Yefei Pang

University of Texas at Austin, Austin, TX, United States

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Publications (30)114.84 Total impact

  • Peter Thomas, Yefei Pang, Jing Dong
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    ABSTRACT: 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 over expression of human PGRMC1 in progesterone receptor-negative breast cancer cell lines causes increased expression of PGRMC1 and membrane progesterone receptor alpha (mPRα) on cell membranes which 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 co-immunoprecipitation 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 which were abolished after co-transfection with siRNA 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 beta (ERβ) was also observed in the membranes of PGRMC1-transfected cells, suggesting PGRMC1 can act as an adaptor protein for multiple classes of steroid receptors.
    Endocrinology 01/2014; · 4.72 Impact Factor
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    ABSTRACT: Prior studies have linked renoprotective effects of estrogens to G-protein-coupled estrogen receptor-1 (GPER-1) and suggest that aldosterone may also activate GPER-1. Here, the role of GPER-1 in murine renal tissue was further evaluated by examining its anatomical distribution, subcellular distribution and steroid binding specificity. Dual immunofluorescent staining using position-specific markers showed that GPER-1 immunoreactivity primarily resides in distal convoluted tubules and the Loop of Henle (stained with Tamm-Horsfall Protein-1). Lower GPER-1 expression was observed in proximal convoluted tubules marked with megalin, and GPER-1 was not detected in collecting ducts. Plasma membrane fractions prepared from whole kidney tissue or HEK293 cells expressing recombinant human GPER-1 (HEK-GPER-1) displayed high-affinity, specific [3H]-17β-estradiol ([3H]-E2) binding, but no specific [3H]-aldosterone binding. In contrast, cytosolic preparations exhibited specific binding to [3H]-aldosterone but not to [3H]-E2, consistent with the subcellular distribution of GPER-1 and mineralocorticoid receptor (MR) in these preparations. Aldosterone and MR antagonists, spironolactone and eplerenone, failed to compete for specific [(3)H]-E2 binding to membranes of HEK-GPER-1 cells. Furthermore, aldosterone did not increase [(35)S]-GTP-γS binding to membranes of HEK-GPER-1 cells, indicating that it is not involved in G protein signaling mediated through GPER-1. During the secretory phases of the estrus cycle, GPER-1 is upregulated on cortical epithelia and localized to the basolateral surface during proestrus and redistributed intracellularly during estrus. GPER-1 is down-modulated during luteal phases of the estrus cycle with significantly less receptor on the surface of renal epithelia. Our results demonstrate that GPER-1 is associated with specific estrogen binding and not aldosterone binding and that GPER-1 expression is modulated during the estrus cycle which may suggest a physiological role for GPER-1 in the kidney during reproduction.
    Molecular and Cellular Endocrinology 11/2013; · 4.04 Impact Factor
  • Peter Thomas, Yefei Pang
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    ABSTRACT: The protective functions of progesterone in the cardiovascular system have received little attention even though evidence has accumulated that progesterone lowers blood pressure, inhibits coronary hyperactivity and has powerful vasodilatory and natriuretic effects. One possible reason why potential beneficial actions of progesterone on cardiovascular functions have not been extensively studied is that divergent effects to those of progesterone have been observed in many clinical trials with synthetic progestins such as medroxyprogesterone acetate which are associated with increased risk of coronary disease. Evidence that progesterone exerts protective effects on cardiovascular functions is briefly reviewed. The finding that progesterone administration decreases blood vessel vasoconstriction in several animal models within a few minutes suggests that rapid, nongenomic progesterone mechanisms are of physiological importance in regulating vascular tone. Rapid activation of second messenger pathways by progesterone has been observed in vascular endothelial and smooth muscle cells, resulting in alterations in endothelial nitric oxide synthase (eNOS) activity and calcium influx, respectively. Both nuclear progesterone receptors (PRs) and novel membrane progesterone receptors (mPRs) are candidates for the intermediaries in these rapid, cell-surface initiated progesterone actions in endothelial and smooth muscle vascular cells. PRs have been detected in both cell types. New data are presented showing mPRα, mPRβ and mPRγ are also present in human endothelial and smooth muscle vascular cells. Preliminary evidence suggests mPRs mediate rapid progestin signaling in these endothelial cells, resulting in down-regulation of cAMP production and increased nitric oxide synthesis. The role of mPRs in progesterone regulation of cardiovascular functions warrants further investigation.
    Steroids 01/2013; · 2.80 Impact Factor
  • Yefei Pang, Jing Dong, Peter Thomas
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    ABSTRACT: Three members of the progestin and adipoQ receptor (PAQR) family, PAQR-7, PAQR-8, and PAQR-5 [membrane progesterone (P4) receptor (PR) (mPR)α, mPRβ, and mPRγ], function as plasma mPRs coupled to G proteins in mammalian cells, but the characteristics of two other members, PAQR6 and PAQR9 (mPRδ and mPRε), remain unclear, because they have only been investigated in yeast expression systems. Here, we show that recombinant human mPRδ and mPRε expressed in MDA-MB-231 breast cancer cells display specific, saturable, high-affinity [(3)H]-P4 binding on the plasma membranes of transfected cells with equilibrium dissociation constants (K(d)s) of 2.71 and 2.85 nm, respectively, and low affinity for R5020, characteristics typical of mPRs. P4 treatment increased cAMP production as well as [(35)S]-guanosine 5'-triphosphate (GTP)γS binding to transfected cell membranes, which was immunoprecipitated with a stimulatory G protein antibody, suggesting both mPRδ and mPRε activate a stimulatory G protein (Gs), unlike other mPRs, which activate an inhibitory G protein (Gi). All five mPR mRNAs were detected in different regions of the human brain, but mPRδ showed greatest expression in many regions, including the forebrain, hypothalamus, amygdala, corpus callosum, and spinal cord, whereas mPRε was abundant in the pituitary gland and hypothalamus. Allopregnanolone and other neurosteroids bound to mPRδ and other mPRs and acted as agonists, activating second messengers and decreased starvation-induced cell death and apoptosis in mPRδ-transfected cells and in hippocampal neuronal cells at low nanomolar concentrations. The results suggest that mPRδ and mPRε function as mPRs coupled to G proteins and are potential intermediaries of nonclassical antiapoptotic actions of neurosteroids in the central nervous system.
    Endocrinology 11/2012; · 4.72 Impact Factor
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    ABSTRACT: Although several studies have reported the localization of membrane progesterone (P(4)) receptors (mPR) in various tissues, few have attempted to describe the distribution and regulation of these receptors in the brain. In the present study, we investigated expression of two mPR subtypes, mPRα and mPRβ, within regions of the brain, known to express estradiol (E(2))-dependent [preoptic area (POA) and hypothalamus] and independent (cortex) classical progestin receptors. Saturation binding and Scatchard analyses on plasma membranes prepared from rat cortex, hypothalamus, and POA demonstrated high-affinity, specific P(4)-binding sites characteristic of mPR. Using quantitative RT-PCR, we found that mPRβ mRNA was expressed at higher levels than mPRα, indicating that mPRβ may be the primary mPR subtype in the rat brain. We also mapped the distribution of mPRβ protein using immunohistochemistry. The mPRβ-immunoreactive neurons were highly expressed in select nuclei of the hypothalamus (paraventricular nucleus, ventromedial hypothalamus, and arcuate nucleus), forebrain (medial septum and horizontal diagonal band), and midbrain (oculomotor and red nuclei) and throughout many areas of the cortex and thalamus. Treatment of ovariectomized female rats with E(2) benzoate increased mPRβ immunoreactivity within the medial septum but not the medial POA, horizontal diagonal band, or oculomotor nucleus. Together, these findings demonstrate a wide distribution of mPRβ in the rodent brain that may contribute to functions affecting behavioral, endocrine, motor, and sensory systems. Furthermore, E(2) regulation of mPRβ indicates a mechanism through which estrogens can regulate P(4) function within discrete brain regions to potentially impact behavior.
    Endocrinology 07/2012; 153(9):4432-43. · 4.72 Impact Factor
  • Peter Thomas, Yefei Pang
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    ABSTRACT: Membrane progesterone receptors (mPRs) are novel G protein-coupled receptors belonging to the progestin and adipoQ receptor family (PAQR) that mediate a variety of rapid cell surface-initiated progesterone actions in the reproductive system involving activation of intracellular signaling pathways (i.e. nonclassical actions). The mPRs are highly expressed in the brain, but research on their neural functions has only been conducted in a single neuronal cell line, GT1-7 cells, which have negligible nuclear progesterone receptor (PR) expression. GT1-7 cells express mPRα and mPRβ on their plasma membranes which is associated with the presence of high-affinity, specific [(3)H]-progesterone receptor binding. The neurosteroid, allopregnanolone, is an effective ligand for recombinant mPRα with a relative binding affinity of 7.6% that of progesterone. Allopregnanolone acts as a potent mPR agonist on GT1-7 cells, mimicking the progesterone-induced decrease in cAMP accumulation and its antiapoptotic actions at low nanomolar concentrations. The decrease in cAMP levels is associated with rapid progesterone-induced downregulation of GnRH pulsatile secretion from perifused GT1-7 cells. The recent suggestion that mPRs are alkaline ceramidases and mediate sphingolipid signaling is not supported by empirical evidence that TNFα does not bind to mPRs overexpressed in human cells and that exogenous sphingomyelinase is ineffective in mimicking progestin actions through mPRs to induce meiotic maturation of fish oocytes. Taken together, these recent studies indicate that mPRs mediate neuroprotective effects of progesterone and allopregnanolone and are also the likely intermediaries in progesterone-induced inhibition of pulsatile GnRH secretion in GT1-7 cells.
    Neuroendocrinology 06/2012; 96(2):162-71. · 3.54 Impact Factor
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    ABSTRACT: Oocyte maturation (OM) in goldfish is induced by the maturation inducing hormone (MIH) via its membrane receptor. Previously, we described the cloning of the membrane progesterone receptor alpha (mPRα or paqr7b) cDNA from a goldfish ovarian cDNA library and obtained experimental evidence that the mPRα protein is an intermediary in MIH induction of OM in goldfish. Three mPR subtypes have been identified in fish by cDNA cloning or by in silico analysis of genome sequence databases. In order to investigate the potential roles of the mPR subtypes in oocyte maturation, we cloned additional mPRs from a goldfish ovarian cDNA library. RACE amplification, and screening of the cDNA library identified one β (paqr8) and two γ subtypes (paqr5) (hereafter referred to as γ-1 and γ-2), respectively. Tissue distribution of mPR subtypes showed differential expression pattern. However, in addition to mPRα, the β, γ-1 and γ-2 subtypes were also expressed in follicle-enclosed oocytes. Cell lines expressing the β, γ-1 and γ-2 genes were established and their steroid binding properties compared. The β subtype exhibited higher binding affinity than the γ subtypes for 17,20β-DHP, the MIH in goldfish. Microinjection of goldfish oocytes with a morpholino antisense oligonucleotide to mPRβ blocked the induction of oocyte maturational competence, whereas injection of antisense oliogonucleotides to mPRγ-1 and γ-2 were ineffective. These results suggest that the goldfish mPRβ protein acts as an intermediary during MIH induction of OM in goldfish, in a manner similar to that described previously for mPRα.
    General and Comparative Endocrinology 03/2012; 177(1):168-76. · 2.82 Impact Factor
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    ABSTRACT: Currently, preterm labour is associated with increased fetal mortality and morbidity and is often associated with elevated levels of inflammatory cytokines. However, the exact mechanisms that lead to this pathology are not fully elucidated. In the present study evidence was obtained using a specific membrane progesterone receptor (mPR) agonist, Org OD 02-0, that the progestin antagonism of apoptotic effects of a cytokine, IL-1β, on human placental BeWo cells is mediated through mPRs. Therefore the aim of this study was to determine whether the gene expression of mPRs and all other known progesterone receptors changes in human placentas at term and during labour. Quantitative PCR (qPCR) in clinical samples revealed a 2.8 fold decrease of mPRβ in labouring comparing to non-labouring tissues and 4.6 fold higher levels of mPRγ in preterm mPRγ compared to term placentas. The ratio of mPRα to PR-B was increased in term compared to preterm samples, whereas it was decreased in labour compared to non-labour placentas. There was also a high correlation between mPRα and PGRMC1 expression irrespective of pathologies. Collectively, our data indicates that changes in the ratios of progesterone receptors rather than individual fluctuations might affect progesterone signalling at the placental level.
    Placenta 02/2012; 33(5):367-72. · 3.12 Impact Factor
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    ABSTRACT: Membrane progesterone receptors (mPRs) have been detected in breast cancer cells and tissues, but their roles in cancer progression remain unclear. Here, we demonstrate the localization, signaling, and antiapoptotic actions of mPRs in two nuclear progesterone receptor (PR)-negative breast cancer cell lines, SKBR3 and MDA-MB-468 (MB468), and mPR expression in human breast tumor biopsies. mPRα, mPRβ, and mPRγ subtypes were detected in both cell lines as well as in breast tumor tissues from 13 individuals irrespective of nuclear steroid receptor expression. Competitive receptor binding studies with a selective PR ligand, R5020, and an mPR agonist, Org OD 02-0 confirmed the presence of functional mPRs on both cancer cell lines. Progesterone treatment of either cell line caused rapid activation of an inhibitory G protein, as well as activation of p42/44 MAP kinase. Treatment with progesterone or Org OD 02-0 significantly decreased cell death and apoptosis in response to serum starvation, whereas testosterone, 17β-estradiol, dexamethasone, and R5020 and RU486 were ineffective. Progesterone treatment of MB468 cells also increased mitochondrial membrane potential and Akt activity, but no decrease in caspase 3 activity was observed. Knockdown of mPRα expression in MB468 cells by siRNA transfection blocked the inhibitory effects of progesterone on cell death. The results indicate that progesterone can act through mPRs to inhibit apoptosis in breast cancer cells. The involvement of mPRs in the development or progression of breast tumor growth through inhibition of cell death is an intriguing possibility and requires further investigation.
    Hormones and Cancer 02/2012; 3(3):101-12.
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    ABSTRACT: The endocrine pancreas has emerged as a target for estrogens. The functions of pancreatic α-, β- and δ-cells are modulated by the endogenous hormone, 17β-estradiol (E2). Low physiological concentrations (100pM-1nM) of E2 rapidly decrease the activity of the ATP-sensitive potassium channel (K(ATP)) and enhance glucose-induced insulin release in β-cells in an estrogen receptor β (ERβ)-dependent manner. In addition to the insulinotropic action of ERβ, the newly described estrogen receptor, GPR30, is involved in the insulinotropic effects of high doses of E2 (100nM-5μM). The specific GPR30 agonist G1 also increases insulin secretion in β-cells. Low glucose-induced calcium oscillations and glucagon secretion are suppressed by E2. The effects on glucagon secretion may be mediated by GPR30. Somatostatin release is also decreased by E2 and G1. In this review we summarize all the data published up to date on the rapid insulinotropic effects of estrogens in the endocrine pancreas and propose a model to integrate the estrogen actions mediated through both receptors.
    Steroids 01/2012; 77(10):951-8. · 2.80 Impact Factor
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    Yefei Pang, Peter Thomas
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    ABSTRACT: The functional characteristics of membrane progesterone receptors (mPRs) have been investigated using recombinant mPR proteins over-expressed in MDA-MB-231 breast cancer cells. Although these cells do not express the full-length progesterone receptor (PR), it is not known whether they express N-terminally truncated PR isoforms which could possibly account for some progesterone receptor functions attributed to mPRs. In the present study, the presence of N-terminally truncated PR isoforms was investigated in untransfected and mPR-transfected MDA-MB-231 cells, and in MDA-MB-468 breast cancer cells. PCR products were detected in PR-positive T47D Yb breast cancer cells using two sets of C-terminus PR primers, but not in untransfected and mPR-transfected MDA-MB-231 cells, nor in MDA-MB-468 cells. Western blot analysis using a C-terminal PR antibody, 2C11F1, showed the same distribution pattern for PR in these cell lines. Another C-terminal PR antibody, C-19, detected immunoreactive bands in all the cell lines, but also recognized α-actinin, indicating that the antibody is not specific for PR. High affinity progesterone receptor binding was identified on plasma membranes of MDA-MB-468 cells which was significantly decreased after treatment with siRNAs for mPRα and mPRβ. Plasma membranes of MDA-MB-468 cells showed very low binding affinity for the PR agonist, R5020, ≤1% that of progesterone, which is characteristic of mPRs. Progesterone treatment caused G protein activation and decreased production of cAMP in MDA-MB-468 cells, which is also characteristic of mPRs. The results indicate that the progestin receptor functions in these cell lines are mediated through mPRs and do not involve any N-terminally truncated PR isoforms.
    Steroids 02/2011; 76(9):921-8. · 2.80 Impact Factor
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    ABSTRACT: Although there is substantial evidence that membrane progestin receptors (mPRs) perform a critical physiological role in meiotic maturation of fish oocytes, it is unknown whether they are also intermediaries in progestin signaling in the surrounding follicular cells. Here, we show that mPRα protein is located on the plasma membranes of both granulosa and theca cells (G/T cells) isolated from Atlantic croaker ovaries and is associated with the presence of a single high affinity, limited capacity, pertussis toxin-sensitive, specific progestin [17,20β,21-trihydroxy-4-pregnen-3-one (20β-S)] membrane binding site with the characteristics of mPRα. Treatment of G/T cells with 20β-S caused rapid G protein activation and a transient, pertussis toxin-sensitive, decrease in cAMP levels, whereas the selective nuclear progesterone receptor agonist, R5020, did not cause G protein activation, consistent with previous reports on mPRα signaling. 20β-S treatment decreased serum starvation-induced cell death in both G/T cells and in seatrout mPRα-transfected MDA-MB-231 cells, whereas R5020 was ineffective. Moreover, a selective mPRα agonist, 10-ethenyl-19-norprogesterone, mimicked the protective action of 20β-S against cell death, which was lost upon knockdown of mPRα protein but not after progesterone receptor knockdown, further demonstrating an involvement of mPRα. Signaling molecules involved in inhibition of apoptosis, Erk and serine-threonine kinase, were activated in G/T cells by 20β-S, which suggests a potential mechanism for mPRα inhibition of apoptosis. This is the first study to demonstrate endogenous mPR signaling in the ovarian follicle and to suggest a novel physiological role for mPRα in mediating the antiapoptotic actions of progestins in ovarian follicle cells.
    Endocrinology 10/2010; 151(12):5916-26. · 4.72 Impact Factor
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    Yefei Pang, Peter Thomas
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    ABSTRACT: Estrogen inhibition of oocyte maturation (OM) and the role of GPER (formerly known as GPR30) were investigated in zebrafish. Estradiol-17beta (E2) and G-1, a GPER-selective agonist, bound to zebrafish oocyte membranes suggesting the presence of GPER which was confirmed by immunocytochemistry using a specific GPER antibody. Incubation of follicle-enclosed oocytes with an aromatase inhibitor, ATD, and enzymatic and manual removal of the ovarian follicle cell layers significantly increased spontaneous OM which was partially reversed by co-treatment with either 100 nM E2 or G-1. Incubation of denuded oocytes with the GPER antibody blocked the inhibitory effects of estrogens on OM, whereas microinjection of estrogen receptor alpha (ERalpha) antisense oligonucleotides into the oocytes was ineffective. The results suggest that endogenous estrogens produced by the follicle cells inhibit or delay spontaneous maturation of zebrafish oocytes and that this estrogen action is mediated through GPER. Treatment with E2 and G-1 also attenuated the stimulatory effect of the teleost maturation-inducing steroid, 17,20beta-dihyroxy-4-pregnen-3-one (DHP), on OM. Moreover, E2 and G-1 down-regulated the expression of membrane progestin receptor alpha (mPRalpha), the intermediary in DHP induction of OM. Conversely DHP treatment caused a >50% decline in GPER mRNA levels. The results suggest that estrogens and GPER are critical components of the endocrine system controlling the onset of OM in zebrafish. A model is proposed for the dual control of the onset of oocyte maturation in teleosts by estrogens and progestins acting through GPER and mPRalpha, respectively, at different stages of oocyte development.
    Developmental Biology 04/2010; 342(2):194-206. · 3.87 Impact Factor
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    ABSTRACT: Recent results showing that the binding characteristics of 33 steroids for human membrane progesterone receptor alpha (hu-mPRalpha) differ from those for the nuclear progesterone receptor (nPR) suggest that hu-mPRalpha-specific agonists can be identified for investigating its physiological functions. The binding affinities of an additional 21 steroids for hu-mPRalpha were determined to explore the structure-activity relationships in more detail and to identify potent, specific mPRalpha agonists. Four synthetic progesterone derivatives with methyl or methylene groups on positions 18 or 19, 18a-methylprogesterone (18-CH(3)P4, Org OE 64-0), 13-ethenyl-18-norprogesterone (18-CH(2)P4, Org 33663-0), 19a-methylprogesterone (19-CH(3)P4, Org OD 13-0) and 10-ethenyl-19-norprogesterone (19-CH(2)P4, Org OD 02-0), showed similar or higher affinities than progesterone for hu-mPRalpha and displayed mPRalpha agonist activities in G-protein and MAP kinase activation assays. All four steroids also bound to the nPR in cytosolic fractions of MCF-7 cells. However, two compounds, 19-CH(2)P4 and 19-CH(3)P4, showed no nPR agonist activity in a nPR reporter assay and therefore are selective mPRalpha agonists suitable for physiological investigations. The structure-binding relationships of the combined series of 54 steroids for hu-mPRalpha deviated strikingly from those of a published set of 60 3-keto or 3-desoxy steroids for nPR. Close correlations were observed between the receptor binding affinities of the steroids and their physicochemical properties calculated by comparative molecular field analysis (CoMFA) for both hu-mPRalpha and nPR. A comparison of the CoMFA field graphs for the two receptors revealed several differences in the structural features required for binding to hu-mPRalpha and nPR which could be exploited to develop additional mPR-specific ligands.
    Steroids 04/2010; 75(4-5):314-22. · 2.80 Impact Factor
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    ABSTRACT: The recent molecular cloning of membrane receptors for progesterone (mPRs) has tremendous implications for understanding the multiple actions of the hormone in the nervous system. The three isoforms which have been cloned from several species, mPRalpha, mPRbeta and mPRgamma, have seven-transmembrane domains, are G protein-coupled and may thus account for the rapid modulation of many intracellular signaling cascades by progesterone. However, in order to elucidate the precise functions of mPRs within the nervous system it is first necessary to determine their expression patterns and also to develop new pharmacological and molecular tools. The aim of the present study was to profile mPR expression in the mouse spinal cord, where progesterone has been shown to exert pleiotropic actions on neurons and glial cells, and where the hormone can also be locally synthesized. Our results show a wide distribution of mPRalpha, which is expressed in most neurons, astrocytes, oligodendrocytes, and also in a large proportion of NG2(+) progenitor cells. This mPR isoform is thus likely to play a major role in the neuroprotective and promyelinating effects of progesterone. On the contrary, mPRbeta showed a more restricted distribution, and was mainly present in ventral horn motoneurons and in neurites, consistent with an important role in neuronal transmission and plasticity. Interestingly, mPRbeta was not present in glial cells. These observations suggest that the two mPR isoforms mediate distinct and specific functions of progesterone in the spinal cord. A significant observation was their very stable expression, which was similar in both sexes and not influenced by the presence or absence of the classical progesterone receptors. Although mPRgamma mRNA could be detected in spinal cord tissue by reverse transcriptase-polymerase chain reaction (RT-PCR), in situ hybridization analysis did not allow us to verify and to map its presence, probably due to its relatively low expression. The present study is the first precise map of the regional and cellular distribution of mPR expression in the nervous system, a prior requirement for in vivo molecular and pharmacological strategies aimed to elucidate their precise functions. It thus represents a first important step towards a new understanding of progesterone actions in the nervous system within a precise neuroanatomical context.
    Neuroscience 12/2009; 166(1):94-106. · 3.12 Impact Factor
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    ABSTRACT: Recent studies by several research groups have shown that G protein estrogen receptor-1 (GPER) formerly known as GPR30, mediates 17beta-estradiol (E2) activation of signal transduction pathways in a variety of human cancer cells and displays E2 binding typical of a membrane estrogen receptor. However, the importance of GPER as an estrogen receptor has been questioned by Otto and co-workers. Some of the pitfalls in investigating the functions of recombinant steroid membrane receptors that may explain the negative results of these investigators are discussed. The characteristics of GPER have also been investigated in a teleost fish, Atlantic croaker, where it has been shown to mediate E2 inhibition of oocyte maturation. Investigations on newly discovered homologous proteins from distantly related vertebrate groups are valuable for determining their fundamental, evolutionarily conserved functions. Therefore, the functions of croaker and human GPERs were compared. The comparisons show that croaker and human GPER have very similar estrogen binding characteristics, typical of estrogen membrane receptors, and activate the same estrogen signaling pathways via stimulatory G proteins (Gs) resulting in increased cAMP production. These results suggest that the estrogen binding and estrogen signaling functions of GPER arose early in vertebrate evolution, prior to the divergence of the teleosts from the tetrapods, more than 200 million years ago. The finding that estrogen membrane signaling through GPER has been conserved for such a long period in two distantly related vertebrate groups, mammals and fish, suggests that this is a fundamental function of GPER in vertebrates, and likely its major physiological role.
    Steroids 11/2009; 75(8-9):595-602. · 2.80 Impact Factor
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    ABSTRACT: The zebrafish nuclear progestin receptor (nPR; official symbol PGR) was identified and characterized to better understand its role in regulating reproduction in this well-established teleost model. A full-length cDNA was identified that encoded a 617-amino acid residue protein with high homology to PGRs in other vertebrates, and contained five domains characteristic of nuclear steroid receptors. In contrast to the multiplicity of steroid receptors often found in euteleosts and attributed to probable genome duplication, only a single locus encoding the full-length zebrafish pgr was identified. Cytosolic proteins from pgr-transfected cells showed a high affinity (K(d) = 2 nM), saturable, single-binding site specific for a native progestin in euteleosts, 4-pregnen-17,20 beta-diol-3-one (17,20 beta-DHP). Both 17,20 beta-DHP and progesterone were potent inducers of transcriptional activity in cells transiently transfected with pgr in a dual luciferase reporter assay, whereas androgens and estrogens had little potency. The pgr transcript and protein were abundant in the ovaries, testis, and brain and were scarce or undetectable in the intestine, muscle, and gills. Further analyses indicate that Pgr was expressed robustly in the preoptic region of the hypothalamus in the brain; proliferating spermatogonia and early spermatocytes in the testis; and in follicular cells and early-stage oocytes (stages I and II), with very low levels within maturationally competent late-stage oocytes (IV) in the ovary. The localization of Pgr suggests that it mediates progestin regulation of reproductive signaling in the brain, early germ cell proliferation in testis, and ovarian follicular functions, but not final oocyte or sperm maturation.
    Biology of Reproduction 10/2009; 82(1):112-22. · 4.03 Impact Factor
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    ABSTRACT: Progesterone's (P4) negative feedback actions in the female reproductive axis are exerted in part by suppression of hypothalamic GnRH release. Here we show that P4 can inhibit GnRH release by a mechanism independent of a nuclear P4 receptor (PR(A/B)). Injections of P4, but not vehicle, allopregnanolone, or dexamethasone, acutely suppressed LH levels in both wild-type and P4 receptor knockout ovariectomized mice; pituitary responsiveness to GnRH was retained during P4 treatment, indicating a hypothalamic action. Superfusion of GnRH-producing GT1-7 cells with medium containing 10(-7) m P4 produced a rapid reduction in GnRH release. Incubation with P4 (10(-9) to 10(-7) M) inhibited forskolin-stimulated cAMP accumulation; cotreatment with pertussis toxin prevented this effect. Treatment of GT1-7 cell membranes with P4 caused activation of an inhibitory G protein (G(i)), as shown by immunoprecipitation with a G(i) antibody of most of the increase in membrane-bound [(35)S]GTPgamma-S. Saturation binding analyses demonstrated the presence of a high affinity (K(d) 5.85 nM), limited capacity (Bmax 62.2 nM) binding site for P4. RT-PCR analysis revealed the presence of mRNAs encoding both isoforms of the membrane P4 receptors, mPRalpha and mPRbeta. Western blotting, immunocytochemistry, and flow cytometry experiments similarly revealed expression of mPR proteins in the plasma membranes of GT1-7 cells. Treatment with mPRalpha siRNA attenuated specific P4 binding to GT1-7 cell membranes and reversed the P4 inhibition of cAMP accumulation. Taken together, our results suggest that negative feedback actions of P4 include rapid PR(A/B)-independent effects on GnRH release that may in part be mediated by mPRs.
    Endocrinology 06/2009; 150(8):3833-44. · 4.72 Impact Factor
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    Yefei Pang, Peter Thomas
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    ABSTRACT: The orphan G protein coupled receptor, GPR30, has the characteristics of a high affinity, specific estrogen membrane receptor on Atlantic croaker oocytes and mediates estrogen inhibition of oocyte maturation in this perciform fish. In order to determine the broad applicability of these findings to other teleosts, similar experiments were conducted in a cyprinid fish, zebrafish, in the present study. GPR30 mRNA expression was detected in zebrafish oocytes but not in the ovarian follicular cells. Both spontaneous and 17, 20beta-dihyroxy-4-pregnen-3-one (DHP)-induced maturation of follicle-enclosed zebrafish oocytes was significantly decreased when they were incubated with either estradiol-17beta, or the GPR30 agonists, ICI 182 780 and tamoxifen, or with the GPR30 specific agonist G-1. On the other hand spontaneous oocyte maturation increased two-fold when zebrafish ovarian follicles were incubated with an aromatase inhibitor, ATD. Moreover, the stimulatory effects of ATD on germinal vesicle breakdown (GVBD) were partially reversed by co-treatment with 100 nM of E2 or G-1. These results suggest that endogenous estrogens acting through GPR30 are involved in maintaining meiotic arrest of zebrafish oocytes.
    General and Comparative Endocrinology 11/2008; 161(1):58-61. · 2.82 Impact Factor
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    Yefei Pang, Jing Dong, Peter Thomas
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    ABSTRACT: Human G protein-coupled receptor 30 (GPR30) mediates estradiol-17beta (E2) activation of adenylyl cyclase in breast cancer cells and displays E2 binding typical of membrane estrogen receptors (mERs). We identified a mER in Atlantic croaker ovaries with characteristics similar to those of human GPR30. To confirm the proposed role of GPR30 as a mER in this distantly related vertebrate group, we cloned GPR30 from croaker ovaries and examined its distribution, steroid binding, and signaling characteristics. Western blot analysis showed the GPR30 protein (approximately 40 kDa) is expressed on the plasma membranes of croaker oocytes and HEK293 cells stably transfected with GPR30 cDNA. Plasma membranes prepared from croaker GPR30-transfected cells displayed high-affinity, limited-capacity, and displaceable binding specific for estrogens, characteristic of mERs. Consistent with previous findings with human GPR30, estrogen treatment of plasma membranes from both croaker ovaries and GPR30-transfected cells caused activation of a stimulatory G protein (Gs) resulting in increased cAMP production. Treatment with E2 as well as G-1, a specific GPR30 ligand, significantly reduced both spontaneous and progestin-induced maturation of both croaker and zebrafish oocytes in vitro, suggesting a possible involvement of GPR30 in maintaining oocyte meiotic arrest in these species. Injection of antisense oligonucleotides to GPR30 into zebrafish oocytes blocked the inhibitory effects of estrogen on oocyte maturation, confirming a role for GPR30 in the control of meiotic arrest. These findings further support our previous suggestion that GPR30 is a vertebrate mER. In addition, the results suggest GRP30 may play a critical role in regulating reentry into the meiotic cell cycle in fish oocytes.
    Endocrinology 08/2008; 149(7):3410-26. · 4.72 Impact Factor

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2k Citations
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Institutions

  • 2003–2013
    • University of Texas at Austin
      • Marine Science Institute
      Austin, TX, United States
  • 2012
    • Shizuoka University
      • Faculty of Science
      Japan
  • 2010
    • Radboud University Nijmegen
      Nymegen, Gelderland, Netherlands
  • 2006
    • The University of Warwick
      • Clinical Sciences
      Warwick, ENG, United Kingdom