Robert A. Shapiro

Estrogen Receptor (ER)α and ERβ Exhibit Unique Pharmacologic Properties When Coupled to Activation of the Mitogen-Activated Protein Kinase Pathway1

The rapid, nongenomic effects of estrogen are increasingly recognized as playing an important role in several aspects of estrogen action. Rapid activation of the mitogen-activated protein kinase (MAPK) signaling pathway by estrogen is among the more recently identified of these effects. To explore the role of estrogen receptors (ERs) in mediating these effects, we have transfected ER-negative Rat-2 fibroblasts with complementary DNA clones encoding either human ERalpha or rat ERbeta and examined their ability to couple to activation of MAPK in response to 17beta-estradiol (17beta-E(2)) and other ligands. For both receptors, addition of E(2) resulted in a rapid phosphorylation of MAPK. Activation of MAPK in ERalpha-transfected cells was partially and completely blocked by the antiestrogens tamoxifen and ICI 182,780, respectively. In ERbeta-transfected cells, MAPK activation was less sensitive to inhibition by tamoxifen and ICI 182,780. We have also observed that, in this model system, a membrane-impermeable estrogen (BSA-E(2)) and 17alpha-E(2) were both able to activate MAPK in a manner similar to E(2) alone. Here also, ICI 182,780 blocked the ability of BSA-E(2) to activate MAPK through ERalpha, but failed to block ERbeta-mediated effects. BSA-E(2) treatment, however, failed to activate nuclear estrogen-response-element-mediated gene transcription. These data show that these nuclear ERs are necessary for estrogens effects at the membrane. This model system will be useful in identifying molecular interactions involved in the rapid effects mediated by the ERs.

Prenatal Stress Generates Deficits in Rat Social Behavior: Reversal by Oxytocin

Neurodevelopmental changes induced by environmental stress exposure play a significant but poorly defined role in the etiology of schizophrenia. Exposure of pregnant female rats to a series of unpredictable stresses during the final week of pregnancy generates behavioral deficits and molecular changes in the offspring similar to those observed in schizophrenic individuals. We used this rat prenatal stress preparation to investigate social withdrawal behaviors that may have relevance to the negative symptoms of schizophrenia. The cumulative time adult male offspring of stress-exposed pregnant female rats actively interacted with a weight-matched, same-sex peer was decreased approximately 76% relative to non-stress exposed control rats. Prenatal stress exposure also diminished the quality of the social interaction behavior indicative of reduced social drive. Analysis of the oxytocinergic system in the prenatally stressed male rats revealed significantly less oxytocin mRNA in the paraventricular nucleus and increased oxytocin receptor binding in the central amygdala. Moreover, oxytocin, but not vasopressin, administration into the central amygdala reversed the social incompetence of the prenatally stressed rats without increasing behavior in non-stressed control animals. In addition, cross-fostering pups from prenatally stressed mothers to non-stressed mothers failed to improve the social deficit of the prenatally stressed male offspring. Two behavioral assays designed to measure anxiety did not differentiate the prenatally stressed rats from non-stressed controls. These data indicate that prenatal stress may be an etiologically appropriate animal model for some aspects of schizophrenic social withdrawal. Furthermore, unpredictable prenatal stress exposure selectively degrades social interaction behaviors without increasing anxiety measures.

Social Interaction Deficits Caused by Chronic Phencyclidine Administration are Reversed by Oxytocin

Chronic administration of phencyclidine (PCP) has been advanced as a valid animal model of the social deficit symptoms of schizophrenia. In these studies, the cumulative time that male rats treated once a day for 14 days with PCP actively engaged in social behavior was decreased approximately 75% relative to saline-treated control animals. In addition, these socially impaired rats had an increase in the relative amount of noncontact interactions compared with saline-injected peers. Social behaviors were preferentially affected by PCP treatment because in two anxiety-related behavioral assays, the open field and light/dark emergence tests, there was a failure to differentiate between the PCP-treated rats and saline-injected control rats. Considering the general importance of the neuropeptides oxytocin and vasopressin in male social behaviors, studies of molecular markers related to these neuropeptides were performed. Hypothalamic oxytocin mRNA expression was significantly decreased while oxytocin receptor binding was increased in the central nucleus of the amygdala following chronic PCP treatment. Given the significance of central nucleus of the amygdala in social behavior, oxytocin was infused into the central nucleus of experimental and control male rats, and their postinfusion social interaction and open field behaviors were analyzed. A bilateral infusion of 1 μg of oxytocin into the central amygdala selectively restored the normal quantity and quality of social behavior in chronic PCP-treated male rats without altering open field behaviors. These findings suggest that deficits in the central oxytocinergic system may underlie the social impairment exhibited in this animal model of schizophrenia.

Estrogen‐mediated neuroprotection against β‐amyloid toxicity requires expression of estrogen receptor α or β and activation of the MAPK pathway

It is well documented that estrogen can activate rapid signaling pathways in a variety of cell types. These non‐classical effects of estrogen have been reported to be important for cell survival after exposure to a variety of neurotoxic insults. Since direct evidence of the ability of the estrogen receptors (ERs) α and/or β to mediate such responses is lacking, the hippocampal‐derived cell line HT22 was stably transfected with either ERα (HTERα) or ERβ (HTERβ). In HTERα and HTERβ cells, but not untransfected cells, an increase in ERK2 phosphorylation was measured within 15 min of 17β‐estradiol treatment. The ER antagonist ICI 182, 780 (1 µm) and the MEK inhibitor, PD98059 (50 µm) blocked this increase in ERK2 phosphorylation. Treatment of HT22, HTERα and HTERβ cells with the β‐amyloid peptide (25–35) (10 µm) resulted in a significant decrease in cell viability. Pre‐treatment for 15 min with 10 nm 17β‐estradiol resulted in a 50% increase in the number of living cells in HTERα and HTERβ cells, but not in HT22 cells. Finally, ICI 182, 780 and PD98059 prevented 17β‐estradiol‐mediated protection. This study demonstrates that both ERα and ERβ can couple to rapid signaling events that mediate estrogen‐elicited neuroprotection.

Multiple pathways transmit neuroprotective effects of gonadal steroids

Numerous preclinical studies suggest that gonadal steroids, particularly estrogen, may be neuroprotective against insult or disease progression. This paper reviews the mechanisms contributing to estrogen-mediated neuroprotection. Rapid signaling pathways, such as MAPK, P13K, Akt, and PKC, are required for estrogens ability to provide neuroprotection. These rapid signaling pathways converge on genomic pathways to modulate transcription of E2-responsive genes via ERE-dependent and ERE-independent mechanisms. It is clear that both rapid signaling and transcription are important for estrogens neuroprotective effects. A mechanistic understanding of estrogen-mediated neuroprotection is crucial for the development of therapeutic interventions that enhance quality of life withou deleterious side effects.

Estrogen induces rapid translocation of estrogen receptor β, but not estrogen receptor α, to the neuronal plasma membrane

Estrogen receptors can activate transcription in the nucleus, and activate rapid signal transduction cascades in the cytosol. Multiple reports identify estrogen receptors at the plasma membrane, while others document the dynamic responses of estrogen receptor to ligand binding. However, the function and identity of membrane estrogen receptors remain controversial. We have used confocal microscopy and cell fractionation on the murine hippocampus-derived HT22 cell line and rat primary cortical neurons transfected with estrogen receptor-green fluorescent protein constructs to address the membrane localization of these receptors. We observe translocation of estrogen receptor beta (beta) to the plasma membrane 5 min after exposure to 17beta-estradiol, whereas estrogen receptor alpha (alpha) localization remains unchanged. Membrane localization of estrogen receptor beta is transient, selective for 17beta-estradiol, and is not blocked by ICI182,780. Inhibition of the mitogen-activated protein kinase pathway does not block estrogen-mediated estrogen receptor beta membrane translocation, and in fact prolongs membrane localization. These data suggest that while both estrogen receptor alpha and estrogen receptor beta can be present at the neuronal membrane, their presence is differentially regulated.

Estrogen replacement regimen and brain infusion of lipopolysaccharide differentially alter steroid receptor expression in the uterus and hypothalamus

The regimen of estrogen replacement can alter the consequences of estrogen therapy and stressors. To determine the long-term effects and interaction of these systems on the brain and periphery, adult female rats were infused with lipopolysaccharide (LPS) into the fourth ventricle of the brain for 4 weeks, and ovariectomized rats were administered either constant or pulsed regimens of estrogen replacement (17β-estradiol) until sacrifice at 8 weeks. Constant, but not pulsed, estrogen replacement reduced ERα and increased HSP90, HSP70, and PRB uterine protein levels. Both estrogen regimens increased ERβ, HSP27, and PRA uterine proteins. Both regimens reduced hypothalamic levels of ERα, but not ERβ, HSP, or PR. No changes were observed in the hippocampus. Long-term brain infusion of LPS activated microglia and reduced body weight, but did not alter corticosterone or nitrotyrosine levels. LPS infusion into intact rats suppressed uterine weight, increased ERα and decreased HSP90 in the uterus. LPS did not alter uterine weight in ovariectomized rats treated with constant or pulsed estrogen. Together, these data suggest the timing of estrogen replacement and neuroinflammatory stressors can profoundly affect uterine and hypothalamic steroid receptor expression and may be important parameters to consider in the post-menopausal intervention with estrogen.

Downregulation of M1 and M2 Muscarinic Receptor Subtypes in Y1 Mouse Adrenocarcinoma Cells

Muscarinic acetylcholine receptors are important in mediating neurotransmission in the central and peripheral nervous systems and regulate a broad spectrum of physiologic responses. Chronic exposure of cells to muscarinic agonists results in a decrease in receptor number and diminished coupling to effectors. We examined this phenomenon in 2 of the 5 described receptor subtypes: ml, one of the predominant subtypes expressed in brain, and m2, the major cardiac subtype. DNA clones for these receptors were transfected into the Y1 mouse adrenal cell line and a variant Y1 clone. Kin 8, in which cAMP-dependent protein kinase activity is greatly reduced. Transfected receptors could be expressed at high levels and were functional as determined by their ability to bind muscarinic ligands and stimulate phosphoinositide turnover (ml) or inhibit adenylyl cyclase (m2). We determined the susceptibility of the receptor subtypes to internalization after chronic exposure to the muscarinic agonist, carbachol, and as a consequence of activation of cAMP-dependent protein kinase and protein kinase C. These experiments suggest that the mechanisms for internalization of the m1 and m2 receptor subtypes differ. The ml, but not the m2, receptor was internalized in response to activation of protein kinase C and this internalization was dependent on the presence of a functional cAMP-dependent protein kinase. Activation of either protein kinase C or cAMP-dependent protein kinase did not mimic agonist-induced internalization of either m1 or m2. There was a large surplus of receptors for coupling to the effector enzymes in cells expressing several hundred fmol receptor per mg membrane protein. At low receptor numbers, agonist-induced desensitization was correlated with internalization of receptors.