Fiona O'Mahony

Developmental Phenotype of a Membrane Only Estrogen Receptor α (MOER) Mouse

Estrogen receptors (ERs) α and β exist as nuclear, cytoplasmic, and membrane cellular pools in a wide variety of organs. The relative contributions of each ERα pool to in vivo phenotypes resulting from estrogen signaling have not been determined. To address this, we generated a transgenic mouse expressing only a functional E domain of ERα at the plasma membrane (MOER). Cells isolated from many organs showed membrane only localized E domain of ERα and no other receptor pools. Liver cells from MOER and wild type mice responded to 17-β-estradiol (E2) with comparable activation of ERK and phosphatidylinositol 3-kinase, not seen in cells from ERαKO mice. Mating the MOER female mice with proven male wild type breeders produced no pregnancies because the uterus and vagina of the MOER female mice were extremely atrophic. Ovaries of MOER and homozygous Strasbourg ERαKO mice showed multiple hemorrhagic cysts and no corpus luteum, and the mammary gland development in both MOER and ERαKO mice was rudimentary. Despite elevated serum E2 levels, serum LH was not suppressed, and prolactin levels were low in MOER mice. MOER and Strasbourg female mice showed plentiful abdominal visceral and other depots of fat and increased body weight compared to wild type mice despite comparable food consumption. These results provide strong evidence that the normal development and adult functions of important organs in female mice requires nuclear ERα and is not rescued by membrane ERα domain expression alone.

Estrogen reduces lipid content in the liver exclusively from membrane receptor signaling.

Membrane-localized estrogen receptor can alter metabolism without translocating to the nucleus. No Need for the Nuclear Middle Man Estrogen suppresses the transcription of genes that encode enzymes involved in lipid synthesis in the liver. The estrogen receptor α (ERα) can initiate downstream signaling from the plasma membrane or can translocate to the nucleus to alter gene expression. Using mice engineered with ERα that could not bind DNA or transcriptional activators and could only signal from the plasma membrane (membrane-localized ERα) or that were entirely null for ERα, Pedram et al. showed that activation of only the membrane-localized ERα was sufficient to decrease cholesterol synthesis in murine hepatocytes. Activation of membrane-localized ERα with either estrogen or a pharmacological agonist triggered the phosphorylation of the transcription factor Srebf1 by AMP-activated kinase (AMPK), which prevented Srebf1 cleavage by the protease S1P, nuclear translocation, and the transcription of its target genes. Activation of membrane-localized ERα counteracted the insulin-stimulated synthesis of fatty acids in the liver. Thus, even without the ability to directly regulate transcription, membrane-localized ERα can indirectly inhibit gene expression and influence metabolism. Estrogen induces signal transduction through estrogen receptor α (ERα), which localizes to both the plasma membrane and nucleus. Using wild-type mice, ERα knockout (ERKO) mice, or transgenic mice expressing only the ligand-binding domain of ERα exclusively at the plasma membrane (MOER), we compared the transcriptional profiles of liver tissue extracts after mice were injected with the ERα agonist propyl-pyrazole-triol (PPT). The expression of many lipid synthesis–related genes was comparably decreased in livers from MOER or wild-type mice but was not suppressed in ERKO mice, indicating that only membrane-localized ERα was necessary for their suppression. Cholesterol, triglyceride, and fatty acid content was decreased only in livers from wild-type and MOER mice exposed to PPT, but not in the livers from the ERKO mice, validating the membrane-driven signaling pathway on a physiological level. PPT-triggered activation of ERα at the membrane induced adenosine monophosphate–activated protein kinase to phosphorylate sterol regulatory element–binding factor 1 (Srebf1), preventing its association with and therefore its proteolytic cleavage by site-1 protease. Consequently, Srebf1 was sequestered in the cytoplasm, preventing the expression of cholesterol synthesis–associated genes. Thus, we showed that inhibition of gene expression mediated by membrane-localized ERα caused a metabolic phenotype that did not require nuclear ERα.

Female Gender-specific Inhibition of KCNQ1 Channels and Chloride Secretion by 17β-Estradiol in Rat Distal Colonic Crypts

The estrogen sex steroid 17β-estradiol rapidly inhibits secretagogue-stimulated cAMP-dependent Cl– secretion in the female rat distal colonic crypt by the inhibition of basolateral K+ channels. In Ussing chamber studies, both the anti-secretory response and inhibition of basolateral K+ current was shown to be attenuated by pretreatment with rottlerin, a PKCδ-specific inhibitor. In whole cell patch-clamp analysis, 17β-estradiol inhibited a chromanol 293B-sensitive KCNQ1 channel current in isolated female rat distal colonic crypts. Estrogen had no effect on KCNQ1 channel currents in colonic crypts isolated from male rats. Female distal colonic crypts expressed a significantly higher amount of PKCδ in comparison to male tissue. PKCδ and PKA were activated at 5 min in response to 17β-estradiol in female distal colonic crypts only. Both PKCδ- and PKA-associated with the KCNQ1 channel in response to 17β-estradiol in female distal colonic crypts, and no associations were observed in crypts from males. PKA activation, association with KCNQ1, and phosphorylation of the channel were regulated by PKCδ as the responses were blocked by pretreatment with rottlerin. Taken together, our experiments have identified the molecular targets underlying the anti-secretory response to estrogen involving the inhibition of KCNQ1 channel activity via PKCδ- and PKA-dependent signaling pathways. This is a novel gender-specific mechanism of regulation of an ion channel by estrogen. The anti-secretory response described in this study provides molecular insights whereby estrogen causes fluid retention effects in the female during periods of high circulating plasma estrogen levels.

Estrogen Modulates Metabolic Pathway Adaptation to Available Glucose in Breast Cancer Cells

Most cancers use glucose as substrate for aerobic glycolysis in preference to oxidative phosphorylation. However, variable glucose concentrations within the in-vivo tumor microenvironment may necessitate metabolic plasticity. Furthermore, little information exists on a role for estrogen receptors in modulating possible metabolic adaptations in breast cancer cells. Here we find that MCF-7 cells switch between metabolic pathways depending on glucose availability and 17β-estradiol (E(2)) potentiates adaptation. In high glucose conditions E(2) up-regulates glycolysis via enhanced AKT kinase activity and suppresses tricarboxylic acid cycle activity. After a decrease in extracellular glucose, mitochondrial pathways are activated in preference to glycolysis. In this setting, E(2) suppresses glycolysis and rescues cell viability by stimulating the tricarboxylic acid cycle via the up-regulation of pyruvate dehydrogenase (PDH) activity. E(2) also increases ATP in low glucose-cultured cells, and the novel phosphorylation of PDH by AMP kinase is required for these metabolic compensations. Capitalizing on metabolic vulnerability, knockdown of PDH in the low-glucose state strongly potentiates ionizing radiation-induced apoptosis and reverses the cell survival effects of E(2). We propose that lowering glucose substrate and inhibiting PDH may augment adjuvant therapies for estrogen receptor-positive breast cancer.

Genomic Priming of the Antisecretory Response to Estrogen in Rat Distal Colon throughout the Estrous Cycle

The secretion of Cl(-) across distal colonic crypt cells provides the driving force for the movement of fluid into the luminal space. 17beta-Estradiol (E2) produces a rapid and sustained reduction in secretion in females, which is dependent on the novel protein kinase C delta (PKC delta) isozyme and PKA isoform I targeting of KCNQ1 channels. This sexual dimorphism in the E2 response is associated with a higher expression level of PKC delta in female compared with the male tissue. The present study revealed the antisecretory response is regulated throughout the female reproductive (estrous) cycle and is primed by genomic regulation of the kinases. E2 (1-10 nm) decreased cAMP-dependent secretion in colonic epithelia during the estrus, metestrus, and diestrus stages. A weak inhibition of secretion was demonstrated in the proestrus stage. The expression levels of PKC delta and PKA fluctuated throughout the estrous cycle and correlated with the potency of the antisecretory effect of E2. The expression of PKC delta and PKA were up-regulated by estrogen at a transcriptional level via a PKC delta-MAPK-cAMP response element-binding protein-regulated pathway indicating a genomic priming of the antisecretory response. PK Cdelta was activated by the membrane-impermeant E2-BSA, and this response was inhibited by the estrogen receptor antagonist ICI 182,780. The 66-kDa estrogen receptor-alpha isoform was present at the plasma membrane of female colonic crypt cells with a lower abundance found in male colonic crypts. The study demonstrates estrogen regulation of intestinal secretion both at a rapid and transcriptional level, demonstrating an interdependent relationship between both nongenomic and genomic hormone responses.

Sex and estrous cycle-dependent rapid protein kinase signaling actions of estrogen in distal colonic cells

Previous studies from our laboratory demonstrated that 17beta-estradiol (E2) rapidly inhibits Cl(-) secretion in rat and human distal colonic epithelium. The inhibition has been shown to occur via targeting of a basolateral K(+) channel identified as the KCNQ1 (KvLQT1) channel. E2 indirectly modulates the channel activity via a cascade of second messengers which are rapidly phosphorylated in response to E2. The anti-secretory mechanism may be the manner by which E2 induces fluid retention in the intestine during periods of high circulating plasma E2. Here we review the sex-dependent and estrous cycle regulation of this novel rapid response to E2. The inhibition of KCNQ1 channel activity and Cl(-) secretion will be of interest in the future in the investigation of the retentive effects of estrogen in female tissue and also in the study of secretory disorders and drugable targets of the intestine.

Oestrogen promotes KCNQ1 potassium channel endocytosis and postendocytic trafficking in colonic epithelium

•  Oestrogen (E2) exposure leads to a decrease in both Cl− secretion and KCNQ1 current. This inhibition is maintained by a rapid and sustained retrieval of the channel from the plasma membrane. •  The E2‐stimulated internalization of KCNQ1 occurs via a dynamin‐ and clathrin‐dependent mechanism. •  KCNQ1 is recycled back to the cell membrane via Rab4 and Rab11 rather than being degraded. •  The signalling pathway activated by E2 and leading to KCNQ1 internalization involves a signalling cascade, in which the activation of protein kinase Cδ induces the phosphorylation of AMP‐dependent kinase. Oestrogen stimulated an increase in the association of KCNQ1 with the ubiquitin ligase Nedd4.2. •  The findings provide evidence for a hormone‐stimulated regulation of KCNQ1 surface density in colonic epithelium. Moreover, this study complements the understanding of the mechanisms for E2‐induced inhibition of KCNQ1 previously described, and provides new insights on hormonal regulation of ion channel retrieval from the plasma membrane.

Sexual dimorphism and oestrogen regulation of KCNE3 expression modulates the functional properties of KCNQ1 K⁺ channels.

Non‐Technical Summary  High levels of oestrogen are known to cause fluid retention in fertile females. It is thought that the increase in body fluid volume is necessary for proper implantation of the fertilised egg in the uterus. We show that the activity of a potassium ion channel, which drives salt and water movement across the cell membranes of the intestine, is inhibited by oestrogen and this effect is only found in females and is maximal during the peak phase of oestrogen in the oestrous cycle (when fertilization and implantation occur). These findings help us to understand the molecular mechanisms underlying the fluid retention effects of oestrogen in health and the potential adverse effects this response may have in exacerbating disease where fluid secretion is compromised such as in cystic fibrosis (the so‐called CF ‘gender gap’).

Estrogen inhibits chloride secretion caused by cholera and Escherichia coli enterotoxins in female rat distal colon.

Excessive Cl(-) secretion is the driving force for secretory diarrhea. 17β-Estradiol has been shown to inhibit Cl(-) secretion in rat distal colon through a nongenomic pathway. We examined whether 17β-estradiol inhibits Cl(-) secretion in an animal model of secretory diarrhea and the downstream effectors involved. The effect of 17β-estradiol on cholera toxin and heat-stable enterotoxin induced Cl(-) secretion in rat colonic mucosal sheets was studied by current-voltage clamping. Selective permeabilization of apical or basolateral membranes with amphotericin B or nystatin was used to isolate basolateral K(+) channel and apical Cl(-) channel activity, respectively. 17β-Estradiol dose-dependently inhibited secretory responses to both toxins with IC(50) values of approximately 1nM. This effect was female-gender specific, with no inhibition observed in male tissues. 17β-Estradiol responses were insensitive to the pure anti-estrogen ICI 182,720. 17β-Estradiol exerted its effects downstream of enterotoxin-induced production of second messengers (cAMP and cGMP) but was dependent on PKCδ activation. In nystatin-permeabilized tissues, apical Cl(-) currents were unaffected by 17β-estradiol treatment while basolateral K(+) current was profoundly inhibited by the hormone. This current was sensitive to the specific KCNQ1 channel inhibitors chromanol 293B and HMR-1556. In conclusion, 17β-estradiol inhibits enterotoxin-induced Cl(-) secretion via a PKCδ-dependent mechanism involving inhibition of basolateral KCNQ1 channels. These data elucidate mechanisms of 17β-estradiol inhibition of Cl(-) secretion induced by enterotoxins in intestinal epithelia, which may be relevant for the treatment of diarrheal diseases.

Berberine Reduces cAMP-Induced Chloride Secretion in T84 Human Colonic Carcinoma Cells through Inhibition of Basolateral KCNQ1 Channels

Berberine is a plant alkaloid with multiple pharmacological actions, including antidiarrhoeal activity and has been shown to inhibit Cl− secretion in distal colon. The aims of this study were to determine the molecular signaling mechanisms of action of berberine on Cl− secretion and the ion transporter targets. Monolayers of T84 human colonic carcinoma cells grown in permeable supports were placed in Ussing chambers and short-circuit current measured in response to secretagogues and berberine. Whole-cell current recordings were performed in T84 cells using the patch-clamp technique. Berberine decreased forskolin-induced short-circuit current in a concentration-dependent manner (IC50 80 ± 8 μM). In apically permeabilized monolayers and whole-cell current recordings, berberine inhibited a cAMP-dependent and chromanol 293B-sensitive basolateral membrane K+ current by 88%, suggesting inhibition of KCNQ1 K+ channels. Berberine did not affect either apical Cl− conductance or basolateral Na+–K+-ATPase activity. Berberine stimulated p38 MAPK, PKCα and PKA, but had no effect on p42/p44 MAPK and PKCδ. However, berberine pre-treatment prevented stimulation of p42/p44 MAPK by epidermal growth factor. The inhibitory effect of berberine on Cl− secretion was partially blocked by HBDDE (∼65%), an inhibitor of PKCα and to a smaller extent by inhibition of p38 MAPK with SB202190 (∼15%). Berberine treatment induced an increase in association between PKCα and PKA with KCNQ1 and produced phosphorylation of the channel. We conclude that berberine exerts its inhibitory effect on colonic Cl− secretion through inhibition of basolateral KCNQ1 channels responsible for K+ recycling via a PKCα-dependent pathway.