Estrogen, K+ Channels and Homeostasis
The gonadal steroid estrogen is a pleiotropic hormone that has widespread actions not only on reproductive tissues but also has pronounced effects in the central nervous system (CNS). For over twenty-five years, my laboratory has been studying the actions of estrogen in the brain, specifically in hypothalamic neurons that control homeostasis and behavior, using molecular, electrophysiological and behavioral techniques. We have discovered a novel estrogenic signaling pathway that can better explain the differences between females and males, not only in terms of the reproductive cycle but also differences in stress responses, motivation and mood. These actions of estrogen are similar to the effects of neurotransmitters like serotonin in the CNS. We use an in vitro slice preparation to do whole-cell patch recording in hypothalamic neurons followed by single cell reverse transcription-polymerase chain reaction (RT-PCR) to identify changes in the expression of receptors and K+ channel transcripts during different physiological states.
In collaboration with Drs. Oline Ronnekleiv and Tom Scanlan in the Department, we have developed a new non-steroidal compound that mimics the actions of estrogen in proopiomelanocortin (POMC) neurons called STX (Figure 1). We have discovered that estrogen acts directly on these neurons via a novel estrogen G protein-coupled receptor to alter their activity. This unique membrane-associated estrogen receptor (mER) is Gq-coupled to activation of a phospholipase C-protein kinase C-protein kinase A pathway leading to desensitization of m-opioid and GABAB receptors in POMC and GABA neurons (Figure 2). We use several transgenic mouse models in which POMC, neuropeptide Y or kisspeptin neurons are tagged with green fluorescence protein in order to selectively target these neurons in the brain slice preparation using visualized patch recording. Using these transgenic mice we are identifying other channels which are targets for the mER signaling pathway and its cross-talk with other neurotransmitter (e.g., serotonin) and hormones (e.g., leptin) signaling pathways.
As proof of principle, estrogen is known to control feeding behavior, energy homeostasis and core body temperature. STX appears to mimic all of these CNS actions of estrogen in the female guinea pig, whose reproductive cycle mimics that of the human (Figures 3 & 4). Moreover, we are involved in collaborative studies with Drs. Steve Kohama, Martha Neuringer and Henryk Urbanski at the Oregon National Primate Research Center, to measure the effects of STX on core body temperature in aged female rhesus monkeys. In addition, estrogen has been known to have "neuroprotective" effects on higher cognitive functions. Indeed, in collaboration with colleagues (Drs. Anne Etgen, Diane Lebesque, and Suzanne Zukin) at Albert Einstein College of Medicine, we have found that acute STX is as efficacious as estrogen to prevent hippocampal neuron loss following global ischemia in middle aged rats (Lebesgue et al., PLoS One 5:e8642, 2010). Therefore, STX appears to mimic many of the CNS actions of estrogen in rodents and sub-human primates.
Finally, we have utilized a custom brain-specific guinea pig microarray chip, developed in collaboration with Dr. Oline Ronnekleiv, to compare/contrast the actions of STX and 17β-estradiol on gene regulation in relevant hypothalamic nuclei. We have used this gene chip to identify genes important for neuronal excitability (Ca2+, K+ channels) and signal transduction molecules (Figure 2) that are regulated by estrogen treatment in female guinea pigs (Figure 5).
Figure 1. STX, a membrane-estrogen receptor selective ligand (US 7,196,119 Patent), is more potent than 17β-estradiol (E2) to desensitize the GABAB response in hypothalamic proopiomelanocortin (POMC) neurons. Using whole-cell patch recording techniques, composite dose–response curves were generated to measure the difference in potency between E2 and STX to desensitize the baclofen (GABAB) response in POMC neurons. Data are presented as mean ± SEM (n = 4–11 cells/data point. The EC50 for STX was 2.6 nM, which is 17-fold lower than that found for E2 (46.0 nM). Insert: Molecular structure of STX. From Qiu et al., J. Neurosci. 26:5649, 2006.
Figure 2. A cellular model of the rapid signaling of estrogen in hypothalamic neurons.
Besides the classical ER-mediated gene activation via an estrogen response element (ERE), E2 can activate a membrane-associated ER (mER) that is Gαq -coupled to activation of phospholipase C that catalyzes the hydrolysis of membrane-bound phosphatidylinositol 4,5-biphosphate (PIP2) to inositol 1,4,5 triphosphate (IP3) and diacylglycerol (DAG). Calcium is released from intracellular stores (endoplasmic reticulum) by IP3, and DAG activates protein kinase Cδ (PKCδ). Through phosphorylation, adenylyl cyclase (AC) activity is upregulated by PKC. The generation of cAMP activates PKA, which can rapidly uncouple GABAB and μ-opioid (μ) receptors from their effector system through phosphorylation of a downstream effector molecule (e.g., G protein-coupled, inwardly rectifying K+ (GIRK) channel). The mER-mediated modulation of kinase pathways reduces the capacity of neuromodulators such as GABA and β-endorphin (β-End) to inhibit POMC neuronal excitability. Finally, mER-mediated activation of PKA can lead to phosphorylation of cAMP-response element binding protein (pCREB), which can then alter gene transcription through its interaction with a CREB response element (CRE). These signaling pathways were elucidated using whole-cell patch recording techniques with selective agonists and antagonists and single-cell RT-PCR measurements of transcripts in POMC neurons. From Kelly & Ronnekleiv, Molecular & Cellular Endocrinology 308:17, 2009.
Figure 3. Estrogen and STX significantly attenuate the body weight gain in female guinea pigs after ovariectomy. Female guinea pigs were ovariectomized (on day 0) and allowed to recover for 1 week before being given bi-daily subcutaneous injections of oil (OIL), estradiol benzoate (EB), or STX (see Materials and Methods). A two-way ANOVA (repeated measures) revealed an overall significant effect of both estrogen and STX (p < 0.001), and post hoc Newman–Keuls analysis revealed daily significant differences between estrogen and oil-treated, and STX and oil-treated groups (**p < 0.01). Bars represent the mean ± SEM of six and four animals per group for EB and STX treatment, respectively. From Roepke et al., Endocrinology149:6113,2008.
Figure 4. STX and 17β-estradiol benzoate (EB) decrease the core body temperature of ovariectomized female guinea pigs. The mER selective ligand STX (6mg/kg bi-daily injections; n=8) decreased the core body temperature compared to the vehicle (n=6) but was not significantly different than the EB group (20 mg/kg bi-daily injection; n=6). The data are presented as the mean ± SEM for each hour of the day averaged over the last 21 days of the temperature probe recordings (one week after the first injection). The data were analyzed using a two-way ANOVA (Drug factor p-value = 0.017) with post-hoc Newman-Kuehl's Pairwise comparisons. All data points for both STX and EB were significantly different from vehicle (p<0.01).From Roepke et al. Endocrinology 151:4926, 2010.
Figure 5. Functional categorization of the identified genes from the guinea pig cDNA library. The guinea pig, brain-specific cDNA library of estrogen-regulated genes isolated from the Suppression Subtractive Hybridization (SSH) were sequenced, identified and categorized. The pie chart represents the distribution of identified cDNA transcripts on the guinea pig microarray chip in 10 categories determined by their Gene Ontology function. The number besides the category title denotes the number of genes in that group. A total of 710 unique genes were identified while over 480 transcripts from the SSH were not identified using BLAST (blastn) search engine. From Roepke et al., Endocrinology 149:6113, 2008.