Feedback modulation of cholesterol metabolism by the lipid-responsive non-coding RNA LeXis.

Liver X receptors (LXRs) are transcriptional regulators of cellular and systemic cholesterol homeostasis. Under conditions of excess cholesterol, LXR activation induces the expression of several genes involved in cholesterol efflux, facilitates cholesterol esterification by promoting fatty acid synthesis, and inhibits cholesterol uptake by the low-density lipoprotein receptor. The fact that sterol content is maintained in a narrow range in most cell types and in the organism as a whole suggests that extensive crosstalk between regulatory pathways must exist. However, the molecular mechanisms that integrate LXRs with other lipid metabolic pathways are incompletely understood. Here we show that ligand activation of LXRs in mouse liver not only promotes cholesterol efflux, but also simultaneously inhibits cholesterol biosynthesis. We further identify the long non-coding RNA LeXis as a mediator of this effect. Hepatic LeXis expression is robustly induced in response to a Western diet (high in fat and cholesterol) or to pharmacological LXR activation. Raising or lowering LeXis levels in the liver affects the expression of genes involved in cholesterol biosynthesis and alters the cholesterol levels in the liver and plasma. LeXis interacts with and affects the DNA interactions of RALY, a heterogeneous ribonucleoprotein that acts as a transcriptional cofactor for cholesterol biosynthetic genes in the mouse liver. These findings outline a regulatory role for a non-coding RNA in lipid metabolism and advance our understanding of the mechanisms that coordinate sterol homeostasis.

Enhanced thermogenesis in the blinc of an eye.

Increasing the thermogenic capacity of adipose tissue has been proposed as a strategy for combating obesity and its associated metabolic disorders. In this issue of Molecular Cell, Zhao et al. (2014) identify the long noncoding RNA Blnc1 as a driver of thermogenesis in brown and beige adipocytes.

The macrophage LBP gene is an LXR target that promotes macrophage survival and atherosclerosis.

The liver X receptors (LXRs) are members of the nuclear receptor superfamily that regulate sterol metabolism and inflammation. We sought to identify previously unknown genes regulated by LXRs in macrophages and to determine their contribution to atherogenesis. Here we characterize a novel LXR target gene, the lipopolysaccharide binding protein (LBP) gene. Surprisingly, the ability of LXRs to control LBP expression is cell-type specific, occurring in macrophages but not liver. Treatment of macrophages with oxysterols or loading with modified LDL induces LBP in an LXR-dependent manner, suggesting a potential role for LBP in the cellular response to cholesterol overload. To investigate this further, we performed bone marrow transplant studies. After 18 weeks of Western diet feeding, atherosclerotic lesion burden was assessed revealing markedly smaller lesions in the LBP(-/-) recipients. Furthermore, loss of bone marrow LBP expression increased apoptosis in atherosclerotic lesions as determined by terminal deoxynucleotidyl transferase dUTP nick end labeling staining. Supporting in vitro studies with isolated macrophages showed that LBP expression does not affect cholesterol efflux but promotes the survival of macrophages in the setting of cholesterol loading. The LBP gene is a macrophage-specific LXR target that promotes foam cell survival and atherogenesis.

Role of nuclear receptor corepressor RIP140 in metabolic syndrome.

Obesity and its associated complications, which can lead to the development of metabolic syndrome, are a worldwide major public health concern especially in developed countries where they have a very high prevalence. RIP140 is a nuclear coregulator with a pivotal role in controlling lipid and glucose metabolism. Genetically manipulated mice devoid of RIP140 are lean with increased oxygen consumption and are resistant to high-fat diet-induced obesity and hepatic steatosis with improved insulin sensitivity. Moreover, white adipocytes with targeted disruption of RIP140 express genes characteristic of brown fat including CIDEA and UCP1 while skeletal muscles show a shift in fibre type composition enriched in more oxidative fibres. Thus, RIP140 is a potential therapeutic target in metabolic disorders. In this article we will review the role of RIP140 in tissues relevant to the appearance and progression of the metabolic syndrome and discuss how the manipulation of RIP140 levels or activity might represent a therapeutic approach to combat obesity and associated metabolic disorders. This article is part of a Special Issue entitled: Translating nuclear receptors from health to disease.

The SUMO E3-ligase PIAS1 couples reactive oxygen species-dependent JNK activation to oxidative cell death.

Human endometrial stromal cells (HESCs) exposed to reactive oxygen species (ROS) mount a hypersumoylation response in a c-Jun N-terminal kinase (JNK)-dependent manner. The mechanism that couples JNK signaling to the small ubiquitin-related modifier (SUMO) pathway and its functional consequences are not understood. We show that ROS-dependent JNK activation converges on the SUMO pathway via PIAS1 (protein inhibitor of activated STAT1). Unexpectedly, PIAS1 knockdown not only prevented ROS-dependent hypersumoylation but also enhanced JNK signaling in HESCs. Conversely, PIAS overexpression increased sumoylation of various substrates, including c-Jun, yet inhibited basal and ROS-dependent JNK activity independently of its SUMO ligase function. Expression profiling demonstrated that PIAS1 knockdown enhances and profoundly modifies the transcriptional response to oxidative stress signals. Using a cutoff of 2-fold change or more, a total of 250 ROS-sensitive genes were identified, 97 of which were not dependent on PIAS1. PIAS1 knockdown abolished the regulation of 43 genes but also sensitized 110 other genes to ROS. Importantly, PIAS1 silencing was obligatory for the induction of several cellular defense genes in response to oxidative stress. In agreement, PIAS1 knockdown attenuated ROS-dependent caspase-3/7 activation and subsequent apoptosis. Thus, PIAS1 determines the level of JNK activity in HESCs, couples ROS signaling to the SUMO pathway, and promotes oxidative cell death.

Silencing of the JNK pathway maintains progesterone receptor activity in decidualizing human endometrial stromal cells exposed to oxidative stress signals.

Survival of the conceptus is dependent on continuous progesterone signaling in the maternal decidua but how this is achieved under conditions of oxidative stress that characterize early pregnancy is unknown. Using primary cultures, we show that modest levels of reactive oxygen species (ROS) increase sumoylation in human endometrial stromal cells (HESCs), leading to enhanced modification and transcriptional inhibition of the progesterone receptor (PR). The ability of ROS to induce a sustained hypersumoylation response, or interfere with PR activity, was lost upon differentiation of HESCs into decidual cells. Hypersumoylation in response to modest levels of ROS requires activation of the JNK pathway. Although ROS-dependent JNK signaling is disabled on decidualization, the cells continue to mount a transcriptional response, albeit distinct from that observed in undifferentiated HESCs. We further show that attenuated JNK signaling in decidual cells is a direct consequence of altered expression of key pathway modulators, including induction of MAP kinase phosphatase 1 (MKP1). Overexpression of MKP1 dampens JNK signaling, prevents hypersumoylation, and maintains PR activity in undifferentiated HESCs exposed to ROS. Thus, JNK silencing uncouples ROS signaling from the SUMO conjugation pathway and maintains progesterone responses and cellular homeostasis in decidual cells under oxidative stress conditions imposed by pregnancy.

Transforming growth factor-beta1 attenuates expression of both the progesterone receptor and Dickkopf in differentiated human endometrial stromal cells.

TGFbeta1 is thought to be intimately involved in cyclic tissue remodeling and inflammatory events associated with menstruation. Menstruation is initiated by progesterone withdrawal; however, the underlying mechanisms are not well understood. In the present study, we have tested the hypothesis that locally produced TGFbeta1 may influence expression of progesterone receptor (PR) or the Wnt antagonist Dickkopf-1 (DKK) with consequential impact on regulation of menstruation. Endometrial stromal cells (ESC) were isolated from endometrial biopsy samples collected from patients undergoing gynecological procedures for benign indications. Treatment of differentiated ESC with TGFbeta1 (10 ng/ml) significantly inhibited the expression of mRNAs encoding PR and DKK. TGFbeta1 also attenuated the protein expression of PR and secretion of DKK proteins in culture supernatants. Neutralization of endogenous TGFbeta1 signaling abolished the TGFbeta1-induced effects, significantly increased expression of PR, and increased DKK protein release levels to that of differentiated ESCs, confirming the specificity of the TGFbeta1 effect. Additionally, in vitro decidualization of ESCs significantly augmented DKK protein release. Moreover, although TGFbeta1 was capable of signaling via the Sma- and mothers against decapentaplegic (MAD)-related protein (SMAD) pathway, the inhibitory effect on DKK was SMAD independent. Conversely, the inhibitory effect of TGFbeta1 on PR was dependent on SMAD signal transduction. In conclusion, these results suggest that local TGFbeta1 signaling can potentiate progesterone withdrawal by suppressing expression of PR and may coordinate tissue remodeling associated with menstruation by inducing Wnt-signaling via inhibition of DKK, which we found to be up-regulated as a consequence of decidualization of ESCs.

The androgen and progesterone receptors regulate distinct gene networks and cellular functions in decidualizing endometrium.

Progesterone is indispensable for differentiation of human endometrial stromal cells (HESCs) into decidual cells, a process that critically controls embryo implantation. We now show an important role for androgen receptor (AR) signaling in this differentiation process. Decreased posttranslational modification of the AR by small ubiquitin-like modifier (SUMO)-1 in decidualizing cells accounted for increased responsiveness to androgen. By combining small interfering RNA technology with genome-wide expression profiling, we found that AR and progesterone receptor (PR) regulate the expression of distinct decidual gene networks. Ingenuity pathway analysis implicated a preponderance of AR-induced genes in cytoskeletal organization and cell motility, whereas analysis of AR-repressed genes suggested involvement in cell cycle regulation. Functionally, AR depletion prevented differentiation-dependent stress fiber formation and promoted motility and proliferation of decidualizing cells. In comparison, PR depletion perturbed the expression of many more genes, underscoring the importance of this nuclear receptor in diverse cellular functions. However, several PR-dependent genes encode for signaling intermediates, and knockdown of PR, but not AR, compromised activation of WNT/beta-catenin, TGFbeta/SMAD, and signal transducer and activator of transcription (STAT) pathways in decidualizing cells. Thus, the nonredundant function of the AR in decidualizing HESCs, centered on cytoskeletal organization and cell cycle regulation, implies an important role for androgens in modulating fetal-maternal interactions. Moreover, we show that PR regulates HESC differentiation, at least in part, by reprogramming growth factor and cytokine signal transduction.

Transcriptional regulation of macrophage cholesterol efflux and atherogenesis by a long noncoding RNA.

Nuclear receptors regulate gene expression in response to environmental cues, but the molecular events governing the cell type specificity of nuclear receptors remain poorly understood. Here we outline a role for a long noncoding RNA (lncRNA) in modulating the cell type-specific actions of liver X receptors (LXRs), sterol-activated nuclear receptors that regulate the expression of genes involved in cholesterol homeostasis and that have been causally linked to the pathogenesis of atherosclerosis. We identify the lncRNA MeXis as an amplifier of LXR-dependent transcription of the gene Abca1, which is critical for regulation of cholesterol efflux. Mice lacking the MeXis gene show reduced Abca1 expression in a tissue-selective manner. Furthermore, loss of MeXis in mouse bone marrow cells alters chromosome architecture at the Abca1 locus, impairs cellular responses to cholesterol overload, and accelerates the development of atherosclerosis. Mechanistic studies reveal that MeXis interacts with and guides promoter binding of the transcriptional coactivator DDX17. The identification of MeXis as a lncRNA modulator of LXR-dependent gene expression expands understanding of the mechanisms underlying cell type-selective actions of nuclear receptors in physiology and disease.

FoxO3a and BCR-ABL regulate cyclin D2 transcription through a STAT5/BCL6-dependent mechanism.

Cell cycle arrest by FoxO transcription factors involves transcriptional repression of cyclin D, although the exact mechanism remains unclear. In this study, we used the BCR-ABL-expressing cell line BV173 as a model system to investigate the mechanisms whereby FoxO3a regulates cyclin D2 expression. Inhibition of BCR-ABL by STI571 results in down-regulation of cyclin D2 expression, activation of FoxO3a activity, and up-regulation of BCL6 expression. Using reporter gene assays, we demonstrate that STI571, FoxO3a, and BCL6 can repress cyclin D2 transcription through a STAT5/BCL6 site located within the cyclin D2 promoter. We propose that BCR-ABL inhibition leads to FoxO3a activation, which in turn induces the expression of BCL6, culminating in the repression of cyclin D2 transcription through this STAT5/BCL6 site. This process was verified by mobility shift and chromatin immunoprecipitation analyses. We find that conditional activation of FoxO3a leads to accumulation of BCL6 and down-regulation of cyclin D2 at protein and mRNA levels. Furthermore, silencing of FoxO3a and BCL6 in BCR-ABL-expressing cells abolishes STI571-mediated effects on cyclin D2. This report establishes the signaling events whereby BCR-ABL signals are relayed to cyclin D2 to mediate cell cycle progression and defines a potential mechanism by which FoxO proteins regulate cyclin D2 expression.

Differential expression of FOXO1 and FOXO3a confers resistance to oxidative cell death upon endometrial decidualization.

The integrity of the feto-maternal interface is critical for survival of the conceptus. This interface, consisting of the maternal decidua and the invading placental trophoblast, is exposed to profound changes in oxygen tension during pregnancy. We demonstrate that human endometrial stromal cells become extraordinarily resistant to oxidative stress-induced apoptosis upon decidualization in response to cAMP and progesterone signaling. This differentiation process is associated with the induction of the forkhead transcription factor FOXO1, which in turn increases the expression of the mitochondrial antioxidant manganese superoxide dismutase. However, silencing of FOXO1 did not increase the susceptibility of decidualized cells to oxidative cell death. Comparative analysis demonstrated that hydrogen peroxide, a source of free radicals, strongly induces FOXO3a mRNA and protein expression in undifferentiated human endometrial stromal cells but not in decidualized cells. Expression of a constitutively active FOXO3a mutant elicited apoptosis in decidualized cells. Furthermore, silencing of endogenous FOXO3a in undifferentiated cells abrogated apoptosis induced by hydrogen peroxide. These results suggest that the induction of FOXO1 may enhance the ability of decidualized cells to prevent oxidative damage while the simultaneous repression of FOXO3a expression disables the signaling pathway responsible for oxidative cell death. The differential regulation of FOXO expression provides the decidua with a robust system capable of coping with prolonged episodes of oxidative stress during pregnancy.

Progestins regulate the expression and activity of the forkhead transcription factor FOXO1 in differentiating human endometrium.

Menstruation, or cyclic shedding of nonpregnant endometrial tissue with associated bleeding, occurs only in humans and a few other species. This breakdown of the endometrium in response to falling ovarian progesterone levels is a complex process, characterized by local leukocyte infiltration, expression and activation of matrix metalloproteinases, and apoptosis. Spontaneous decidualization (differentiation) of the stromal compartment precedes the cyclic shedding of the endometrium in various menstruating species but the mechanisms that link these processes are not understood. In this study, we identified FOXO1 as a key transcription factor responsible for mediating apoptosis of decidualized human endometrial stromal cells (HESCs) in response to progesterone withdrawal. We demonstrate that medroxyprogesterone acetate (MPA, a synthetic progestin) enhances the expression of FOXO1 in differentiating HESCs while simultaneously inducing cytoplasmic retention and inactivation of FOXO1. Withdrawal of MPA from decidualized HESCs results in rapid nuclear accumulation of FOXO1, increased BIM expression, a proapoptotic FOXO1 target gene, and cell death. Conversely, silencing of FOXO1 expression completely abolishes cell death induced by MPA withdrawal. In summary, the observation that differentiating HESCs become dependent on progesterone signaling for survival through induction and reversible inactivation of FOXO1 suggests a novel mechanism that links decidualization of the endometrium to menstruation.

Convergence of interferon-gamma and progesterone signaling pathways in human endometrium: role of PIASy (protein inhibitor of activated signal transducer and activator of transcription-y).

All cardinal events during the reproductive cycle, including ovulation, implantation, and menstruation, are characterized by a profound tissue remodeling and an associated local inflammatory response. The ovarian hormone progesterone is a key modulator of inflammatory signals in reproductive tissues, but the underlying mechanisms are not well understood. In this study, we report that differentiating human endometrial stromal cells (ESCs) acquire resistance to interferon-gamma (IFNgamma)-dependent signal transducers and activators of transcription (STAT) 1 signaling, although phosphorylation, nuclear translocation, and binding of STAT1 to DNA, are unaffected. These observations prompted an investigation into the role of nuclear repressors of STAT1 signaling. We demonstrate that protein inhibitor of activated STAT-y is complexed to the progesterone receptor (PR) in human ESCs and that its ability to repress STAT1 signaling is dependent upon activation of PR in response to hormone binding. Conversely, IFNgamma and protein inhibitor of activated STAT-y synergistically inhibited PR-dependent transcription, demonstrating that the progesterone and IFNgamma signaling pathways engage in reciprocal transcriptional antagonism in human endometrium.

Progesterone promotes focal adhesion formation and migration in breast cancer cells through induction of protease-activated receptor-1.

Progesterone and progestins have been demonstrated to enhance breast cancer cell migration, although the mechanisms are still not fully understood. The protease-activated receptors (PARs) are a family of membrane receptors that are activated by serine proteases in the blood coagulation cascade. PAR1 (F2R) has been reported to be involved in cancer cell migration and overexpressed in breast cancer. We herein demonstrate that PAR1 mRNA and protein are upregulated by progesterone treatment of the breast cancer cell lines ZR-75 and T47D. This regulation is dependent on the progesterone receptor (PR) but does not require PR phosphorylation at serine 294 or the PR proline-rich region mPRO. The increase in PAR1 mRNA was transient, being present at 3  h and returning to basal levels at 18  h. The addition of a PAR1-activating peptide (aPAR1) to cells treated with progesterone resulted in an increase in focal adhesion (FA) formation as measured by the cellular levels of phosphorylated FA kinase. The combined but not individual treatment of progesterone and aPAR1 also markedly increased stress fiber formation and the migratory capacity of breast cancer cells. In agreement with in vitro findings, data mining from the Oncomine platform revealed that PAR1 expression was significantly upregulated in PR-positive breast tumors. Our observation that PAR1 expression and signal transduction are modulated by progesterone provides new insight into how the progestin component in hormone therapies increases the risk of breast cancer in postmenopausal women.

TGFß1 attenuates expression of prolactin and IGFBP-1 in decidualized endometrial stromal cells by both SMAD-dependent and SMAD-independent pathways.

BACKGROUND: Decidualization (differentiation) of the endometrial stromal cells during the secretory phase of the menstrual cycle is essential for successful implantation. Transforming Growth Factor ß1 (TGFß1) canonically propagates its actions via SMAD signalling. A role for TGFß1 in decidualization remains to be established and published data concerning effects of TGFß1 on markers of endometrial decidualization are inconsistent. METHODOLOGY/PRINCIPAL FINDINGS: Non-pregnant endometrial stromal cells (ESC) and first trimester decidual stromal cells (DSC) were cultured in the presence or absence of a decidualizing stimulus. Incubation of ESCs with TGFß1 (10 ng/ml) down-regulated the expression of transcripts encoding the decidual marker proteins prolactin (PRL), insulin-like growth factor binding protein-1 (IGFBP-1) and tissue factor (TF). TGFß1 also inhibited secretion of PRL and IGFBP-1 proteins by ESCs and surprisingly this response preceded down-regulation of their mRNAs. In contrast, DSCs were more refractory to the actions of TGFß1, characterized by blunted and delayed down-regulation of PRL, IGFBP-1, and TF transcripts, which was not associated with a significant reduction in secretion of PRL or IGFBP-1 proteins. Addition of an antibody directed against TGFß1 increased expression of IGFBP-1 mRNA in decidualised cells. Knockdown of SMAD 4 using siRNAs abrogated the effect of TGFß1 on expression of PRL in ESCs but did not fully restore expression of IGFBP-1 mRNA and protein. CONCLUSIONS/SIGNIFICANCE: TGFß1 inhibits the expression and secretion of decidual marker proteins. The impact of TGFß1 on PRL is SMAD-dependent but the impact on IGFBP1 is via an alternative mechanism. In early pregnancy, resistance of DSC to the impact of TGFß1 may be important to ensure tissue homeostasis.

TBX22 missense mutations found in patients with X-linked cleft palate affect DNA binding, sumoylation, and transcriptional repression.

The T-box transcription factor TBX22 is essential for normal craniofacial development, as demonstrated by the finding of nonsense, frameshift, splice-site, or missense mutations in patients with X-linked cleft palate (CPX) and ankyloglossia. To better understand the function of TBX22, we studied 10 different naturally occurring missense mutations that are phenotypically equivalent to loss-of-function alleles. Since all missense mutations are located in the DNA-binding T-box domain, we first investigated the preferred recognition sequence for TBX22. Typical of T-box proteins, the resulting sequence is a palindrome based around near-perfect copies of AGGTGTGA. DNA-binding assays indicate that missense mutations at or near predicted contact points with the DNA backbone compromise stable DNA-protein interactions. We show that TBX22 functions as a transcriptional repressor and that TBX22 missense mutations result in impaired repression activity. No effect on nuclear localization of TBX22 was observed. We find that TBX22 is a target for the small ubiquitin-like modifier SUMO-1 and that this modification is required for TBX22 repressor activity. Although the site of SUMO attachment at the lysine at position 63 is upstream of the T-box domain, loss of SUMO-1 modification is consistently found in all pathogenic CPX missense mutations. This implies a general mechanism linking the loss of SUMO conjugation to the loss of TBX22 function. Orofacial clefts are well known for their complex etiology and variable penetrance, involving both genetic and environmental risk factors. The sumoylation process is also subject to and profoundly affected by similar environmental stresses. Thus, we suggest that SUMO modification may represent a common pathway that regulates normal craniofacial development and is involved in the pathogenesis of both Mendelian and idiopathic forms of orofacial clefting.

Regulation of the SUMO pathway sensitizes differentiating human endometrial stromal cells to progesterone.

cAMP is required for differentiation of human endometrial stromal cells (HESCs) into decidual cells in response to progesterone, although the underlying mechanism is not well understood. We now demonstrate that cAMP signaling attenuates ligand-dependent sumoylation of the progesterone receptor (PR) in HESCs. In fact, decidualization is associated with global hyposumoylation and redistribution of small ubiquitin-like modifier (SUMO)-1 conjugates into distinct nuclear foci. This altered pattern of global sumoylation was not attributable to impaired maturation of SUMO-1 precursor or altered expression of E1 (SAE1/SEA2) or E2 (Ubc9) enzymes but coincided with profound changes in the expression of E3 ligases and SUMO-specific proteases. Down-regulation of several members of the protein inhibitors of activated STAT (PIAS) family upon decidualization pointed toward a role of these E3 ligases in PR sumoylation. We demonstrate that PIAS1 interacts with the PR and serves as its E3 SUMO ligase upon activation of the receptor. Furthermore, we show that silencing of PIAS1 not only enhances PR-dependent transcription but also induces expression of prolactin, a decidual marker gene, in progestin-treated HESCs without the need of simultaneous activation of the cAMP pathway. Our findings demonstrate how dynamic changes in the SUMO pathway mediated by cAMP signaling determine the endometrial response to progesterone.