roles in the establishment of the hyperplastic phenotype in PR-A transgenic mice

IntroductionExpression of the A and B forms of progesterone receptor (PR) in an appropriate ratio is critical for mammary development. Mammary glands of PR-A transgenic mice, carrying an additional A form of PR as a transgene, exhibit morphological features associated with the development of mammary tumors. Our objective was to determine the roles of estrogen (E) and progesterone (P) in the genesis of mammary hyperplasias/preneoplasias in PR-A transgenics.MethodsWe subjected PR-A mice to hormonal treatments and analyzed mammary glands for the presence of hyperplasias and used BrdU incorporation to measure proliferation. Quantitative image analysis was carried out to compare levels of latency-associated peptide and transforming growth factor beta 1 (TGFβ1) between PR-A and PR-B transgenics. Basement membrane disruption was examined by immunofluorescence and proteolytic activity by zymography.ResultsThe hyperplastic phenotype of PR-A transgenics is inhibited by ovariectomy, and is reversed by treatment with E + P. Studies using the antiestrogen ICI 182,780 or antiprogestins RU486 or ZK 98,299 show that the increase in proliferation requires signaling through E/estrogen receptor alpha but is not sufficient to give rise to hyperplasias, whereas signaling through P/PR has little impact on proliferation but is essential for the manifestation of hyperplasias. Increased proliferation is correlated with decreased TGFβ1 activation in the PR-A transgenics. Analysis of basement membrane integrity showed loss of laminin-5, collagen III and collagen IV in mammary glands of PR-A mice, which is restored by ovariectomy. Examination of matrix metalloproteases (MMPs) showed that total levels of MMP-2 correlate with the steady-state levels of PR, and that areas of laminin-5 loss coincide with those of activation of MMP-2 in PR-A transgenics. Activation of MMP-2 is dependent on treatment with E and P in ovariectomized wild-type mice, but is achieved only by treatment with P in PR-A mice.ConclusionsThese data establish a link between hormonal response, proliferation, modulation of MMP activity and maintenance of basement membrane integrity that depend on a balance in the expression levels of PR-A and PR-B isoforms. Notably, concomitant increased proliferation, due to inhibition of TGFβ1 activation, and loss of basement membrane integrity, via increased MMP-2 activity, appear to be prerequisites for the PR-A hyperplastic phenotype.

Ovarian stimulation with FSH reduces phosphorylation of gonadotrope progesterone receptor and LH secretion in the rat

Administration of human FSH (hFSH) to cyclic rats during the dioestrous phase attenuates progesterone receptor (PR)-dependent events of the preovulatory LH surge in pro-oestrus. The increased bioactivity of the putative ovarian gonadotropin surge inhibiting/attenuating factor induced by hFSH treatment is not associated with a decrease in PR protein expression, and the possibility of its association at a PR posttranslational effect has been raised. The present experiments aimed to analyse PR phosphorylation status in the gonadotrope of rats with impaired LH secretion induced by in vivo hFSH injection. Two experimental approaches were used. First, incubated pro-oestrous pituitaries from hFSH-injected cycling and oestrogen-treated ovariectomized (OVX) rats were used to analyze the effect of calyculin, an inhibitor of intracellular phosphatases, on PR-dependent LH release, which was measured in the incubation medium by RIA. Second, pituitaries taken from hFSH-injected intact cycling and OVX rats and later incubated with P or GNRH1 were used to assess the phosphorylation rate of gonadotrope. The latter was analysed in formalin-fixed, paraffin-embedded tissue sections by immunohistochemistry using a MAB that recognizes the phosphorylated (p) form of PR at Ser294. Calyculin reduced the ovary-mediated inhibition of hFSH in GNRH1-stimulated LH secretion. In addition, the immunohistochemical expression of pSer294 PR was significantly reduced after ovarian stimulation with hFSH in pituitaries from pro-oestrous rats incubated with P or GNRH1. Altogether, these results suggested that the ovarian-dependent inhibitory effect of FSH injection on the preovulatory LH secretion in the rat may involve an increase in dephosphorylation of PR.

The knockdown of progesterone receptor isoform B (PR-B) promotes proliferation in immortalized endometrial stromal cells

The knockdown of PR-B by siRNA resulted in a significant increase in proliferation in an immortalized endometrial stromal cell line. This suggests that PR-B knockdown might be responsible, at least in part, for increased proliferation and resistance to apoptosis as seen in the eutopic and ectopic endometrium of women with endometriosis.

Distinct Temporal and Spatial Activities of RU486 on Progesterone Receptor Function in Reproductive Organs of Ovariectomized Mice

RU486 is an incomplete progesterone receptor (PR) antagonist due to its partial agonist activity. To investigate the tissue-specific effects of RU486 on PR function in an ovariectomized mouse model, we used the progesterone receptor activity indicator mouse and evaluated the key determinants of progesterone-dependent gene activity: PR, coregulators, and kinases. As might be expected, acute RU486 treatment (6 h after injection) reduced PR transcriptional activity in the uterus, compared with vehicle or progesterone (P4) treatments. Chronic RU486 treatment (3 d) had a distinctly different effect on PR-mediated transcription, elevating PR activity in both the uterus and mammary gland, whereas chronic P4 treatment reduced PR activity in both tissues. This elevated uterine PR activity was associated with increased modified forms of PR and total protein levels of steroid receptor coactivator (SRC)-1 without affecting SRC-2 or SRC-3 protein levels. In addition to increased levels of coactivators, chronic RU486 treatment activated the ERK-1/2 and c-Jun N-terminal kinase signaling pathways in the uterus in a manner comparable with P4 treatment. In contrast to our observations in the uterus, chronic RU486 treatment increased modified forms of PR and the SRC-3 protein levels (but not SRC-1 and SRC-2 levels) in luminal epithelial cells of the mammary gland. Chronic RU486 also activated the c-Jun N-terminal kinase, but not ERK-1/2, signaling pathways in mammary luminal epithelial cells. This report suggests that in comparison with chronic natural hormone (P4), a mixed antagonist/agonist (RU486) induces a distinct temporal and spatial pattern of cellular genetic regulators that accompany ligand-specific gene expression.

Progestins and breast cancer

Progestins exert their progestational activity by binding to the progesterone receptor (form A, the most active and form B, the less active) and may also interact with other steroid receptors (androgen, glucocorticoid, mineralocorticoid, estrogen). They can have important effects in other tissues besides the endometrium, including the breast, liver, bone and brain. The biological responses of progestins cover a very large domain: lipids, carbohydrates, proteins, water and electrolyte regulation, hemostasis, fibrinolysis, and cardiovascular and immmunological systems. At present, more than 200 progestin compounds have been synthesized, but the biological response could be different from one to another depending on their structure, metabolism, receptor affinity, experimental conditions, target tissue or cell line, as well as the biological response considered.There is substantial evidence that mammary cancer tissue contains all the enzymes responsible for the local biosynthesis of estradiol (E2) from circulating precursors. Two principal pathways are implicated in the final steps of E2 formation in breast cancer tissue: the ‘aromatase pathway’, which transforms androgens into estrogens, and the ‘sulfatase pathway’, which converts estrone sulfate (E1S) into estrone (E1) via estrone sulfatase. The final step is the conversion of weak E1 to the potent biologically active E2 via reductive 17β-hydroxysteroid dehydrogenase type 1 activity. It is also well established that steroid sulfotransferases, which convert estrogens into their sulfates, are present in breast cancer tissues.It has been demonstrated that various progestins (e.g. nomegestrol acetate, medrogestone, promegestone) as well as tibolone and their metabolites can block the enzymes involved in E2 bioformation (sulfatase, 17β-hydroxysteroid dehydrogenase) in breast cancer cells. These substances can also stimulate the sulfotransferase activity which converts estrogens into the biologically inactive sulfates. The action of progestins in breast cancer is very controversial; some studies indicate an increase in breast cancer incidence, others show no difference and still others a significant decrease. Progestin action can also be a function of combination with other molecules (e.g. estrogens). In order to clarify and better understand the response of progestins in breast cancer (incidence, mortality), as well as in hormone replacement therapy or endocrine dysfunction, new clinical trials are needed studying other progestins as a function of the dose and period of treatment.

FK506-Binding Protein 52 Is Essential to Uterine Reproductive Physiology Controlled by the Progesterone Receptor A Isoform

FK506-binding protein 52 (FKBP52) is a tetratricopeptide repeat protein that associates with steroid receptors in complexes containing heat shock protein 90. To investigate the role of FKBP52 in steroid-regulated physiology, we generated FKBP52-deficient mice. FKBP52 (-/-) females are sterile due to a complete failure of implantation, a process that requires estrogen (ER) and progesterone receptors (PR). Because the uterus expresses two forms of PR, PR-A and PR-B, we investigated all three receptors as potential targets of FKBP52 action. FKBP52 (-/-) uteri showed a normal growth response to estradiol, and unaltered expression of genes controlled by ER and PR-B. In contrast, FKBP52 (-/-) uteri were neither able to express two PR-A-regulated genes, nor undergo decidualization in response to progesterone, suggesting that FKBP52 specifically regulates PR-A at this organ. Analysis of uterine PR heterocomplexes showed preferential association of FKBP52 with PR-A compared with PR-B. Loss of FKBP52 neither disrupted the PR-A/heat shock protein 90 interaction, nor impaired uterine PR-A hormone-binding function, demonstrating the essential role of FKBP52 in PR-A action to be downstream of the hormone-binding event. Transcription studies in +/+ and -/- mouse embryonic fibroblast cells showed a near-complete loss of PR-A activity at mouse mammary tumor virus and synthetic progesterone response element promoters, although partial reductions of ER and PR-B were also observed. Partial disruptions of ovulation and mammary development were also found in FKBP52 (-/-) females. Taken as a whole, our results show FKBP52 to be an essential regulator of PR-A action in the uterus, while being a nonessential but contributory regulator of steroid receptors in the mammary and ovary. These data may now provide the basis for selective targeting of steroid-regulated physiology through tetratricopeptide repeat proteins.

Differential expression of glucocorticoid receptor in human breast tissues and related neoplasms

Glucocorticoid receptor (GR) is a steroid hormone receptor that has been shown to play important roles in mammary development and differentiation, and has been implicated in breast tumourigenesis, but its precise biological significance in mammary pathophysiology remains unclear. In order to generate a comprehensive expression profile for GR in normal versus neoplastic breast tissues, GR expression was investigated in situ in 400 human breast tissue samples, comprising normal tissue and a range of benign, pre-invasive, and invasive lesions, using immunohistochemical assays. The novel expression of GR in myoepithelium, not observed in luminal epithelium, not only demonstrates expression patterns exclusive to the alpha form of oestrogen receptor and progesterone receptor and suggests distinctive functions between GR and these two important steroid hormone receptors in the breast, but may also indicate unique physiological and perhaps pathological roles for the myoepithelium in mediating the effects of glucocorticoid hormones in the breast. The strong expression of GR in metaplastic carcinomas (94.4%) and malignant phyllodes tumours (92.3%) suggests a pathogenetic role for GR, and implies that targeting GR in these tumours may have potential therapeutic application. However, studies on the roles of GR in mammary carcinogenesis should be interpreted with great caution, based on the lack of GR expression in cancer cells in the great majority (98.2%) of non-metaplastic carcinomas, which has gone unnoticed in previous studies. This marked discrepancy warrants a re-examination of the biological roles of GR in the pathophysiology of breast malignancy. The lack of methylation in the promoter region of the GR gene in all 118 non-metaplastic carcinomas, as demonstrated by methylation-specific PCR and bisulphite DNA sequencing analysis, indicates that methylation is less likely to play a role in the reduction of GR expression in non-metaplastic carcinoma of the breast.

Making Progress on Breast Cancer

For the last two years, scientists across the country have been working together in a highly collaborative effort to uncover the links between exposure to environmental pollutants, puberty, and development of breast cancer. On 10–11 November 2005 the Breast Cancer and the Environment Research Centers (BCERCs), a joint effort of the NIEHS and the National Cancer Institute, came together in Michigan to share what they have accomplished to date. Each center reported on advances made over the past year in their two research components: 1) research on the basic biology of mammary gland development, and 2) epidemiologic studies of how environmental factors affect puberty in girls. The impetus for creation of the centers came from appeals from advocates, who urged the NIH to support a more comprehensive approach to research on the environmental causes of breast cancer. “We wanted a research approach that focused on environmental causes, and we wanted more involvement from the advocates’ perspective,” said Dale Eastman, vice president of the Alamo Breast Cancer Foundation. “The centers represent a great opportunity to conduct transdisciplinary science to explore the problem of breast cancer from many different perspectives, including the valuable perspective of [breast cancer survivor] advocates,” said Robert Hiatt, principal investigator for the BCERC at the University of California, San Francisco, which collaborates with Lawrence Berkeley National Laboratory and serves as the coordinating center for the BCERC network. The other three centers are housed at the Fox Chase Cancer Center in Philadelphia (which collaborates with Mount Sinai School of Medicine in New York and the University of Alabama at Birmingham), the University of Cincinnati in Ohio (which collaborates with Cincinnati Children’s Hospital Medical Center), and Michigan State University in East Lansing. Three important features characterize the BCERC program. First, the four centers work together as a network in which experimental methods are coordinated in order to maximize the pooling and comparison of the data generated. Second, to allow adequate time to track the subjects and collect comprehensive information on the onset and progress of puberty, the BCERCs will be funded for seven years, an unusually long time, given that most NIH grants are funded for a maximum of five years. Third, representatives of breast cancer survivor advocacy organizations are integral members of the centers. The advocate members participate in many aspects of the decision-making processes, collaborate with the Community Outreach and Translation Cores of the centers, and added much to the discussion at the annual meeting.

Trafficking of peripheral blood CD56 bright cells to the decidualizing uterus—new tricks for old dogmas?

CD56 bright lymphocytes become abundant in the human uterus during every menstrual cycle, following the surge in pituitary-derived luteinizing hormone (LH), which initiates final oocyte maturation. While the uterus is host to some CD56 bright cells prior to ovulation, the rapid increase is thought to be due to proliferation of the resident population, accompanied by recruitment of CD56 bright lymphocytes from the circulation. The rapid increase in CD56 bright cells is concurrent with the onset of decidualization, the transformation of uterine stromal cells into secretory decidual cells. Uterine CD56 bright cells proliferate and differentiate to become the predominant lymphocytes of the post-ovulatory uterus. These distinct, tissue-specific natural killer (NK) cells either die prior to menses or increase in number during early pregnancy, and then decline toward the end of the first trimester. Since lymphocytes home to tissues from the circulation, we investigated mechanisms of NK cell traffic over the course of natural menstrual cycles by measuring functional interactions between CD56+ cells from blood and endothelial cells using the Stamper–Woodruff assay of lymphocyte adhesion to frozen tissue sections. While a baseline level of adhesion was maintained throughout the cycle, elevated l-selectin-dependent adhesion of peripheral blood CD56 bright cells occurred during a peri-ovulatory window. However, there were no significant menstrual cycle-induced changes in the transcription of l-selectin, alpha 4 integrin or LFA-1, or in expression of these proteins by NK cells, suggesting that the enhanced adhesion was due to post-translational modifications of these molecules. Quantitative RT-PCR failed to amplify the message for LH receptor or the alpha or beta forms of progesterone or estrogen receptors from blood NK cell subsets. Thus, we conclude that the actions of LH, E 2, and P 4 on NK cells that promote interactions with endothelium and potential uterine homing are indirectly mediated through the responsiveness of other cell types.

Overexpressed progesterone receptor form B inhibit invasive activity suppressing matrix metalloproteinases in endometrial carcinoma cells

In this study, we focused on the influence of progesterone and its receptor in invasion and MMPs on endometrial carcinoma cells. The growth of Ishikawa cells, to which an progesterone receptor form B (PR-B) expressing vector was transfected, was inhibited by progesterone as was the inhibition of the expression of cyclin D1. By invasion assay, in conditions with progesterone, the invasiveness of Ishikawa cells was inhibited as well as the expression of (metalloproteinase) MMP-1, -2, -7 and -9 and Ets-1 decreased. These results suggest that activation of PR-B by progesterone results in tumor suppression by inhibiting cell growth and invasiveness via suppression of the expression of MMPs.

Progesterone Receptor Deficient in Chromatin Binding Has an Altered Cellular State

Our previous work has shown that the progesterone receptor (PR) can exist in two distinct functional states in mammary adenocarcinoma cells. The differences in function included the ability to activate a promoter in organized chromatin, sensitivity to ligand, and ligand-independent activation. To determine whether these functional differences were because of altered cellular processing, we carried out biochemical analyses of the functionally distinct PRs. Although the majority of PR is localized to the nucleus, biochemical partitioning resulted in a loosely bound (cytosolic) fraction, and a tightly bound (nuclear) fraction. In the absence of progestins, the functionally distinct PRs differed significantly in partitioning between the two fractions. To characterize these fractions further, we analyzed interactions of unliganded PR with chaperones by coimmunoprecipitation. We determined that PR in the cytosolic fraction associated with hsp90 and p23. In contrast, PR in the nuclear fraction consisted of complexes containing hsp90, p23, and FKBP51 as well as PR that was dimerized and highly phosphorylated. Hormone treatment significantly reduced the formation of all PR-chaperone complexes. The hsp90 inhibitor, geldanamycin, similarly blocked transcriptional activity of both functionally distinct receptors. However, the two forms of the PR differed in their ability to associate with the mouse mammary tumor virus promoter in organized chromatin. These findings provide new information about the composition and distribution of mature progesterone receptor complexes in mammary adenocarcinoma cells, and suggest that differences in receptor subcellular distribution have a significant impact on their function. These findings also reveal that transiently expressed steroid receptors may not always be processed like their endogenous counterparts.