The Bcl6-SMRT/NCoR cistrome represses inflammation to attenuate atherosclerosis

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Highlights? Bcl6 is a critical antiatherogenic regulator ? Inhibition of Bcl6-SMRT/NCoR interactions induces atherogenic gene expression ? SMRT and NCoR cistromes each exceed 30,000 sites, with a nearly 50% overlap ? Bcl6 recruits SMRT and NCoR to NF-κB-driven inflammatory and tissue remodeling genes     

Disorder in a target for the smad2 mad homology 2 domain and its implications for binding and specificity

The Smad2 Mad homology 2 (MH2) domain binds to a diverse group of proteins which do not share a common sequence motif. We have used NMR to investigate the structure of one of these interacting proteins, the Smad binding domain (SBD) of Smad anchor for receptor activation (SARA). Our results indicate that the unbound SBD is highly disordered and forms no stable secondary or tertiary structures. Additionally we have used fluorescence binding studies to study the interaction between the MH2 domain and SBD and find that no region of the SBD dominates the interaction between the MH2 and the SBD. Our results are consistent with a series of hydrophobic patches on the MH2 that are able to recognize disordered regions of proteins. These findings elucidate a mechanism by which a single domain (MH2) can specifically recognize a diverse set of proteins which are unrelated by sequence, lead to a clearer picture of how MH2 domains function in the transforming growth factor-beta-signaling pathway and suggest possible mechanisms for controlling interactions with MH2 domains.     

Imaging of caffeine-inducible release of intracellular calcium in cultured embryonic mouse telencephalic neurons

To gain a better understanding of Ca²⁺-induced Ca²⁺ release in central neurons, we have studied the increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) induced by application of caffeine to cells cultured from embryonic mouse telencephalon (hippocampus or cortex). The magnitudes and distributions of changes in [Ca²⁺]_i in neuron somata were measured by quantitative video microscopy. We observed that application of caffeine to pyramidally shaped neurons typically initiated an increase in [Ca²⁺]_i in the cytoplasmic region between the nucleus and the base of a major dendrite. [Ca²⁺] in this region increased over a period of 3 to 6 s and was followed by, with a slight delay, a surge of Ca²⁺ that moved across the soma and into or over the nucleus. Similar Ca²⁺ that moved across the soma and into or over the nucleus. Similar Ca²⁺ responses to caffeine were observed in Ca²⁺-containing and nominally Ca²⁺-free external solutions, suggesting that caffeine was inducing Ca²⁺ release from intracellular stores. Ca²⁺ responses to caffeine were potentiated by inducing a tonic Ca²⁺ influx through N-methyl-D-aspartate (NMDA)-type glutamate receptors activated by 0.3 μM glutamate and multiple responses to caffeine could be elicited by using this Ca²⁺ influx to refill the intracellular stores. Ryanodine inhibition of caffeine-induced Ca²⁺ release was use- and concentration-dependent; the median effective concentration EC₅₀ for ryanodine declined from 22 μM for the first application of caffeine to 20 nM for the fourth. We conclude, based on these responses to caffeine, that ryanodine-sensitive mechanisms of intracellular Ca²⁺ release are active in hippocampal and cortical neurons and may be involved in generation of directed Ca²⁺ waves that engulf the nucleus. © 1995 John Wiley & Sons, Inc.     

Novel genes underlying beta cell survival in metabolic stress

Relative insulin deficiency, in response to increased metabolic demand (obesity, genetic insulin resistance, pregnancy and aging) lead to Type2 diabetes. Susceptibility of the type 2 diabetes has a genetic basis, as a subset of people with risk factors (obesity, Insulin Resistance, pregnancy), develop Type2 Diabetes. We aimed to identify ‘cluster’ of overexpressed genes, underlying increased beta cell survival in diabetes resistant C57BL/6J ob/ob mice (compared to diabetes susceptible BTBR ob/ob mice). We used ‘consensus’ overexpression status to identify ‘cluster’ of 11 genes consisting of Aldh18a1, Rfc4, Dynlt3, Prom1, H13, Psen1, Ssr4, Dad1, Anpep, Fam111a and Plk1. Information (biological processes, molecular functions, cellular components, protein-protein interactions/associations, gene deletion/knockout/inhibition studies) of all the genes in ‘cluster’ were collected by text mining using different literature search tools, gene information databases and protein-protein interaction databases. Beta cell specific function of these genes were also inferred using meta analysis tool of Beta Cell Biology Consortium, by studying the expression pattern of these genes in microarray studies related to beta-cell stimulation/injury, pancreas development and growth and cell differentiation. In the ‘clusters’, 6 genes (Dad1, Psen1, Ssr4, Rfc4, H13, Plk1) have a role in cell survival. Only Psen1 was previously identified to have role in successful beta cell compensation. We advocate these genes to be potentially involved in successful beta cell compensation and prevent T2D in humans, by conferring protection against diabetogenic insults.     

Insights into Negative Regulation by the Glucocorticoid Receptor from Genome-wide Profiling of Inflammatory Cistromes

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Highlights? TLR4 activation dramatically remodels the GR cistrome in macrophages ? Both negative and positive GR enhancers include GREs, NF-κB, and AP-1 sites ? Classical models fail to predict positive or negative polarity of GR regulation ? Hormone treatment marks negative enhancers by histone deacetylation     

Research resource: Comparative nuclear receptor atlas: basal and activated peritoneal B-1 and B-2 cells

Na?ve murine B cells are typically divided into three subsets based on functional and phenotypic characteristics: innate-like B-1 and marginal zone B cells vs. adaptive B-2 cells, also known as follicular or conventional B cells. B-1 cells, the innate-immune-like component of the B cell lineage are the primary source of natural antibodies and have been shown to modulate autoimmune diseases, human B-cell leukemias, and inflammatory disorders such as atherosclerosis. On the other hand, B-2 cells are the principal mediators of the adaptive humoral immune response and represent an important pharmacological target for various conditions including rheumatoid arthritis, lupus erythematosus, and lymphomas. Using the resources of the Nuclear Receptor Signaling Atlas program, we used quantitative real-time PCR to assess the complement of the 49 murine nuclear receptor superfamily expressed in quiescent and toll-like receptor (TLR)-stimulated peritoneal B-1 and B-2 cells. We report the expression of 24 nuclear receptors in basal B-1 cells and 25 nuclear receptors in basal B-2 cells, with, in some cases, dramatic changes in response to TLR 4 or TLR 2/1 stimulation. Comparative nuclear receptor profiling between B-1 and peritoneal B-2 cells reveals a highly concordant expression pattern, albeit at quantitatively dissimilar levels. We also found that splenic B cells express 23 nuclear receptors. This catalog of nuclear receptor expression in B-1 and B-2 cells provides data to be used to better understand the specific roles of nuclear receptors in B cell function, chronic inflammation, and autoimmune disease.     

Thyroid hormone receptor repression is linked to type I pneumocyte-associated respiratory distress syndrome

Although the lung is a defining feature of air-breathing animals, the pathway controlling the formation of type I pneumocytes, the cells that mediate gas exchange, is poorly understood. In contrast, the glucocorticoid receptor and its cognate ligand have long been known to promote type II pneumocyte maturation; prenatal administration of glucocorticoids is commonly used to attenuate the severity of infant respiratory distress syndrome (RDS). Here we show that knock-in mutations of the nuclear co-repressor SMRT (silencing mediator of retinoid and thyroid hormone receptors) in C57BL/6 mice (SMRTmRID) produces a previously unidentified respiratory distress syndrome caused by prematurity of the type I pneumocyte. Though unresponsive to glucocorticoids, treatment with anti-thyroid hormone drugs (propylthiouracil or methimazole) completely rescues SMRT-induced RDS, suggesting an unrecognized and essential role for the thyroid hormone receptor (TR) in lung development. We show that TR and SMRT control type I pneumocyte differentiation through Klf2, which, in turn, seems to directly activate the type I pneumocyte gene program. Conversely, mice without lung Klf2 lack mature type I pneumocytes and die shortly after birth, closely recapitulating the SMRTmRID phenotype. These results identify TR as a second nuclear receptor involved in lung development, specifically type I pneumocyte differentiation, and suggest a possible new type of therapeutic option in the treatment of RDS that is unresponsive to glucocorticoids.     

Analysis of heart rate deflection points to predict the anaerobic threshold by a computerized method

Many studies have used the heart rate deflection points (HRDPs) during incremental exercise tests, because of their strong correlation with the anaerobic threshold. The aim of this study was to evaluate the profile of the HRDPs identified by a computerized method and compare them with ventilatory and lactate thresholds. Twenty-four professional soccer players (age, 22 ± 5 years; body mass, 74 ± 7 kg; height 177 ± 7 cm) volunteered for the study. The subjects completed a Bruce-protocol incremental treadmill exercise test to volitional fatigue. Heart rate (HR) and alveolar gas exchange were recorded continuously at ≥1 Hz during exercise testing. Subsequently, the time course of the HR was fit by a computer algorithm, and a set of lines yielding the lowest pooled residual sum of squares was chosen as the best fit. This procedure defined 2 HRDPs (HRDP1 and HRDP2). The HR break points averaged 43.9 ± 5.9 and 89.7 ± 7.5% of the VO2peak. The HRDP1 showed a poor correlation with ventilatory threshold (VT; r = 0.50), but HRDP2 was highly correlated to the respiratory compensation (RC) point (r = 0.98). Neither HRDP1 nor HRDP2 was correlated with LT1 (at VO2 = 2.26 ± 0.72 L·min(-1); r = 0.26) or LT2 (2.79 ± 0.59 L·min(-1); r = 0.49), respectively. LT1 and LT2 also were not well correlated with VT (2.93 ± 0.68 L·min(-1); r = 0.20) or RC (3.82 ± 0.60 L·min(-1); r = 0.58), respectively. Although the HR deflection points were not correlated to LT, HRDP2 could be identified in all the subjects and was strongly correlated with RC, consistent with a relationship to cardiorespiratory fatigue and endurance performance.     

Changes in membrane hydrogen and sodium conductances during progesterone-induced maturation of Ambystoma oocytes

A voltage-gated hydrogen ion-selective conductance has been previously described in the immature oocyte of the urodele amphibian Ambystoma. The present study was prompted by reports that changes in membrane voltage and internal pH, as well as in internal sodium ion concentration, occur during the hormone-induced maturation of oocytes from other amphibians. As activation of membrane currents might mediate changes in internal ion concentrations in addition to altering the membrane voltage, microelectrode recording techniques have been employed to examine changes in membrane conductances which occur during maturation of Ambystoma oocytes. It was observed that during the first 5 hr of maturation the magnitude of the hydrogen ion conductance gradually decreased, and that subsequently there was an increase in the amplitude of a voltage-dependent noninactivating sodium conductance. After 6 to 7 hr, after the loss of the hydrogen conductance and at about the time of germinal vesicle breakdown, the resting potential of the oocyte spontaneously shifted from approximately -10 mV to approximately +30 mV, where it remained until at least 24 hr after the initiation of maturation. This voltage transition was due to the appearance of mechanisms generating inward current in the oocyte membrane; part of this inward current was due to the tonic activation of the sodium conductance. Changes in internal pH and internal sodium ion concentration occurred during maturation, as judged from shifts in the reversal potentials of both hydrogen and sodium currents. A gradual decrease in internal hydrogen ion concentration was observed up until the time of disappearance of the hydrogen conductance (change in internal pH from about 7.15 in immature oocytes to about 7.40 by 3 hr after application of progesterone). This was followed, as sodium conductance increased, by an apparent rise in the internal sodium ion concentration (from about 6 mM to about 17 mM by 10 hr postprogesterone).