Meeting Report: 2009 Jensen Symposium on Nuclear Receptors - Day 3
Day 3 of 3
Day 3 of the 2009 Jensen Symposium on Nuclear Receptors featured talks by Ronald Evans, Pierre Chambon, Mitchell Lazar, Bruce Spiegelman and John Baxter (Signals regrets that it was unable to report on the talk by Tom Burris). Finally, Elwood Jensen himself added some closing remarks for Signals' coverage of the meeting.
The question of whether physiology and the circadian rhythm are intrinsically linked and, if so, how, is one that Ronald Evans (Salk Institute) has been investigating, and formed the basis of the first talk of the final day. The central clock of the suprachiasmatic nucleus in the central nervous system communicates with peripheral clocks in a variety of tissues, such as pancreas, liver and muscle, with biological endpoints in the co-ordination of metabolism as well as in diseases states such as inflammation. The mechanistic basis of the clock is a series of interlocking transcriptional feedback loops, involving BmalI, Per, Cry1 and several nuclear receptors, alternately activating and repressing transcription to attain homeostatic control over the system. Evans turned the spotlight on the role of AMPK (5’-AMP-activated protein kinase) in this system. This sensor of cellular energy status tweaks the clock through a series of targeted, destabilizing phosphorylations of Cry1, and Evans showed that its activation by low glucose levels in culture alters circadian rhythms in fibroblasts, in part by relieving Cry1 inhibition of RevErbα (this could be recapitulated in vivo in mouse liver).
To integrate these studies with physiology, Evans shifted gear to discuss whether exercise mimetics could substitute for exercise. He had previously shown that a skeletal muscle-targeted transgenic PPARδ mouse had enhanced exercise performance and endurance, but wanted to know if this was feasible in the adult, where the muscle fiber type identity is already specified. He found that the PPARδ modulator GW1516 did not promote endurance in adult mice unless there was a training regimen added to the protocol. Evans then showed that activation of AMPK by aminoimidazole carboxamide ribonucleotide (AICAR) activated PPARδ and increased running endurance without training, and that AMPK activation of oxidative genes such as SCD1, FAS, PGC1 and PDK4 was PPARδ-dependent. He concluded by stressing that AMPK, previously thought to be a cytoplasmic enzyme involved in the circadian clock, has endpoints in the nucleus which influence transcription and, ultimately, metabolism.
Pierre Chambon (IGBMC) opened with a review of work over the past twenty years centered around the creation in his laboratory of a cell-specific, temporally-controlled somatic mutagenesis system based on the Cre-ERT2 recombinase, which can be used to explore the function of any given gene throughout the life of the mouse. He described how it was first used to investigate the role of RXRα and RXRβ in epidermal keratinocyes (EK). Mouse with EK-specific knockouts of these genes spontaneously developed a skin syndrome mimicking atopic dermatitis (AD), a skin disease with systemic abnormalities such as asthma. AD in most cases has a family history and is typically treated with glucocorticoids. Chambon and his colleagues found widespread evidence of systemic abnormalities in the RXRα/β EK knockout mice, and found that a mouse overexpressing the epithelial-derived pro-inflammatory cytokine thymic stromal lymphopoietin (TSLP) had a similar phenotype. Interestingly, mice lacking only the C-terminal AF-2 of RXRα/β did not develop the AD-1 like skin and inflammatory abnormalities. Chambon and colleagues next painted the skin of mice with a series of nuclear receptor ligands and found that ligands for RARγ and VDR induced TSLP expression in keratinocytes. In sum, this suggested a model for TSLP regulation involving repression by unliganded VDR and RARγ, and synergistic activation by liganded VDR and RARγ. Nuclear run-on assays using EK nuclei showed that TSLP is controlled at the transcriptional level by topical skin treatment with vitamin D analogs and retinoic acid. Examination of the TSLP promoter using ChIP showed that VDR and RXRα bound to a specific DR3 element in the TSLP promoter in EK cells and that RARγ, but not α, bound to a specific DR2 in both the presence and absence of ligand. Chambon concluded by showing that a negative GRE present in the TSLP promoter was functional in vivo when bound to glucocorticoid receptor (GR)-activated fluocinolone acetonide, a synthetic GR agonist, and was sufficient per se to repress the basal level of TSLP.
Mitchell Lazar’s (U Penn) talked introduced the GR as an early player in adipogenesis, a process more commonly associated with PPARγ, at least where nuclear receptors are concerned. He made reference first to the widespread obesity epidemic in the United States, as well as highlighting the fact that obesity is a risk factor for cancer. As a preface to his work mapping the adipocyte PPARγ cistrome, Lazar stressed the essential role of PPARγ in co-ordinating the adipogenic gene program, and its role as a target for the thiazolidinedione class of drugs. The cistrome analysis showed that PPARγ bound preferentially to DR1s, confirming previous results, but was also commonly found to sit down in the vicinity of C/EBPα binding sites at genes encoding enzymes involved in cellular lipid metabolism, fatty acid metabolism and carboxylic acid metabolism. Turning to epigenetics, Lazar probed the role of intergenic PPARγ binding sites distant from gene promoters, and showed that there was an association between these sites and histone acetylation – chromatin remodeling – activity. Using a ChIP-chip strategy to search for histone modifications, he found an adipocyte-specific enhancer in the resistin gene that was bound by both PPARγ and C/EBPα. Lazar next described his laboratory’s efforts to understand regulation of the PPARγ gene itself, and described how a genome-wide location analysis (GWLA) of H3K9Ac found transient chromatin marks at the PPARG locus that occurred early in adipogenesis. These marks overlay a site previously shown to be bound by both CEBPα in the mature adipocyte. Intriguingly, they also showed that a GRE was also in the region, and that knockdown of GR ablated adipogenesis. This was expanded to ChIP-seq analyses for GR and CEBPβ in adipocyte differentiation, which showed that GREs and CEBP binding sites are commonly found near each other, and that GR and CEBPβ often bind in tandem early in adipogenesis on a large number of genes, including PPARγ. Lazar concluded that GR collaborates with CEBPs early in adipogeneis to induce PPARγ and other genes which drive the adipogenic program forward.
The metabolic syndrome is a constellation of illnesses associated with disrupted energy homeostasis, including hyperlipidemia, obesity and diabetes, and much of its mechanistic basis is grounded in abnormal function of white (WAT) and brown (BAT) adipose tissue. Bruce Spiegelman (Dana Farber Cancer Institute) painted a picture of the brown adipocyte as an anti-obesity cell, high in mitochondria and designed to dissipate energy through heat production. He posed the basic question: what makes BAT brown? He noted that although PGC-1α is absolutely required for thermogenesis in BAT, it was not required for brown adipocyte differentiation. The key molecules is in fact PRDM16, a zinc finger protein that is enriched in BAT and absent in WAT, and that induces the brown fat cell phenotype (elevated cidea, UCP1) in white fat cell precursors. Loss of function studies showed that shRNA of BRDM16 in immortalized brown fat cells causes them to lose their phenotype, however, when he carried out the experiment in primary brown fat cells, they acquired the characteristics of skeletal muscle cells. Conversely, primary muscle cells, when shown PRDM16, develop into brown fat cells. PRDM16 is a coactivator of PPARγ, and PPARγ binding is required for the adipogenic functions of PRDM16. Myoblasts, however, express very little PPARγ, which begged the question, what is the partner for PRDM that drives the transition from myoblast to brown fat cell? It turned out to be C/EBPβ, and Spiegelman showed that he was able to drive brown fat cell identity in naïve mouse embryonic fibroblasts with the addition of CEBPβ and PRDM16, inducing PPARγ, UCP-1, PGC-1 and cidea; these cells could then be transplanted into mice as functioning BAT depots. Spiegelman closed with a reference to the possible therapeutic implications of PRDM16, showing that transgenic mice expressing modest amounts of PRDM16 under the control of the aP2 promoter in WAT cells gained less weight, had more less fat mass, higher lean mass, elevated energy expenditure and improved glucose tolerance and insulin sensitivity.
John Baxter (Methodist Hospital) began with a summary of the substantial health care costs of cardiovascular disease and obesity. One of his main interests has been in developing thyroid receptor (TR) agonists which retain beneficial effects on lipid profiles but dial out adverse effects in the cardiovascular system. One such ligand, KB2115, is emerging as a promising candidate in primary dyslipidemia and is currently in a Phase II trial for this disease. Its liver effects include induction of the LDL receptor and down regulation of sterol regulatory binding element protein 1 (SREBP1c), a key transcription factor in fatty acid synthesis, both of which lead to improved serum lipid profiles and ultimately beneficial effects on atherosclerosis. Baxter compared this to the statin class of drugs, which have a substantial number of undesirable side effects, and pointed out that there is synergy between TR ligands and statins in reducing LDL cholesterol. Reviewing further work in his lab, he showed that KB2115 also reduced serum levels of lipoprotein (a), a risk factor for heart attacks, and that KB3495, another TRβ-selective ligand, reduces atherosclerosis in apoE knockout mice on a high cholesterol diet for 24 weeks.
In closing, Elwood Jensen provided the following personal remarks for Signals' report:
"As we come to the end of this series of impressive presentations, I would like to express my appreciation and admiration to the speakers for sharing their findings with us. I also suggest that we give a standing ovation to Sohaib Khan and his colleagues for assembling this all-star cast who have both entertained and informed us during the past three days. I would also like to thank the audience for their interest and astute questions, some of which I am sure have suggested new topics for investigation. Finally, for those of you who have not met her, I would like to introduce my German/Swiss wife, Hiltrud, who, although she is an actress and singer, has a personal inerest in the science described since she is a breast cancer survivor.
In closing, I would like to express my own awe at the progress that has been made during the period since we tried with little success to receive acceptance of the following claim:
The first step in estrogen action
Is a strong, non-covalent attraction
For a receptor site
Where it snuggles up tight
Without metabolic reaction.
Bon-Voyage and Komm Gut Nach Hause."
Elwood Jensen