Mark Christian

RIP140-Targeted Repression of Gene Expression in Adipocytes

ABSTRACT Ligand-dependent repression of nuclear receptor activity forms a novel mechanism for regulating gene expression. To investigate the intrinsic role of the corepressor RIP140, we have monitored gene expression profiles in cells that express or lack the RIP140 gene and that can be induced to undergo adipogenesis in vitro. In contrast to normal white adipose tissue and in vitro-differentiated wild-type adipocytes, RIP140-null cells show elevated energy expenditure and express high levels of the uncoupling protein 1 gene (Ucp1), carnitine palmitoyltransferase 1b, and the cell-death-inducing DFF45-like effector A. Conversely, all these changes are abrogated by the reexpression of RIP140. Analysis of the Ucp1 promoter showed RIP140 recruitment to a key enhancer element, demonstrating a direct role in repressing gene expression. Therefore, reduction in the levels of RIP140 or prevention of its recruitment to nuclear receptors may provide novel mechanisms for the control of energy expenditure in adipose cells.

A Functional Interaction between RIP140 and PGC-1α Regulates the Expression of the Lipid Droplet Protein CIDEA

ABSTRACT Nuclear receptors activate or repress target genes depending on the recruitment of coactivators or corepressors. The corepressor RIP140 and the PPAR coactivator 1α (PGC-1α) both play key roles in the regulated transcription of genes involved in energy homeostasis. We investigated the roles of RIP140 and PGC-1α in controlling the expression of CIDEA, an important regulatory factor in adipose cell function and obesity. Ectopically expressed CIDEA surrounded lipid droplets in brown adipocytes and induced the formation of lipid droplets in nonadipogenic cell lines. The expression and promoter activity of CIDEA was repressed by RIP140 and induced by PGC-1α, mediated through the binding of estrogen-related receptor α and NRF-1 to their cognate binding sites. Importantly, we demonstrate that RIP140 interacts directly with PGC-1α and suppresses its activity. The direct antagonism of PGC-1α by RIP140 provides a mechanism for regulating target gene transcription via nuclear receptor-dependent and -independent pathways.

Brown and white adipose tissues: intrinsic differences in gene expression and response to cold exposure in mice

Brown adipocytes dissipate energy, whereas white adipocytes are an energy storage site. We explored the plasticity of different white adipose tissue depots in acquiring a brown phenotype by cold exposure. By comparing cold-induced genes in white fat to those enriched in brown compared with white fat, at thermoneutrality we defined a “brite” transcription signature. We identified the genes, pathways, and promoter regulatory motifs associated with “browning,” as these represent novel targets for understanding this process. For example, neuregulin 4 was more highly expressed in brown adipose tissue and upregulated in white fat upon cold exposure, and cell studies showed that it is a neurite outgrowth-promoting adipokine, indicative of a role in increasing adipose tissue innervation in response to cold. A cell culture system that allows us to reproduce the differential properties of the discrete adipose depots was developed to study depot-specific differences at an in vitro level. The key transcriptional events underpinning white adipose tissue to brown transition are important, as they represent an attractive proposition to overcome the detrimental effects associated with metabolic disorders, including obesity and type 2 diabetes.

Deregulation of the serum- and glucocorticoid-inducible kinase SGK1 in the endometrium causes reproductive failure

Infertility and recurrent pregnancy loss (RPL) are prevalent but distinct causes of reproductive failure that often remain unexplained despite extensive investigations. Analysis of midsecretory endometrial samples revealed that SGK1, a kinase involved in epithelial ion transport and cell survival, is upregulated in unexplained infertility, most prominently in the luminal epithelium, but downregulated in the endometrium of women suffering from RPL. To determine the functional importance of these observations, we first expressed a constitutively active SGK1 mutant in the luminal epithelium of the mouse uterus. This prevented expression of certain endometrial receptivity genes, perturbed uterine fluid handling and abolished embryo implantation. By contrast, implantation was unhindered in Sgk1−/− mice, but pregnancy was often complicated by bleeding at the decidual-placental interface and fetal growth retardation and subsequent demise. Compared to wild-type mice, Sgk1−/− mice had gross impairment of pregnancy-dependent induction of genes involved in oxidative stress defenses. Relative SGK1 deficiency was also a hallmark of decidualizing stromal cells from human subjects with RPL and sensitized these cells to oxidative cell death. Thus, depending on the cellular compartment, deregulated SGK1 activity in cycling endometrium interferes with embryo implantation, leading to infertility, or predisposes to pregnancy complications by rendering the feto-maternal interface vulnerable to oxidative damage.

Metabolic regulation by the nuclear receptor corepressor RIP140

Whereas the importance of activating gene expression in metabolic pathways to control energy homeostasis is well established, the contribution of transcriptional inhibition is less well defined. In this review we highlight a crucial role of RIP140, a transcriptional corepressor for nuclear receptors, in the regulation of energy expenditure. Mice devoid of the RIP140 gene are lean, exhibit resistance to high-fat-diet-induced obesity, and have increased glucose tolerance and insulin sensitivity. Consistent with these observations, RIP140 suppresses the expression of gene clusters that are involved in lipid and carbohydrate metabolism, including fatty acid oxidation, oxidative phosphorylation and mitochondrial uncoupling. Therefore, the functional interplay between transcriptional activators and the corepressor RIP140 is an essential process in metabolic regulation.

Coactivator function of RIP140 for nf{kappa}b/rela-dependent cytokine gene expression

Inflammatory responses represent a hallmark of numerous pathologies including sepsis, bacterial infection, insulin resistance, and malign obesity. Here we describe an unexpected coactivator function for the nuclear receptor interacting protein 140 (RIP140) for nuclear factor kappaB (NFkappaB), a master transcriptional regulator of inflammation in multiple tissues. Previous work has shown that RIP140 suppresses the expression of metabolic gene networks, but we have found that genetic as well as acute deficiency of RIP140 leads to the inhibition of the proinflammatory program in macrophages. The ability of RIP140 to function as a coactivator for cytokine gene promoter activity relies on direct protein-protein interactions with the NFkappaB subunit RelA and histone acetylase cAMP-responsive element binding protein (CREB)-binding protein (CBP). RIP140-dependent control of proinflammatory gene expression via RelA/CBP may, therefore, represent a molecular rational for the cellular integration of metabolic and inflammatory pathways.

Uterine Selection of Human Embryos at Implantation

Human embryos frequently harbor large-scale complex chromosomal errors that impede normal development. Affected embryos may fail to implant although many first breach the endometrial epithelium and embed in the decidualizing stroma before being rejected via mechanisms that are poorly understood. Here we show that developmentally impaired human embryos elicit an endoplasmic stress response in human decidual cells. A stress response was also evident upon in vivo exposure of mouse uteri to culture medium conditioned by low-quality human embryos. By contrast, signals emanating from developmentally competent embryos activated a focused gene network enriched in metabolic enzymes and implantation factors. We further show that trypsin, a serine protease released by pre-implantation embryos, elicits Ca2+ signaling in endometrial epithelial cells. Competent human embryos triggered short-lived oscillatory Ca2+ fluxes whereas low-quality embryos caused a heightened and prolonged Ca2+ response. Thus, distinct positive and negative mechanisms contribute to active selection of human embryos at implantation.

Disordered IL-33/ST2 Activation in Decidualizing Stromal Cells Prolongs Uterine Receptivity in Women with Recurrent Pregnancy Loss

Decidualization renders the endometrium transiently receptive to an implanting blastocyst although the underlying mechanisms remain incompletely understood. Here we show that human endometrial stromal cells (HESCs) rapidly release IL-33, a key regulator of innate immune responses, upon decidualization. In parallel, differentiating HESCs upregulate the IL-33 transmembrane receptor ST2L and other pro-inflammatory mediators before mounting a profound anti-inflammatory response that includes downregulation of ST2L and increased expression of the soluble decoy receptor sST2. We demonstrate that HESCs secrete factors permissive of embryo implantation in mice only during the pro-inflammatory phase of the decidual process. IL-33 knockdown in undifferentiated HESCs was sufficient to abrogate this pro-inflammatory decidual response. Further, sequential activation of the IL-33/ST2L/sST2 axis was disordered in decidualizing HESCs from women with recurrent pregnancy loss. Signals from these cultures prolonged the implantation window but also caused subsequent pregnancy failure in mice. Thus, Il-33/ST2 activation in HESCS drives an autoinflammatory response that controls the temporal expression of receptivity genes. Failure to constrain this response predisposes to miscarriage by allowing out-of-phase implantation in an unsupportive uterine environment.

RIP140 directs histone and DNA methylation to silence Ucp1 expression in white adipocytes

Nuclear receptors control the function of cells by regulating transcription from specific gene networks. The establishment and maintenance of epigenetic gene marks is fundamental to the regulation of gene transcription and the control of cell function. RIP140 is a corepressor for nuclear receptors that suppresses transcription from a broad programme of metabolic genes and thereby controls energy homoeostasis in vivo. Here we show by analysis of Ucp1, a gene which is typically expressed in brown but not white adipocytes, that RIP140 is essential for both DNA and histone methylation to maintain gene repression. RIP140 expression promotes the assembly of DNA and histone methyltransferases (HMTs) on the Ucp1 enhancer and leads to methylation of specific CpG residues and histones as judged by bisulphite genomic sequencing and chromatin immunoprecipitation assays. Our results suggest that RIP140 serves as a scaffold for both DNA and HMT activities to inhibit gene transcription by two key epigenetic repression systems.

Regulated expression of signal transducer and activator of transcription, Stat5, and its enhancement of PRL expression in human endometrial stromal cells in vitro.

Differentiation of human endometrium during the secretory phase of the menstrual cycle is characterized by expression of a variety of genes implicated in the establishment and maintenance of pregnancy. An increased abundance of signal transducers and activators of transcription (Stats) in the secretory phase suggests Stat5 as a component of the differentiation of endometrium in response to ovarian hormone stimulation in vivo. Decidualization is initiated in a subset of endometrial stromal cells (ESC) in vivo during the secretory phase, but it is unclear whether regulated expression of Stat5 is a feature of these cells. Here, therefore, the abundance and subcellular distribution of Stat5 in ESC after a decidualization stimulus of cAMP plus medroxyprogesterone acetate (MPA) has been investigated in vitro. Western blotting revealed an increase in the apparent abundance of Stat5a and Stat5b, in the cytosolic and nuclear fractions, at 2, 3, and 4 d after stimulation. The potential functional relevance of this increase in Stat5 is suggested by the ability of transiently transfected Stat5a or Stat5b to significantly enhance the response of the decidual PRL promoter to cAMP/MPA and attenuation of the response to cAMP/MPA by dominant negative Stat5. Recent evidence suggests endometrial differentiation, including PRL production, as a possible target of antiphospholipid antibodies (aPL) prevalent in recurrent miscarriage. Monoclonal antibody, ID2, which has similar reactivity as human aPL, significantly decreased the apparent abundance of nuclear Stat5b in response to cAMP/MPA and was associated with decreased decidual PRL promoter activation and PRL secretion. Regulated expression of Stat5 is therefore a component of decidual differentiation of human ESC and contributes significantly to activation of the decidual PRL promoter. Alteration of this process by an aPL component suggests decidual differentiation as a potential clinical target in recurrent early miscarriages.