Mark Leid

Sirt1 promotes fat mobilization in white adipocytes by repressing PPAR-gamma.

Calorie restriction extends lifespan in organisms ranging from yeast to mammals. In yeast, the SIR2 gene mediates the life-extending effects of calorie restriction. Here we show that the mammalian SIR2 orthologue, Sirt1 (sirtuin 1), activates a critical component of calorie restriction in mammals; that is, fat mobilization in white adipocytes. Upon food withdrawal Sirt1 protein binds to and represses genes controlled by the fat regulator PPAR-γ (peroxisome proliferator-activated receptor-γ), including genes mediating fat storage. Sirt1 represses PPAR-γ by docking with its cofactors NCoR (nuclear receptor co-repressor) and SMRT (silencing mediator of retinoid and thyroid hormone receptors). Mobilization of fatty acids from white adipocytes upon fasting is compromised in Sirt1+/- mice. Repression of PPAR-γ by Sirt1 is also evident in 3T3-L1 adipocytes, where overexpression of Sirt1 attenuates adipogenesis, and RNA interference of Sirt1 enhances it. In differentiated fat cells, upregulation of Sirt1 triggers lipolysis and loss of fat. As a reduction in fat is sufficient to extend murine lifespan, our results provide a possible molecular pathway connecting calorie restriction to life extension in mammals.

International Union of Pharmacology. LXIII. Retinoid X Receptors

The physiological effects of retinoic acids (RAs) are mediated by members of two families of nuclear receptors, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs), which are encoded by three distinct human genes, RXRα, RXRβ, and RXRγ. RARs bind both all-trans- and 9-cis-RA, whereas only the 9-cis-RA stereoisomer binds to RXRs. As RXR/RAR heterodimers, these receptors control the transcription of RA target genes through binding to RA-response elements. This review is focused on the structure, mode of action, ligands, expression, and pharmacology of RXRs. Given their role as common partners to many other members of the nuclear receptor superfamily, these receptors have been the subject of intense scrutiny. Moreover, and despite numerous studies since their initial discovery, RXRs remain enigmatic nuclear receptors, and there is still no consensus regarding their role. Indeed, multiple questions about the actual biological role of RXRs and the existence of an endogenous ligand have still to be answered.

International Union of Pharmacology. LX. Retinoic Acid Receptors

Retinoid is a term for compounds that bind to and activate retinoic acid receptors (RARα, RARβ, and RARγ), members of the nuclear hormone receptor superfamily. The most important endogenous retinoid is all-trans-retinoic acid. Retinoids regulate a wide variety of essential biological processes, such as vertebrate embryonic morphogenesis and organogenesis, cell growth arrest, differentiation and apoptosis, and homeostasis, as well as their disorders. This review summarizes the considerable amount of knowledge generated on these receptors.

P300 FUNCTIONS AS A COACTIVATOR FOR THE PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR ALPHA

The integrator protein, p300, was demonstrated to interact with mouse peroxisome proliferator-activated receptor α in a ligand-enhanced manner. The PPARα-interacting domain of p300 was mapped to amino acids 39–117 which interacted strongly with PPARα but did not interact with retinoic acid receptor-γ or retinoid X receptor-α. Amino acids within the carboxyl terminus of PPARα as well as residues within the hinge region were required for ligand-dependent interaction with p300. p300 enhanced the transcriptional activation properties of PPARα and, therefore, can be considered a bona fide coactivator for this nuclear receptor. These observations extend the group of p300-interacting proteins to include mPPARα and further characterize the molecular mechanisms of PPARα-mediated transcriptional regulation.

Identification of nuclear receptor corepressor as a peroxisome proliferator-activated receptor alpha interacting protein.

Nuclear receptor corepressor (NCoR) was demonstrated to interact strongly with peroxisome proliferator-activated receptor α (PPARα), and PPARα ligands suppressed this interaction. In contrast to the interaction of PPARα with the coactivator protein, p300, association of the receptor with NCoR did not require any part of the PPARα ligand binding domain. NCoR was found to suppress PPARα-dependent transcriptional activation in the context of a PPARα·retinoid X receptor α (RXRα) heterodimeric complex bound to a peroxisome proliferator-responsive element in human embryonic kidney 293 cells. This repression was reversed agonists of either receptor demonstrating a functional interaction between NCoR and PPARα·RXRα heterodimeric complexes in mammalian cells. NCoR appears to influence PPARα signaling pathways and, therefore, may modulate tissue responsiveness to peroxisome proliferators.

Involvement of the Histone Deacetylase SIRT1 in Chicken Ovalbumin Upstream Promoter Transcription Factor (COUP-TF)-interacting Protein 2-mediated Transcriptional Repression

Chicken ovalbumin upstream promoter transcription factor (COUP-TF)-interacting proteins 1 and 2 (CTIP1 and CTIP2) enhance transcriptional repression mediated by COUP-TF II and have been implicated in hematopoietic cell development and malignancies. CTIP1 and CTIP2 are also sequence-specific DNA-binding proteins that repress transcription through direct, COUP-TF-in-dependent binding to a GC-rich response element. CTIP1- and CTIP2-mediated transcriptional repression is insensitive to trichostatin A, an inhibitor of known class I and II histone deacetylases. However, chromatin immunoprecipitation assays revealed that expression of CTIP2 in mammalian cells resulted in deacetylation of histones H3 and/or H4 that were associated with the promoter region of a reporter gene. CTIP2-mediated transcriptional repression, as well as deacetylation of promoter-associated histones H3/H4 in CTIP2-transfected cells, was reversed by nicotinamide, an inhibitor of class III histone deacetylases such as the mammalian homologs of yeast Silent Information Regulator 2 (Sir2). The human homolog of yeast Sir2, SIRT1, was found to interact directly with CTIP2 and was recruited to the promoter template in a CTIP2-dependent manner. Moreover, SIRT1 enhanced the deacetylation of template-associated histones H3/H4 in CTIP2-transfected cells, and stimulated CTIP2-dependent transcriptional repression. Finally, endogenous SIRT1 and CTIP2 co-purified from Jurkat cell nuclear extracts in the context of a large (1–2 mDa) complex. These findings implicate SIRT1 as a histone H3/H4 deacetylase in mammalian cells and in transcriptional repression mediated by CTIP2.

COUP-TF (chicken ovalbumin upstream promoter transcription factor)-interacting protein 1 (CTIP1) is a sequence-specific DNA binding protein

Chicken ovalbumin upstream promoter transcription factor (COUP-TF)-interacting proteins 1 and 2 [CTIP1/Evi9/B cell leukaemia (Bcl) l1a and CTIP2/Bcl11b respectively] are highly related C(2)H(2) zinc finger proteins that are abundantly expressed in brain and the immune system, and are associated with immune system malignancies. A selection procedure was employed to isolate high-affinity DNA binding sites for CTIP1. The core binding site on DNA identified in these studies, 5-GGCCGG-3 (upper strand), is highly related to the canonical GC box and was bound by a CTIP1 oligomeric complex(es) in vitro. Furthermore, both CTIP1 and CTIP2 repressed transcription of a reporter gene harbouring a multimerized CTIP binding site, and this repression was neither reversed by trichostatin A (an inhibitor of known class I and II histone deacetylases) nor stimulated by co-transfection of a COUP-TF family member. These results demonstrate that CTIP1 is a sequence-specific DNA binding protein and a bona fide transcriptional repressor that is capable of functioning independently of COUP-TF family members. These findings may be relevant to the physiological and/or pathological action(s) of CTIPs in cells that do not express COUP-TF family members, such as cells of the haematopoietic and immune systems.

Ligand-induced Peroxisome Proliferator-activated Receptor α Conformational Change

Structurally diverse peroxisome proliferators and related compounds that have been demonstrated to induce the ligand-dependent transcriptional activation function of mouse peroxisome proliferator-activated receptor α (mPPARα) in transfection experiments were tested for the ability to induce conformational changes within mPPARα in vitro. WY-14,643, 5,8,11,14-eicosatetraynoic acid, LY-171883, and clofibric acid all directly induced mPPARα conformational changes as evidenced by a differential protease sensitivity assay. Carboxyl-terminal truncation mutagenesis of mPPARα differentially affected the ability of these ligands to induce conformational changes suggesting that PPAR ligands may make distinct contacts with the receptor. Direct interaction of peroxisome proliferators and related compounds with, and the resulting conformational alteration(s) in, mPPARα may facilitate interaction of the receptor with transcriptional intermediary factors and/or the general transcription machinery and, thus, may underlie the molecular basis of ligand-dependent transcriptional activation mediated by mPPARα.

Behavioral, pharmacological, and molecular characterization of an amphibian cannabinoid receptor

Abstract : Investigation of cannabinoid pharmacology in a vertebrate with a phylogenetic history distinct from that of mammals may allow better understanding of the physiological significance of cannabinoid neurochemistry. Taricha granulosa, the roughskin newt, was used here to characterize an amphibian cannabinoid receptor. Behavioral experiments demonstrated that the cannabinoid agonist levonantradol inhibits both newt spontaneous locomotor activity and courtship clasping behavior. Inhibition of clasping was dose‐dependent and potent (IC50 = 1.2 μg per animal). Radioligand binding studies using [3H]CP‐55940 allowed identification of a specific binding site (KD = 6.5 nM, Bmax = 1,853 fmol/mg of protein) in brain membranes. Rank order of affinity of several ligands was consistent with that reported for mammalian species (KD, nM) : CP‐55940 (3.8) > levonantradol (13.0) > WIN55212‐2 (25.7) ▴ anandamide (1,665) >> anandamide + 100 μM phenylmethylsulfonyl fluoride (2,398). The cDNA encoding the newt CB1 cannabinoid receptor was cloned, and the corresponding mRNA of 5.9 kb was found to be highly expressed in brain. A nonclonal Chinese hamster ovary cell line stably expressing the newt CB1 cannabinoid receptor was prepared that allowed demonstration of cannabinoid‐mediated inhibition of adenylate cyclase (EC 4.6.1.1) activity. This inhibition was dose‐dependent and occurred at concentrations consistent with affinities determined through radioligand binding experiments. The behavioral, pharmacological, and molecular cloning results demonstrate that a CB1 cannabinoid receptor is expressed in the CNS of the roughskin newt. This amphibian CB1 is very similar in density, ligand binding affinity, ligand binding specificity, and amino acid sequence to mammalian CB1. The high degree of evolutionary conservation of cannabinoid signaling systems implies an important physiological role in vertebrate brain function.

Detection of Apoptosis by TUNEL Assay

Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) assay has been designed to detect apoptotic cells that undergo extensive DNA degradation during the late stages of apoptosis. The method is based on the ability of TdT to label blunt ends of double-stranded DNA breaks independent of a template. This chapter describes an assay for detection of apoptotic cells during mouse odontogenesis using a colorimetric TUNEL system.