Nuclear Receptor-Interacting Protein 1 (NRIP1)
Determining the regulatory mechanisms of nuclear receptor action was one major focal topic of research in the 1990s. During this period, many nuclear receptor-associated proteins were identified as transcriptional coregulators, which were broadly categorized as coactivators and corepressors. Human receptor-interacting protein 140 (RIP140) was identified as a ligand-dependent interacting protein of estrogen receptor α (ERα) by far-Western blotting, and mouse RIP140 was isolated as a corepressor of orphan receptor TR2 from a yeast two-hybrid screening and later found to also interact with retinoic acid receptor (RAR) in a ligand-enhanced manner (Lee et al. 1998). An official gene name nrip1 was established for RIP140 by the HUGO gene nomenclature committee. The mouse gene is located on chromosome 16, in region C3.1, whereas the human gene is located on q11.2 of chromosome 21. DNA sequence analyses conclude that this gene is conserved in all vertebrate species examined. Experimental data have validated its interaction and gene coregulatory activity for all the nuclear receptors examined, as well as several transcription factors. The list includes AR, ER, GR, RARα/β, RXRα/β, PPARα/γ/δ, PXR, LXRα/β, VDR, AhR, ERRα/β/γ, RORβ, HNF4α, TR2, TR4, c-jun, SF-1, RelA, and GRIP1. Most experimental data in the past have demonstrated its ligand-enhanced gene repressive activity (Wei 2004). Recent studies have begun to elucidate other functions of RIP140 beyond the nucleus. These extranuclear activities of RIP140 are needed for certain specialized signaling pathways and/or functions of fully differentiated cells such as adipocytes, neurons, and macrophages (see later cytoplasmic RIP140).
Expression of RIP140 and Its Regulation
Posttranslational Modifications of RIP140
RIP140 can be phosphorylated at nine serine and two threonine residues. Phosphorylation at Thr202 and Thr207 by ERK2 facilitates its recruiting p300 which in turn acetylates RIP140 at Lys158 and Lys287. These sequential PTMs enhance RIP140’s gene repressive activity by enhancing the recruitment of HDAC3. Phosphorylation at Ser102 and Ser1003 is catalyzed by protein kinase C epsilon (PKCε), which promotes PRMT1-mediated methylation at Arg240, Arg650, and Arg948. These sequential PTMs lead to the recruitment of exportin 1, which would facilitate its nuclear export (see following). Interestingly, a high-fat diet can initiate this nuclear export pathway in adipocytes by activating nuclear PKCε activity.
Nine lysine residues on RIP140 can be modified by acetylation. Acetylation at Lys482, Lys 529, and Lys607 in repressive domain 2 (RD2)/RD3 region promotes nuclear export and reduces its gene repressive activity, whereas acetylation at Lys158 and Lys287 in RD1 region leads to a stronger gene repressive activity and nuclear retention. Acetylation at Lys158 and Lys287 is catalyzed by p300 following ERK2-mediated phosphorylation (see above). Interestingly, this is elevated in the adipocyte differentiation process of the 3T3-L1 model indicating that RIP140’s gene repressive activity is increasingly needed in later stages of adipocyte differentiation.
Methylation can occur at either arginine or lysine residues of RIP140. Arginine methylation at Arg240, Arg650, and Arg946 is mediated by PRMT1, which promotes the interaction of RIP140 with the exportin 1 subunit CRM1, thereby stimulating RIP140’s nuclear export and reducing its nuclear activity in transcriptional repression. On the other hand, lysine methylation at Lys591, Lys653, and Lys757 elevates its gene repressive activity by unknown mechanisms. Interestingly, demethylation at these three lysine residues of RIP140 is required for its methylation at the three arginine residues. This suggests a signal cross talk among various protein methylation enzyme machineries in the nucleus in order to coordinate RIP140’s PTMs and, as a result, its interacting partners, subcellular localization, and biological activity.
Pyridoxal 5′-phosphate (PLP) conjugation is found at Lys613 of RIP140, which elevates its gene repressive activity. This modification is regulated by the cellular status of PLP level, the active form of vitamin B6. This finding suggests that RIP140 may sense PLP or other nutritional factors through PTMs, which then modulates its property and biological activity.
Sumo-1 conjugation is found for the human protein at Lys 756 and Lys 1154 (Lys757 and Lys 1157 for the mouse RIP140), which may regulate its gene repressive activity and nuclear distribution. The sumoylation enzyme for RIP140 has not been identified. Interestingly, Lys757 on the mouse protein can be methylated, but its relationship with potential Lys757 sumoylation is unclear.
Functional Roles of RIP140
The ubiquitous expression profile of RIP140 suggests its role in many biological processes. Based on the phenotype of RIP140-null mice, this gene is essential for normal ovulation in female animals, and metabolism in general. At the molecular level, RIP140 is known, mostly, for its corepressive activity in gene transcription through its four RDs which mediate its interaction with HDAC, CtBP, and other chromatin remodeling proteins (Wei 2004). Recent studies indicate that RIP140 can also function as a coactivator for transcription of certain genes, including fatty acid synthase (FAS) in hepatocytes and several proinflammatory genes in macrophages. The demonstrated opposing activities, with regard to gene transcriptional control, of RIP140 would indicate that the coregulatory function of RIP140 in transcription might be gene, transcription factor, and/or cell context specific. A hypothesis for its diverse functions was first proposed based upon its extensive PTMs. This hypothesis has been validated in several studies which examined the functional significance of specific PTMs of RIP140 as detailed above (Huq et al. 2008). In addition, its tightly regulated nuclear export and cytoplasmic distribution strongly suggest other functional roles for RIP140 outside the nucleus. This has also begun to be established in more recent studies, including its activity in modulating insulin-stimulated glucose uptake in adipocytes and adipokine secretion (Ho et al. 2009; Ho and Wei 2012), interacting with IP3R to modulate calcium signaling in modulating neuronal stress response (Feng et al. 2014), and interacting with CAPNS1 to activate calpain for suppressing STAT6 activation in macrophage M2 polarization (Lin et al. 2015a).
It is clear that the biological activity of RIP140 is tightly regulated by its PTMs in response to certain extracellular/intracellular cues or stimuli. The functional roles of RIP140 in animals have been demonstrated using, primarily, genetically manipulated mice. It is important to note that phenotypes of whole-body gene manipulation in animals, either gene knockout or overexpression, are the manifestation of multiple defects in various tissues/organs and therefore should not be simply interpreted as the direct consequence of a single gene defect. Moreover, cytoplasmic functions of RIP140 will complicate the phenotypes of these genetically altered animals. Therefore, in interpreting these animal data, it is critically important to take into consideration the physiological or pathological context where RIP140 can possibly function in the nucleus and/or cytoplasm and how RIP140 protein may be modified. In the following, its functional roles deduced from studying whole animals or cell cultures are summarized.
Role in Ovulation
Depletion of RIP140 in mouse revealed its essential role in female fertility, particularly for the control of ovulation. The absence of RIP140 impacts many steps including follicular rupture, cumulus cell-oocyte complex expansion, and oocyte release (White et al. 2000). Further studies indicate that RIP140 is also critical for the expression of EGF-like factors which are essential in cumulus expansion and, possibly, follicular rupture.
Role in Adipocyte
Role in Hepatocytes
In liver cells, RIP140 can function as a corepressor or coactivator for LXR. As a corepressor of LXR, RIP140 suppresses PEPCK expression to reduce gluconeogenesis. As a coactivator of LXR, RIP140 enhances SREBP-1c and FAS expression to increase lipogenesis. Interestingly, RIP140-null mice are resistant to the development of hepatic steatosis under a high-fat diet. In studying these animals, RIP140 has also been identified as an important regulator for triglyceride storage in sepsis, starvation, and cancer cachexia. These results indicate a potential role for RIP140 in liver lipogenesis and triglyceride storage.
Role in Muscle Cells
RIP140 is differentially expressed in muscle cells: glycolytic fibers express a higher level, but oxidative fibers express a lower level, of RIP140. Microarray and metabolic analyses of muscle cells reveal that depleting RIP140 enhances the expression of genes involved in oxidative phosphorylation, fatty acid oxidation, and mitochondria biogenesis. This study demonstrates the role of RIP140 in controlling muscle metabolism and provides a clue for the defect of cardiac function in RIP140 overexpressed transgenic mice (see later sections). RIP140 regulates fatty acid oxidation via the control of Akt signaling in skeletal muscle cells. RIP140 also negatively regulates GLUT4 trafficking and glucose uptake in oxidative skeletal muscle through UCP1-associated AMPK activation. These studies indicate RIP140 as an important player in skeletal muscle where it can modulate mitochondrial function (Constantinescu et al. and Fritah et al. See citations in Fig. 4).
Role in Macrophages
For the monocyte-macrophage lineage, nuclear RIP140 functions as a coregulator for NF-κB/CBP complex in classic (M1) macrophage activation. This is important for LPS-stimulated transcription of several inflammatory genes. Depleting RIP140 in macrophages impairs LPS-stimulated proinflammatory cytokine production, because NF-κB transcriptional activity is reduced (Zschiedrich et al.). These studies reveal that RIP140 is a principal regulator of inflammation and/or inflammation-related diseases. Additionally, LPS-activated Syk-mediated RIP140 protein phosphorylation and then ubiquitination/degradation contribute to resolution of inflammatory responses and endotoxin tolerance (Ho et al. 2012). Related to cholesterol metabolism and inflammation, RIP140 mRNA and protein levels can be regulated by cholesterol via miRNA-33 that targets 3′UTR of RIP140 mRNA (Ho et al. 2011). RIP140 also coregulates LXR to modulate cholesterol levels in foam cells derived from macrophages (Lin et al. 2015b). Lowering RIP140 expression not only prevents M1 activation but also activates M2 activation through its cytosolic function that suppresses STAT6 (master regulator of M2 polarization), indicating that RIP140 plays a critical role in M1-M2 phenotype switch (Lin et al. 2015a). The relevance to metabolism is validated in a macrophage-specific RIP140 knockdown (mϕRIP140KD) mouse model which showed improved systemic insulin sensitivity and white adipose tissue browning under a high-fat diet challenge (Liu et al. 2014). As a proof of concept, Liu et al. successfully developed a therapeutic strategy, using designer macrophages where RIP140 level was greatly reduced, to manage (both prevent and treat) diet-induced metabolic diseases (Liu et al. 2015b, 2015a). When bred to atherosclerosis-prone ApoE mice, mϕRIP140KD also reduced foam cell formation because of reduced RIP140-mediated suppression of cholesterol transport genes ABCA1 and ABCG1, significantly alleviating western diet-induced plaque burden and atherosclerosis (Lin et al. 2015b). Recently, RIP140 was shown to act as a corepressor of orphan receptor TR4 which suppressed osteoclastogenesis. This pathway was also validated in the mϕRIP140KD mouse model, which indeed exhibited an osteopenia phenotype (in prep). These recent studies demonstrate RIP140’s pleiotropic activities in the monocyte-macrophage lineage, all are critically relevant to various pathophysiological conditions including septic shock, wound healing, diabetes, atherosclerosis, and osteopenia. However, the exact mechanism that regulates RIP140’s intracellular localization in macrophages and cells derived from this lineage remains to be examined.
Role in Cardiomyocytes
In studying whole-body RIP140-overexpressing transgenic mice, a role for RIP140 in cardiac hypertrophy and functions was identified. This finding suggests a need to control the expression level of RIP140 in cardiomyocytes. However, it remains unclear if the cardiac defects are results directly from the expression of RIP140 in cardiomyocytes or that may be caused by systemic changes in whole-body metabolism. To this end, RIP140 overexpression induces NF-kB-mediated inflammatory responses and metabolic dysfunction in neonatal rat cardiomyocytes, also supporting RIP140’s role as a coactivator of NF-kB in certain types of cells (Zhang et al.; see citation in Fig. 4).
Role in Brain Cells
RIP140 has been implicated in animal behaviors related with memory, stress, and emotional regulation. RIP140-deficient mice showed long-term learning and memory deficits and stress response indicating its role in the neurophysiological development (see citations in Fig. 4). Feng et al. uncovered RIP140’s function in neurons, in response to neuronal damage/stress, by translocating to the cytoplasm to bind IP3R, thereby modulating calcium release to protect neurons from apoptotic death (Feng et al. 2014). RIP140’s role in microglia is demonstrated in mϕRIPKD mice which showed reduced RIP140 expression in the ventromedial hypothalamus. These mice exhibited increased depressive and anxiety-like behavior. Mechanistically, this study reported that RIP140 depletion in macrophages reduced NPY production in astrocytes (Flaisher-Grinberg et al. 2014). Importantly, RIP140 expression can be dampened by behavioral stress, which is also associated with an elevated brain cholesterol level, indicting RIP140’s additional role in brain cholesterol homeostasis (Feng et al. 2015). In embryonic stem cell differentiation, RIP140 is a potent repressor of genes in retinoic acid-induced early differentiation such as Oct4 (Wu et al. 2014), and it further suppresses neuronal differentiation by forming a complex with LSD1 to specifically regulate certain retinoic acid target genes, such as Pax6 (Wu et al. 2016). These studies suggest that RIP140 plays important roles in regulating different brain cells, including neuron stem cells, microglia, astrocytes, and neurons, to affect brain functions as reflected in various types of neurological disorders such as memory, stress response, and emotional disorder.
Role in Cancer Cells
RIP140 was first identified in human breast cancer cells and has been suggested to play important roles in oncogenic signaling pathways in various tumors including breast, ovary, colon, and liver tumors. Docquier et al. reported that RIP140 enhances ERβ’s suppressive effect on 17 β-estradiol-induced transactivation and cell proliferation in ovarian cancer cells. In colorectal cancer, RIP140 was found to repress proliferation of intestinal epithelial cells and human colon cancer cells through the APC/β-catenin signaling pathway, supporting a tumor suppressor role of RIP140. RIP140 is also shown to inhibit hepatocellular carcinoma cell proliferation and migration via inactivating β-catenin/TCF signaling. Clinically, RIP140 was first identified as a chronic lymphocytic leukemia (CLL) prognostic factor, and low RIP140 expression was associated with poor prognosis for overall survival, but the molecular mechanism of RIP140’s action in CLL has not been studied. These results suggest that RIP140 acts as a tumor suppressor and can be a potential biomarker for specific tumors (see citations in Fig. 4).
Cytoplasmic RIP140: Player in Pathophysiology of Metabolic Diseases, Inflammation, and Neurological Disorders
The findings that cytoplasmic accumulation of RIP140 in adipocytes may contribute to their dysfunctions such as impaired glucose uptake, adipokine secretion, and lipolysis would strongly support its functional role in regulating systemic metabolism as demonstrated in whole-body knockout mice, which, in part, is attributable to the cytoplasmic RIP140. In animals, a high-fat diet promotes RIP140’s nuclear export in adipocytes. From a clinical point of view, targeting cytoplasmic RIP140, or blocking signaling pathways that promote RIP140’s cytoplasmic accumulation, may provide a more specific, beneficial/protective effect in the management of metabolic disorders (Ho and Wei 2012).
Cytoplasmic function of RIP140 has also been found in IL-4-stimulated macrophage in M2 polarization. Upon IL-4 stimulation, RIP140 is translocated to the cytoplasm to interact with CAPNS1 to activate calpain1/2, which cleaves PTP1B, a negative regulator of STAT6 (Lin et al. 2015a). Through this pathway, cytoplasmic RIP140 ultimately suppresses STAT6 activity in M2 polarization. Of a physiological relevance, it was found that wound healing indeed was more efficient in mϕRIP140KD mice, which have significantly reduced M1 proinflammatory macrophages and enhanced M2 anti-inflammatory macrophages. Therefore, RIP140 not only acts as a positive regulator of M1 activation but also acts as a negative regulator of M2 activation, indicating a pivotal functional role for RIP140 in maintaining the balance between M1 and M2 polarization in activated macrophages. These studies further support the notion that RIP140 can be a potential therapeutic target for metabolic and inflammatory pathological conditions (Liu et al. 2015b).
The third type of cell where RIP140 also plays a significant role in the cytoplasm is neuron. In stressed neurons, unfolded protein response induces RIP140’s translocation to the endoplasmic reticulum (ER) to form complex with IP3R. Through this interaction, increasingly ER localized RIP140 attenuates IP3R-mediated Ca2+ release, thereby protecting stressed neurons from apoptotic death (Feng et al. 2014). This study uncovers a new function of RIP140 in neurons and sheds lights into an important physiological/pathological context where cytoplasmic RIP140 can modulate various neurological diseases.
RIP140 was first identified as a universal gene transcriptional corepressor that acts, primarily, to regulate the activities of nuclear receptors in a ligand-enhanced manner. Its gene repressive activity is mediated by its four RDs that recruit various corepressive factors, histone-modifying enzymes, and chromatin remodelers. This was complicated by the fact that RIP140 can be extensively modified by PTMs, which drastically affect its ability to recruit interacting partners and alter its subcellular distribution, stability, and functions. Studies of whole-body gene knockout, overexpression of transgenic mice, and lineage-specific KD transgenic mice provide insights into its multiple physiological roles in regulating metabolism, inflammation, reproduction, and behavior in animals. While most of these activities have been associated with its nuclear functions, it has become clear that some might have been affected by its extranuclear activities.
In the nucleus, the canonical activity of RIP140 is to suppress hormone-activated transcription activation, such as by antagonizing coactivators PGC-1, PCAF, and p300 in a hormone-dependent manner. However, nuclear RIP140 can also function as a coactivator for those without hormone ligands, such as NF-κB in macrophages. It is likely that nuclear RIP140 provides the principal gatekeeper to dampen hormonal activation of most nuclear receptors in hormone-targeted cells, which would be representative of the canonical activity of nuclear RIP140. For certain transcription factors, such as NF-κB, nuclear RIP140 can function as a coactivator, which is likely gene or context dependent. In the context of system biology, RIP140 seems to provide, primarily, a counteracting force to maintain homeostasis in gene expression and cellular responses in the face of challenges such as hormones, cytokines, and stress. Most of these activities are manifested in a cell autonomous fashion and as a negative feedback control both in the nucleus and the cytoplasm (see the following).
Studies of RIP140’s PTMs reveal its cytoplasmic distribution and novel functions in multiple specialized cell types, including adipocytes, neurons, and macrophages. In adipocytes, cytoplasmic RIP140 interacts with AS160 and perilipin to modulate glucose homeostasis, lipid metabolism, and vesicle trafficking. In macrophages, cytoplasmic RIP140 interacts with CAPNS1 to dampen STAT6 activation (and M2 polarization). In neurons, cytoplasmic RIP140 is primarily localized on the ER to form a complex with IP3R (ER stress-induced neurons), which attenuates calcium release thereby protecting cells from apoptosis.
Because of its highly specialized function and modification in response to stimuli/inputs presented to certain specific cell types, RIP140 can be a potential therapeutic target. Therapeutics targeting RIP140, either the nuclear or the cytoplasmic form, can be developed for numerous diseases including metabolic disorders, inflammation, cancers, and neurological disorders. As a proof of concept, targeting RIP140 in specialized cell types to manage diseased conditions has been demonstrated in several recent studies of mouse models. Future challenges reside at the identification of specific agents that can specifically augment RIP140’s expression and/or PTM in order to target specialized pathways/cells that are most specific to disease progression. This would drastically reduce side effects and toxicity as seen in most therapeutics developed to target molecules/pathways common to multiple organs/tissues/cell types. This is particularly important because many of these medical conditions are chronic in nature, and therefore toxicity of therapeutics is of most concern.
This work was supported by DK54733, DK60521, DK54733-11S, DK60521-12S1, Dean’s Commitment, and the Distinguished McKnight University Professorship of University of Minnesota to LNW.
- Lin Y-W, Lee B, Liu P-S, Wei L-N. Receptor-interacting protein 140 orchestrates the dynamics of macrophage M1/M2 polarization. J Innate Immun. 2016;8(1):97–107.Google Scholar
- Liu P-S, Lin Y-W, Lee B, McCrady-Spitzer SK, Levine JA, Wei L-N. Reducing RIP140 expression in macrophage alters ATM infiltration, facilitates white adipose tissue browning, and prevents high-fat diet-induced insulin resistance. Diabetes. 2014;63(12):4021–31.PubMedPubMedCentralCrossRefGoogle Scholar