Membrane estrogen receptor α is essential for estrogen signaling in the male skeleton.

The importance of estrogen receptor α (ERα) for the regulation of bone mass in males is well established. ERα mediates estrogenic effects both via nuclear and membrane-initiated ERα (mERα) signaling. The role of mERα signaling for the effects of estrogen on bone in male mice is unknown. To investigate the role of mERα signaling, we have used mice (Nuclear-Only-ER; NOER) with a point mutation (C451A), which results in inhibited trafficking of ERα to the plasma membrane. Gonadal-intact male NOER mice had a significantly decreased total body areal bone mineral density (aBMD) compared to WT littermates at 3, 6 and 9 months of age as measured by dual-energy X-ray absorptiometry (DEXA). High-resolution microcomputed tomography (µCT) analysis of tibia in 3-month-old males demonstrated a decrease in cortical and trabecular thickness in NOER mice compared to WT littermates. As expected, estradiol (E2) treatment of orchidectomized (ORX) WT mice increased total body aBMD, trabecular BV/TV and cortical thickness in tibia compared to placebo treatment. E2 treatment increased these skeletal parameters also in ORX NOER mice. However, the estrogenic responses were significantly decreased in ORX NOER mice compared with ORX WT mice. In conclusion, mERα is essential for normal estrogen signaling in both trabecular and cortical bone in male mice. Increased knowledge of estrogen signaling mechanisms in the regulation of the male skeleton may aid in the development of new treatment options for male osteoporosis.

The role of membrane ERα signaling in bone and other major estrogen responsive tissues.

Estrogen receptor α (ERα) signaling leads to cellular responses in several tissues and in addition to nuclear ERα-mediated effects, membrane ERα (mERα) signaling may be of importance. To elucidate the significance, in vivo, of mERα signaling in multiple estrogen-responsive tissues, we have used female mice lacking the ability to localize ERα to the membrane due to a point mutation in the palmitoylation site (C451A), so called Nuclear-Only-ER (NOER) mice. Interestingly, the role of mERα signaling for the estrogen response was highly tissue-dependent, with trabecular bone in the axial skeleton being strongly dependent (>80% reduction in estrogen response in NOER mice), cortical and trabecular bone in long bones, as well as uterus and thymus being partly dependent (40-70% reduction in estrogen response in NOER mice) and effects on liver weight and total body fat mass being essentially independent of mERα (<35% reduction in estrogen response in NOER mice). In conclusion, mERα signaling is important for the estrogenic response in female mice in a tissue-dependent manner. Increased knowledge regarding membrane initiated ERα actions may provide means to develop new selective estrogen receptor modulators with improved profiles.

Tamoxifen regulates cell fate through mitochondrial estrogen receptor beta in breast cancer.

Tamoxifen (TAM) has both cytostatic and cytotoxic properties for breast cancer. TAM engaged mitochondrial estrogen receptor beta (ERß) as an antagonist in MCF7-BK cells, increasing reactive oxygen species (ROS) concentrations from the mitochondria that were required for cytotoxicity. In part, this derived from TAM downregulating manganese superoxide dismutase (MnSOD) activity by causing the nitrosylation of tyrosine 34, thereby increasing ROS. ROS-activated protein kinase C delta and c-jun N-terminal kinases, resulting in the mitochondrial translocation of Bax and cytochrome C release. Interestingly, TAM failed to cause high ROS levels or induce cell death in MCF7-BK-TR cells due to stimulation of MnSOD activity through agonistic effects at mitochondrial ERß. In several mouse xenograft models, lentiviral shRNA-induced knockdown of MnSOD caused tumors that grew in the presence of TAM to undergo substantial apoptosis. Tumor MnSOD and mitochondrial ERß are therefore targets for therapeutic intervention to reverse TAM resistance and enhance a cell death response.

Cell localization, physiology, and nongenomic actions of estrogen receptors.

The existence of binding proteins for the female sex steroid, 17beta-estradiol, has been known for almost 50 years. Presently, two estrogen receptors (ERs), ER-alpha and ER-beta, have been cloned in mammals, and they are expressed in many cell types of metazoans. ERs act primarily as nuclear transcription factors, and this effect is enhanced by ligand binding. Emerging data have identified a separate pool of receptors for this steroid in the plasma membrane, but the mechanisms of action and cellular functions of these proteins are just beginning to be defined. In this review, the known details of the nuclear and plasma membrane ER functions will be discussed. A particular focus will be to define the signaling pathways from the membrane that lead to important cell physiology effects of estrogen. The potential interactions of membrane ER with other local proteins will also be discussed, and the unique but often complementary roles of the receptor pools will be highlighted. These details may be of additional relevance to other steroid receptors, since there is evidence of their existence in the cell membrane.

Rapid actions of plasma membrane estrogen receptors.

Functional evidence for the existence of plasma membrane estrogen receptors in a variety of cell types continues to accumulate. Many of these functions originate from rapid signaling events, transduced in response to 17beta-estradiol (E(2)). It has been convincingly shown that E(2) activates phosphoinositol 3-kinase and protein kinase B/AKT, and stimulates ERK and p38 MAP kinases. In part, this stems from G-protein activation and the resulting calcium flux. As a result, the link between E(2) action at the cell membrane and discrete biological actions in the cell has been strengthened. There is now convincing in vitro evidence that E(2) can modulate the functions of neural and vascular cells via non-genomic actions. Thus, the actions of discrete pools of E(2) receptors are likely to contribute to the overall effects of the sex steroids.

Natriuretic peptides suppress vascular endothelial cell growth factor signaling to angiogenesis.

Vascular endothelial cell growth factor (VEGF) is essential for angiogenesis. Atrial natriuretic peptide (ANP) inhibits the production of VEGF, but whether this important vascular peptide also inter- rupts VEGF signaling to angiogenesis is unknown. In cultured bovine aortic endothelial cells, VEGF significantly stimulated extracellular signal-regulated protein kinase activity and phosphorylation, which was inhibited 60% by coincubation with ANP or a natriuretic peptide clearance receptor specific ligand (NPRC), C-type NAP-(4-23) [C-ANP-(4-23)]. VEGF also stimulated c-Jun N-terminal kinase (JNK) and p38 activities/phosphorylation that were prevented by the two natriuretic peptides (NP). A specific NP guanylate cyclase (GC) receptor antagonist, HS-142-1, blocked the actions of ANP [but not those of C-ANP-(4-23)], supporting the involvement of both GC and NPRC receptors. VEGF and expression of constituitively active JNK each stimulated the synthesis of cyclin D1 and increased the activity of the cyclin-dependent kinase-4, which was inhibited 55% by ANP. VEGF induced endothelial cell proliferation and migration, which was significantly blocked by NP or by expressing a dominant negative JNK-1. VEGF stimulated human microvascular endothelial cells to form capillary tubes, which was significantly inhibited by expressing dominant negative JNK-1 and by NP. Therefore, VEGF induction of critical steps in angiogenesis is enhanced through JNK activation. The actions are significantly prevented by NP, which act through both the NPRC and GC receptors to block growth factor signaling. Thus, NP are candidate antiangiogenesis factors that inhibit both the synthesis and function of VEGF.

Plasma membrane estrogen receptors signal to antiapoptosis in breast cancer.

Chemotherapy or irradiation treatment induces breast cancer cell apoptosis, but this can be limited by estradiol (E2) through unknown mechanisms. To investigate this, we subjected estrogen receptor-expressing human breast cancer cells (MCF-7 and ZR-75-1) to paclitaxel (taxol) or to UV irradiation. Marked increases in cell apoptosis were induced, but these were significantly reversed by incubation with E2. Taxol or UV stimulated c-Jun N-terminal kinase (JNK) activity, which was inhibited by E2. Expression of a dominant-negative Jnk-1 protein strongly prevented taxol- or UV-induced apoptosis, whereas E2 inhibition of apoptosis was reversed by expression of constituitively active Jnk-1. As targets for participation in apoptosis, Bcl-2 and Bcl-xl were phosphorylated in response to JNK activation by taxol or UV; this was prevented by E2. Taxol or UV activated caspase activity in a JNK-dependent fashion and caused the cleavage of procaspase-9 to caspase-9, each inhibited by E2. Independently, the steroid also activated extracellular signal-regulated protein kinase activity, which contributed to the antiapoptotic effects. We report novel and rapid mechanisms by which E2 prevents chemotherapy or radiation-induced apoptosis of breast cancer, probably mediated through the plasma membrane estrogen receptor.

Estrogen signals to the preservation of endothelial cell form and function.

Estrogen is important for the primary prevention of vascular disease in young women, but the mechanisms of protection at the vascular cell are still largely unknown. Although traditionally thought of as a nuclear transcription factor, the estrogen receptor has also been identified in the cell plasma membrane to signal but serve largely undefined roles. Here we show that estradiol (E2) rapidly activates p38beta mitogen-activated protein kinase in endothelial cells (EC), which activates the mitogen-activated protein kinase-activated protein kinase-2 and the phosphorylation of heat shock protein 27. The sex steroid preserves the EC stress fiber formation and actin and membrane integrity in the setting of metabolic insult. E2 also prevents hypoxia-induced apoptosis and induces both the migration of EC and the formation of primitive capillary tubes. These effects are reversed by the inhibition of p38beta, by the expression of a dominant-negative mitogen-activated protein kinase-activated protein kinase-2 protein, or by the expression of a phosphorylation site mutant heat shock protein 27. E2 signaling from the membrane helps preserve the EC structure and function, defining potentially important vascular-protective effects of this sex steroid.

Nuclear receptor versus plasma membrane oestrogen receptor.

The co-existence of both plasma membrane and nuclear oestrogen receptors has changed our thinking about the mechanisms of the actions of this sex steroid. To date, however, the plasma membrane receptor has not been isolated. However, many emerging data implicate this receptor in the rapid, non-genomic effects of oestrogen, and this is seen when the membrane receptor effects a variety of signal transduction events. Although discrete actions of oestradiol could be mediated through the plasma membrane receptor, there is probably often a coordination of effects mediated through both receptors, perpetuating and magnifying the cell biological effects of the steroid.

Natriuretic peptides inhibit G protein activation. Mediation through cross-talk between cyclic GMP-dependent protein kinase and regulators of G protein-signaling proteins.

Atrial natriuretic peptide (ANP) inhibits the proliferation of many cells, in part through interfering with signal transduction enacted by G protein-coupled growth factor receptors. Signaling interactions between ANP and the G protein-coupled growth factor receptor ligand, endothelin-3 (ET-3), regulate astrocyte proliferation at a very proximal but undefined point. Here, we find that ANP inhibits the ability of ET-3 to activate Galpha(q) and Galpha(i) in these cells. ANP stimulated the translocation of endogenous regulators of G protein-signaling (RGS) proteins 3 and 4 from the cytosol to the cell membrane, and enhanced their association with Galpha(q) and Galpha(i). ANP effects were significantly blocked by HS-142-1, an inhibitor of guanylate cyclase activation, or by ET-3. KT5823, an inhibitor of cyclic GMP-dependent protein kinase (PKG) reversed the RGS translocation induced by ANP; conversely, expression of an active catalytic subunit of PKG-I, or 8-bromo-cyclic GMP stimulated RGS translocation. ANP caused the phosphorylation of both RGS proteins in a PKG-dependent fashion, and the expressed PKG (in the absence of ANP) also stimulated RGS phosphorylation. A novel cross-talk between PKG and RGS proteins is stimulated by ANP and leads to the increased translocation and association of RGS proteins with Galpha. The rapid inactivation of G proteins provides a mechanism by which ANP inhibits downstream signaling to the cell proliferation program.

Cell membrane and nuclear estrogen receptors (ERs) originate from a single transcript: studies of ERalpha and ERbeta expressed in Chinese hamster ovary cells.

The existence of a putative membrane estrogen receptor (ER) has been supported by studies accomplished over the past 20 yr. However, the origin and functions of this receptor are not well defined. To study the membrane receptor, we transiently transfected cDNAs for ERalpha or ERbeta into Chinese hamster ovary (CHO) cells. Transfection of ERalpha resulted in a single transcript by Northern blot, specific binding of labeled 17beta-estradiol (E2), and expression of ER in both nuclear and membrane cell fractions. Competitive binding studies in both compartments revealed near identical dissociation constants (K(d)S) of 0.283 and 0.287 nM, respectively, but the membrane receptor number was only 3% as great as the nuclear receptor density. Transfection of ERbeta3 also yielded a single transcript and nuclear and membrane receptors with respective Kd values of 1.23 and 1.14 nM; the membrane receptor number was only 2% compared with expressed nuclear receptors. Estradiol binding to CHO-ERalpha or CHO-ERbeta activated Galphaq and G(alpha)s proteins in the membrane and rapidly stimulated corresponding inositol phosphate production and adenylate cyclase activity. Binding by 17-beta-E2 to either expressed receptor comparably enhanced the nuclear incorporation of thymidine, critically dependent upon the activation of the mitogen-activated protein kinase, ERK (extracellular regulated kinase). In contrast, c-Jun N-terminal kinase activity was stimulated by 17-beta-E2 in ERbeta-expressing CHO, but was inhibited in CHO-ERalpha cells. In summary, membrane and nuclear ER can be derived from a single transcript and have near-identical affinities for 17-beta-E2, but there are considerably more nuclear than membrane receptors. This is also the first report that cells can express a membrane ERbeta. Both membrane ERs activate G proteins, ERK, and cell proliferation, but there is novel differential regulation of c-Jun kinase activity by ERbeta and ERalpha.

Role of vascular endothelial cell growth factor in Ovarian Hyperstimulation Syndrome.

Controlled ovarian hyperstimulation with gonadotropins is followed by Ovarian Hyperstimulation Syndrome (OHSS) in some women. An unidentified capillary permeability factor from the ovary has been implicated, and vascular endothelial cell growth/permeability factor (VEGF) is a candidate protein. Follicular fluids (FF) from 80 women who received hormonal induction for infertility were studied. FFs were grouped according to oocyte production, from group I (0-7 oocytes) through group IV (23-31 oocytes). Group IV was comprised of four women with the most severe symptoms of OHSS. Endothelial cell (EC) permeability induced by the individual FF was highly correlated to oocytes produced (r2 = 0.73, P < 0.001). Group IV FF stimulated a 63+/-4% greater permeability than FF from group I patients (P < 0. 01), reversed 98% by anti-VEGF antibody. Group IV fluids contained the VEGF165 isoform and significantly greater concentrations of VEGF as compared with group I (1,105+/-87 pg/ml vs. 353+/-28 pg/ml, P < 0. 05). Significant cytoskeletal rearrangement of F-actin into stress fibers and a destruction of ZO-1 tight junction protein alignment was caused by group IV FF, mediated in part by nitric oxide. These mechanisms, which lead to increased EC permeability, were reversed by the VEGF antibody. Our results indicate that VEGF is the FF factor responsible for increased vascular permeability, thereby contributing to the pathogenesis of OHSS.

Extracellular signal-regulated protein kinase/Jun kinase cross-talk underlies vascular endothelial cell growth factor-induced endothelial cell proliferation.

Ligand binding to vascular endothelial cell growth factor (VEGF) receptors activates the mitogen-activated protein kinases extracellular signal-regulated kinase (ERK) and c-Jun N-terminal protein kinase (JNK). Possible cross-communication of ERK and JNK effecting endothelial cell (EC) actions of VEGF is poorly understood. Incubation of EC with PD 98059, a specific mitogen-activated protein kinase kinase inhibitor, or transfection with Y185F, a dominant negative ERK2, strongly inhibited VEGF-activated JNK. JNK was also activated by ERK2 expression in the absence of VEGF, inhibited 82% by co-transfection with dominant negative SEK-1, indicating upstream activation of JNK by ERK. VEGF-stimulated JNK activity was also reversed by dominant negative SEK-1. Other EC growth factors exhibited similar cross-activation of JNK through ERK. VEGF stimulated the nuclear incorporation of thymidine, reversed 89% by PD 98059 and 72% by Y185F. Dominant negative SEK-1 or JNK-1 also significantly reduced VEGF-stimulated thymidine incorporation. Expression of wild type Jip-1, which prevents JNK nuclear translocation, inhibited VEGF-induced EC proliferation by 75%. VEGF stimulated both cyclin D1 synthesis and Cdk4 kinase activity, inhibited by PD 98059 and dominant negative JNK-1. Important events for VEGF-induced G1/S progression and cell proliferation are enhanced through a novel ERK to JNK cross-activation and subsequent JNK action.

Astrocyte progression from G1 to S phase of the cell cycle depends upon multiple protein interaction.

The proliferation of cultured astrocytes is positively and negatively regulated, respectively, by the endogenous neuropeptides, endothelin-3 (ET-3) and atrial natriuretic peptide (ANP). Here, we determined the important steps for the modulation by ET and ANP of G1 to S phase cell cycle progression. ET-3 stimulated an increased number of fetal rat diencephalic astrocytes to progress through G1/S, and this was blocked significantly by ANP. ET augmented the gene expression and/or protein production of D-type, A and E cyclins, whereas ANP inhibited these events significantly. ET also stimulated the activation of the cyclin-dependent kinases Cdk2, Cdk4, and Cdk6, directed against the retinoblastoma protein pRb, and this was inhibited by as much as 80% by ANP. As an additional mechanism of cell cycle restraint, ANP stimulated the production of multiple cyclin-dependent kinase inhibitory (CKI) proteins, including p16, p27, and p57. This was critical because antisense oligonucleotides to each CKI reversed ANP-induced inhibition of ET-stimulated DNA synthesis by as much as 85%. CKI antisense oligonucleotides also reversed the ANP inhibition of Cdk phosphorylation of pRb. In turn, ET inhibited ANP-stimulated production of the CKIs, thereby promoting cell cycle progression. Specific and changing associations of the CKI with Cdk2 and Cdk4 were stimulated by ANP and inhibited by ET. Our findings identify several mechanisms by which endogenous modulators of astrocyte proliferation can control the G1-S progression and indicate that multiple CKIs are necessary to restrain cell cycle progression in these cells.

Oestrogen and progesterone inhibit the stimulated production of endothelin-1.

Important vascular proteins such as endothelin-1 (ET-1) promote the development of cardiovascular diseases. Oestrogen, and perhaps progesterone, prevent the development of vascular disease in women through incompletely understood cellular mechanisms. We hypothesized that oestradiol or progesterone might regulate the production of ET-1 as a potential novel mechanism. We found that serum and angiotensin II (AII) significantly stimulated ET-1 secretion from cultured bovine aortic endothelial cells, inhibited 50-75% by oestradiol or by progesterone. Serum and AII stimulated ET-1 mRNA levels, inhibited at least 70% by oestradiol and by progesterone. Serum stimulated ET-1 transcription mainly through the first 43 nucleotides of the ET-1 promoter, but oestradiol and progesterone did not inhibit this. In contrast, AII stimulated ET-1 transcription through nucleotides -143 to -98, specifically involving an activator protein-1 (AP-1) site at -102. Oestradiol and progesterone caused a 60-70% inhibition of AII-stimulated wild-type construct -. 143ET-1/CAT activity (CAT is chloramphenicol acyltransferase). AII-stimulation of ET-1 transcription was critically dependent on stimulation of mitogen-activated protein kinase (erk) activity, inhibited by oestradiol and progesterone. In summary, we found that sex steroids inhibit AII-induced erk signalling to the ET-1 transcriptional programme. This novel mechanism of negative transcriptional regulation by oestradiol and progesterone decreases the production of ET-1, potentially contributing to the vascular protective effects of these steroids.

IGF I induction of p53 requires activation of MAP kinase in cardiac muscle cells.

The aim of this study was to investigate whether IGF I induction of p53 expression and p21 promoter require activation of MAP kinase in cardiac muscle cells. Compared to cardiomyocytes transfected with control vector, activation of MAP kinase by IGF I was decreased by approximately 60-70% in the cells transfected with dominant negative MAP kinase Y185. Transfection with Y185 also resulted in decreased induction of p53 mRNA by IGF I (70% reduction). In the cells transfected with a wildtype p21WAF1/CIP1 promoter construct, activation of luciferase reporter gene by IGF I was decreased in the cells co-transfected with Y185. To further confirm these findings, cells were preincubated with PD98059, a specific MAP kinase kinase inhibitor. As expected, PD98059 inhibited induction of p53 mRNA and p21WAF1/CIP1 promoter by IGF I. These data indicate that transcriptional activation of p53 and p21WAF1/CIP1 by IGF I involves MAP kinase pathway in cardiomyocytes, and thus link MAP kinase to negative modulation of the cell cycle in cardiac muscle cells.

Estrogen and progesterone inhibit vascular smooth muscle proliferation.

Estrogen (E) has been identified in epidemiologic and prospective studies to protect against the development of cardiovascular disease in women. It is unclear whether progesterone (P) is similarly beneficial. The mechanisms by which E or P might act are incompletely defined. One possibility is that sex steroids inhibit the proliferation of vascular smooth muscle, an early/important event in vascular pathology. We examined the ability of E and P to inhibit the growth of human umbilical vein smooth muscle cells (hUVSMC) in culture, when stimulated by serum or the mitogen, endothelin-1 (ET-1). Serum and ET-1 stimulated hVSMC cell numbers by approximately 110% and 43% respectively, compared with control, after 3 days in culture. This stimulation was maximally reversed 75% by E and 64% by P. No synergistic or additive effects of the two steroids were found. ET-1 and serum stimulated mitogen-activated protein kinase (MAP-K) and MAP-kinase kinase activities, and these were critical for mitogenesis. Mitogen-stimulated MAP-kinase kinase and MAP-K activities were significantly inhibited by either E or P. The steroids also inhibited mitogen-stimulated c-fos and c-myc, downstream targets for MAP-K action. Critical signaling and molecular events through which mitogens stimulate VSMC proliferation can be significantly inhibited by E or P, providing a potential cellular mechanism for their vascular protective actions.

Vasoactive peptides modulate vascular endothelial cell growth factor production and endothelial cell proliferation and invasion.

The proliferation of vascular endothelial cells (EC) is an important event in angiogenesis. The synthesis of the EC growth factor, vascular endothelial cell growth factor (VEGF), is stimulated by a variety of activators; but the effects of important vasoactive peptides are not well understood, and there are no known natural inhibitors of VEGF production. We found that the vasoactive peptides endothelin (ET)-1 and ET-3 stimulated the synthesis of VEGF protein 3-4-fold in cultured human vascular smooth muscle cells, comparable in magnitude to hypoxia. ET-1 and ET-3 acted through the ETA and ETB receptors, respectively, and signaling through protein kinase C was important. Atrial natriuretic peptide (ANP), C-type natriuretic peptide, and C-ANP-(4-23), a ligand for the natriuretic peptide clearance receptor, equipotently inhibited production of VEGF by as much as 88% and inhibited ET- or hypoxia-stimulated VEGF transcription. EC proliferation and invasion of matrix were stimulated by VEGF secreted into the medium by ET-incubated vascular smooth muscle cells. This was inhibited by ANP. Our results identify the natriuretic peptides as the first peptide inhibitors of VEGF synthesis and indicate a novel mechanism by which vasoactive peptides could modulate angiogenesis.