Progesterone Receptor Membrane Research Articles

Progesterone/Org inhibits lung adenocarcinoma cell growth via membrane progesterone receptor alpha.

BackgroundThe aim of this study was to determine whether progesterone could inhibit the growth of lung adenocarcinoma cells via membrane progesterone receptor alpha (mPRα) and elucidate its potential mechanism. The relationship between mPRα expression and the survival prognosis of lung adenocarcinoma patients was studied.MethodsA mPRα knockdown lung adenocarcinoma cell line was constructed and treated with P4 and Org (a derivative of P4 and specific agonist of mPRα). Cell proliferation was assessed using CCK‐8 and plate colony formation assays. Protein expression was detected by western blotting. A nude mouse model of lung adenocarcinoma was established to assess the antitumor effect of P4/Org in vivo.ResultsWe initially determined that mPRα could promote the development of lung adenocarcinoma through the following lines of evidence. High expression of mPRα both at the mRNA and protein level was significantly associated with the poor prognosis of lung adenocarcinoma patients. The downregulation of mPRα inhibited the proliferation of lung adenocarcinoma cells. We further showed that mPRα mediates the ability of P4 to inhibit the growth of lung adenocarcinoma cells through the following lines of evidence: P4/Org inhibited the proliferation of lung adenocarcinoma cells; mPRα mediated the ability of P4/Org to inhibit lung adenocarcinoma cell proliferation; mPRα mediated the ability of P4/Org to inhibit the PKA (cAMP‐dependent protein kinase)/CREB (cAMP responsive element binding protein) and PKA/β‐catenin signaling pathways; and P4/Org inhibited the growth of a lung adenocarcinoma tumor model in vivo.ConclusionsIn summary, the results of our study show that progesterone can inhibit lung adenocarcinoma cell growth via mPRα.

Expression of nuclear progesterone receptors (nPRs), membrane progesterone receptors (mPRs) and progesterone receptor membrane components (PGRMCs) in the human endometrium after 6 months levonorgestrel low dose intrauterine therapy

The classical steroid receptors (nuclear receptors), including those for progesterone (nPRs), are thoroughly characterized. The knowledge about so-called non-genomic effects, which are mediated by extra-nuclear initiated signals, has increased immensely the last decades. In a previous clinical study of endometrial hyperplasia, we observed that the antiproliferative progestin effect persisted after 3 months treatment with levonorgestrel (LNG) intrauterine system (IUS) even with a complete downregulation of nPRs. This raised the question of what other mechanisms than signaling through nPRs could explain such an observation. In the present study, RT-qPCR was employed to characterize mRNA expression for nPRs, membrane progesterone receptors (mPRs) and progesterone receptor membrane components (PGRMCs) in women (n = 42) with endometrial hyperplasia that received intrauterine low dose LNG for 6 months. At the end of this period endometrial tissue showed that nPRs were virtually completely downregulated (≈ 10 % of baseline) whereas the levels of remaining mPRs, subtype-α, -β and -γ were 76 %, 59 % and 73 % of baseline, respectively. PGRMC1 was downregulated to 15 % of baseline, in contrast to PGRMC2, which was upregulated to about 30 % above baseline. We used human cancer cells from uterine cervix (C-4I cells) as control. Progesterone caused a concentration-dependent antiproliferative effect but in several and separate studies, we were unable to detect nPRs (immunocytochemistry) in the C-4I cells. The use of RT-qPCR showed that nPRs were undetectable in C-4I cells, in contrast to mPRs and PGRMCs with a distinct mRNA expression. The present study suggests that mPRs and/or PGRMCs preserve the antiproliferative effect of LNG in the human endometrium and are responsible for the concentration-dependent antiproliferative effect of progesterone in C-4I cells.

SAT-739 Membrane Progesterone Receptors (mPRs/PAQRs) Promote Proliferation, Migration and Neurotrophin Expression in Schwann Cell-like Adipose Stem Cells, Potentially Improving Peripheral Nerve Regeneration

Peripheral nerve injury is a problem affecting millions of people worldwide, causing significant disability and a reduction in the quality of life. The neurons of the peripheral nervous system are characterized by a significant regeneration capability. However, the functional recovery is unsatisfactory in most cases, making the identification of new strategies to improve the regenerative outcome a major medical need. Schwann cells (SCs), the main neuroglial cells of the peripheral nervous system, trans-differentiate after nerve injury towards a phenotype, known as the repair phenotype, that promotes nerve regeneration. Nonetheless, adult human SCs have poor expansion capability in vitro, undermining their potential clinical development. Several studies have shown that mesenchymal stem cells, especially when differentiated into a Schwann cell-like (SCL) phenotype, may represent an alternative to primary SCs, promoting pro-regenerative effects both in vitro and in vivo. Recently, membrane progesterone receptors (mPRs), members of the progestin and adipoQ receptor (PAQR) family, have been shown to be present and active in Schwann cells. Progesterone activates mPRs in SCs to promote cell migration and proliferation, change cell morphology, and modulate the expression of key SC differentiation factors. These findings suggest a possible role for mPRs in nerve repair. The goal of the present project is to study the role of mPRs in the promotion of pro-regenerative effects in mesenchymal adipose stem cells (ASC), both undifferentiated (uASC) and after differentiation toward the SCL phenotype (SCL-ASC). We first characterized uASC and SCL-ASC, confirming that the latter showed increased expression of SC markers like S100B, Sox10, GFAP, P0 and Krox20, alongside a more pronounced spindle-like shape leading to an increased aspect ratio. The differentiation protocol also increased mPR gene expression in SCL-ASC compared to uASC, especially mPRα (PAQR7) and mPRβ (PAQR8). Treatment with the specific mPR agonist Org OD 02-0 (02-0) caused cell elongation in SCL-ASC, mimicking what happens to SCs in vivo when they trans-differentiate towards the repair phenotype. 02-0 treatment also led to increased cell migration and cell proliferation in both uASC and SCL-ASC, consistent with a pro-regenerative role of mPRs. The effect was more rapid and evident in SCL-ASC. Lastly, we analyzed the effect of mPR activation on neutrophin production and release in uASC and SCL-ASC. A significant increase was observed in the expression and release of the brain derived neurotrophic factor (BDNF), whose role in promoting neurogenesis is well established. Our results support the hypothesis that mPRs have an important role in the modulation of uASC and SCL-ASC physiology and their activation may promote nerve regeneration.

SUN-559 Involvement of Sarco/Endoplasmic Reticulum Ca- ATPase (SERCA) in Membrane Progesterone Receptor Alpha (PAQR7)-Mediated Progesterone Induction of Vascular Smooth Muscle Relaxation

Progesterone (P4) exerts multiple beneficial effects on the human cardiovascular system through its actions on vascular endothelial cells and also by acting directly on vascular smooth muscle cells (VSMCs). Membrane progesterone receptor alpha (mPRα) has been shown to mediate the rapid P4-induction of human VSMC relaxation through activation of MAPK, Akt/Pi3k and RhoA/ROCK signaling pathways and the resulting reduction of calcium influx through calcium channels. In this study, we demonstrate that treatment of cultured human VSMCs with P4 for 1-2 hours increases both the mRNA and protein expression of sarco/endoplasmic reticulum Ca- ATPase (SERCA), the major transporter of calcium from the cytosol into the sarcoplasmic reticulum (SR) during muscle relaxation. Knockdown of mPRα with siRNA completely blocked this stimulatory effect of P4 as well as that of OD 02-0, a mPR selective agonist, on SERCA protein expression. In contrast, expression levels of phospholamban (PLB), a SR protein that reversibly inhibits SERCA were downregulated by this P4 treatment, and mRNA expression of a channel that releases calcium from the SR, inositol trisphosphate receptor (IP3R), was unaltered after treatment with P4. Moreover, treatments with P4 and OD 02-0, but not with R5020, a nuclear PR agonist, increased PLB phosphorylation, which would result in disinhibition of SERCA function. P4 and OD 02-0 significantly increased calcium levels in the SR detected with Fluo-5N, a specific SR calcium indicator, and caused VSMC relaxation. These effects were blocked by cyclopiazonic acid (CPA, a SERCA inhibitor), suggesting that SERCA plays a critical role in P4 induction of VSMC relaxation. Similarly, the effects of P4 and OD 02-0 on relaxation of umbilical artery rings measured with a myograph were significantly attenuated by CPA, which confirms the critical role of SERCA in the rapid action of P4 and 02-0 on vascular muscle relaxation. P4 has previously been shown to activate MAPK and Akt signaling pathways to induce VSMC relaxation. The P4- and OD 02-0-induced increases in calcium in the SR were blocked by MAPK and Akt/Pi3k signaling inhibitors, AZD6244 and wortmannin. Taken together, these results suggest that the direct, rapid effects of P4 on relaxation of VSMCs through mPRα involves regulation of the expression and function of the SR proteins SERCA and PLB through MAPK and Akt signaling pathways.

Impaired oocyte maturation and ovulation in membrane progestin receptor (mPR) knockouts in zebrafish

Accumulating evidence suggest that membrane progestin receptor α (mPRα) is the membrane receptor mediating nongenomic progestin signaling that induces oocyte maturation in teleost. However, the involvement of other members of mPR family in oocyte maturation is still unclear. In this study, we found impaired oocyte maturation in zebrafish lacking mPRα1, mPRα2, mPRβ, or mPRγ2. In contrast, no difference was observed in oocyte maturation in the single knockout of mPRγ1, mPRδ, or mPRε. To study possible redundant functions of different mPRs in oocyte maturation, we generated a zebrafish line lacking all seven kinds of mPRs (mprs−/−). We found oocyte maturation was further impaired in mprs−/−. In addition, oocyte ovulation delay was observed in mprs−/− females, which was associated with low levels of nuclear progestin receptor (Pgr), a key regulator for ovulation. We also found reduced fertility in mprs−/− female zebrafish. Furthermore, eggs spawned by mprs−/− females were of poor quality.

Traumatic Brain Injury Alters PAQR7‐induced Vasorelaxation in Pressurized Cerebral Arteries

Traumatic brain injury (TBI) induces acute changes in cerebrovascular function, including alterations to cerebral artery contractility, which ultimately increase the incidence of both ischemic and hemorrhagic events. Cerebrovascular tone is controlled by hormonal, neurogenic, endothelial, and myogenic responses. Progesterone, a steroid hormone, has been evaluated as a potential treatment to improve TBI outcome; however, progesterone has failed to translate to patient benefits in clinical trials, potentially due to its pleiotropic effects on multiple receptors. Progestin & adipoQ receptor 7 (PAQR7) is a plasma membrane progesterone receptor involved in the vasorelaxation of human blood vessels. However, little is known about the smooth muscle‐specific role of PAQR7 in resistance size cerebral arteries following TBI. Here, we examined PAQR7 function in pressurized endothelium‐denuded cerebral resistance size arteries 24 hours following moderately severe TBI (controlled cortical impact). Baseline myogenic tone of middle cerebral arteries (MCA) was attenuated following TBI. PAQR7‐induced vasorelaxation in pressurized MCA from TBI rats was attenuated in a concentration‐dependent manner as compared to sham controls (10−9M‐10−7M). Moreover, pre‐incubation with inhibitors of adenylyl cyclase (AC), PKA, or PKG attenuated PAQR7‐induced vasorelaxation in both groups. In conclusion, smooth muscle‐specific PAQR7 vasodilation involves an AC‐PKA/PKG pathway in cerebral resistance size arteries, and moderately severe TBI attenuates cerebral artery myogenic tone, which reduces overall arterial vasodilatory capacity.Support or Funding InformationSupported by The Florida State University Graduate School Dissertation Award Grant.

Nuclear and membrane progesterone receptors expression in placenta from early to late pregnancy in sheep: Effects of restricted nutrition and realimentation

Nutrient restriction and/or realimentation may affect several placental functions, such as expression of selected regulatory factors, blood flow and other processes in sheep and other species. To determine the effects of the plane of nutrition, nulliparous white face ewes (6–8 months) carrying singletons on day 50 of gestation were randomly assigned to two dietary treatments receiving 100% of National Research Council recommendations (control; C) or 60% of C (restricted; R). Two groups remained on C or R diets from day 50 until day 130. From day 90–130 another group of C fed ewes was switched to the R diet, and another group of R fed ewes was switched to the C diet. This resulted in 7 groups (n = 5–6 ewes/group): C (day 50, 90 and 130), R (day 90 and 130), CR (day 130) and RC (day 130). At these time points, placental tissues were collected for the evaluation of progesterone receptor (PGR) protein expression (whole tissue), and mRNA expression in maternal (caruncular, CAR) and fetal (cotyledon, COT) (separated tissues). Data were statistically analyzed using analysis of variance (SAS 9.4). Protein for PGRAB and PGRB isoforms was detected using immunohistochemistry in all placental tissues, but the pattern of expression differed depending on pregnancy stage and placental compartment (e.g., CAR vs COT). PGRAB protein expression, quantified using image analysis, was greater (P < 0.04) on day 50 than 90 or 130, and was not affected by plane of nutrition. In CAR and COT, PGRAB mRNA expression was greater (P < 0.05) on day 50 than 90 or 130. PGRB mRNA expression was greater (P < 0.03) in CAR on day 50 than 90 and 130, and was greatest (P < 0.02) in COT on day 50, less on day 130, and least on day 90. For the membrane progesterone receptors, PAQR7 (membrane PGR alpha) mRNA expression was greater (P < 0.05) on days 50 and 90 than 130 in CAR, and greater (P < 0.01) on days 50 than 90 and 130 in COT; PAQR8 (membrane PGR beta) was similar throughout pregnancy in CAR and COT, and PAQR5 (membrane PGR gamma) was greatest (P < 0.0001) on day 130 in COT, but similar throughout pregnancy in CAR. Plane of nutrition affected (P < 0.05) mRNA expression for all genes in CAR and COT throughout pregnancy. These data indicate that expression of PGR in ovine placenta is dependent on stage of pregnancy and plane of nutrition in sheep. The mechanisms of how diet and stage of pregnancy influences placental PGR expression and function remains to be elucidated.

The Role of mPRδ and mPRε in Human Glioblastoma Cells: Expression, Hormonal Regulation, and Possible Clinical Outcome.

Glioblastomas (GBM) are the most frequent and aggressive primary tumor of the central nervous system. In recent years, it has been proposed that sex hormones such as progesterone play an essential role in GBM biology. Membrane progesterone receptors (mPRs) are a group of G protein-coupled receptors with a wide distribution and multiple functions in the organism. There are five mPRs subtypes described in humans: mPRα, mPRβ, mPRγ, mPRδ, and mPRε. It has been reported that human-derived GBM cells express the mPRα, mPRβ, and mPRγ subtypes, and that progesterone promotes GBM progression in part by mPRα specific activation; however, it is still unknown if mPRδ and mPRε are also expressed in this type of tumor cells. In this study, we characterized the expression and hormonal regulation of mPRδ and mPRε in human GBM cells. We also analyzed a set of biopsies from TCGA. We found that the expression of these receptors is dependent on the tumor's grade and that mPRδ expression is directly correlated to patients' survival while the opposite is observed for mPRε. By RT-qPCR, Western blot, and immunofluorescence, the expression of mPRδ and mPRε was detected for the first time in human GBM cells. An in silico analysis showed possible progesterone response elements in the promoter regions of mPRδ and mPRε, and progesterone treatments downregulated the expression of these receptors. Our results suggest that mPRδ and mPRε are expressed in human GBM cells and that they are relevant to GBM biology.

Sertoliform Endometrioid Carcinoma of the Ovary in a Young Woman: Diagnostic Challenges for the Pathologist

Sertoliform endometrioid carcinoma (SEC) is a rare variant of ovarian endometrioid carcinoma commonly affecting older women. The morphology of this tumour closely resembles that of Sertoli and Sertoli–Leydig cell tumours. Microscopically, the tumour is composed of anastomosing cords, nests, trabeculae and tubules lined by pseudostratified columnar epithelium with elongated nuclei. We present a case of Sertoliform endometrioid carcinoma in a 24-year-old woman. A 24-year-old woman presented with lower abdominal pain and bleeding per vagina for the past 6 months associated with loss of weight and appetite. Ultrasound abdomen showed a complex pelvic mass. Positron emission tomography–computed tomography scan showed complex abdominopelvic mass with metastatic peritoneal and omental nodules, moderate ascites and right pleural thickening. Complete cytoreduction was performed. On microscopy, both ovaries showed infiltration by a malignant neoplasm composed of cells arranged as sheets, trabeculae, nests and focal tubules lined by moderately pleomorphic cells with elongated ovoid hyperchromatic nuclei, inconspicuous nucleoli and scant cytoplasm. Focally, cribriform pattern was noted with tall columnar lining epithelium showing nuclear stratification. The tumour cells were positive for cytokeratin, epithelial membrane antigen, oestrogen receptor and progesterone receptor and negative for p53, synaptophysin, chromogranin, CD99, inhibin and calretinin. A diagnosis of Sertoliform endometrioid carcinoma of the ovaries was rendered. To our knowledge, only a single case report has been published so far where SEC presented in a woman in her twenties. We would like to emphasise with this case the need to include SEC as a differential diagnosis for tumours with sex cord stromal components seen in younger women and to reiterate the importance of adequate and thorough sampling of the tumour to look for conventional endometrioid carcinoma areas.

MPRα mediates P4/Org OD02-0 to improve the sensitivity of lung adenocarcinoma to EGFR-TKIs via the EGFR-SRC-ERK1/2 pathway.

The discovery of epidermal growth factor receptor (EGFR) mutations has made EGFR tyrosine kinase inhibitors (EGFR-TKIs) a milestone in the treatment for advanced non-small cell lung cancer (NSCLC). However, patients lacking EGFR mutations are not sensitive to EGFR-TKI treatment and the emergence of secondary resistance poses new challenges for the targeted therapy of lung cancer. In this study, we identified that the expression of membrane progesterone receptor α (mPRα) was associated with EGFR mutations in lung adenocarcinoma patients and subsequently affected the efficacy of EGFR-TKIs. Progesterone (P4) or its derivative Org OD02-0 (Org), which is mediated by mPRα, increases the function of EGFR-TKIs to suppress the proliferation, migration, and invasion of lung adenocarcinoma cells in vitro and in vivo. In addition, the mPRα pathway triggers delayed resistance to EGFR-TKIs. Mechanistic investigations demonstrated that the mPRα pathway can crosstalk with the EGFR pathway by activating nongenomic effects to inhibit the EGFR-SRC-ERK1/2 pathway, thereby promoting antitumorigenic effects. In conclusion, our data describe an essential role for mPRα in improving sensitivity to EGFR-TKIs, thus rationalizing its potential as a therapeutic target for lung adenocarcinomas.

Membrane Progesterone Receptors (mPRs/PAQRs) Differently Regulate Migration, Proliferation, and Differentiation in Rat Schwann Cells.

Several studies in the last decade demonstrated that progestogens play an important role in the biology of Schwann cells, the main neuroglial cells of the peripheral nervous system. Since a recent study showed that the S42 rat Schwann cell line expressed membrane progesterone receptors (mPRs), members of the PAQR family, in this study, we examined mPR expression in a more physiological model, primary rat Schwann cells. We demonstrated that primary rat Schwann cells show a different pattern of mPR expression compared to the previously studied model; mPRα (PAQR7) and β (PAQR8) isoforms were the major mPR members identified, with different sub-cellular localizations. Activation of the nuclear progesterone receptor (PR) with the specific agonist R5020 upregulated mPR expression, while activation of mPRs with the specific agonist Org OD 02-0 changed their sub-cellular localization. An in-depth analysis revealed additional effects of mPR activation, such as AKT activation, reduced expression of the myelin-associated glycoprotein (MAG), morphological changes, altered expression of several Schwann cell differentiation markers, and increased Schwann cell migration and proliferation. In conclusion, we identified mPRα and mPRβ in primary rat Schwann cells, and our findings suggest a putative role for mPRs in the regulation of Schwann cell migration, proliferation, and differentiation. Therefore, mPRs are a potential pharmacological target for Schwann cell-related disorders and neurodegenerative diseases, especially those in which Schwann cell-mediated axon remyelination is desirable.

Vitellogenesis in Blue Gourami is Accompanied by Brain Transcriptome Changes

The blue gourami (Trichogaster trichopterus) is a model for hormonal control of reproduction in Anabantidae fish, but also relevant to other vertebrates. We analyzed the female blue gourami brain transcriptome in two developmental stages: pre-vitellogenesis (PVTL) before yolk accumulation in the oocytes, and high vitellogenesis (HVTL) at the end of yolk accumulation in the oocytes. RNA sequencing of whole-brain transcriptome identified 34,368 unique transcripts, 23,710 of which could be annotated by homology with other species. We focused on the transcripts showing significant differences between the stages. Seventeen and fourteen annotated genes were found to be upregulated in PVTL and HVTL, respectively. Five nuclear transcripts, three of which contain the homeobox domain (ARX, DLX5, CERS6), were upregulated in PVTL. Additionally, several receptors previously known to be involved in reproduction were identified, and three of these, G-protein coupled receptor 54, Membrane progesterone receptor epsilon, and Gonadotropin-releasing hormone II receptor (GPCR, mPR, and GnRHR) were measured by quantitative RT-PCR in brain, pituitary, and ovary samples from PVTL and HVTL stage females. Of these, GPCR was highly expressed in the brain and pituitary as compared to the ovary in both PVTL and HVTL. GnRHR was highly expressed in the ovary compared to the brain and pituitary, and its levels in the brain were significantly higher in PVTL than HVTL. Brain mPR mRNA levels were likewise higher in PVTL than HVTL. In conclusion, this study details changes in the female blue gourami brain transcriptome through yolk accumulation in the oocytes and identifies key genes that may mediate this process.

Role of mPRα (PAQR7) in progesterone-induced Ca2+ decrease in human vascular smooth muscle cells.

We have shown progesterone exerts a direct action on vascular smooth muscle cells (VSMCs) to induce relaxation through activation of membrane progesterone receptor alpha (mPRα)-dependent signaling pathways, but information on downstream events is lacking. Progesterone-induced changes in calcium concentrations in human umbilical artery VSMCs through mPRα-dependent signaling pathways and the involvement of Rho/ROCK signaling were investigated. Acute in vitro treatment with progesterone and the selective mPRα agonist 10-ethenyl-19-norprogesterone (Org OD 02-0, 02-0) blocked the rapid prostaglandin F2α-induced calcium increase. This inhibitory progesterone action was prevented by knockdown of mPRα but not by knockdown of the nuclear progesterone receptor, confirming it is mediated through mPRα. The decrease in calcium levels and VSMC relaxation were abolished by treatment with FPL64176 (Ca2+ channel activator), supporting a role for decreased calcium channel activity in this progesterone action. The reduction in calcium was attenuated by pretreatment with pertussis toxin, 8-Bromo-cAMP and forskolin, indicating this progesterone action involves activation of an inhibitory G protein and downregulation of cAMP-dependent signaling. Inhibition of MAPK and Akt signaling with PD98059 and ML-9, respectively, prevented the progesterone-induced calcium concentration decrease and VSMC relaxation. Forskolin decreased progesterone-induced MAPK and Akt phosphorylation which suggests that the cAMP status influences calcium levels indirectly through altering these signaling pathways. Progesterone and 02-0 treatments decreased RhoA activity and ROCK phosphorylation, which suggests that reduced RhoA/ROCK signaling is a component of the mPRα-mediated progesterone actions on VSMCs. The results suggest that progesterone induces VSMC relaxation by reducing cellular calcium levels through mPRα-induced alterations in multiple signaling pathways.

Downregulation of nuclear progestin receptor (Pgr) and subfertility in double knockouts of progestin receptor membrane component 1 (pgrmc1) and pgrmc2 in zebrafish

The progestin receptor membrane components (Pgrmcs) contain two paralogs, Pgrmc1 and Pgrmc2. Our previous research into single knockout of Pgrmc1 or Pgrmc2 suggests that Pgrmc1 and Pgrmc2 regulate membrane progestin receptor or steroid synthesis and therefore female fertility in zebrafish. Additional roles of Pgrmcs may not be determined in using single Pgrmc knockouts due to compensatory roles between Pgrmc1 and Pgrmc2. To address this question, we crossed single knockout pgrmc1 (pgrmc1−/−) with pgrmc2 (pgrmc2−/−), and generated double knockouts for both pgrmc1 and pgrmc2 (pgrmc1/2−/−) in a vertebrate model, zebrafish. In addition to the delayed oocyte maturation and reduced female fertility, significant reduced ovulation was found in double knockout (pgrmc1/2−/−) in vivo, though not detected in either single knockout of Pgrmc (pgrmc1−/− or pgrmc2−/−). We also found significant down regulation of nuclear progestin receptor (Pgr) protein expression only in pgrmc1/2−/−, which was most likely the cause of reduced ovulation. Lower protein expression of Pgr also resulted in reduced expression of metalloproteinase in pgrmc1/2−/−. With this study, we have provided new evidence for the physiological functions of Pgrmcs in the regulation of female fertility by regulation of ovulation, likely via regulation of Pgr, which affects regulation of metalloproteinase expression and oocyte ovulation.

Expression of membrane progestin receptors (mPRs) α, β and γ in the bovine uterus during the oestrous cycle and pregnancy

Progesterone (P4) affects cell function through the nuclear progesterone receptor and membrane-bound progesterone binding proteins, including the membrane progestin receptors (mPRs) alpha (mPRα), beta (mPRβ) and gamma (mPRγ), which belong to the progestin and adipoQ receptor family (PAQR7, 8 and 5, respectively). The aim of this study was to determine the mRNA and protein expression levels of mPRα, mPRβ and mPRγ through real-time PCR and Western blot analyses, respectively, and to determine the cellular localization of these proteins in the bovine endometrium and myometrium on days 2–5, 6–10, 11–16 and 17–20 of the oestrous cycle and weeks 3–5, 6–8 and 9–12 of pregnancy (n = 5/each time period). The resulting data showed the highest (P < 0.05) mPRα and mPRβ mRNA expression in the endometrium on days 11–16 of the oestrous cycle compared to the other stages. In the myometrium, the level of mPRα mRNA was the lowest (P < 0.05) on days 6–16 of the oestrous cycle, while mPRβ was the lowest on days 11–16. There were no changes (P > 0.05) in mPRγ mRNA expression in the endometrium and myometrium during the oestrous cycle. During pregnancy, in the endometrium and myometrium, the levels of mPRα and mPRβ mRNA were comparable with those observed during the oestrous cycle. However, mPRγ mRNA expression was the highest (P < 0.001) during all stages of pregnancy compared with that observed during the oestrous cycle in both uterine tissues. The mPRα protein level only changed in the myometrium and was the highest (P < 0.05) during weeks 9–12 of pregnancy. However, in the endometrium, the expression of mPRβ protein was higher (P < 0.05) on days 6–10 of the oestrous cycle than during weeks 6–8 of pregnancy. Strong positive immunoreactions for all mPR proteins were observed in the luminal and glandular epithelium but were less evident in the stromal cells and myocytes. In addition, all proteins were also localized in the endothelial cells of blood vessels in the uterus, suggesting that P4 may affect blood flow in this organ through mPRs. The presence of mPR receptors in the uterus indicates their participation in the regulation of uterine functions.

Metabotropic, but not allosteric, effects of neurosteroids on GABAergic inhibition depend on the phosphorylation of GABAA receptors

Neuroactive steroids (NASs) are synthesized within the brain and exert profound effects on behavior. These effects are primarily believed to arise from the activities of NASs as positive allosteric modulators (PAMs) of the GABA-type A receptor (GABAAR). NASs also activate a family of G protein-coupled receptors known as membrane progesterone receptors (mPRs). Here, using surface-biotinylation assays and electrophysiology techniques, we examined mPRs' role in mediating the effects of NAS on the efficacy of GABAergic inhibition. Selective mPR activation enhanced phosphorylation of Ser-408 and Ser-409 (Ser-408/9) within the GABAAR β3 subunit, which depended on the activity of cAMP-dependent protein kinase A (PKA) and protein kinase C (PKC). mPR activation did not directly modify GABAAR activity and had no acute effects on phasic or tonic inhibition. Instead, mPR activation induced a sustained elevation in tonic current, which was blocked by PKA and PKC inhibition. Substitution of Ser-408/9 to alanine residues also prevented the effects of mPR activation on tonic current. Furthermore, this substitution abolished the effects of sustained NAS exposure on tonic inhibition. Interestingly, the allosteric effects of NAS on GABAergic inhibition were independent of Ser-408/9 in the β3 subunit. Additionally, although allosteric effects of NAS on GABAergic inhibition were sensitive to a recently developed "NAS antagonist," the sustained effects of NAS on tonic inhibition were not. We conclude that metabotropic effects of NAS on GABAergic inhibition are mediated by mPR-dependent modulation of GABAAR phosphorylation. We propose that this mechanism may contribute to the varying behavioral effects of NAS.

Abstract 846: 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: The role of progesterone and its receptors in breast cancer progression continue to be studied but remain controversial. Progesterone membrane receptors with the ability to regulate kinase signals, mediating breast cancer proliferation have been demonstrated. Increased expression of the progesterone receptor membrane component 1 (PGRMC1), a heme - binding protein with the ability to interact and stabilize epidermal growth factor receptor (EGFR) is frequently found in breast cancer tissue. The basis of the signaling mechanisms of progesterone membrane receptors remain largely unknown. Both the nuclear and membrane progesterone receptors could play a significant role in the development and progression of breast cancers and both could become viable therapeutic options. We, aim to investigate the molecular circuitry of PGRMC1 in both ER-positive and Triple Negative Breast Cancers (TNBCs). Materials and Methods: Human breast tissues were utilized to identify the expression of PGRMC1 along with a panel of normal and breast cancer cell lines. PGRMC1 overexpressing ZR-75-1 and MDA-MB-468 breast cancer cell lines were selected and treated with AG-205 (PGRMC1 inhibitor) and PGRMC1 siRNAs, while PGRMC1 was overexpressed in MCF10A non-malignant breast epithelial cells. MTS, qRT-PCR, Western blot, immunofluorescence, immunohistochemistry and flow cytometry were performed to study cell proliferation, apoptosis and key markers involved in these processes. In silico analysis utilizing publicly available gene expression datasets were also performed. Results: Immunohistochemistry demonstrated strong staining for PGRMC1 in human breast cancer tissue compared to normal breast tissue. Increased PGRMC1 expression was observed specifically in ZR-75-1 and MDA-MB-468 cells by qRT-PCR, Western blot and immunofluorescence, these results were validated and compared to microarray-based gene expression analysis of breast cell lines and breast tumor data sets. Both AG-205 and PGRMC1 targeted siRNAs decreased cell proliferation in ZR-75-1 and MDA-MB-468. Minimal effects of AG-205 were observed in MCF10A non-malignant breast epithelial cells. AG-205 treatment and silencing of PGRMC1 induced apoptosis in both cancer cell lines. Furthermore, PGRMC1 overexpression transformed MCF10A cells into a malignant phenotype. Key markers of cell proliferation (pAKT, CCND1, pEGFR, pmTOR) and apoptosis (PTEN, Bcl2, Bax,) revealed that PGRMC1 inhibition decreases proliferation while overexpression promotes tumorigenesis. Conclusion: Our data demonstrates that PGRMC1 plays a prominent role in both ER-positive and TNBCs. These initial findings uncover the potential of PGRMC1 as an oncogene and therapeutic target for breast cancer patients who overexpress this gene. Citation Format: Diego A. Pedroza, Ramadevi Subramani Reddy, Adriana Galvez, 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 American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 846.

Genomic sequence analyses of classical and non-classical lamprey progesterone receptor genes and the inference of homologous gene evolution in metazoans

BackgroundNuclear progesterone receptor (nPR) is an evolutionary innovation in vertebrates that mediates genomic responses to progesterone. Vertebrates also respond to progesterone via membrane progesterone receptors (mPRs) or membrane associated progesterone receptors (MAPRs) through rapid nongenomic mechanisms. Lampreys are extant agnathan vertebrates, residing at the evolutionary juncture where vertebrates diverged from invertebrates. A survey of the progesterone receptor (PR) gene sequences in lamprey genomes would inform PR gene evolutionary events during the transition from invertebrates to vertebrates.ResultsIn this study, we annotated sequences of one nPR, four mPR (β, γ, δ and ε) and four MAPR genes from genomes of two lamprey species (Petromyzon marinus and Lethenteron japonicum). To infer the origin and evolutionary history of PR genes, we constructed phylogenetic trees of PR homologous sequences across representative species of metazoans. Phylogenetic analyses revealed that the mPRγ gene first appeared in non-bilaterians, and the mPRβ gene likely arose from a duplication of mPRγ. On the other hand, the mPRγ gene gave rise to the mPRδ and ε genes much later in the vertebrate lineage. In addition, the mPRα gene first appeared in cartilaginous fishes, likely derived from duplication of mPRβ after the agnathan-gnathostome divergence. All known MAPR genes were present in the lamprey genomes. Progesterone receptor membrane component 1 (PGRMC1), neudesin and neuferricin genes probably evolved in parallel in non-bilaterians, whereas two copies of PGRMC genes probably derived from duplication of ancestral PGRMC1 sequence and appeared before the speciation of lampreys.ConclusionsNon-classical mPR and MAPR genes first evolved in non-bilaterians and classical nPR genes evolved later in basal vertebrates. Sequence repertoires for membrane progesterone receptor genes in vertebrates likely originated from an ancestral metazoan sequence and expanded via several duplication events.

Characterization, relative abundances of mRNA transcripts, and subcellular localization of two forms of membrane progestin receptors (mPRs) in the common Chinese cuttlefish, Sepiella japonica

The membrane progestin receptor (mPR) family has been characterized in several species, including fish, frogs, rats, and humans. Results of previous studies indicate mPRs mediate the rapid, nongenomic action of progestins. In this study, the full-length cDNA of Sepiella japonica mPR-beta (mPRβ) and mPR-gamma (mPRγ) were characterized. Furthermore, sjmPR mRNA relative abundances were assessed for different tissues. There was also determination of the subcellular localization of mPRs, and investigation of the effect of sjmPRs on ovarian development via proximate actions on the brain and ovary of S. japonica. Results of tissue distribution assays indicated mPRβ and mPRγ transcripts were present predominantly in the brain and ovary. As ovaries developed, the abundance of SjmPRs mRNA transcripts increased and peaked during the interstitial growth phase (III), followed by a marked decrease afterward in both the brain and ovary. In addition, confocal microscopy evaluations of HEK293 T cells expressing the mPRs-EGFP gene indicated both SjmPRβ and SjmPRγ were localized in the plasma membrane of HEK293 T cells. Taken together, these findings indicate S. japonica protein is a membrane progestin receptor capable of inducing ovary maturation in cephalopods.

Arterial Smooth Muscle Cell PAQR7 Activation Attenuates Myogenic Tone In Pressurized Cerebral Arteries

Sex steroid hormones (estrogen, progesterone) are regulators of arterial contractility. Specifically, progesterone exerts rapid, nongenomic vasodilation via membrane receptors termed membrane progesterone receptors (mPR) belonging to the GPCR-like family of Progestin & Adiponectin Q Receptors (PAQR). Moreover, PAQR7 (mPRα) is reportedly expressed in smooth muscle cells (SMC) of human conduit vessels and has a putative role in vasodilation. However, the expression and function of PAQR7 in pressurized resistance size arteries, arteries that regulate local blood flow and blood pressure, have not been investigated. PURPOSE: To examine the expression and physiological function of PAQR7 in cerebral resistance size arteries. METHODS: RT-PCR was performed on whole cerebral arteries and isolated arterial SMCs to detect PAQR7 mRNA expression. Western blotting was performed on whole cerebral arteries to examine PAQR7 protein expression. Pressurized artery myography was performed on endothelium-intact and -denuded cerebral arteries to examine the role of SMC PAQR7 in vasodilation using the PAQR7-specific agonist, Org OD 02-0. RESULTS: PAQR7 mRNA (whole artery and isolated arterial SMCs) and protein were detected in resistance size cerebral arteries (n=4). Interestingly, there were no significant differences in vasodilation between endothelium-intact and -denuded arteries at any concentration (10-9M to 10-5M, n=4). CONCLUSION: These data suggest that arterial smooth muscle PAQR7 activation elicits vasodilation in pressurized resistance size arteries.