Receptor-interacting Protein 140 mRNA Research Articles

Behavioral stress reduces RIP140 expression in astrocyte and increases brain lipid accumulation.

Receptor-interacting protein 140 (RIP140) is highly expressed in the brain, and acts in neurons and microglia to affect emotional responses. The present study reveals an additional function of RIP140 in the brain, which is to regulate brain lipid homeostasis via its action in astrocytes. We found forced swim stress (FSS) significantly reduces the expression level of RIP140 and elevates cholesterol content in the brain. Mechanistically, FSS elevates endoplasmic reticulum stress, which suppresses RIP140 expression by increasing microRNA 33 (miR33) that targets RIP140 mRNA's 3'-untranslated region. Consequentially, cholesterol biosynthesis and export are dramatically increased in astrocyte, the major source of brain cholesterol. These results demonstrate that RIP140 plays an important role in maintaining brain cholesterol homeostasis through, partially, regulating cholesterol metabolism in, and mobilization from, astrocyte. Altering RIP140 levels can disrupt brain cholesterol homeostasis, which may contribute to behavioral stress-induced neurological disorders.

Low RIP140 expression rescues basal FA uptake via differential expression of FATP1 and CD36 in skeletal muscle cells exposed to high palmitate

We have shown in L6 skeletal muscle cells that high fatty acid (FA) exposure decreases basal FA uptake and oxidation and that low receptor interacting protein 140 (RIP140) increases FA uptake. To provide mechanistic insights for this adaptation, we measured the expression of proteins and genes involved in the regulation of FA metabolism under high FA availability. L6 myotubes were silenced using RNAi sequences for either RIP140 (RIP) or a negative control (NC) and incubated ± palmitate (HF: 400μM) in the basal state. siRNA treatment reduced (P<0.05) RIP140 mRNA (55%) and protein (75%) levels. HF treatment reduced (P<0.05) mRNA expression of markers of oxidative capacity by 48–64% (COX4, MCAD, CPT1, PGC‐1α) but increased CPT1 protein levels (56%) in NC cells. Low RIP140 in HF‐treated cells restored COX4 mRNA and CPT1 protein levels back to control levels. HF treatment increased (P<0.05) FATP1 mRNA (100%) and total CD36 protein content (95%) but decreased (P<0.05) CD36 mRNA (63%) and FATP1 protein (59%) expression. Low RIP140 in HF‐treated cells restored CD36 mRNA and protein content back to control levels but did not affect the already high FATP1 mRNA levels. Plasma membrane CD36 content was not affected (P>0.05) by either treatment. HF treatment increased (P<0.05) basal pAKTser473 (443%), pAKTthr308 (126%) and pPKC‐ζ (96%) in NC cells. Low RIP140 in HF‐treated cells restored pPKC‐ζ back to control levels and reduced (P<0.05) pAKTthr308. Our data suggest that the rise in basal FA uptake induced by low RIP140 expression in HF‐treated cells is partially mediated by an increase in FATP1 mRNA expression.USC Women in Science and Engineering, USC Integrative and Evolutionary Biology and USC Zumberge Research Innovation Fund

Modulation of Clock Gene Expression by the Transcriptional Coregulator Receptor Interacting Protein 140 (RIP140)

Circadian rhythms are generated in central and peripheral tissues by an intracellular oscillating timing mechanism known as the circadian clock. Several lines of evidence show a strong and bidirectional interplay between metabolism and circadian rhythms. Receptor interacting protein 140 (RIP140) is a coregulator for nuclear receptors and other transcription factors that represses catabolic pathways in metabolic tissues. Although RIP140 functions as a corepressor for most nuclear receptors, mounting evidence points to RIP140 as a dual coregulator that can repress or activate different sets of genes. Here, we demonstrate that RIP140 mRNA and protein levels are under circadian regulation and identify RIP140 as a modulator of clock gene expression, suggesting that RIP140 can participate in a feedback mechanism affecting the circadian clock. We show that the absence of RIP140 disturbs the basal levels of BMAL1 and other clock genes, reducing the amplitude of their oscillations. In addition, we demonstrate that RIP140 is recruited to retinoid-related orphan receptor (ROR) binding sites on the BMAL1 promoter, directly interacts with RORα, and increases transcription from the BMAL1 promoter in a RORα-dependent manner. These results indicate that RIP140 is not only involved in metabolic control but also acts as a coactivator for RORα, influencing clock gene expression.

Cholesterol regulation of receptor‐interacting protein 140 via microRNA‐33 in inflammatory cytokine production

Receptor interacting protein 140 (RIP140) is a nuclear receptor coregulator that affects a wide spectrum of biological processes. It is unclear whether and how the expression level of RIP140 can be modulated and whether RIP140 is involved in inflammatory diseases. Here, we examine how intracellular cholesterol regulates RIP140 expression, and we evaluate the effect of RIP140 expression on macrophage proinflammatory potential. Macrophages treated with modified low-density lipoprotein express higher RIP140 mRNA and protein levels. Consistently, simvastatin reduces RIP140 levels, which can be reversed by mevalonate. Moreover, a high-fat diet elevates RIP140 but lowers miR-33 levels in peritoneal macrophages, and increases the production of IL-1β and TNF-α in macrophages. Mechanistically, miR-33 targets RIP140 mRNA by recognizing its target located in a highly conserved sequence of the 3'-untranslated region (3'-UTR) of RIP140 mRNA. Consequentially, miR-33 reduces RIP140 coactivator activity for NF-κB, which is supported by the reduction in NF-κB reporter activity and the inflammatory potential in macrophages. This study uncovers a cholesterol-miR-33-RIP140 regulatory pathway that modulates the proinflammatory potential in macrophages in response to an alteration in the intracellular cholesterol status, and identifies RIP140 as a direct target of miR-33 that mediates simvastatin-triggered anti-inflammation.

Role of Receptor-Interacting Protein 140 in human fat cells

BackgroundMice lacking Receptor-interacting protein 140 (RIP140) have reduced body fat which at least partly is mediated through increased lipid and glucose metabolism in adipose tissue. In humans, RIP140 is lower expressed in visceral white adipose tissue (WAT) of obese versus lean subjects. We investigated the role of RIP140 in human subcutaneous WAT, which is the major fat depot of the body.MethodsMessenger RNA levels of RIP140 were measured in samples of subcutaneous WAT from women with a wide variation in BMI and in different human WAT preparations. RIP140 mRNA was knocked down with siRNA in in vitro differentiated adipocytes and the impact on glucose transport and mRNA levels of target genes determined.ResultsRIP140 mRNA levels in subcutaneous WAT were decreased among obese compared to lean women and increased by weight-loss, but did not associate with mitochondrial DNA copy number. RIP140 expression increased during adipocyte differentiation in vitro and was higher in isolated adipocytes compared to corresponding pieces of WAT. Knock down of RIP140 increased basal glucose transport and mRNA levels of glucose transporter 4 and uncoupling protein-1.ConclusionsHuman RIP140 inhibits glucose uptake and the expression of genes promoting energy expenditure in the same fashion as the murine orthologue. Increased levels of human RIP140 in subcutaneous WAT of lean subjects may contribute to economize on energy stores. By contrast, the function and expression pattern does not support that RIP140 regulate human obesity.

Elevated expression of the metabolic regulator receptor-interacting protein 140 results in cardiac hypertrophy and impaired cardiac function

Receptor-interacting protein 140 (RIP140) is a ligand-dependent cofactor for nuclear receptors that regulate networks of genes involved in cellular processes, including metabolism. An important role for RIP140 in metabolic control has been identified in RIP140 null mice, whose phenotypes include derepression of genes involved in energy mobilization or catabolism in adipocytes and a switch to more oxidative fibres in skeletal muscle. We hypothesized that ubiquitous expression of RIP140 would suppress metabolic processes, leading to defects in development or cellular function. The primary effect of exogenous expression of RIP140 mRNA (real-time PCR) and protein (western blotting) in transgenic mice is impaired postnatal heart function. There was rapid onset of cardiac hypertrophy and ventricular fibrosis, detected microscopically, in male RIP140 transgenic mice from 4 weeks of age, resulting in 25% mortality by 5 months. RIP140 exogenous expression in the heart leads to decreased mitochondria state III and state IV membrane potential and oxygen consumption. Quantitative PCR showed more than 50% reduced expression of genes involved in mitochondrial activity and fatty acid metabolism, including mitochondrial transcription factor A, cytochrome oxidase VIIa, cytochrome XII, CD36, medium-chain acyl dehydrogenase, and fatty acid transport protein, many of which are known targets for nuclear receptors, including peroxisome proliferator-activated receptors PPARalpha and PPARdelta and oestrogen-related receptors ERRalpha and ERRgamma. This study demonstrates that RIP140 is an important cofactor in postnatal cardiac function and that inhibition of the action of RIP140 may provide a model system to investigate specific interventions designed to prevent or delay the onset of cardiac disease.

MicroRNA mir-346 targets the 5′-untranslated region of receptor-interacting protein 140 (RIP140) mRNA and up-regulates its protein expression

RIP140 (receptor-interacting protein 140) is a transcriptional co-repressor that regulates diverse genes such as those responsive to hormones and involved in metabolic processes. The expression of RIP140 is regulated by multiple hormonal activities in adipose tissue and cancer cell lines. However, it is unclear whether and how RIP140 is regulated post-transcriptionally. Using 5'RACE (rapid amplification of 5' cDNA ends), we have identified a novel 5' splice variant of RIP140 mRNA in mouse brain and P19 cells. A target sequence for miRNA (microRNA) mir-346 was found in the 5'UTR (5'-untranslated region) of RIP140 mRNA; this miRNA is also expressed endogenously in mouse brain and P19 cells. Gain- and loss-of-function studies demonstrated that mir-346 elevates RIP140 protein levels by facilitating association of its mRNA with the polysome fraction. Furthermore, the activity of mir346 does not require Ago-2 (Argonaute 2). The expression of mir-346 enhances the gene repressive activity of RIP140. This is the first report demonstrating post-transcriptional regulation of RIP140 mRNA, involving the enhancing effect of a specific miRNA that targets RIP140's 5'UTR.

RIP140 Gene and Protein Expression Levels are Downregulated in Visceral Adipose Tissue in Human Morbid Obesity

Receptor-interacting protein 140 (RIP140) is a corepressor for nuclear receptors with an important role in the inhibition of energy expenditure. Rip140-knockout mice are lean and resistant to diet-induced obesity due to an increase in mitochondrial biogenesis, fatty acid oxidation, and oxidative phosphorylation. The aim of the present work was to evaluate the effect of morbid obesity on gene and protein expression levels of RIP140 in visceral adipose tissue (VAT). VAT biopsies obtained from 17 subjects were used in the study. Patients were classified as lean (body mass index [BMI]=21.8+/-1.3 kg/m2) or obese (BMI=48.2+/-2.6 kg/m2). Reverse transcription polymerase chain reaction and Western blot analyses were performed to quantify the expression levels of RIP140 in VAT. We also analyzed glucose and lipid profile as well as some inflammatory factors. Obese patients exhibited significantly lower RIP140 mRNA expression levels compared to lean subjects (lean=1.00+/-0.17 arbitrary units, obese=0.65+/-0.18 arbitrary units; P<0.05). Protein expression of RIP140 followed the same trend, being significantly higher in lean volunteers (lean=1.00+/-0.18 arbitrary units, obese=0.45+/-0.11 arbitrary units; P<0.05). Furthermore, a significant negative correlation was found between RIP140 protein levels and both BMI (rho=-0.85; P<0.001) and body fat percentage (rho=-0.88; P<0.001). The lower gene and protein expression levels of RIP140 in obese subjects may suggest a compensatory mechanism in order to favor energy expenditure and reduce fat accumulation in obesity states.

Gonadotropin regulation of RIP140 messenger ribonucleic acid expression in the rat ovary

Female mice null for receptor-interacting protein 140 (RIP140) are infertile because of the failure of follicle rupture. The present study examined gonadotropin regulation of RIP140 expression in immature rat ovary. Treatment with PMSG increased ovarian RIP140 mRNA and protein levels. In contrast, hCG treatment rapidly inhibited RIP140 mRNA and protein levels within 1–3 h. RIP140 mRNA was detected in theca cells of growing follicles in untreated ovary and in granulosa cells in PMSG-treated ovary. Interestingly, hCG treatment reduced RIP140 mRNA levels in granulosa cells of preovulatory follicles, but not of growing follicles. Neither treatment of immature rats with diethylstilbestrol in vivo nor of immature granulosa cells with FSH in vitro affected RIP140 mRNA levels. Treatment of immature granulosa cells with 17β-estradiol in vitro, however, stimulated RIP140 mRNA levels. In cultured preovulatory granulosa cells, RIP140 mRNA levels were stimulated at 1 h and then declined to below control levels by 3 h after LH treatment. Treatment with MDL-12,330A, an inhibitor of adenylate cyclase, or chelerythrine chloride, an inhibitor of protein kinase C (PKC), inhibited LH-stimulated RIP140 gene expression. Furthermore, forskolin or TPA treatment for 1 h mimicked the stimulatory action of LH, indicating the involvement of both adenylate cyclase and PKC pathways. These results demonstrate the stimulation by PMSG and inhibition by hCG of RIP140 expression in granulosa cells of preovulatory follicles in the rat ovary.

Receptor-Interacting Protein 140 Is a Repressor of the Androgen Receptor Activity

The androgen receptor (AR) is a ligand-activated transcription factor that controls growth and survival of prostate cancer cells. In the present study, we investigated the regulation of AR activity by the receptor-interacting protein 140 (RIP140). We first showed that RIP140 could be coimmunoprecipitated with the receptor when coexpressed in 293T cells. This interaction appeared physiologically relevant because chromatin immunoprecipitation assays revealed that, under R1881 treatment, RIP140 could be recruited to the prostate-specific antigen encoding gene in LNCaP cells. In vitro glutathione S-transferase pull-down assays provided evidence that the carboxy-terminal domain of AR could interact with different regions of RIP140. By means of fluorescent proteins, we demonstrated that ligand-activated AR was not only able to translocate to the nucleus but also to relocate RIP140 from very structured nuclear foci to a diffuse pattern. Overexpression of RIP140 strongly repressed AR-dependent transactivation by preferentially targeting the ligand binding domain-dependent activity. Moreover, disruption of RIP140 expression induced AR overactivation, thus revealing RIP140 as a strong AR repressor. We analyzed its mechanism of transrepression and first demonstrated that different regions of RIP140 could mediate AR-dependent repression. We then showed that the carboxy-terminal end of RIP140 could reverse transcriptional intermediary factor 2-dependent overactivation of AR. The use of mutants of RIP140 allowed us to suggest that C-terminal binding protein played no role in RIP140-dependent inhibition of AR activity, whereas histone deacetylases partly regulated that transrepression. Finally, we provided evidence for a stimulation of RIP140 mRNA expression in LNCaP cells under androgen treatment, further emphasizing the role of RIP140 in androgen signaling.

Transcriptional Regulation of the Human NRIP1/RIP140 Gene by Estrogen Is Modulated by Dioxin Signalling

Receptor interacting protein 140 (RIP140) is a negative transcriptional regulator of nuclear hormone receptors that is required for the maintenance of energy homeostasis and ovulation. In this study, we investigated the mechanisms by which RIP140 expression is controlled by estrogens in breast cancer cells. We first analyzed by real time reverse transcription-polymerase chain reaction the regulation of RIP140 mRNA accumulation by estrogen receptor (ER) ligands in MCF-7 cells. We showed that the induction by estradiol (E2) was rapid and did not affect the apparent stability of the mRNA, suggesting a direct transcriptional regulation. To further study the underlying regulatory mechanisms, we then characterized the human RIP140 gene. We identified several noncoding exons with alternative splicing and localized the promoter region more than 100 kilobases upstream from the coding exon. Although we mapped a perfect consensus estrogen response element able to bind ERalpha in gel shift and in chromatin immunoprecipitation experiments, the effect of E2 on RIP140 gene transcription was very modest. This might result at least in part from the presence of an overlapping aryl hydrocarbon receptor (AhR) binding site, which interfered with the E2 response on both the transiently transfected reporter construct and the accumulation of the endogenous RIP140 mRNA. Altogether, our data indicate that the RIP140 gene exhibits a complex structure with several noncoding exons and supports transcriptional cross-talk and feedback involving the ERalpha and AhR nuclear receptors.

Transcriptional Activation of the Nuclear Receptor Corepressor RIP140 by Retinoic Acid: A Potential Negative-Feedback Regulatory Mechanism

Through the use of microarray analysis it was discovered that the nuclear receptor coregulator, receptor interacting protein 140 (RIP140), was induced early during all-trans retinoic acid (RA)-induced differentiation of human embryonal carcinoma cells. A rapid, fourfold induction of RIP140 mRNA was detected within 3 h of RA treatment in human embryonal carcinoma and MCF-7 human breast cancer cells. RIP140 protein levels were induced within 6 h of RA treatment. The RA induction of RIP140 mRNA did not require de novo protein synthesis, consistent with RIP140 being a direct transcriptional target of retinoid receptors. Promoter/enhancer elements directly upstream of the RIP140 coding region supported RA-induced transcription of a luciferase gene. In addition the ability of overexpressed RIP140 to repress ligand activated retinoid receptors was confirmed. The finding that RIP140 is a direct transcriptional target of RA is one of the first examples of acute transcriptional regulation of a nuclear receptor coactivator or corepressor. These data are consistent with a model by which RA induction of RIP140 supplies a negative feedback signal toward ligand-activated retinoid receptors.

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Female mice null for receptor-interacting protein 140 (RIP140) are infertile because of the failure of follicle rupture. The present study examined gonadotropin regulation of RIP140 expression in immature rat ovary. Treatment with PMSG increased ovarian RIP140 mRNA and protein levels. In contrast, hCG treatment rapidly inhibited RIP140 mRNA and protein levels within 1–3 h. RIP140 mRNA was detected in theca cells of growing follicles in untreated ovary and in granulosa cells in PMSG-treated ovary. Interestingly, hCG treatment reduced RIP140 mRNA levels in granulosa cells of preovulatory follicles, but not of growing follicles. Neither treatment of immature rats with diethylstilbestrol in vivo nor of immature granulosa cells with FSH in vitro affected RIP140 mRNA levels. Treatment of immature granulosa cells with 17β-estradiol in vitro, however, stimulated RIP140 mRNA levels. In cultured preovulatory granulosa cells, RIP140 mRNA levels were stimulated at 1 h and then declined to below control levels by 3 h after LH treatment. Treatment with MDL-12,330A, an inhibitor of adenylate cyclase, or chelerythrine chloride, an inhibitor of protein kinase C (PKC), inhibited LH-stimulated RIP140 gene expression. Furthermore, forskolin or TPA treatment for 1 h mimicked the stimulatory action of LH, indicating the involvement of both adenylate cyclase and PKC pathways. These results demonstrate the stimulation by PMSG and inhibition by hCG of RIP140 expression in granulosa cells of preovulatory follicles in the rat ovary.