Using U0126 to dissect the role of the extracellular signal-regulated kinase 1/2 (ERK1/2) cascade in the regulation of gene expression by endothelin-1 in cardiac myocytes

The hypertrophic agonist endothelin-1 rapidly but transiently activates the extracellular signal-regulated kinase 1/2 (ERK1/2) cascade (and other signalling pathways) in cardiac myocytes, but the events linking this to hypertrophy are not understood. Using Affymetrix rat U34A microarrays, we identified the short-term (2-4 h) changes in gene expression induced in neonatal myocytes by endothelin-1 alone or in combination with the ERK1/2 cascade inhibitor, U0126. Expression of 15 genes was significantly changed by U0126 alone, and expression of an additional 78 genes was significantly changed by endothelin-1. Of the genes upregulated by U0126, four are classically induced through the aryl hydrocarbon receptor (AhR) by dioxins suggesting that U0126 activates the xenobiotic response element in cardiac myocytes potentially independently of effects on ERK1/2 signalling. The 78 genes showing altered expression with endothelin-1 formed five clusters: (i) three clusters showing upregulation by endothelin-1 according to time course (4 h > 2 h; 2 h > 4 h; 2 h approximately 4 h) with at least partial inhibition by U0126; (ii) a cluster of 11 genes upregulated by endothelin-1 but unaffected by U0126 suggesting regulation through signalling pathways other than ERK1/2; (iii) a cluster of six genes downregulated by endothelin-1 with attenuation by U0126. Thus, U0126 apparently activates the AhR in cardiac myocytes (which must be taken into account in protracted studies), but careful analysis allows identification of genes potentially regulated acutely via the ERK1/2 cascade. Our data suggest that the majority of changes in gene expression induced by endothelin-1 are mediated by the ERK1/2 cascade.

Characterisation of chicken riboflavin carrier protein gene structure and promoter regulation by estrogen

Estrogen is an important steroid hormone that mediates most of its effects on regulation of gene expression by binding to intracellular receptors. The consensus estrogen response element (ERE) is a 13 bp palindromic inverted repeat with a three nucleotide spacer. However, several reports suggest that many estrogen target genes are regulated by diverse elements, such as imperfect EREs and ERE half sites (ERE 1/2),which are either the proximal or the distal half of the palindrome. To gain more insight into ERE half site-mediated gene regulation, we used a region from the estrogen-regulated chicken riboflavin carrier protein (RCP) gene promoter that contains ERE half sites. Using moxestrol, an analogue of estrogen and transient transfection of deletion and mutation containing RCP promoter/reporter constructs in chicken hepatoma (LMH2A) cells, we identified an estrogen response unit (ERU) composed of two consensus ERE 1/2 sites and one non-consensus ERE 1/2 site. Mutation of any of these sites within this ERU abolishes moxestrol response. Further, the ERU is able to confer moxestrol responsiveness to a heterologous promoter. Interestingly, RCP promoter is regulated by moxestrol in estrogen responsive human MCF-7 cells, but not in other cell lines such as NIH3T3 and HepG2 despite estrogen receptor-alpha (ER-�) co transfection. Electrophoretic mobility shift assays (EMSAs) with promoter regions encompassing the half sites and nuclear extracts from LMH2A cells show the presence of a moxestrol-induced complex that is abolished by a polyclonal anti-ER� antibody. Surprisingly, estrogen receptor cannot bind to these promoter elements in isolation. Thus, there appears to be a definite requirement for some other factor(s) in addition to estrogen receptor, for the generation of a suitable response of this promoter to estrogen. Our studies therefore suggest a novel mechanism of gene regulation by estrogen, involving ERE half sites without direct binding of ER to the cognate elements.

Differential interaction of estrogen receptor and thyroid hormone receptor isoforms on the rat oxytocin receptor promoter leads to differences in transcriptional regulation

Both the estrogen receptor (ER) and thyroid hormone receptor (TR) are members of the nuclear receptor superfamily. Two isoforms of the ER, alpha and beta, exist. The TRalpha and beta isoforms are products of two distinct genes that are further differentially spliced to give TRalpha1 and alpha2, TRbeta1 and beta2. The TRs have been shown to interfere with ER-mediated transcription from both the consensus estrogen response element (ERE) and the rat preproenkephalin (PPE) promoter, possibly by competing with ER binding to the ERE or by squelching coactivators essential for ER-mediated transcription. The rat oxytocin receptor (OTR) gene is thought to be involved in several facets of reproductive and affiliative behaviors. 17beta-Estradiol-bound ERs upregulate the OTR gene in the ventromedial hypothalamus, a region critical for the induction of lordosis behavior in several species. We investigated the effects of the ligand-binding TR isoforms on the ER-mediated transcription from a physiological promoter of a behaviorally relevant gene such as the OTR. Only ERalpha could induce the OTR gene in two cell lines tested, the CV-1 and the SK-N-BE2C neuroblastoma cell lines. ERbeta was incapable of inducing the gene in either cell line. ERalpha is therefore not equivalent to ERbeta on this physiological promoter. Indeed, in the neural cell line, ERbeta can inhibit ERalpha-mediated induction from the OTR promoter. While the TRalpha1 isoform inhibited ERalpha-mediated induction in the neural cell line, the TRbeta1 isoform stimulated induction, thus demonstrating isoform specificity in the interaction. The use of a DNA-binding mutant, the TR P box mutant, showed that inhibition of ERalpha-mediated induction of the rat OTR gene promoter by the TRalpha1 isoform does not require DNA-binding ability. SRC-1 overexpression relieved TRalpha1-mediated inhibition in both cell lines, suggesting that squelching for coactivators is an important molecular mechanism in TRalpha-mediated inhibition. Such interactions between TR and ER isoforms on the rat OTR promoter provide a mechanism to achieve neuroendocrine integration.

Differential crosstalk between estrogen receptor (ER) alpha and ER beta and the thyroid hormone receptor isoforms results in flexible regulation of the consensus ERE

Crosstalk between nuclear receptors is important for conversion of external and internal stimuli to a physiologically meaningful response by cells. Previous studies from this laboratory have demonstrated crosstalk between the estrogen (ER) and thyroid hormone receptors (TR) on two estrogen responsive physiological promoters, the preproenkephalin and oxytocin receptor gene promoter. Since ERa and ERb are isoforms possessing overlapping and distinct transactivation properties, we hypothesized that the interaction of ERa and b with the various TR isoforms would not be equivalent. To explore this hypothesis, the consensus estrogen response element (ERE)derived from the Xenopus vitellogenin gene is used to investigate the differences in interaction between ERa and b isoforms and the different TR isoforms in fibroblast cells. Both the ER isoforms transactivate from the consensus ERE, though ERa transactivates to a greater extent than ERb. Although neither of the TRb isoforms have an effect on ERa transactivation from the consensus ERE, the liganded TRa1 inhibits the ERa transactivation from the consensus ERE. In contrast, the liganded TRa1 facilitates ERb-mediated transactivation. The crosstalk between the TRb isoforms with the ERa isoform, on the consensus ERE, is different from that with the ERb isoform. The use of a TRa1 mutant, which is unable to bind DNA, abolishes the ability of the TRa1 isoform to interact with either of the ER isoforms. These differences in nuclear receptor crosstalk reveal an important functional difference between isoforms, which provides a novel mechanism for neuroendocrine integration.

Isoform specificity for oestrogen receptor and thyroid hormone receptor genes and their interactions on the NR2D gene promoter

Oestrogens are critical for the display of lordosis behaviour and, in recent years, have also been shown to be involved in synaptic plasticity. In the brain, the regulation of ionotropic glutamate receptors has consequences for excitatory neurotransmission. Oestrogen regulation of the N-methyl-d-aspartate receptor subunit 2D (NR2D) has generated considerable interest as a possible molecular mechanism by which synaptic plasticity can be modulated. Since more than one isoform of the oestrogen receptor (ER) exists in mammals, it is possible that oestrogen regulation via the ERalpha and ERbeta isoforms on the NR2D oestrogen response element (ERE) is not equivalent. In the kidney fibroblast (CV1) cell line, we show that in response to 17beta-oestradiol, only ERalpha, not ERbeta, could upregulate transcription from the ERE which is in the 3' untranslated region of the NR2D gene. When this ERE is in the 5' position, neither ERalpha nor ERbeta showed transactivation capacity. Thyroid hormone receptor (TR) modulation of ER mediated induction has been shown for other ER target genes, such as the preproenkephalin and oxytocin receptor genes. Since the various TR isoforms exhibit distinct roles, we hypothesized that TR modulation of ER induction may also be isoform specific. This is indeed the case. The TRalpha1 isoform stimulated ERalpha mediated induction from the 3'-ERE whereas the TRbeta1 isoform inhibited this induction. This study shows that isoforms of both the ER and TR have different transactivation properties. Such flexible regulation and crosstalk by nuclear receptor isoforms leads to different transcriptional outcomes and the combinatorial logic may aid neuroendocrine integration.

Integration of steroid hormone initiated membrane action to genomic function in the brain

Estrogen is a ligand for the estrogen receptor (ER), which on binding 17beta-estradiol, functions as a ligand-activated transcription factor and regulates the transcription of target genes. This is the slow genomic mode of action. However, rapid non-genomic actions of estrogen also exist at the cell membrane. Using a novel two-pulse paradigm in which the first pulse rapidly initiates non-genomic actions using a membrane-limited estrogen conjugate (E-BSA), while the second pulse promotes genomic transcription from a consensus estrogen response element (ERE), we have demonstrated that rapid actions of estrogen potentiate the slower transcriptional response from an ERE-reporter in neuroblastoma cells. Since rapid actions of estrogen activate kinases, we used selective inhibitors in the two-pulse paradigm to determine the intracellular signaling cascades important in such potentiation. Inhibition of protein kinase A (PKA), PKC, mitogen activated protein kinase (MAPK) or phosphatidylinositol 3-OH kinase (PI-3K) in the first pulse decreases potentiation of transcription. Also, our data with both dominant negative and constitutive mutants of Galpha subunits show that Galpha(q) initiates the rapid signaling cascade at the membrane in SK-N-BE(2)C neuroblastoma cells. We discuss two models of multiple kinase activation at the membrane Pulses of estrogen induce lordosis behavior in female rats. Infusion of E-BSA into the ventromedial hypothalamus followed by 17beta-estradiol in the second pulse could induce lordosis behavior, demonstrating the applicability of this paradigm in vivo. A model where non-genomic actions of estrogen couple to genomic actions unites both aspects of hormone action.

Calcium Flux in Neuroblastoma Cells Is a Coupling Mechanism between Non-Genomic and Genomic Modes of Estrogens

Estrogens have been demonstrated to rapidly modulate calcium levels in a variety of cell types. However, the significance of estrogen-mediated calcium flux in neuronal cells is largely unknown. The relative importance of intra- and extracellular sources of calcium in estrogenic effects on neurons is also not well understood. Previously, we have demonstrated that membrane-limited estrogens, such as E-BSA given before an administration of a 2-hour pulse of 17beta-estradiol (E(2)), can potentiate the transcription mediated by E(2) from a consensus estrogen response element (ERE)-driven reporter gene. Inhibitors to signal transduction cascades given along with E-BSA or E(2) demonstrated that calcium flux is important for E-BSA-mediated potentiation of transcription in a transiently transfected neuroblastoma cell line. In this report, we have used inhibitors to different voltage-gated calcium channels (VGCCs) and to intracellular store receptors along with E-BSA in the first pulse or with E(2) in the second pulse to investigate the relative importance of these channels to estrogen-mediated transcription. Neither L- nor P-type VGCCs seem to play a role in estrogen action in these cells; while N-type VGCCs are important in both the non-genomic and genomic modes of estrogen action. Specific inhibitors also showed that the ryanodine receptor and the inositol trisphosphate receptor are important to E-BSA-mediated transcriptional potentiation. This report provides evidence that while intracellular stores of calcium are required to couple non-genomic actions of estrogen initiated at the membrane to transcription in the nucleus, extracellular sources of calcium are also important in both non-genomic and genomic actions of estrogens. Copyright (c) 2005 S. Karger AG, Basel.

Estrogens and thyroid hormone: Non-genomic effects are coupled to transcription.

Estrogens and thyroid hormones are regulators of important diverse physiological processes such as reproduction, thermogenesis, neural development, neural differentiation and cardiovascular functions. Both are ligands for receptors in the nuclear receptor superfamily, which act as ligand-dependent transcription factors, regulating transcription. However, estrogens and thyroid hormones also rapidly (within minutes or seconds) activate kinase cascades and calcium increases, presumably initiated at the cell membrane. We discuss the relevance of both modes of hormone action, including the membrane estrogen receptor, to physiology, with particular reference to lordosis behavior. We first showed that estrogen restricted to the membrane can, in fact, lead to subsequent increases in transcription from a consensus estrogen response element-based reporter in the neuroblastoma cell line, SK-N-BE(2)C. Using a novel hormonal paradigm, we also showed that the activation of protein kinase A, protein kinase C, mitogen activated protein kinase and increases in calcium were important in the ability of the membrane-limited estrogen to potentiate transcription. We discuss the source of calcium important in transcriptional potentiation. Since estrogens and thyroid hormones have common effects on neuroprotection, cognition and mood, we also hypothesized that crosstalk could occur between the rapid actions of thyroid hormones and the genomic actions of estrogens. In neural cells, we showed that triiodothyronine acting rapidly via MAPK can increase transcription by the nuclear estrogen receptor ERa from a consensus estrogen response element, possibly by the phosphorylation of the ERa. Novel mechanisms that link signals initiated by hormones from the membrane to the nucleus are physiologically relevant and can achieve neuroendocrine integration

Estrogen receptor-mediated transcription involves the activation of multiple kinase pathways in neuroblastoma cells

While many physiological effects of estrogens (E) are due to regulation of gene transcription by liganded estrogen receptors (ERs), several effects are also mediated, at least in part, by rapid non-genomic actions of E. Though the relative importance of rapid versus genomic effects in the central nervous system is controversial, we showed previously that membrane-limited effects of E, initiated by an estradiol bovine serum albumin conjugate (E2-BSA), could potentiate transcriptional effects of 17beta-estradiol from an estrogen response element (ERE)-reporter in neuroblastoma cells. Here, using specific inhibitors and activators in a pharmacological approach, we show that activation of phosphatidylinositol-3-phosphate kinase (PI3K) and mitogen activated protein kinase (MAPK) pathways, dependent on a Galphaq coupled receptor signaling are important in this transcriptional potentiation. We further demonstrate, using ERalpha phospho-deficient mutants, that E2-BSA mediated phosphorylation of ERalpha is one mechanism to potentiate transcription from an ERE reporter construct. This study provides a possible mechanism by which signaling from the membrane is coupled to transcription in the nucleus, providing an integrated view of hormone signaling in the brain.

Estrogen regulation of chicken riboflavin carrier protein gene is mediated by ERE half sites without direct binding of estrogen receptor

Estrogen is an important steroid hormone that mediates most of its effects on regulation of gene expression by binding to intracellular receptors. The consensus estrogen response element (ERE) is a 13 bp palindromic inverted repeat with a three nucleotide spacer. However, several reports suggest that many estrogen target genes are regulated by diverse elements, such as imperfect EREs and ERE half sites (ERE 1/2), which are either the proximal or the distal half of the palindrome. To gain more insight into ERE half site-mediated gene regulation, we used a region from the estrogen-regulated chicken riboflavin carrier protein (RCP) gene promoter that contains ERE half sites. Using moxestrol, an analogue of estrogen and transient transfection of deletion and mutation containing RCP promoter/reporter constructs in chicken hepatoma (LMH2A) cells, we identified an estrogen response unit (ERU) composed of two consensus ERE 1/2 sites and one non-consensus ERE 1/2 site. Mutation of any of these sites within this ERU abolishes moxestrol response. Further, the ERU is able to confer moxestrol responsiveness to a heterologous promoter. Interestingly, RCP promoter is regulated by moxestrol in estrogen responsive human MCF-7 cells, but not in other cell lines such as NIH3T3 and HepG2 despite estrogen receptor-alpha (ER-�) co transfection. Electrophoretic mobility shift assays (EMSAs) with promoter regions encompassing the half sites and nuclear extracts from LMH2A cells show the presence of a moxestrol-induced complex that is abolished by a polyclonal anti-ER� antibody. Surprisingly, estrogen receptor cannot bind to these promoter elements in isolation. Thus, there appears to be a definite requirement for some other factor(s) in addition to estrogen receptor, for the generation of a suitable response of this promoter to estrogen. Our studies therefore suggest a novel mechanism of gene regulation by estrogen, involving ERE half sites without direct binding of ER to the cognate elements.

Unification of the probe and singular sources methods for the inverse boundary value problem by the no-response test

In this article, we use the no-response test idea, introduced in Luke and Potthast (2003) and Potthast (Preprint) and the inverse obstacle problem, to identify the interface of the discontinuity of the coefficient gamma of the equation del (.) gamma(x)del + c(x) with piecewise regular gamma and bounded function c(x). We use infinitely many Cauchy data as measurement and give a reconstructive method to localize the interface. We will base this multiwave version of the no-response test on two different proofs. The first one contains a pointwise estimate as used by the singular sources method. The second one is built on an energy (or an integral) estimate which is the basis of the probe method. As a conclusion of this, the probe and the singular sources methods are equivalent regarding their convergence and the no-response test can be seen as a unified framework for these methods. As a further contribution, we provide a formula to reconstruct the values of the jump of gamma(x), x is an element of partial derivative D at the boundary. A second consequence of this formula is that the blow-up rate of the indicator functions of the probe and singular sources methods at the interface is given by the order of the singularity of the fundamental solution.

Myocyte remodeling in response to hypertrophic stimuli requires nucleocytoplasmic shuttling of muscle LIM protein

CSRP3 or muscle LIM protein (MLP) is a nucleocytoplasmic shuttling protein and a mechanosensor in cardiac myocytes. MLP regulation and function was studied in cultured neonatal rat myocytes treated with pharmacological or mechanical stimuli. Either verapamil or BDM decreased nuclear MLP while phenylephrine and cyclic strain increased it. These results suggest that myocyte contractility regulates MLP subcellular localization. When RNA polymerase II was inhibited with alpha-amanitin, nuclear MLP was reduced by 30%. However, when both RNA polymerase I and II were inhibited with actinomycin D, there was a 90% decrease in nuclear MLP suggesting that its nuclear translocation is regulated by both nuclear and nucleolar transcriptional activity. Using cell permeable synthetic peptides containing the putative nuclear localization signal (NLS) of MLP, nuclear import of the protein in cultured rat neonatal myocytes was inhibited. The NLS of MLP also localizes to the nucleolus. Inhibition of nuclear translocation prevented the increased protein accumulation in response to phenylephrine. Furthermore, cyclic strain of myocytes after prior NLS treatment to remove nuclear MLP resulted in disarrayed sarcomeres. Increased protein synthesis and brain natriuretic peptide expression were also prevented suggesting that MLP is required for remodeling of the myo filaments and gene expression. These findings suggest that nucleocytoplasmic shuttling MLP plays an important role in the regulation of the myocyte remodeling and hypertrophy and is required for adaptation to hypertrophic stimuli. (C) 2009 Elsevier Inc. All rights reserved.

Designs for an adaptive tuned vibration absorber with variable shape stiffness element

An adaptive tuned vibration absorber (ATVA) with a smart variable stiffness element is capable of retuning itself in response to a time-varying excitation frequency., enabling effective vibration control over a range of frequencies. This paper discusses novel methods of achieving variable stiffness in an ATVA by changing shape, as inspired by biological paradigms. It is shown that considerable variation in the tuned frequency can be achieved by actuating a shape change, provided that this is within the limits of the actuator. A feasible design for such an ATVA is one in which the device offers low resistance to the required shape change actuation while not being restricted to low values of the effective stiffness of the vibration absorber. Three such original designs are identified: (i) A pinned-pinned arch beam with fixed profile of slight curvature and variable preload through an adjustable natural curvature; (ii) a vibration absorber with a stiffness element formed from parallel curved beams of adjustable curvature vibrating longitudinally; (iii) a vibration absorber with a variable geometry linkage as stiffness element. The experimental results from demonstrators based on two of these designs show good correlation with the theory.

Structure and spectroelectrochemical response of arene− ruthenium and arene−osmium complexes with potentially hemilabile noninnocent ligands

Nine of the compounds [M(L2−)(p-cymene)] (M = Ru, Os, L2− = 4,6-di-tert-butyl-N-aryl-o-amidophenolate) were prepared and structurally characterized (Ru complexes) as coordinatively unsaturated, formally 16 valence electron species. On L2−-ligand based oxidation to EPR-active iminosemiquinone radical complexes, the compounds seek to bind a donor atom (if available) from the N-aryl substituent, as structurally certified for thioether and selenoether functions, or from the donor solvent. Simulated cyclic voltammograms and spectroelectrochemistry at ambient and low temperatures in combination with DFT results confirm a square scheme behavior (ECEC mechanism) involving the Ln ligand as the main electron transfer site and the metal with fractional (δ) oxidation as the center for redox-activated coordination. Attempts to crystallize [Ru(Cym)(QSMe)](PF6) produced single crystals of [RuIII(QSMe •−)2](PF6) after apparent dissociation of the arene ligand.