Differential TGF-ß Signaling in Glial Subsets Underlies IL-6-Mediated Epileptogenesis in Mice.

TGF-ß1 is a master cytokine in immune regulation, orchestrating both pro- and anti-inflammatory reactions. Recent studies show that whereas TGF-ß1 induces a quiescent microglia phenotype, it plays a pathogenic role in the neurovascular unit and triggers neuronal hyperexcitability and epileptogenesis. In this study, we show that, in primary glial cultures, TGF-ß signaling induces rapid upregulation of the cytokine IL-6 in astrocytes, but not in microglia, via enhanced expression, phosphorylation, and nuclear translocation of SMAD2/3. Electrophysiological recordings show that administration of IL-6 increases cortical excitability, culminating in epileptiform discharges in vitro and spontaneous seizures in C57BL/6 mice. Intracellular recordings from layer V pyramidal cells in neocortical slices obtained from IL-6 -: treated mice show that during epileptogenesis, the cells respond to repetitive orthodromic activation with prolonged after-depolarization with no apparent changes in intrinsic membrane properties. Notably, TGF-ß1 -: induced IL-6 upregulation occurs in brains of FVB/N but not in brains of C57BL/6 mice. Overall, our data suggest that TGF-ß signaling in the brain can cause astrocyte activation whereby IL-6 upregulation results in dysregulation of astrocyte -: neuronal interactions and neuronal hyperexcitability. Whereas IL-6 is epileptogenic in C57BL/6 mice, its upregulation by TGF-ß1 is more profound in FVB/N mice characterized as a relatively more susceptible strain to seizure-induced cell death.

Preferential lentiviral targeting of astrocytes in the central nervous system.

The ability to visualize and genetically manipulate specific cell populations of the central nervous system (CNS) is fundamental to a better understanding of brain functions at the cellular and molecular levels. Tools to selectively target cells of the CNS include molecular genetics, imaging, and use of transgenic animals. However, these approaches are technically challenging, time consuming, and difficult to control. Viral-mediated targeting of cells in the CNS can be highly beneficial for studying and treating neurodegenerative diseases. Yet, despite specific marking of numerous cell types in the CNS, in vivo selective targeting of astrocytes has not been optimized. In this study, preferential targeting of astrocytes in the CNS was demonstrated using engineered lentiviruses that were pseudotyped with a modified Sindbis envelope and displayed anti-GLAST IgG on their surfaces as an attachment moiety. Viral tropism for astrocytes was initially verified in vitro in primary mixed glia cultures. When injected into the brains of mice, lentiviruses that displayed GLAST IgG on their surface, exhibited preferential astrocyte targeting, compared to pseudotyped lentiviruses that did not incorporate any IgG or that expressed a control isotype IgG. Overall, this approach is highly flexible and can be exploited to selectively target astrocytes or other cell types of the CNS. As such, it can open a window to visualize and genetically manipulate astrocytes or other cells of the CNS as means of research and treatment.

TGF-ß signaling through SMAD2/3 induces the quiescent microglial phenotype within the CNS environment.

Microglia are myeloid-derived cells that colonize the central nervous system (CNS) at early stages of development and constitute up to 20% of the glial populations throughout life. While extensive progress has been recently made in identifying the cellular origin of microglia, the mechanism whereby the cells acquire the unique ramified and quiescent phenotype within the CNS milieu remains unknown. Here, we show that upon co-culturing of either CD117(+) /Lin(-) hematopoietic progenitors or CD11c(+) bone marrow derived cells with organotypic hippocampal slices or primary glia, the cells acquire a ramified morphology concomitant with reduced levels of CD86, MHCII, and CD11c and up-regulation of the microglial cell-surface proteins CX(3) CR1 and Iba-1. We further demonstrate that the transforming growth factor beta (TGF-ß) signaling pathway via SMAD2/3 phosphorylation is essential for both primary microglia and myeloid-derived cells in order to acquire their quiescent phenotype. Our study suggests that the abundant expression of TGF-ß within the CNS during development and various inflammatory processes plays a key role in promoting the quiescent phenotype of microglia and may thus serve as a target for therapeutic strategies aimed at modulating the function of microglia in neurodegenerative diseases such as Alzheimer's and prion.

Unliganded estrogen receptor-beta regulation of genes is inhibited by tamoxifen.

Tamoxifen can stimulate the growth of some breast tumors and others can become resistant to tamoxifen. We previously showed that unliganded ERbeta inhibits ERalpha-mediated proliferation of MCF-7 cells. We investigated if tamoxifen might have a potential negative effect on some breast cancer cells by blocking the effects of unliganded ERbeta on gene regulation. Gene expression profiles demonstrated that unliganded ERbeta upregulated 196 genes in MCF-7 cells. Tamoxifen significantly inhibited 73 of these genes by greater than 30%, including several growth-inhibitory genes. To explore the mechanism whereby unliganded ERbeta activates genes and how tamoxifen blocks this effect, we used doxycycline-inducible U2OS-ERbeta cells to produce unliganded ERbeta. Doxycycline produced a dose-dependent activation of the NKG2E, MSMB and TUB3A genes, which was abolished by tamoxifen. Unliganded ERbeta recruitment of SRC-2 to the NKG2E gene was blocked by tamoxifen. Our findings suggest that tamoxifen might exert a negative effect on ERbeta expressing tumors due to its antagonistic action on unliganded ERbeta.

Cell type- and estrogen receptor-subtype specific regulation of selective estrogen receptor modulator regulatory elements.

Selective estrogen receptor modulators (SERMs), such as tamoxifen and raloxifene can act as estrogen receptor (ER) antagonists or agonists depending on the cell type. The antagonistic action of tamoxifen has been invaluable for treating breast cancer, whereas the agonist activity of SERMs also has important clinical applications as demonstrated by the use of raloxifene for osteoporosis. Whereas the mechanism whereby SERMs function as antagonists has been studied extensively very little is known about how SERMs produce agonist effects in different tissues with the two ER types; ERalpha and ERbeta. We examined the regulation of 32 SERM-responsive regions with ERalpha and ERbeta in transiently transfected MCF-7 breast, Ishikawa endometrial, HeLa cervical and WAR-5 prostate cancer cells. The regions were regulated by tamoxifen and raloxifene in some cell types, but not in all cell lines. Tamoxifen activated similar number of regions with ERalpha and ERbeta in the cell lines, whereas raloxifene activated over twice as many regions with ERbeta compared to ERalpha. In Ishikawa endometrial cancer cells, tamoxifen activated 17 regions with ERalpha, whereas raloxifene activated only 2 regions, which might explain their different effects on the endometrium. Microarray studies also found that raloxifene regulated fewer genes than tamoxifen in U2OS bone cancer cells expressing ERalpha, whereas tamoxifen was equally effective at regulating genes with ERalpha and ERbeta. Our studies indicate that tamoxifen is a non-selective agonist, whereas raloxifene is a relative ERbeta-selective agonist, and suggest that ERbeta-selective SERMs might be safer for treating clinical conditions that are dependent on the agonist property of SERMs.

Liquiritigenin is a plant-derived highly selective estrogen receptor beta agonist.

After the Women's Health Initiative found that the risks of hormone therapy outweighed the benefits, a need for alternative drugs to treat menopausal symptoms has emerged. We explored the possibility that botanical agents used in Traditional Chinese Medicine for menopausal symptoms contain ERbeta-selective estrogens. We previously reported that an extract containing 22 herbs, MF101 has ERbeta-selective properties. In this study we isolated liquiritigenin, the most active estrogenic compound from the root of Glycyrrhizae uralensis Fisch, which is one of the plants found in MF101. Liquiritigenin activated multiple ER regulatory elements and native target genes with ERbeta but not ERalpha. The ERbeta-selectivity of liquiritigenin was due to the selective recruitment of the coactivator steroid receptor coactivator-2 to target genes. In a mouse xenograph model, liquiritigenin did not stimulate uterine size or tumorigenesis of MCF-7 breast cancer cells. Our results demonstrate that some plants contain highly selective estrogens for ERbeta.

Differential regulation of native estrogen receptor-regulatory elements by estradiol, tamoxifen, and raloxifene.

Estrogen receptors (ERs) regulate gene transcription by interacting with regulatory elements. Most information regarding how ER activates genes has come from studies using a small set of target genes or simple consensus sequences such as estrogen response element, activator protein 1, and Sp1 elements. However, these elements cannot explain the differences in gene regulation patterns and clinical effects observed with estradiol (E(2)) and selective estrogen receptor modulators. To obtain a greater understanding of how E(2) and selective estrogen receptor modulators differentially regulate genes, it is necessary to investigate their action on a more comprehensive set of native regulatory elements derived from ER target genes. Here we used chromatin immunoprecipitation-cloning and sequencing to isolate 173 regulatory elements associated with ERalpha. Most elements were found in the introns (38%) and regions greater than 10 kb upstream of the transcription initiation site (38%); 24% of the elements were found in the proximal promoter region (<10 kb). Only 11% of the elements contained a classical estrogen response element; 23% of the elements did not have any known response elements, including one derived from the naked cuticle homolog gene, which was associated with the recruitment of p160 coactivators. Transfection studies found that 80% of the 173 elements were regulated by E(2), raloxifene, or tamoxifen with ERalpha or ERbeta. Tamoxifen was more effective than raloxifene at activating the elements with ERalpha, whereas raloxifene was superior with ERbeta. Our findings demonstrate that E(2), tamoxifen, and raloxifene differentially regulate native ER-regulatory elements isolated by chromatin immunoprecipitation with ERalpha and ERbeta.

The ovarian endothelin network: an evolving story.

The endothelin (ET) system consists of three ET isopeptides, several converting enzyme isoforms and two G-protein-coupled receptors, ETA and ETB, which are linked to multiple signaling pathways. Less than 20 years after the initial detection of ET-1 in granulosa cells, the ovarian ET network continues to expand with the discovery of new members and functions. ETs influence a broad range of essential reproductive processes, such as ovulation, steroidogenesis and luteolysis. Therefore, a more comprehensive understanding of the ovarian ET network might provide new strategies for controlling reproduction. This review presents up-to-date findings on the ET network in the ovary.

Multiple transcription factor elements collaborate with estrogen receptor alpha to activate an inducible estrogen response element in the NKG2E gene.

Estrogen receptors (ERs) regulate transcription by interacting with regulatory elements in target genes. However, known ER regulatory elements cannot explain the expression profiles of genes activated by estradiol (E2) and selective estrogen receptor modulators (SERMs). We previously showed that the killer cell lectin-like receptor (NKG2E) gene is regulated by E2, tamoxifen, and raloxifene. Here we used the NKG2E gene as a model to investigate the mechanism whereby target genes are regulated by E2 and SERMs with ERalpha. The ER regulatory element in the NKG2E promoter was mapped to the -1825 and -1686 region. Full activation of the NKG2E promoter required the collaboration between a transcription factor cluster containing c-jun, heat-shock factor 2, and CCAAT/enhancer-binding protein beta and a unique variant estrogen response element (ERE) that has only a two nucleotide spacer between half sites. The cluster elements and the variant ERE were inactive on their own, but the regulation by E2 and SERMs was restored when the c-jun, heat-shock factor-2, and CCAAT/enhancer-binding protein beta cluster was placed upstream of the variant ERE. The activation of the NKG2E gene by E2 and selective ER modulators was associated with the recruitment of the p160 coactivators glucocorticoid receptor-interacting protein 1 and amplified in breast cancer 1 but not steroid receptor coactivator 1. These studies identified one of the most complex ER regulatory units thus far reported and demonstrate that a cluster of flanking transcription factors collaborate with ER to induce a functional ERE in the NKG2E promoter.

Identification of a novel alternatively spliced variant endothelin converting enzyme-1 lacking a transmembrane domain.

Endothelin-converting enzyme (ECE)-1 cleaves big endothelins, as well as bradykinin and beta-amyloid peptide. Several isoforms of ECE-1 (ECE-1a, 1b, 1c, and 1d) have been identified to date, they differ only in their amino terminus and share the catalytic domain located in the C-terminal end. In addition to full-length ECE-1 forms, we identified novel, alternatively spliced messenger RNAs (mRNAs) of ECE-1b, 1c, and 1d. These splice variants (SVs) lack exon 3', which codes for the transmembrane (TM) region and is present in full-length forms. SV mRNAs were highly expressed in endothelial cells (EC) derived from macrovascular and microvascular beds. Analyses of ECE-1d and its SV forms in stably transfected human embryonic kidney (HEK)-293 cells revealed that both proteins were recognized by antibodies to C-terminal ECE-1, but an antibody to the N-terminal only bound ECE-1d. The novel protein, designated ECE-1sv, has an apparent molecular weight of 75 kDa. ECE-1sv lacks the TM sequence (or signal peptide) and, therefore, is expected to remain cytosolic. Presence of ECE-1sv in different cellular compartments than the full-length forms of the ECE-1 may suggest a distinct physiologic role for these proteins.