Progesterone Receptor Membrane Component 1 Protein Research Articles

Computational Identification and Validation of Non-Synonymous SNPs in Progesterone Receptor Membrane Complex 1 Linked to Lung Cancer

Numerous gene polymorphisms have been attributed to Lung cancer, but PGRMC1 (Progesterone receptor membrane component 1) is a lesser-known candidate among them. However, emerging research is slowly suggesting the role of polymorphisms in PGRMC1 gene-associated tumorigenesis. Nevertheless, phenotypic changes still need to be studied. The main aim of this study is to identify the most deleterious nsSNPs (non-synonymous single nucleotide polymorphisms) in PGRMC1 that can potentially increase the susceptibility to lung cancer progression. In this work, we scrutinized highly detrimental nsSNPs for PGRMC1 from the available dbSNP database. We further categorized using the FATHMM server to enlist the nsSNPs, which are driver mutants capable of affecting the function of the PGRMC1 protein. We employed clinical evidence from the COSMIC database for further evaluation and confirming the presence of nsSNPs in lung cancer patients. There are 12 nsSNPs reported in lung cancer patients, which are L32M, R47C, D141N, G20W, S57C, R70P, I89V, G118V, A191D, G95V, E157K, and G168V predicted to damage the functions. Conclusively, through a comprehensive comparison of the outcomes obtained through these computational methods, we identified novel I89V, D120E, G95V and G168V nsSNPs that pose substantial risks to the functionality of the PGRMC1 protein. Focusing on the importance of PGRMC1 in lung cancer, analyzing its function was conceded to unveil the interlink between genetic mutation and phenotypic changes. Thus, this study provides insights into the influence of PGRMC1variants in Lung cancer. The evaluated nsSNPs can significantly aid future research on the gene and its association with lung cancer progression in large population distribution frequencies of genotypes among different subgroups.

Expression of PGRMC1 in patients with polycystic ovary syndrome and its molecular mechanism for regulating ovarian granulosa cell apoptosis and glucolipid metabolism.

Polycystic ovary syndrome (PCOS) is one of the most common endocrine diseases in women with reproductive age, which is associated with hyperandrogenism, insulin resistance, and ovulatory dysfunction. Progesterone receptor membrane component 1 (PGRMC1) can mediate progesterone to inhibit the apoptosis of ovarian granulosa cells and the growth of follicles, and to induce glucolipid metabolism disorder in ovarian granulosa cells, which is closely related to the occurrence and development of PCOS. This study aims to determine the expression of PGRMC1 in serum, ovarian tissue, ovarian granulosa cells, and follicular fluid in PCOS patients and non-PCOS patients, analyze the value of PGRMC1 in diagnosis and prognosis evaluation of PCOS, and investigate its molecular mechanism on ovarian granulosa cell apoptosis and glucolipid metabolism. A total of 123 patients were collected from the Department of Obstetrics and Gynecology in Guangdong Women and Children Hospital (hereinafter referred to as "our hospital") from August 2021 to March 2022 and divided into 3 groups: a PCOS pre-treatment group (n=42), a PCOS treatment group (n=36), and a control group (n=45). The level of PGRMC1 in serum was detected by enzyme linked immunosorbent assay (ELISA). The diagnostic and prognostic value of PGRMC1 was evaluated in patients with PCOS by receiver operating characteristic (ROC) curve. Sixty patients who underwent a laparoscopic surgery from the Department of Obstetrics and Gynecology in our hospital from January 2014 to December 2016 were collected and divided into a PCOS group and a control group (n=30). The expression and distribution of PGRMC1 protein in ovarian tissues were detected by immunohistochemical staining. Twenty-two patients were collected from Reproductive Medicine Center in our hospital from December 2020 to March 2021, and they divided into a PCOS group and a control group (n=11). ELISA was used to detect the level of PGRMC1 in follicular fluid; real-time RT-PCR was used to detect the expression level of PGRMC1 mRNA in ovarian granulosa cells. Human ovarian granular cell line KGN cells were divided into a scrambled group which was transfected with small interfering RNA (siRNA) without interference and a siPGRMC1 group which was transfected with specific siRNA targeting PGRMC1. The apoptotic rate of KGN cells was detected by flow cytometry. The mRNA expression levels of PGRMC1, insulin receptor (INSR), glucose transporter 4 (GLUT4), very low density lipoprotein receptor (VLDLR), and low density lipoprotein receptor (LDLR) were determined by real-time RT-PCR. The serum level of PGRMC1 in the PCOS pre-treatment group was significantly higher than that in the control group (P<0.001), and the serum level of PGRMC1 in the PCOS treatment group was significantly lower than that in the PCOS pre-treatment group (P<0.001). The areas under curve (AUC) of PGRMC1 for the diagnosing and prognosis evaluation of PCOS were 0.923 and 0.893, respectively, and the cut-off values were 620.32 and 814.70 pg/mL, respectively. The positive staining was observed on both ovarian granulosa cells and ovarian stroma, which the staining was deepest in the ovarian granulosa cells. The average optical density of PGRMC1 in the PCOS group was significantly increased in ovarian tissue and ovarian granulosa cells than that in the control group (both P<0.05). Compared with the control group, the PGRMC1 expression levels in ovarian granulosa cells and follicular fluid in the PCOS group were significantly up-regulated (P<0.001 and P<0.01, respectively). Compared with the scrambled group, the apoptotic rate of ovarian granulosa cells was significantly increased in the siPGRMC1 group (P<0.01), the mRNA expression levels of PGRMC1 and INSR in the siPGRMC1 group were significantly down-regulated (P<0.001 and P<0.05, respectively), and the mRNA expression levels of GLUT4, VLDLR and LDLR were significantly up-regulated (all P<0.05). Serum level of PGRMC1 is increased in PCOS patients, and decreased after standard treatment. PGRMC1 could be used as molecular marker for diagnosis and prognosis evaluation of PCOS. PGRMC1 mainly localizes in ovarian granulosa cells and might play a key role in regulating ovarian granulosa cell apoptosis and glycolipid metabolism.

The role of progesterone and the progesterone receptor in cancer: progress in the last 5 years

ABSTRACT Introduction Patients with various advanced cancers devoid of nuclear progesterone receptors (nPR) have demonstrated increased quality and length of life when treated with the PR modulator mifepristone, which likely works by interacting with membrane PRs (mPR). Areas covered Two immunomodulatory proteins are discussed that seem to play a role in cancers that proliferate whether the malignant tumor is positive or negative for the nPR. These two proteins are the progesterone receptor membrane component-1 (PGRMC-1) and the progesterone-induced blocking factor (PIBF). Both PGRMC-1 and the parent form of PIBF foster increased tumor aggressiveness, whereas splice variants of the 90 kDa form of PIBF inhibit immune response against cancer cells. Expert opinion The marked clinical improvement following 200–300 mg of mifepristone is likely related to blocking PIBF. In the low dosage used, mifepristone likely acts as an agonist for PGRMC-1 protein. Mifepristone may be less effective for cancers positive for the nPR because the nPR may be protective and blocking it may have detrimental effects. Based on this hypothetical model, the development of other potential treatment options to provide even greater efficacy for treating cancer are discussed.

New Insights as to Why Progesterone Receptor Modulators, such as Mifepristone, Seem to Be More Effective in Treating Cancers that Are Devoid of the Classical Nuclear Progesterone Receptor.

Mifepristone treatment for advanced cancer has demonstrated considerable improvement in both length and quality of life in patients who no longer have any other treatment options. The target is the progesterone induced blocking factor (PIBF), which helps the tumor to invade the normal tissue and proliferate and suppress cellular immunity. Most of the benefit has been observed in cancers not associated with the classical nuclear progesterone receptor (nPR). There are data showing that the presence of a nPR may be associated with a better prognosis. Membrane PRs seem to be responsible for PIBF secretion. Mifepristone, possibly fails to block another P associated protein that enables the tumor to proliferate, e.g., the progesterone receptor membrane component-1 (PGRMC-1) protein. One hypothesis is that the nPR helps to inhibit tumor production of PGRMC-1 protein. Thus, mifepristone may inhibit tumor spread by suppressing PIBF, but this may be negated by blocking the nPR, allowing PGRMC-1 levels to increase.

Loss of β-Arrestins or six Gα proteins in HEK293 cells caused Warburg effect and prevented progesterone-induced rapid proteasomal degradation of progesterone receptor membrane component 1

Hormonal dysregulation plays a significant role in the metabolic switching during malignant transformation. Progesterone Receptor Membrane Component 1 (PGRMC1) is a single-pass transmembrane receptor activated by the binding of progesterone (P4), a sex hormone. In a previous study, P4 treatment caused rapid (within 30 min) induction of aerobic glycolysis in transformed HEK293 cells, a hallmark malignant phenotype known as the Warburg effect. This metabolic reprogramming was associated with the proteasomal degradation of a 70 kilodalton (kDa) PGRMC1. PGRMC1 interacts with a variety of proteins, including G protein-coupled receptors (GPCRs) and P4-PGRMC1 signaling modulates cyclic adenosine monophosphate (cAMP) production. Therefore, we hypothesized that the P4-induced Warburg effect and proteasomal degradation of PGRMC1 involve G proteins and β-Arrestins (ARRBs). In the present study, we investigated P4-induced aerobic glycolysis, proteasomal degradation of p70 PGRMC1, as well as abundance and subcellular translocation of PGRMC1 along with two key glycolytic enzymes Hexokinase 1 (HK1) and Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) in six Gα subunit (Gsix) proteins or ARRB1/2-deficient HEK293 cells. Loss of ARRB1/2 or Gsix proteins inhibited P4-induced p70 PGRMC1 degradation but failed to prevent the P4-induced Warburg effect. Also, deficiency of ARRB1/2 or Gsix proteins differentially affected the basal as well as P4-induced abundance and subcellular translocation of PGRMC1, HK1, and GAPDH proteins. Overall, the findings indicate that P4-PGRMC1-mediated metabolic reprogramming in HEK293 cells depends on β-Arrestins and Gα proteins suggesting the involvement of an underlying GPCR signal transduction pathway.

Crosstalk between progesterone receptor membrane component 1 and estrogen receptor α promotes breast cancer cell proliferation

Progesterone (P4) and estradiol (E2) have been shown to stimulate and regulate breast cancer proliferation via classical nuclear receptor signaling through progesterone receptor (PR) and estrogen receptor α (ERα), respectively. However, the basis of communication between PR/ERα and membrane receptors remains largely unknown. Here, we aim to identify classical and nonclassical endocrine signaling mechanisms that can alter cell proliferation through a possible crosstalk between PR, ERα, and progesterone receptor membrane component 1 (PGRMC1), a membrane receptor frequently observed in breast cancer cells. While P4 and E2 treatment increased cell proliferation of ER+/PR+/PGRMC1 overexpressing breast cancer cells, silencing ERα and PR or treatment with selective estrogen receptor modulator (SERM) tamoxifen, or (PR-antagonist) RU-486 decreased cell proliferation. All four treatments rapidly altered PGRMC1 mRNA levels and protein expression. Furthermore, P4 and E2 treatments rapidly activated EGFR a known interacting partner of PGRMC1 and its downstream signaling. Interestingly, downregulation of ERα by tamoxifen and ERα silencing decreased the expression levels of PGRMC1 with no repercussions to PR expression. Strikingly PGRMC1 silencing decreased ERα expression irrespective of PR. METABRIC and TCGA datasets further demonstrated that PGRMC1 expression was comparable to that of ERα in Luminal A and B breast cancers. Targeting of PR, ERα, and PGRMC1 confirmed that a crosstalk between classical and nonclassical signaling mechanisms exists in ER+ breast cancer cells that could enhance the growth of ER+/PR+/PGRMC1 overexpressing tumors.

Abstract P6-05-08: Progesterone receptor membrane component 1 controls cellular proliferation and plays a key role in the molecular circuitry of both ER positive and triple negative breast cancers

Abstract Introduction: Increased expression of progesterone receptor membrane component 1 (PGRMC1), has been linked to breast cancer cell proliferation and tumor growth via epidermal growth factor receptor (EGFR) dependent interaction. As estrogen receptor positive breast cancers receive endocrine therapy and triple negative breast cancers (TNBC) rely on chemotherapeutic agents and or small molecule inhibitors. PGRMC1 could be a potential therapeutic target for both estrogen receptor positive and TNBCs, however its mechanism of action remains elusive. We, aim to understand the signaling mechanism behind PGRMC1 and its potential as viable target for the treatment of breast cancers. Materials and Methods: A panel of non-malignant and malignant breast cell lines were cultured and screened for PGRMC1 expression. PGRMC1 overexpressing breast cancer cell lines were treated with AG-205 (PGRMC1 inhibitor) and PGRMC1 targeting siRNAs. MTS, qRT-PCR, Western blot, immunofluorescence, flow cytometry assays and phospho explorer antibody array analysis were performed. Results: Increased PGRMC1 mRNA and protein levels were observed in estrogen receptor positive ZR-75-1 and TNBC MDA-MB-468 cells, these results were validated and compared to online RNA-seq based gene expression analysis of breast cell lines and breast tumor data sets. Data from online databases also demonstrated PGRMC1 overexpression in multiple breast cancer subtypes (Luminal, Basal B and Basal A). Immunohistochemistry demonstrated strong staining for PGRMC1 in human breast cancer tissue compared to normal tissue. Both AG-205 treatment and PGRMC1 silencing decreased cell proliferation, induced cell cycle arrest at the G0/G1 phase, promoted apoptosis and reduced the capability of the cells to migrate and invade. Phospho-specific antibody array data demonstrated overall downregulation of the EGFR/PI3K/AKT signaling mechanisms following AG-205 and PGRMC1 silencing. Alteration in the expression of key markers of cell proliferation, apoptosis and cell cycle revealed that PGRMC1 inhibition decreases breast cancer proliferation. Conclusion: Our data demonstrate that PGRMC1 plays a prominent role in regulating breast cancer growth and progression by altering the EGFR/PI3K/AKT signaling mechanisms. Citation Format: Diego A Pedroza, Alejandra Bencomo, Adriana Galvez, Ramadevi Subramani, Venkatesh Rajamanickam, Rajkumar Lakshmanaswamy. Progesterone receptor membrane component 1 controls cellular proliferation and plays a key role in the molecular circuitry of both ER positive and triple negative breast cancers [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-05-08.

Progesterone induced Warburg effect in HEK293 cells is associated with post-translational modifications and proteasomal degradation of progesterone receptor membrane component 1

Progesterone (P4) is a major steroid hormone that has important effects on metabolism. The progesterone receptor membrane component 1 (PGRMC1) is a non-canonical P4 binding protein. The biological functions affected by PGRMC1 include cholesterol/steroid biosynthesis and metabolism, iron homeostasis and heme trafficking, autophagy, regulation of cell cycle and proliferation, cell migration and invasion. PGRMC1 has been an attractive target for therapeutic intervention in cancer and neurodegenerative disorders due to its biological role in promoting cell survival. P4 has been used in a number of clinical applications and is considered neuroprotective. The involvement of PGRMC1 in P4-mediated regulation of cellular glucose metabolism is not well studied. PGRMC1 is a 21 kDa protein but complex post-translational modifications (PTMs) lead to the existence of several high molecular mass proteins whose molecular function, intracellular distribution, and physiological relevancies are not fully known. Therefore, in this study, P4-PGRMC1-mediated cellular glucose metabolism and PTMs of PGRMC1 were studied using wild-type and CRISPR/Cas9 mediated PGRMC1 knockout (KO) human embryonic kidney-derived (HEK293) cell lines. A 70 kDa (p70) and 100 kDa (p100) PGRMC1 proteins were identified that are predominantly associated with endoplasmic reticulum/mitochondria and nuclear fractions in the cells, respectively. Phosphorylation, acetylation, ubiquitination, and sumoylation of native PGRMC1 under serum starvation were identified which provided an explanation for the higher molecular masses. This study indicates that P4-PGRMC1 signaling caused a rapid increase in glycolysis in the presence of oxygen (aerobic glycolysis) and a corresponding decrease in cellular respiration, known as the Warburg effect. Further, it was demonstrated that the P4-induced increase in glycolysis is associated with rapid proteasomal degradation of the p70 and reduction of the nuclear p100 protein level. P4 treatment also caused significant alteration in the dynamics of PGRMC1 PTMs and its association with potential interacting proteins. Overall, this study provides a hitherto unknown aspect of P4-PGRMC1 mediated signaling that changes basic cellular metabolism in HEK293 cells.

Ca2+/Calmodulin‐Dependent Protein Kinase Kinase β Negatively Regulates Progesterone Mediated PGRMC1 Signaling and the Warburg Effect

ObjectivesCa2+/calmodulin‐dependent protein kinase kinase β (CaMKKβ) signaling cascades directly regulate a variety of physiological processes and have been implicated in several human diseases, including cancer and neurodegenerative disorders. We discovered progesterone‐receptor membrane component 1 (PGRMC1) is differentially phosphorylated in CaMKKβ knockout (KO) HEK293 cells. PGRMC1 is a member of the membrane‐associated progesterone receptor (MAPR) family with a cytochrome b5‐like heme‐binding region. The gene is known to be involved in diverse functions, including regulation of cytochrome P450, steroidogenesis, vesicle trafficking, progesterone signaling and mitotic spindle and cell cycle regulation. PGRMC1 is associated with multiple progesterone‐dependent effects in diverse cell types. However, direct progesterone binding to PGRMC1 is yet to be demonstrated. The lack of credible ligand binding to PGRMC1 may be due to the fact that bacterially expressed PGRMC1 preparations may not possess the necessary post‐translational modifications (PTMs) required for progesterone binding. Therefore, we hypothesized that CaMKKβ is an upstream kinase that mediates phosphorylation of PGRMC1 and, thereby, regulates progesterone signaling.MethodologyWe used TiO2 column enrichment followed by mass spectrometry to identify differentially expressed phosphopeptides derived from CaMKKβ KO versus wild type HEK293 cells. We interrogated cellular metabolism by measuring oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) as an indicator of mitochondrial respiration and glycolysis, respectively. Further, we used isolectric focusing (IEF) followed by SDS‐PAGE and immunoblotting to identify different charged fractions of PGRMC1.ResultsLoss of CaMKKβ significantly decreased PGRMC1 protein expression in multiple CaMKKβ KO cell lines. TiO2 column enriched phosphopeptide analysis revealed the presence of phosphorylated S57, T178, Y180 and S181 peptides in CaMKKβ KO cells whereas only S181 phosphorylation was found in wild type cells. In addition, IEF analysis revealed multiple charged fractions of PGRMC1. The ~pI/pH‐3 fraction was significantly higher in CaMKKβ KO cells which may correspond to increased phosphorylation. Loss of CaMKKβ significantly increased the rate of glycolysis but reduced mitochondrial OCR. Treatment with 10μM progesterone significantly increased the rate of glycolysis within 30 mins in wild type cells; however it failed to show a similar effect in CaMKKβ KO cells. In contrast, progesterone treatment significantly lowered OCR in both wild type and CaMKKβ KO cells within 30 mins.ConclusionCaMKKβ negatively regulates PRGMC1 phosphorylation which may, in turn, control the relative turnover of the protein. CaMKKβ negatively regulates the progesterone‐mediated Warburg effect which may explain the role of CaMKKβ in tumorigenesis and neurodegenerative disease. Our study identifies a link between CaMKKβ and progesterone signaling which may be used for therapeutic targeting.Support or Funding InformationSupported by CIHR grant # MOP‐130282 (PF)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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) 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.

Abstract 3481: Sigma-2 ligands sensitize triple negative breast cancer cells to PARP inhibitor treatment

Abstract Triple-negative breast cancer (TNBC) accounts for 15-25% of breast cancer cases. Effective targeted treatment for TNBC is lacking. Poly (ADP-ribose) polymerase (PARP) is a key enzyme involved in single-strand DNA break repair. PARP inhibitors have been used in combination with various chemotherapeutic agents in TNBC with limited success. The sigma-2 receptor is associated with cellular proliferation and quiescence and has been used as the target for imaging solid tumors by positron emission tomography. The sigma-2 receptor has been identified as progesterone receptor membrane component 1 (PGRMC1) protein complex. Recent work has suggested that the sigma-2 receptor may also be an effective therapeutic target. In this study we looked for potential synergy between PARP inhibitors and sigma-2 ligands using a triple negative breast cancer cell line, MDA-MB231. We showed that the sigma-2 ligand, SW43, and a PGRMC1 ligand, AG205, induced cell toxicity and caspase 3 activation in a dose-dependent manner. The combination of SW43 and PARP inhibitor, Olaparib, resulted in additive cytotoxicity by cell viability assay and colony forming assay. Olaparib inhibited PARP activity in MDA-MB231 cell line, suggesting that the single-strand DNA break repair may be inhibited. SW43 and AG205 treatment decreased PGRMC1 and EGFR protein levels, suggesting that sigma-2/PGRMC1 ligands reduce growth factor signaling. In conclusion, SW43 and Olaparib induce cell death by separate signaling pathways. Sigma-2 ligands may selectively sensitize tumors to PARP inhibitor treatment in triple negative breast cancer patients. Citation Format: Chenbo Zeng, Julia Mankoff, Robert H. Mach. Sigma-2 ligands sensitize triple negative breast cancer cells to PARP inhibitor treatment. [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 3481. doi:10.1158/1538-7445.AM2015-3481

Elements in support of the ‘non-identity’ of the PGRMC1 protein with the σ2 receptor

σ2 Receptor subtype is overexpressed in a variety of human tumors, with σ2 agonists showing antiproliferative effects towards tumor cells through multiple pathways that depend both on the tumor cell type and on the molecule type. Therefore, σ2 receptor is an intriguing target for tumor diagnosis and treatment despite the fact that that it has not yet been cloned. One of the last attempts to characterize σ2 receptors led to identify it as the progesterone receptor membrane component 1 (PGRMC1). Although still controversial, such identity appears to have been accepted. We the aim of contributing to solve this controversy, in this work we stably silenced or overexpressed PGRMC1 protein in human MCF7 adenocarcinoma cells. Western blotting analyses were performed to quantify the presence of PGRMC1 protein on each of the three MCF7 cell lines variants, while scatchard analyses with radioligand were performed in order to determine the expression of the σ2 receptors. In order to correlate the antiproliferative effect of σ2 receptor agonist with PGRMC1 density, some σ2 ligands were administered to each of the three MCF7 cells variants. The results suggested that PGRMC1 and σ2 receptors are two different molecular entities.

Spectroscopic and Mutagenesis Studies of Human PGRMC1

Progesterone receptor membrane component 1 (PGRMC1) is a 25 kDa protein with an N-terminal transmembrane domain and a putative C-terminal cytochrome b5 domain. Heme-binding activity of PGRMC1 has been shown in various homologues of PGRMC1. Although the general definition of PGRMC1 is as a progesterone receptor, progesterone-binding activity has not been directly demonstrated in any of the purified PGRMC1 proteins fully loaded with heme. Here, we show that the human homologue of PGRMC1 (hPGRMC1) binds heme in a five-coordinate (5C) high-spin (HS) configuration, with an axial tyrosinate ligand, likely Y95. The negatively charged tyrosinate ligand leads to a relatively low redox potential of approximately -331 mV. The Y95C or Y95F mutation dramatically reduces the ability of the protein to bind heme, supporting the assignment of the axial heme ligand to Y95. On the other hand, the Y95H mutation retains ∼90% of the heme-binding activity. The heme in Y95H is also 5CHS, but it has a hydroxide axial ligand, conceivably stabilized by the engineered-in H95 via an H-bond; CO binding to the distal ligand-binding site leads to an exchange of the axial ligand to a histidine, possibly H95. We show that progesterone binds to hPGRMC1 and introduces spectral changes that manifest conformational changes to the heme. Our data offer the first direct evidence supporting progesterone-binding activity of PGRMC1.

Alzheimer's therapeutics targeting amyloid beta 1-42 oligomers II: Sigma-2/PGRMC1 receptors mediate Abeta 42 oligomer binding and synaptotoxicity.

Amyloid beta (Abeta) 1–42 oligomers accumulate in brains of patients with Mild Cognitive Impairment (MCI) and disrupt synaptic plasticity processes that underlie memory formation. Synaptic binding of Abeta oligomers to several putative receptor proteins is reported to inhibit long-term potentiation, affect membrane trafficking and induce reversible spine loss in neurons, leading to impaired cognitive performance and ultimately to anterograde amnesia in the early stages of Alzheimer's disease (AD). We have identified a receptor not previously associated with AD that mediates the binding of Abeta oligomers to neurons, and describe novel therapeutic antagonists of this receptor capable of blocking Abeta toxic effects on synapses in vitro and cognitive deficits in vivo. Knockdown of sigma-2/PGRMC1 (progesterone receptor membrane component 1) protein expression in vitro using siRNA results in a highly correlated reduction in binding of exogenous Abeta oligomers to neurons of more than 90%. Expression of sigma-2/PGRMC1 is upregulated in vitro by treatment with Abeta oligomers, and is dysregulated in Alzheimer's disease patients' brain compared to age-matched, normal individuals. Specific, high affinity small molecule receptor antagonists and antibodies raised against specific regions on this receptor can displace synthetic Abeta oligomer binding to synaptic puncta in vitro and displace endogenous human AD patient oligomers from brain tissue sections in a dose-dependent manner. These receptor antagonists prevent and reverse the effects of Abeta oligomers on membrane trafficking and synapse loss in vitro and cognitive deficits in AD mouse models. These findings suggest sigma-2/PGRMC1 receptors mediate saturable oligomer binding to synaptic puncta on neurons and that brain penetrant, small molecules can displace endogenous and synthetic oligomers and improve cognitive deficits in AD models. We propose that sigma-2/PGRMC1 is a key mediator of the pathological effects of Abeta oligomers in AD and is a tractable target for small molecule disease-modifying therapeutics.

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.

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 Modulates Human Cytochrome P450 Activities in an Isoform-Dependent Manner

Cytochromes P450 (P450s) catalyze the metabolism of a wide spectrum of compounds. Recently, progesterone receptor membrane component 1 (PGRMC1), which shares a key structural motif with cytochrome b(5), has been reported to bind to sterol- or steroid-synthesizing P450s, enhancing their activities. In this study, we investigated whether PGRMC1 affects human drug-metabolizing P450 activities. Using coexpression systems for PGRMC1 and P450s (CYP3A4, CYP2C9, or CYP2E1) in HepG2 cells, we found that PGRMC1 decreased the V(max) values and increased the K(m) values of the CYP3A4 activities, and it decreased the V(max) values but did not affect the K(m) values of the CYP2C9 activities. In contrast, PGRMC1 hardly affected the CYP2E1 activities. These results suggest that PGRMC1 negatively modulates the drug-metabolizing activities of P450, although it was isoform but not substrate dependent. It is worth noting that coimmunoprecipitation analysis using coexpression systems for FLAG-PGRMC1 and Myc-P450s in human embryonic kidney 293 cells revealed that PGRMC1 interacts with all three P450s, although the affinity seemed to vary. In 29 human liver microsomes (HLMs), there was a 5-fold variability in the PGRMC1 protein levels. By the correlation analyses using the P450 activities and the PGRMC1 levels, we could neither observe the contribution of PGRMC1 to the P450 activities in HLMs nor that of the NADPH-cytochrome P450 reductase or cytochrome b(5). In conclusion, in contrast to sterol- or steroid-synthesizing P450s, we found that PGRMC1 negatively modulates the human drug-metabolizing activities of P450 through direct interaction. Further studies are needed to determine the clinical significance of PGRMC1 in the pharmacokinetics of drugs.