Progesterone Receptor Membrane Component 1 Expression Research Articles

Sigma-2 receptor and progesterone receptor membrane component 1 (PGRMC1) are two different proteins: Proofs by fluorescent labeling and binding of sigma-2 receptor ligands to PGRMC1

A controversial relationship between sigma-2 and progesterone receptor membrane component 1 (PGRMC1) proteins, both representing promising targets for the therapy and diagnosis of tumors, exists since 2011, when the sigma-2 receptor was reported to be identical to PGRMC1. Because a misidentification of these proteins will lead to biased future research hampering the possible diagnostic and therapeutic exploitation of the two targets, there is the need to solve the debate on their identity. With this aim, we have herein investigated uptake and distribution of structurally different fluorescent sigma-2 receptor ligands by flow cytometry and confocal microscopy in MCF7 cells, where together with intrinsic sigma-2 receptors, PGRMC1 was constitutively present or alternatively silenced or overexpressed. HCT116 cells, with constitutive or silenced PGRMC1, were also studied. These experiments showed that the fluorescent sigma-2 ligands bind to their receptor irrespective of PGRMC1 expression. Furthermore, isothermal titration calorimetry was conducted to examine if DTG and PB28, two structurally distinct nanomolar affinity sigma-2 ligands, bind to purified PGRMC1 proteins that have recently been revealed to form both apo-monomeric and heme-mediated dimeric forms. While no binding to apo-PGRMC1 monomer was detected, a micromolar affinity to heme-mediated dimerized PGRMC1 was demonstrated in DTG but not in PB28. The current data provide evidence that sigma-2 receptor and PGRMC1 are not identical, paving the pathway for future unbiased research in which these two attractive targets are treated as different proteins while the identification of the true sigma-2 protein further needs to be pursued.

Progesterone, Inflammatory Cytokine (TNF-α), and Oxidative Stress (H2O2) Regulate Progesterone Receptor Membrane Component 1 Expression in Fetal Membrane Cells.

Progesterone receptor membrane component 1 (PGRMC1) is an important novel mediator of progesterone (P4) function in fetal membrane cells. We demonstrated previously that PGRMC1 is differentially expressed in fetal membranes among pregnancy subjects and diminished in preterm premature rupture of membrane subjects. In the current study, we aim to elucidate whether PGRMC1 expression is regulated by P4, tumor necrosis factor α (TNF-α), and H2O2 in fetal membrane cells. Primary cultured membrane cells were serum starved for 24 hours followed by treatments of P4, 17 hydroxyprogesterone caproate, and medroxyprogesterone 17 acetate (MPA) at 10(-7) mol/L with ethanol as vehicle control; TNF-α at 10, 20, and 50 ng/mL with phosphate-buffered saline (PBS) as control; and H2O2 at 10 and 100 μmol/L with culture media as control for 24, 48, and 72 hours. The messenger RNA (mRNA) and protein expression of PGRMC1 was quantified using polymerase chain reaction and Western blotting, respectively. We found that PGRMC1 protein expression was regulated by MPA, TNF-α, and H2O2 in a dose-dependent manner. This regulation is also specific to the type of cell (amnion, chorion, or decidua). The upregulation of PGRMC1 by MPA might be mediated through glucocorticoid receptor (GR) demonstrated using amnion and chorion cells model with GR knockdown by specific small interfering RNA transfection. The mRNA expression of PGRMC1 was decreased by H2O2 (100 μmol/L) treatment in amnion cells, which might ultimately result in downregulation of PGRMC1 protein as our data demonstrated. None of other treatments changed PGRMC1 mRNA level in these cells. We conclude that these stimuli act as regulatory factors of PGRMC1 in a cell-specific manner.

Progesterone Receptor Membrane Component-1 (PGRMC1) nucleolar localization in bovine granulosa cells and its putative interaction with nucleolin.

PGRMC1 is a multifunctional protein that is found in multiple subcellular compartments, suggesting a specific function at each site. Among the several subcellular sites of expression, PGRMC1 was found in the nucleolus of human cells (Ahmad et al. 2009) and bovine zygotes (Luciano et al. 2010). However, the role at this site is not clear. Therefore, the aim of this study is to assess whether PGRMC1 modulates nucleolar function. Immunofluorescence experiments confirmed nucleolar localization in rat spontaneously immortalized granulosa cells, bovine granulosa cells (bGC) and bovine oocytes. Moreover, in bGC PGRMC1 co-localizes with nucleolin, a well-known nucleolar marker exerting important functions within the nucleolus. Additionally, siRNA mediated gene knockdown experiments showed that when PGRMC1 expression is silenced, nucleolin localization shifts from the nucleolus to the nucleoplasm, suggesting a PGRMC1/nucleolin functional association. However, in situ proximity ligation assay did not detect a direct interaction between these two proteins, suggesting the involvement of additional molecules that could mediate PGRMC1/nucleolin interaction. In conclusion, these studies suggest a function for PGRMC1 in nucleolar activity and set the stage for further investigations aimed at dissecting PGRMC1’s molecular mechanisms of action in the nuclear compartment.

Characterization of Progesterone Receptor Membrane Component 1 in Peripheral Blood Mononuclear Cells and Plasma during Pregnancy

Objective: Progesterone receptor membrane component 1 (PGRMC1) is a novel membrane progesterone receptor that is highly expressed and actively regulated in gestational tissues. Research suggests that PGRMC1 plays a role in mediating progesterone function in gestational tissues and maintaining pregnancy. It has also been reported that PGRMC1 is detectable in the plasma and peripheral blood mononuclear cells (PBMCs) in patients with cancer and may serve as a biomarker. Therefore, our objective is to identify whether PGRMC1 is present in plasma and PBMCs during pregnancy and thus may be investigated as a candidate biomarker for obstetric pathology including preterm birth. Study design: Whole blood samples were collected from pregnant patients. Plasma and PBMCs were isolated. Western blot and immunoprecipitation- mass spectrometry (IP-MS) were used to investigate the presence of PGRMC1 in plasma. Flow cytometry and Western blot were used to detect PGRMC1 expression in PBMCs collected from pregnant subjects. Results: Flow cytometry and Western blot results confirmed that PGRMC1 is differentially expressed in different cell types of PBMCs. However, Western blot and IP-MS results demonstrated that PGRMC1 is undetectable in plasma samples from pregnant women. Conclusions: This pilot study characterized the presence of PGRMC1 in the bloodstream during pregnancy. PGRMC1 is differentially expressed in different cell types of PBMCs of pregnant subjects but is not detectable in the plasma of these subjects. These data provide a foundation for future evaluation of PGRMC1 in PBMCs as a potential novel biomarker for PTB.

Progesterone Receptor Membrane Component 1 (PGRMC1) Expression in Murine Retina

ABSTRACTPurpose: Sigma receptors 1 (σR1) and 2 (σR2) are thought to be two distinct proteins which share the ability to bind multiple ligands, several of which are common to both receptors. Whether σR1 and σR2 share overlapping biological functions is unknown. Recently, progesterone receptor membrane component 1 (PGRMC1) was shown to contain the putative σR2 binding site. PGRMC1 has not been studied in retina. We hypothesize that biological interactions between σR1 and PGRMC1 will be evidenced by compensatory upregulation of PGRMC1 in σR1−/− mice.Methods: Immunofluorescence, RT-PCR, and immunoblotting methods were used to analyze expression of PGRMC1 in wild-type mouse retina. Tissues from σR1−/− mice were used to investigate whether a biological interaction exists between σR1 and PGRMC1.Results: In the eye, PGRMC1 is expressed in corneal epithelium, lens, ciliary body epithelium, and retina. In retina, PGRMC1 is present in Müller cells and retinal pigment epithelium. This expression pattern is similar, but not identical to σR1. PGRMC1 protein levels in neural retina and eye cup from σR1−/− mice did not differ from wild-type mice. Nonocular tissues, lung, heart, and kidney showed similar Pgrmc1 gene expression in wild-type and σR1−/− mice. In contrast, liver, brain, and intestine showed increased Pgrmc1 gene expression in σR1−/− mice.Conclusion: Despite potential biological overlap, deletion of σR1 did not result in a compensatory change in PGRMC1 protein levels in σR1−/− mouse retina. Increased Pgrmc1 gene expression in organs with high lipid content such as liver, brain, and intestine indicates a possible tissue-specific interaction between σR1 and PGRMC1. The current studies establish the presence of PGRMC1 in retina and lay the foundation for analysis of its biological function.

The PGRMC1 Protein Level Correlates with the Binding Activity of a Sigma-2 Fluorescent Probe (SW120) in Rat Brain Cells.

We examined the progesterone receptor membrane component 1 (PGRMC1) protein expression in rat brain cells as the prerequisite step to understand the biology of PGRMC1 in the central nervous system (CNS). We also performed correlation studies between the PGRMC1 protein level and the binding activity of a sigma-2 fluorescent probe, SW120, in order to explore the possibility of using sigma-2 radiotracer of positron emission tomography (PET) to noninvasively image the CNS. Embryonic primary neurons, astrocytes, oligodendrocytes, and microglia cells were cultured. Immunocytochemistry, Western blot, and SW120 staining were performed in these cells. The protein expression of PGRMC1 determined by immunocytochemistry and SW120 staining is prominent in neurons and relatively low in astrocytes, oligodendrocytes, and microglia cells. The PGRMC1 expression level correlates with the binding activity of SW120 in rat brain cells. The sigma-2 receptor PET radiotracer can be potentially used to noninvasively image neuron/synapse densities in the CNS.

Expression and localization of progesterone receptor membrane component 1 (PGRMC1) in the rhesus macaque cervix

Progestogens can have rapid actions on cervix epithelium to alter mucus secretion (1). PGRMC1 is a member of a group of progesterone binding proteins that may mediate rapid non-classical progesterone (P) effects in target cells (2). PGRMC1 could also mediate the action of P in cells that lack nuclear progesterone receptor (PGR) and represent a novel pharmaceutical target for new contraceptives. Little is known, however, regarding the expression and localization of PGRMC1 in the primate cervix. The goal of this study was to compare the expression of PGR and PGRMC1 in the rhesus macaque cervix during the proliferative and secretory phase of the menstrual cycle.

May progesterone receptor membrane component 1 (PGRMC1) predict the risk of breast cancer?

Objective: Our and other studies have pointed on an important role of progesterone receptor membrane component 1 (PGRMC1) in development of breast cancer, especially in hormone therapy. To investigate if PGRMC1 could be used to predict the risk for getting breast cancer, we assessed in tissues of patients with primary invasive breast cancer, if the expression of PGRMC1 may be associated with the expression of estrogen receptor alpha (ERα), progesterone receptor (PR), and ki67.Methods: Samples from 109 patients with breast cancer between the years 2008 and 2014 were obtained with the patients’ consent. Each sample was evaluated for the ERα, PR, Ki67, and PGRMC1 expression by immunohistochemistry using serial sections from the ame paraffin block comparing malignant tissue to benign tissue.Results: Expression of PGRMC1 is increased in tumor area compared with non-cancerous tissue and positively correlates with ERα expression (OR = 1.42 95%CI 1.06–1.91, p = 0.02). No association was obtained between expression of PGRMC1 and PR or Ki67.Conclusion: It can be suggested that women with breast epithelium highly expressing PGRMC1 and in interaction with ERα may have an increased risk to develop breast cancer, especially when treated with hormone therapy.

Abstract 3791: PGRMC1 expression is significantly up-regulated in triple negative breast cancer in vivo with associated activation of cyclin D1 target genes: Analysis of 1019 invasive breast cancers in the TCGA database

Abstract Background: A major gap in our understanding of cancer biology and treatment is how subpopulations of tumor cells escape chemotherapeutic toxicity. One mechanism of resistance is for a cancer cell to temporarily stop proliferating and enter the G0 or dormant phase of the cell cycle. The sigma-2 receptor (s2R) is expressed at different levels in both proliferating and dormant tumor cells, and as such holds great potential for imaging both proliferation and dormancy for the goal of identifying therapeutic response and resistance. Mach and colleagues have validated s2R imaging compounds as receptor-based biomarkers of tumor proliferation in vitro. s2R is a binding site on progesterone receptor membrane component 1 (PGRMC1), but little is known about its expression in breast cancer. The goal of this study was to explore expression of PGRMC1 in normal breast tissue and breast cancer, as a whole and stratified by tumor subtype to provide more information about the role of s2R in breast cancer. Methods: The TCGA database was queried for invasive breast cancers with available RNA sequencing data (RNA Seq V2 RSEM). In total, data was available from 1019 invasive breast cancers: 738 ER+, 215 ER-, 643 PR+, 307 PR-, 149 HER2+, 508 HER2-, 102 TNBC. PGRMC1 expression was compared between all groups and plotted according to subtype. mRNA expression was tested for correlation with 20,531 genes and 461 genes correlating with PGRMC1 (Pearson r>0.25, p<10−15) within tumor samples were identified. A heatmap of expression levels for the top positively and negatively correlated genes was generated. These were then analyzed for enrichment of pathways, functions, networks, and upstream regulators using Ingenuity Pathway Analysis. The analysis was done using functions from Matlab R2012b. Results: PGRMC1 mRNA expression was significantly upregulated in TNBC compared to both normal tissue (1.3 fold; p = 6 × 10−6) and other breast cancer subtypes (1.33 fold; p = 6 × 10−11). Additionally, direction of changes of target genes for regulators suggested activation of cyclin D1 (CCND1, z = 2.45, p = 8 × 10−5) and Myc (z = 1.95, p = 1 × 10−6), and inhibition of RICTOR (z = -4, p = 5 × 10−5) when PGRMC1 was more expressed. Measurement of mRNA levels of cyclin D1 demonstrated that expression was negatively correlated with PGRMC1, despite target gene activation with an average Pearson r = -0.22. Conclusion: PGRMC1, the target of a putative novel marker of cancer cell proliferation and resistance, is upregulated in TNBC compared to normal breast tissue and other tumor subtypes and this is associated with cyclin D1 target gene activation and RICTOR target gene inhibition. These data demonstrate differential expression of s2R in breast cancer and an association between s2R and key cell cycle markers. Citation Format: Elizabeth S. McDonald, Chenbo Zeng, David A. Mankoff, Andrew Kossenkov, Robert H. Mach. PGRMC1 expression is significantly up-regulated in triple negative breast cancer in vivo with associated activation of cyclin D1 target genes: Analysis of 1019 invasive breast cancers in the TCGA database. [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 3791. doi:10.1158/1538-7445.AM2015-3791

Progesterone Receptor Membrane Component-1 in cell division: its role in bovine granulosa cells mitosis.

The present studies were aimed to assess Progesterone Receptor Membrane Component-1 (PGRMC1) role in regulating bovine granulosa cells (bGC) mitosis. First, we performed immunofluorescence studies on in vitro cultured bGC collected from antral follicles, which showed that PGRMC1 localizes to the spindle apparatus in mitotic cells. Then, to evaluate PGRMC1 effect on cell proliferation we silenced its expression with RNA interference technique (RNAi). Quantitative RT-PCR and immunoblotting confirmed down-regulation of PGRMC1 expression, when compared to CTRL-RNAi treated bGC (p<0.05). After 72h of culture, PGRMC1 silencing determined a lower growth rate (p<0.05) and a higher percentage of cells arrested at G2/M phase as assessed by flowcytometry (p<0.05). Accordingly, live imaging studies revealed more aberrant mitosis and a delayed M-phase in PGRMC1-RNAi treated cells compared to CTRL-RNAi group (p<0.05). These data confirmed that PGRMC1 is directly involved in bGC mitosis and ongoing preliminary studies are aimed to elucidate its putative mechanisms of action. Since PGRMC1 is a membrane protein, we hypothesize its possible involvement in vesicular trafficking and endocytosis, which is in turn an important process to assure proper cell division. To assess this hypothesis, we have preliminarily conducted immunofluorescence and in situ proximity ligation assay experiments that showed PGRMC1 co-localization and direct interaction with clathrin. This is important since clathrin is an essential protein for both endosomes formation, and cell division acting directly on the spindle apparatus. Thus our studies set the stage for analysis aimed to further characterize PGRMC1’s mechanism of action in mitotic cell.

Expression and cellular distribution of estrogen and progesterone receptors and the real-time proliferation of porcine cumulus cells

Although the expression of estrogen and progesterone receptors within porcine ovary and cumulus-oocyte complexes (COCs) is well recognized, still little information is known regarding expression of the progesterone receptor (PGR), PGR membrane component 1 (PGRMC1) and of estrogen-related receptors (ERRγ and ERRβ/γ) in separated cumulus cells in relation to real-time proliferation. In this study, a model of oocytes-separated cumulus cells was used to analyze the cell proliferation index and the expression PGR, PGRMC1 and of ERRγ and ERRβ/γ during 96-h cultivation in vitro using real-time quantitative PCR (qRT-PCR) and confocal microscopic observation. We found that PGR protein expression was increased at 0 h, compared with PGR protein expression after 96 h of culture (P < 0.001). The expression of PGRMC1, ERRγ and ERRβ/γ was unchanged. After using qRT-PCR we did not found statistical differences in expression of PGR, PGRMC1, ERRγ and ERRβ/γ during 96 h of cumulus cells in vitro culture (IVC). We supposed that the differential expression of the PGR protein at 0 h and after 96 h is related to a time-dependent down-regulation, which may activate a negative feedback. The distribution of PGR, PGRMC1 proteins may be linked with the translocation of receptors to the cytoplasm after the membrane binding of respective agonists and intra-cytoplasmic signal transduction. Furthermore, cumulus cells analyzed at 0 h were characterized by decreased proliferation index, whereas those after 96 h of culture revealed a significant increase of proliferation index, which may be associated with differentiation/luteinization of these cells during real-time proliferation.

Hyaluronic acid promotes the expression of progesterone receptor membrane component 1 via epigenetic silencing of miR-139-5p in human and rat granulosa cells.

Primary ovarian insufficiency (POI) is a serious reproductive dysfunction in which the follicle pool is reduced and depleted. Abnormal apoptosis of ovarian granulosa cells (GCs) is believed to result in follicle loss. Progesterone receptor membrane component 1 (PGRMC1), which is critical for GC survival, was reported to be reduced in POI patients, but the mechanism is unknown. In the present study, we found that PGRMC1 expression was correlated with the level of hyaluronic acid (HA) in POI patients. HA up-regulated PGRMC1 expression in GCs via suppression of miR-139-5p, which was proven by Western blotting and luciferase reporter assays to target PGRMC1. Consistent with these findings, levels of miR-139-5p were significantly increased and presented an inverse correlation with PGRMC1 in POI patients. Noticeably, HA inhibited CD44-mediated miR-139-5p expression but had no effect on luciferase activity after insertion of miR-139 promoter into luciferase plasmid. Interestingly, miR-139-5p was significantly up-regulated in KGN cells (GC tumor cell line) by the histone deacetylase inhibitor trichostatin A, indicating that HA down-regulated miR-139-5p expression via histone deacetylation. Taken together, we report an unrecognized mechanism of HA in the promotion of PGRMC1 expression, suggesting that HA may be a potential molecule for the prevention and treatment of POI.

Deciphering the proteomic signature of human endometrial receptivity.

Are there any proteomic differences between receptive (R) and non-receptive (NR) endometrial receptivity array (ERA)-diagnosed endometria obtained on the same day of a hormonal replacement therapy (HRT) treatment cycle? There is a different proteomic signature between R and NR ERA-diagnosed endometrium obtained on the same day of HRT cycles. The human endometrial transcriptome has been extensively investigated in the last decade resulting in the development of a new diagnostic test based on the transcriptomic signature of the window of implantation (WOI). Much less is known about the proteomics derived from the transcripts present during the WOI. This study was a basic proteomic analysis of human endometrial biopsies taken from twelve IVF patients. Human endometrial biopsies were collected during HRT cycles after 5 days of progesterone (P) administration, and diagnosed as receptive (R; n = 6) or non-receptive (NR; n = 6) by the ERA test. Endometrial proteins were extracted, labelled and separated using differential in-gel electrophoresis (DIGE). Proteins were identified using mass spectrometry, followed up by in silico analysis. Validation studies using western blots and immunolocalization were performed for the progesterone receptor membrane component 1 (PGRMC1) and annexin A6 (ANXA6) proteins. DIGE analysis followed by protein identification by MALDI-MS and database searches revealed 24 differentially expressed proteins in R versus NR samples. In silico analysis showed two pathways which were significantly different between R and NR samples. Expression of PGRMC1 and ANXA6 was validated and localized by western blots and immunohistochemistry. These results highlight these proteins as key targets likely to be important in the comprehension of human endometrial receptivity. This was mainly a descriptive study with no functional studies on the proteins found. We also used a low number of human endometrial samples for the DIGE analysis. This study identified the proteomic profile associated with receptive or non-receptive human endometria. Our findings suggest that although histological dating indicates a putative 'receptive' status within the WOI, a different transcriptomic and proteomic profile is observed in these samples. We should move towards using more personalized WOIs, where identification of the correct endometrial receptivity status, and consequently the success of IVF, relies on individual molecular signatures rather than traditional endometrial dating. F.D.'s participation in this work was supported by the Spanish Ministry of Economy and Competitiveness, through the Miguel Servet Programme (CP13/00075) co-founded by FEDER. The project was also supported by a grant from the Spanish Ministry of Economy and Competitiveness, through the FIS Programme (PI12/00450). The authors have no financial/commercial conflicts of interest to declare.

Expression and localization of progesterone receptor membrane component 1 and 2 and serpine mRNA binding protein 1 in the bovine corpus luteum during the estrous cycle and the first trimester of pregnancy

The aim of this study was to evaluate the mRNA and protein expression and the localization of progesterone receptor membrane component 1 (PGRMC1), PGRMC2, and the PGRMC1 partner serpine mRNA binding protein 1 (SERBP1) in the bovine CL on Days 2 to 5, 6 to 10, 11 to 16, and 17 to 20 of the estrous cycle as well as during Weeks 3 to 5, 6 to 8, and 9 to 12 of pregnancy (n = 5–6 per each period). The highest levels of PGRMC1 and PGRMC2 mRNA expression were found on Days 6 to 16 (P < 0.05) and 11 to 16, respectively, of the estrous cycle and during pregnancy (P < 0.001). The level of PGRMC1 protein was the highest (P < 0.05) on Days 11 to 16 of the estrous cycle compared with the other stages of the estrous cycle and pregnancy, whereas PGRMC2 protein expression (P < 0.001) was the highest on Days 17 to 20 and also during pregnancy. The mRNA expression of SERBP1 was increased (P < 0.05) on Days 11 to 16, whereas the level of its protein product was decreased (P < 0.05) on Days 6 to 10 of the estrous cycle and was at its lowest (P < 0.001) on Days 17 to 20. In pregnant cows, the patterns of SERBP1 mRNA and protein expression remained constant and were comparable with those observed during the estrous cycle. Progesterone receptor membrane component 1 and PGRMC2 localized to both large and small luteal cells, whereas SERBP1 was observed mainly in small luteal cells and much less frequently in large luteal cells. All proteins were also localized in the endothelial cells of blood vessels. The data obtained indicate the variable expression of PGRMC1, PGRMC2, and SERBP1 mRNA and protein in the bovine CL and suggest that progesterone may regulate CL function via its membrane receptors during both the estrous cycle and pregnancy.

Progesterone receptor membrane component 1 (PGRMC1) expression in fetal membranes among women with preterm premature rupture of the membranes (PPROM)

PGRMC1 function is implicated in maintaining fetal membrane (FM) integrity. PGRMC1 was detectable primarily in the cytoplasm of FM cells and was actively regulated in FMs and relevant for PGRMC1-mediated progesterone action. By cell type, PGRMC1 expression was higher in amnion and chorion compared with decidua. By clinical phenotype, PGRMC1 expression was higher among preterm-no-labor and term-no-labor subjects compared to PPROM. PGRMC1 expression appears to be diminished in PPROM subjects.

Progesterone Receptor Membrane Component 1 as the Mediator of the Inhibitory Effect of Progestins on Cytokine-Induced Matrix Metalloproteinase 9 Activity In Vitro

Progesterone (P4) and the progestin, 17α-hydroxyprogesterone caproate, are clinically used to prevent preterm births (PTBs); however, their mechanism of action remains unclear. Cytokine-induced matrix metalloproteinase 9 (MMP-9) activity plays a key role in preterm premature rupture of the membranes and PTB. We demonstrated that the primary chorion cells and the HTR8/SVneo cells (cytotrophoblast cell line) do not express the classical progesterone receptor (PGR) but instead a novel progesterone receptor, progesterone receptor membrane component 1 (PGRMC1), whose role remains unclear. Using HTR8/SVneo cells in culture, we further demonstrated that 6 hours pretreatment with medroxyprogesterone acetate (MPA) and dexamethasone (Dex) but not P4 or 17α-hydroxyprogesterone hexanoate significantly attenuated tumor necrosis factor α-induced MMP-9 activity after a 24-hour incubation period. The inhibitory effect of MPA, but not Dex, was attenuated when PGRMC1 expression was successfully reduced by PGRMC1 small interfering RNA. Our findings highlight a possible novel role of PGRMC1 in mediating the effects of MPA and in modulating cytokine-induced MMP-9 activity in cytotrophoblast cells in vitro.

Enhancement of Cell Surface Expression and Receptor Functions of Membrane Progestin Receptor α (mPRα) by Progesterone Receptor Membrane Component 1 (PGRMC1): Evidence for a Role of PGRMC1 as an Adaptor Protein for Steroid Receptors

A variety of functions have been proposed for progesterone receptor membrane component 1 (PGRMC1), including acting as a component of a membrane progestin receptor and as an adaptor protein. Here we show that stable overexpression of human PGRMC1 in nuclear progesterone receptor (PR)-negative breast cancer cell lines causes increased expression of PGRMC1 and membrane progesterone receptor α (mPRα) on cell membranes that is associated with increased specific [(3)H]progesterone binding. The membrane progestin binding affinity and specificity were characteristic of mPRα, with a Kd of 4.7 nM and high affinity for the mPR-specific agonist, Org OD 02-0, and low affinity for corticosteroids. Progestin treatment caused activation of G proteins, further evidence for increased expression of functional mPRs on PGRMC1-transfected cell membranes. Immunocytochemical and coimmunoprecipitation studies showed a close association of PGRMC1 with mPRα in cell membranes. Transfection of PGRMC1 into spontaneously immortalized rat granulosa cells was associated with membrane expression of PGRMC1 and mPRα as well as antiapoptotic effects of progestins that were abolished after cotransfection with small interfering RNA for mPRα. These data demonstrate that PGRMC1 can act as an adaptor protein, transporting mPRα to the cell surface, and that the progestin binding and apoptotic functions previously ascribed to PGRMC1 are dependent on cell surface expression of mPRα. Collectively, the results suggest PGRMC1 and mPRα are components of a membrane progesterone receptor protein complex. Increased expression of estrogen receptor β was also observed in the membranes of PGRMC1-transfected cells, suggesting that PGRMC1 can act as an adaptor protein for multiple classes of steroid receptors.

Medroxyprogesterone acetate-driven increase in breast cancer risk might be mediated via cross-talk with growth factors in the presence of progesterone receptor membrane component-1

BackgroundThe WHI trial suggests an increase of breast cancer in postmenopausal women probably according to the progestogenic compound, i.e. medroxyprogesterone acetate (MPA). However, the mechanism for a possible carcinogenic effect of MPA remains unclear so far. Progesterone receptor membrane component-1 (PGRMC1) may be important in tumorigenesis and thus may increase breast cancer risk. We investigated the influence of MPA alone and in combination with growth factors on breast cancer cells overexpressing PGRMC1. MethodsMCF-7 cells were stably transfected with PGRMC1 expression plasmid (WT-12 cells). Cells transfected only with the vector were used as control cells (EVC-cells). Medroxyprogesterone acetate (MPA), norethisterone (NET) and progesterone (P) were tested alone and in combination with a mixture of growth factors. Cell proliferation was measured by MTT assay. ResultsThe growth factor mixture (GF) was able to induce cell proliferation in both cell types, however, the effect was much higher in the WT-12 cells. In WT-12 cells both MPA and NET alone significantly increased cell proliferation with values of 40% and 97%, respectively. Progesterone, however, had no effect. In combination with GF MPA significantly further enhanced cell proliferation as compared to the effect of MPA alone and GF alone in both cell lines. NET showed no further increase as compared to NET alone and P had no effect. ConclusionsWe could demonstrate a significant proliferative effect of MPA when combined with high concentrations of growth factors. This effect was more pronounced in breast cancer cells overexpressing PGRMC1. These results may be of clinical relevance since in the combined WHI trial an increased breast cancer risk was found during treatment with conjugated equine estrogens plus MPA.

Overexpression of progesterone receptor membrane component 1

Clinical trials have demonstrated an increased risk of breast cancer during estrogen/norethisterone (NET) therapy. With this in mind, the effects of estrogen/NET combination on the proliferation of breast cancer cells overexpressing the progesterone receptor membrane component 1 (PGRMC1) were examined. The same combination was used for the first time in a mouse xenograft model to determine its effects on tumor development. MCF-7 cells were stably transfected with PGRMC1 expression plasmid (WT-12 cells) or empty vector control (pcDNA-3HA). NET, medroxyprogesterone acetate (MPA), and progesterone were tested alone and sequentially and continuously combined with estradiol (E2). Six-week-old nude mice were inoculated with E2 pellets 24 hours before the injection of tumor cells into both flanks (n = 5-6 mice per group). After 8 days, animals were inoculated with a NET pellet or with placebo pellets, and tumor volumes were recorded twice a week. NET alone significantly increased the proliferation of WT-12 cells, MPA was effective only at the two highest concentrations, and progesterone had no effect. The twofold to threefold E2-induced increase (10 M) was not significantly influenced by the addition of the various progestogens. In contrast, 10 M E2 had no effect; however, addition of MPA and NET triggered a significant proliferative response. In vivo, a sequential combination of NET and E2 also significantly increased the tumor growth of WT-12 cells; empty vector cells did not respond to NET. We have demonstrated for the first time that an E2/NET combination increases the proliferation of PGRMC1-overexpressing breast cancer cells, both in vivo and in vitro. Our results suggest that undetected tumor cells overexpressing PGRMC1 may be more likely to develop into frank tumor cells in women undergoing E2/NET hormone therapy.

Plasminogen Activator Inhibitor 1 RNA-Binding Protein Interacts with Progesterone Receptor Membrane Component 1 to Regulate Progesterone's Ability to Maintain the Viability of Spontaneously Immortalized Granulosa Cells and Rat Granulosa Cells1

Progesterone receptor membrane component 1 (PGRMC1) mediates the antiapoptotic action of progesterone (P4). PGRMC1 interacts with plasminogen activator inhibitor 1 RNA-binding protein (PAIRBP1), but the functional significance of this interaction is unknown. To examine the function of PGRMC1-PAIRBP1 interaction, PAIRBP1 was depleted from spontaneously immortalized granulosa cells (SIGCs) and the effects on the expression and localization of PGRMC1 as well as P4's ability to bind to SIGCs and prevent apoptosis was assessed. Depleting PAIRBP1 enhanced cellular (3)H-P4 binding and did not alter the expression or cellular localization of PGRMC1 but attenuated P4's antiapoptotic action. Transfection of a PGRMC1-green fluorescent protein (GFP) peptide mimic, which binds PAIRBP1 as demonstrated by in situ proximity assay, doubled the rate at which SIGCs undergo apoptosis compared to cells transfected with either the empty GFP expression vector or Pairbp1 small interfering RNA. Moreover, P4 did not prevent these cells from undergoing apoptosis. Similar studies conducted with granulosa cells isolated from immature rats also showed that PGRMC1 interacts with PAIRBP1 and that transfection of PGRMC1-GFP peptide mimic accelerates the rate of granulosa cell apoptosis by 4-fold even in the presence of serum and P4. These studies support the concept that the interaction between PAIRBP1-PGRMC1 is an essential component of the mechanism through which P4 inhibits apoptosis. Surprisingly, PGRMC1-PAIRBP1 interaction is not required for P4 binding or the cellular localization of PGRMC1 but rather appears to couple PGRMC1 to downstream components of the P4-PGRMC1 signal transduction pathway.