Receptor-interacting Protein 140 Research Articles

RIP140 regulates transcription factor HES1 oscillatory expression and mitogenic activity in colon cancer cells.

The transcription factor receptor-interacting protein 140 (RIP140) regulates intestinal homeostasis and tumorigenesis through Wnt signaling. In this study, we investigated its effect on the Notch/HES1 signaling pathway. In colorectal cancer (CRC) cell lines, RIP140 positively regulated HES1 gene expression at the transcriptional level via a recombining binding protein suppressor of hairless (RBPJ)/neurogenic locus notch homolog protein 1 (NICD)-mediated mechanism. In support of these in vitro data, RIP140 and HES1 expression significantly correlated in mouse intestine and in a cohort of CRC samples, thus supporting the positive regulation of HES1 gene expression by RIP140. Interestingly, when the Notch pathway is fully activated, RIP140 exerted a strong inhibition of HES1 gene transcription controlled by the level of HES1 itself. Moreover, RIP140 directly interacts with HES1 and reversed its mitogenic activity in human CRC cells. In line with this observation, HES1 levels were associated with a better patient survival only when tumors expressed high levels of RIP140. Our data identify RIP140 as a key regulator of the Notch/HES1 signaling pathway, with a dual effect on HES1 gene expression at the transcriptional level and a strong impact on colon cancer cell proliferation.

Histone H3 Acetylation Is Involved in Retinoid Acid-Induced Neural Differentiation through Increasing Mitochondrial Function.

Histone acetylation and mitochondrial function contribute importantly to neural differentiation, which is critically associated with neurodevelopmental disorders such as Down Syndrome (DS). However, whether and how histone acetylation regulates mitochondrial function and further affects neural differentiation has not been well described. In this study, when treated with retinoid acid (RA), the human neuroblastoma SH-SY5Y cell line was used as a neural differentiation model. We found that the acetylation of histone H3, especially H3 lysine 14 acetylation (H3K14ac), and mitochondrial function, including biogenesis and electron transport chain, were enhanced during neural differentiation. Specific inhibition of histone acetyltransferases (HATs) induced neural differentiation deficits, accompanied by downregulation of mitochondrial function. Furthermore, RA receptors (RARs) interacting with HATs were involved in the increased H3K14ac and the enhanced mitochondrial function during the neural differentiation process. Finally, receptor-interacting protein 140 (RIP140), a co-repressor of RARs, was also involved in regulating histone acetylation. RIP140 overexpression inhibited histone acetylation and mediated negative feedback on target genes which are involved in RA signaling. These findings evidenced that when interacting with RARs which had been negatively regulated by RIP140, RA promoted neural differentiation by promoting H3K14ac and enhanced mitochondrial function. This provides a molecular foundation for further investigations into abnormal neural development.

Abstract 13959: A Role for Striated Muscle Nuclear Receptor Co-Regulator RIP140 ( Nrip1 ) in the Control of Endurance Fitness

During the development of heart failure (HF), the capacity for myocardial fatty acid oxidation (FAO) and oxidative phosphorylation is reduced, leading to an energy-starved state. Similar metabolic derangements occur in skeletal muscle during HF and with physical inactivity associated with chronic disease states. Receptor Interacting Protein 140 (RIP140) is a nuclear receptor co-regulator that has been shown to repress the transcription of genes involved in mitochondrial energy transduction in highly metabolic tissues. We recently demonstrated that striated muscle-specific RIP140 KO mice (str-RIP140 -/- ) were protected against the development of HF and had an adaptive form of cardiac hypertrophy (PMID: 36927960). To assess the impact of RIP140 deficiency on skeletal muscle function, we conducted physiological and genomic interrogation of str-RIP140 -/- mice. We found that the str-RIP140 -/- mice display an endurance performance phenotype. Specifically, str-RIP140 -/- mice run significantly longer on an endurance treadmill regimen (str-RIP140 -/- = 118.0 min, WT= 73.38 min; p= <0.0001) with increased VO 2max compared to controls (str-RIP140 -/- = 9779 ml/kg/hr, WT= 7748 ml/kg/hr; p= <0.0001). Respiratory Exchange Ratio (RER) was maintained at a decreased level during exercise in str-RIP140 -/- mice vs controls, indicative of increased utilization and capacity for FAO. The response of str-RIP140 -/- mice to endurance exercise training was also assessed. str-RIP140 -/- mice achieved a greater VO 2max following 8 weeks of training compared to control trained mice (str-RIP140 -/- = 9589 ml/kg/hr, WT= 8366 ml/kg/hr; p= 0.002). RNA-sequence analysis comparing RIP140 -/- and control fast-twitch muscle (EDL) prior to and following exercise training identified differentially upregulated genes involved in an array of pathways activated with endurance training including mitochondrial FAO, triglyceride turnover, the slow-twitch fiber program, angiogenesis, and components of the neuromuscular junction. We conclude that RIP140 serves as a co-repressor of a network of genes involved in skeletal muscle endurance. These results suggest that RIP140 could prove to be a target for novel metabolic therapies aimed at cardiac and skeletal muscle diseases.

Knockdown of receptor interacting protein 140 (RIP140) alleviated lipopolysaccharide-induced inflammation, apoptosis and permeability in pulmonary microvascular endothelial cells by regulating C-terminal binding protein 2 (CTBP2)

ABSTRACT The main pathological feature of acute lung injury (ALI) is pulmonary edema caused by increased permeability of pulmonary microvascular endothelial cells (PMVECs). LPS was has been confirmed to lead to cell damage and barrier dysfunction in PMVECs. Furthermore, receptor interacting protein 140 (RIP140) was discovered to be increased in LPS-induced human pulmonary microvascular endothelial cells (HPMECs), but the mechanism of RIP140 on LPS-induced HPMECs has not been investigated. In this study, an acute lung injury model was constructed in LPS-induced HPMECs. After RIP140 was downregulated, inflammation, apoptosis and cell permeability levels were detected by RT-qPCR, TUNEL staining and FITC-Dextran, respectively. Western blotting was used to detect the protein levels of related factors. The binding of RIP140 and C-terminal binding protein 2 (CTBP2) was predicted by database and verified by Co-IP. Subsequently, CTBP2 overexpression was transfected into cells and the above experiments were performed again. The results showed that inflammation, apoptosis and permeability levels of LPS-induced HPMECs were remarkably increased compared to the untreated control group. However, these levels were suppressed after RIP140 was silenced compared to the LPS-induced HPMECs group. Notably, the Co-IP study demonstrated that RIP140 and CTBP2 interacted with each other. Moreover, CTBP2 overexpression reversed the inhibitory effects of RIP140 silencing on LPS-induced inflammation, apoptosis and permeability levels in HPMECs. Together, the study found that interference of RIP140 could alleviate LPS-induced inflammation, apoptosis and permeability in HPMECs by regulating CTBP2.

Abstract 113: Targeting Receptor-interacting Protein 140 (RIP140) To Modulate Energy Metabolism In The Failing Heart

During the development of heart failure (HF), the PPAR/ERR complex becomes deactivated resulting in diminished capacity for mitochondrial fatty acid oxidation (FAO) and ATP production leading to an “energy-starved” state that contributes to progression of HF. Receptor-Interacting protein 140 (RIP140) serves as a co-repressor of PPAR/ERR in some extra-cardiac tissues. We hypothesized that inhibition of RIP140 would re-activate PPAR/ERR enhancing capacity for fuel catabolism and ATP production in the failing heart. Heart and skeletal muscle-specific RIP140 knockout mice (strRIP140KO) were resistant to the development of cardiac hypertrophy and diastolic dysfunction in response to chronic pressure overload that mimicked features of HF with preserved ejection fraction (HFpEF). To further evaluate the role of RIP140 in heart, cardiac-specific (cs) RIP140KO mice were generated. 13 C-substrate NMR studies demonstrated that palmitate oxidation and triglyceride turnover rates were significantly accelerated in isolated perfused csRIP140KO hearts. csRIP140KO were subjected to transverse aortic constriction/apical myocardial infarction surgery (TAC/MI), to produce HF with reduced EF (HFrEF). Compared to controls, csRIP140KO exhibited reduced left ventricular remodeling and systolic dysfunction when subjected to TAC/MI. RNA-sequence analysis demonstrated that many genes involved in FAO, branched-chain amino acid catabolism, oxidative phosphorylation, and adult muscle contraction programs were significantly “protected” (less downregulation) by RIP140 deletion in the context of TAC/MI. To identify candidate cardiac RIP140 targets, “CUT&RUN”-sequencing was conducted on cardiomyocytes (CM) from csRIP140KO and controls to identify changes in enhancer regions. Motif analysis of peaks with increased H3K27ac deposition in the csRIP140KO CM identified ERR, PPAR, myocyte enhancer 2 (MEF2), glucocorticoid receptor (GR), and kruppel-like factor (KLF) binding sites. We conclude that RIP140 serves as a global co-repressor of a network of transcription factors that control cardiac energy metabolic and contractile function, and that inhibition of RIP140 could prove to be a novel therapeutic approach for HF.

Regulation of exosome secretion by cellular retinoic acid binding protein 1 contributes to systemic anti-inflammation

BackgroundIntercellular communications are important for maintaining normal physiological processes. An important intercellular communication is mediated by the exchange of membrane-enclosed extracellular vesicles. Among various vesicles, exosomes can be detected in a wide variety of biological systems, but the regulation and biological implication of exosome secretion/uptake remains largely unclear.MethodsCellular retinoic acid (RA) binding protein 1 (Crabp1) knockout (CKO) mice were used for in vivo studies. Extracellular exosomes were monitored in CKO mice and relevant cell cultures including embryonic stem cell (CJ7), macrophage (Raw 264.7) and hippocampal cell (HT22) using Western blot and flow cytometry. Receptor Interacting Protein 140 (RIP140) was depleted by Crispr/Cas9-mediated gene editing. Anti-inflammatory maker was analyzed using qRT-PCR. Clinical relevance was accessed by mining multiple clinical datasets.ResultsThis study uncovers Crabp1 as a negative regulator of exosome secretion from neurons. Specifically, RIP140, a pro-inflammatory regulator, can be transferred from neurons, via Crabp1-regulated exosome secretion, into macrophages to promote their inflammatory polarization. Consistently, CKO mice, defected in the negative control of exosome secretion, have significantly elevated RIP140-containing exosomes in their blood and cerebrospinal fluid, and exhibit an increased vulnerability to systemic inflammation. Clinical relevance of this pathway is supported by patients’ data of multiple inflammatory diseases. Further, the action of Crabp1 in regulating exosome secretion involves its ligand and is mediated by its downstream target, the MAPK signaling pathway.ConclusionsThis study presents the first evidence for the regulation of exosome secretion, which mediates intercellular communication, by RA-Crabp1 signaling. This novel mechanism can contribute to the control of systemic inflammation by transferring an inflammatory regulator, RIP140, between cells. This represents a new mechanism of vitamin A action that can modulate the homeostasis of system-wide innate immunity without involving gene regulation.EB6KXQYbkzhu3R444VcR3RVideo

Receptor-Interacting Protein 140 Enhanced Temozolomide-Induced Cellular Apoptosis Through Regulation of E2F1 in Human Glioma Cell Lines.

Glioblastoma (GBM), a grade IV glioma, is responsible for the highest years of potential life lost among cancers. The poor prognosis is attributable to its high recurrence rate, caused in part by the development of resistance to chemotherapy. Receptor-interacting protein 140 (RIP140) is a very versatile coregulator of nuclear receptors and transcription factors. Although many of the pathways regulated by RIP140 contribute significantly to cancer progression, the function of RIP140 in GBM remains to be determined. In this study, we found that higher RIP140 expression was associated with prolonged survival in patients with newly diagnosed GBM. Intracellular RIP140 levels were increased after E2F1 activation following temozolomide (TMZ) treatment, which in turn modulated the expression of E2F1-targeted apoptosis-related genes. Overexpression of RIP140 reduced glioma cell proliferation and migration, induced cellular apoptosis, and sensitized GBM cells to TMZ. Conversely, knockdown of RIP140 increased TMZ resistance. Taken together, our results suggest that RIP140 prolongs the survival of patients with GBM both by inhibiting tumor cell proliferation and migration and by increasing cellular sensitivity to chemotherapy. This study helps improve our understanding of glioma recurrence and may facilitate the development of more effective treatments.

Abstract 13921: Targeting Receptor-Interacting Protein 140 to Modulate Energy Metabolism in the Failing Heart

The nuclear receptors, peroxisome proliferator-activated receptors (PPARs), estrogen-related receptors (ERRs), and their co-regulator PPARγ coactivator-1α (PGC-1α), control postnatal cardiac mitochondrial biogenesis and energy metabolism. During the development of heart failure (HF), the activity of PGC-1/PPAR/ERR is reduced resulting in diminished capacity for fatty acid oxidation (FAO) and ATP production potentially contributing to an “energy-starved” state that contributes to progression of HF. Receptor-Interacting protein 140 (RIP140) serves as a co-repressor of PGC-1/PPAR/ERR in skeletal muscle and adipose tissue. We hypothesized that RIP140 represses cardiac energy metabolism in the normal and failing heart. Accordingly, we targeted Nrip1 (encoding RIP140) using a muscle creatinine kinase (MCK)-driven Cre recombinase to generate striated muscle-specific RIP140 knockout (msRIP140 KO) mice. msRIP140 KO mice appeared normal at baseline with no difference in survival or cardiac systolic function compared to littermate controls. RNA-sequence analysis demonstrated that the expression of genes involved in a wide array of mitochondrial energy metabolic pathways including FAO, tricarboxylic acid (TCA) cycle, oxidative phosphorylation (OXPHOS), and branched-chain amino acid (BCAA) degradation pathways were upregulated in msRIP140 KO ventricles, and in msRIP140 KO skeletal muscle. msRIP140 KO mice exhibited significantly less cardiac hypertrophy and diastolic dysfunction in response to chronic pressure overload. Next, cardiac-specific (cs) RIP140 KO mice were generated and subjected to transverse aortic constriction/apical myocardial infarction surgery (TAC/MI), an established HF model. csRIP140 KO mice exhibited less cardiac remodeling and systolic dysfunction compared to littermate controls, along with less downregulation of metabolic genes and induction of cardiac stress ( Nppa and Nppb ) and fibrosis response markers ( Tgfb2 and Col3a1 ). We conclude that RIP140 serves as a global co-repressor of cardiac energy metabolic genes in the adult heart and that modulation of RIP140 activity could prove to be a novel therapeutic approach for HF.

Cognitive impairments in adult mice with RIP140 overexpression in neural stem cells

Receptor-interacting protein 140 (RIP140) is a transcription co-regulator of several transcription factors and a signal transduction regulator. RIP140 was recently implicated in the regulation of cognitive functions. The gene that encodes RIP140 is located on chromosome 21. An increase in RIP140 expression was observed in the fetal cerebral cortex and hippocampus in Down syndrome patients who exhibited strong cognitive disabilities. We hypothesized that RIP140 overexpression affects cognitive function in adult neural development. The present study used a Cre-dependent adeno-associated virus to selectively overexpress RIP140 in neural stem cells using nestin-Cre mice. RIP140 overexpression efficiency was evaluated at the subgranular zone (SGZ) of the dorsal dentate gyrus (dDG) and the subventricular zone (SVZ) of the lateral ventricles (LVs). Mice with RIP140 overexpression in the SGZ exhibited deficits in cognitive function and spatial learning and memory, measured in the Morris water maze, object-place recognition test, and novel object recognition test. However, overexpression of RIP140 in SVZ only impaired performance in the Morris water maze and novel object recognition test but not in the object-place recognition test. Altogether, these results indicated defects in cognitive functions that were associated with RIP140 overexpression in neural stem cells and revealed a behavioral phenotype that may be used as a framework for further investigating the neuropathogenesis of Down syndrome.

RIP140 in Stress Response of the Brain

Receptor interacting protein 140 (RIP140), gene named Nuclear receptor interacting protein 1 (Nrip1), is a transcription co-regulator of numerous nuclear receptors and transcription factors that are important for various biological processes

Effects of visfatin on brown adipose tissue energy regulation using T37i cells

The role of brown adipose tissue (BAT) in pathological states of energy homeostasis and impaired adipocyte function, such as obesity has been a major area of research interest in recent years. Herein, we sought to determine the direct effects of adipokines, visfatin and leptin on BAT thermogenesis.The effects of mouse recombinant visfatin, nicotinamide mononucleotide (NMN) and leptin with or without FK866 were studied on differentiated T37i cells. Treated cells were analyzed for key genes and proteins regulating BAT [UCP-1, PRD1-BF1-RIZ1 homologous domain-containing 16 (PRDM-16), PPARgamma-coactivator-1alpha (PGC-1α) and receptor-interacting protein 140 (RIP-140)] using quantitative PCR and western blot analysis. Data is presented as mean P-values.Both visfatin and leptin had significant concentration dependent effects on thermogenesis in brown pre-adipocytes and at physiological levels, increased uncoupling protein-1 (UCP-1) levels in brown adipocytes. These effects of visfatin were similar to that of nicotinamide mononucleotide (NMN), further strengthening the enzymatic role of visfatin. We also showed that leptin induced UCP-1 mRNA expression and protein production appears to be mediated by visfatin. High concentrations of both visfatin and leptin led to a dramatic decrease in UCP-1 protein levels, supporting the notion that visfatin levels are raised in obesity and that obese people have reduced BAT activity, plausibly through a reduction in UCP-1 levels. Additionally, we found differential regulation of key brown adipogenic genes, specifically, PRD1-BF1-RIZ1 homologous domain-containing 16 (PRDM-16), PPARgamma-coactivator-1alpha (PGC-1α) and receptor-interacting protein 140 (RIP-140) by visfatin. Our observations provide novel insights in the potential actions of visfatin in BAT.

Downregulation of RIP140 in triple-negative breast cancer inhibits the growth and proliferation of cancer cells.

The aim of the present study was to investigate the expression of receptor interacting protein 140 (RIP140) in triple-negative breast cancer (TNBC), and its association with the prognosis of patients with TNBC. A total of 179 patients with breast cancer were included in this study, with 41 cases of TNBC and 138 cases of non-TNBC. Immunohistochemical staining and western blotting were used to detect the protein expression of RIP140 in the cancerous and paracancerous tissues, revealing that expression of RIP140 was increased in TNBC tissues compared with non-TNBC tissues. High expression of RIP140 in breast cancer tissue was associated with a poorer survival time than low RIP140 expression. Using lentiviral transfection to downregulate RIP140 expression in MDA-MB-231 cells, the effects of RIP140 on the growth and proliferation of breast cancer cells was analyzed using subcutaneous tumors in BALB/c nude mice. Immunohistochemical staining using a Ki-67 antibody in subcutaneous tumor tissue was used to assess the proliferation of MDA-MB-231 cells. The short hairpin RNA-mediated downregulation of RIP140 in MDA-MB-231 cells suppressed the growth and the proliferation of subcutaneous tumors in BALB/c nude mice. Downregulation of RIP140 in breast cancer cells may therefore inhibit the growth and the proliferation of these cells, and may provide a therapeutic target for TNBC.

Synergistic effect of receptor-interacting protein 140 and simvastatin on the inhibition of proliferation and survival of hepatocellular carcinoma cells.

Hepatocellular carcinoma is the sixth most prevalent malignant tumor and the third most common cause of cancer-associated mortality. Statins have been investigated for carcinoma prevention and treatment. In addition, receptor-interacting protein 140 (RIP140) has been observed to inhibit the Wnt/β-catenin signaling pathway and cell growth. The present study aimed to investigate whether simvastatin (SV) is able to induce SMCC-7721 cell apoptosis through the Wnt/β-catenin signaling pathway. Initially, a cell model of RIP140 overexpression was established, and then cells were treated with SV. The cell growth, viability and apoptosis were measured by cell counting kit-8 and flow cytometry. Furthermore, the expression levels of RIP140, β-catenin, c-myc and cyclin D1 were detected by reverse transcription-quantitative polymerase chain, western blot analysis and immunofluorescence. The results demonstrated that SV significantly increased the expression of RIP140 in SMCC-7721 cells; however, β-catenin, c-myc and cyclin D1 levels were significantly decreased. Furthermore, the immunofluorescence assay of β-catenin confirmed that SV decreased the content of this protein in SMCC-7721 cells. Notably, RIP140 exerted a synergistic effect on the apoptosis rate induced by SV (RIP140 + SV group), while the alteration in RIP140, β-catenin, c-myc and cyclin D1 levels was more evident in the combination group as compared with the RIP140 or SV alone groups. In conclusion, these results suggested that SV is able to induce the apoptosis of SMCC-7721 cells through the Wnt/β-catenin signaling pathway, as well as that RIP140 and SV exert a synergistic effect on the inhibition of cell proliferation and survival.

Glyburide and retinoic acid synergize to promote wound healing by anti-inflammation and RIP140 degradation

Chronic inflammation underlies the development of metabolic diseases and individuals with metabolic disease often also suffer from delayed wound healing due to prolonged inflammation. Resolving inflammation provides a therapeutic strategy in treating metabolic diseases. We previously showed that during an anti-inflammatory response when macrophages were alternatively (M2) polarized, retinoic acid (RA) dramatically activated arginase 1 gene (Arg1), a gene crucial for wound healing. Here we report that a widely used sulfonylurea drug for type 2 diabetes mellitus (T2DM), glyburide, enhances the anti-inflammatory response and synergizes with RA to promote wound healing. Our data also delineate the mechanism underlying glyburide’s anti-inflammatory effect, which is to stimulate the degradation of a pro-inflammatory regulator, Receptor Interacting Protein 140 (RIP140), by activating Ca2+/calmodulin-dependent protein kinase II (CamKII) that triggers specific ubiquitination of RIP140 for degradation. By stimulating RIP140 degradation, glyburide enhances M2 polarization and anti-inflammation. Using a high-fat diet induced obesity mouse model to monitor wound healing effects, we provide a proof-of-concept for a therapeutic strategy that combining glyburide and RA can significantly improve wound healing. Mechanistically, this study uncovers a new mechanism of action of glyburide and a new pathway modulating RIP140 protein degradation that is mediated by CamKII signaling.

Involvement of receptor-interacting protein 140 in palmitate-stimulated macrophage infiltration of pancreatic beta cells.

Receptor-interacting protein 140 (RIP140) in macrophages stimulates the nuclear factor-κB subunit RelA to activate tumor necrosis factor (TNF)-α and interleukin (IL)-6 transcription. However, under lipotoxic conditions, the involvement of RIP140 in the infiltration of beta cells by macrophages remains unknown. In the present study, murine RAW264.7 macrophages were transfected with a RIP140 overexpression plasmid or siRNA prior to macrophage activation with 500 µM palmitate. Palmitate-free conditioned media was then collected and added to murine insulinoma MIN6 cells. Significant decreases were observed in cell viability (P<0.01), glucose-stimulated insulin secretion (P<0.01) and levels of peroxisome proliferator-activated receptor-γ coactivator-1α (P<0.05), phosphoenolpyruvate carboxykinase and proliferating cell nuclear antigen mRNA (P<0.01) in MIN6 cells. In addition, conditioned media from palmitate-treated and RIP140-upregulated macrophages significantly increased the levels of uncoupling protein-2 (P<0.01), inducible nitric oxide synthase 1 (P<0.01) and pancreatic and duodenal homeobox 1 (P<0.05) mRNA and levels of activated Jun N-terminal kinase (JNK) (P<0.01) and extracellular signal-regulated kinase (ERK) 1/2 (P<0.01). In turn, the conditioned media was found to be significantly enriched in TNF-α and IL-6 (both P<0.05). These results were the opposite of those obtained from MIN6 cells treated with conditioned media from palmitate-treated and RIP140-knockdown macrophages. MIN6 cells were transfected with RIP140 overexpression plasmid or siRNA prior to treatment with 500 µM palmitate and supernatant was collected for use in macrophage chemotaxis assays. In the palmitate-activated and RIP140-overexpressing MIN6 cells, TNF-α and IL-6 secretion increased significantly (both P<0.05) and macrophage chemotaxis towards MIN6 cells was enhanced. By contrast, downregulating RIP140 in MIN6 cells had the opposite effect. These data suggest that RIP140 in macrophages mediates the transcription of inflammatory cytokines when concentration of palmitate is high. Macrophage RIP140 may also impair beta cell function by activating the JNK and ERK1/2 signaling pathways and promoting specific gene transcription. Furthermore, expression of RIP140 in pancreatic beta cells may stimulate macrophage chemotaxis, thus triggering local low-grade inflammation.

RIP140 in monocytes/macrophages regulates osteoclast differentiation and bone homeostasis.

Osteolytic bone diseases, such as osteoporosis, are characterized by diminished bone quality and increased fracture risk. The therapeutic challenge remains to maintain bone homeostasis with a balance between osteoclast-mediated resorption and osteoblast-mediated formation. Osteoclasts are formed by the fusion of monocyte/macrophage-derived precursors. Here we report, to our knowledge for the first time, that receptor-interacting protein 140 (RIP140) expression in osteoclast precursors and its protein regulation are crucial for osteoclast differentiation, activity, and coupled bone formation. In mice, monocyte/macrophage-specific knockdown of RIP140 (mϕRIP140KD) resulted in a cancellous osteopenic phenotype with significantly increased bone resorption and reduced bone formation. Osteoclast precursors isolated from mϕRIP140KD mice had significantly increased differentiation potential. Furthermore, conditioned media from mϕRIP140KD primary osteoclast cultures significantly suppressed osteoblast differentiation. This suppressive activity was effectively and rapidly terminated by specific Syk-stimulated RIP140 protein degradation. Mechanistic analysis revealed that RIP140 functions primarily by inhibiting osteoclast differentiation through forming a transcription-suppressor complex with testicular receptor 4 (TR4) to repress osteoclastogenic genes. These data reveal that monocyte/macrophage RIP140/TR4 complexes may serve as a critical transcription regulatory complex maintaining homeostasis of osteoclast differentiation, activity, and coupling with osteoblast formation. Accordingly, we propose a potentially novel therapeutic strategy, specifically targeting osteoclast precursor RIP140 protein in osteolytic bone diseases.

Overexpression of RIP140 suppresses the malignant potential of hepatocellular carcinoma by inhibiting NF‑κB‑mediated alternative polarization of macrophages.

Tumor-associated macrophages (TAMs) and their alternative activation contribute greatly to the development of hepatocellular carcinoma (HCC). Receptor-interacting protein140 (RIP140) is widely expressed in macrophages and regulates macrophage-mediated energy metabolism, the inflammatory response and tumorigenesis. However, whether RIP140 is involved in the activation of TAMs has not been reported. In the present study, we determined the expression of RIP140 in macrophages after treatment with HCC-conditioned medium (HCM) for 24h. We also analyzed the effects of RIP140 overexpression on macrophage polarization, invasion and apoptosis of HepG2 and Huh7 cells. Transwell and apoptosis assays were used to estimated cell invasion and apoptosis. In addition, we investigated the effects of RIP140 overexpression in macrophages on the growth of H22 cells by subcutaneous injection of H22 cells along with macrophages in BALB/c nude mice. Western blotting and qRT-PCR were used to detect protein and mRNA expression associated with the NF-κB/IL-6 axis in TAMs. Immunohistochemical and immunofluorescence staining were used to evaluate the protein expression of RIP140 or F4/80 in human HCC samples. The protein expression of RIP140 in peripheral blood mononuclear cells was detected by western blotting. Kaplan‑Meier survival curve estimation of overall survival for patients with HCC was based on RIP140 or F4/80 expression in HCC samples. We found that HCM inhibited RIP140 expression and fostered the alternative activation of macrophages. RIP140 overexpression in TAMs significantly inhibited the alternative activation of macrophages by inhibiting the NF-κB/IL-6 axis in TAMs, and suppressed HCC cell growth both invitro and invivo. In addition, the protein expression of RIP140 in peripheral blood monocytes was significantly lower in patients with HCC than in healthy people, and this result was consistent with the expression of RIP140 in HCC samples. Furthermore, low RIP140 expression and high F4/80 expression were found to be closely correlated with shorter survival time of the patients with HCC.

Correlation between receptor-interacting protein 140 expression and directed differentiation of human embryonic stem cells into neural stem cells.

Overexpression of receptor-interacting protein 140 (RIP140) promotes neuronal differentiation of N2a cells via extracellular regulated kinase 1/2 (ERK1/2) signaling. However, involvement of RIP140 in human neural differentiation remains unclear. We found both RIP140 and ERK1/2 expression increased during neural differentiation of H1 human embryonic stem cells. Moreover, RIP140 negatively correlated with stem cell markers Oct4 and Sox2 during early stages of neural differentiation, and positively correlated with the neural stem cell marker Nestin during later stages. Thus, ERK1/2 signaling may provide the molecular mechanism by which RIP140 takes part in neural differentiation to eventually affect the number of neurons produced.

Receptor-interacting Protein 140 represses Sirtuin 3 to facilitate hypertrophy, mitochondrial dysfunction and energy metabolic dysfunction in cardiomyocytes.

The transcriptional cofactor receptor-interacting protein 140 (RIP140) is known as a deleterious regulator of cardiac mitochondrial function and energy metabolic homeostasis. This study revealed that RIP140 repressed Sirtuin 3 (SIRT3), a mitochondrial deacetylase that plays an important role in regulating cardiac function. RIP140 was overexpressed by adenovirus infection or was knocked down by RNA interference in neonatal rat cardiomyocytes. RIP140 overexpression repressed, while RIP140 silencing elevated the expression and activity of SIRT3. Ad-RIP140 enhanced the expressions of the cardiac hypertrophic markers and increased cardiomyocyte surface area, whereas SIRT3 overexpression prevented the effect of Ad-RIP140. Additionally, SIRT3 overexpression reversed Ad-RIP140-induced mitochondrial dysfunction and energy metabolic dysfunction, such as increase in oxidative stress, decrease in mitochondrial membrane potential and ATP production, as well as downregulation of mitochondrial DNA-encoded genes and genes related to mitochondrial genome replication and transcription, mitochondrial oxidative phosphorylation and fatty acid oxidation. In contrast, SIRT3 silencing exacerbated RIP140-induced cardiomyocyte hypertrophy and mitochondrial dysfunction. Furthermore, the repression of SIRT3 by RIP140 was dependent on estrogen-related receptor-α (ERRα). The involvement of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) was ruled out of SIRT3 suppression by RIP140. RIP140 and PGC-1α might act as functional antagonists on the regulation of SIRT3. This study indicates that suppression of SIRT3 by RIP140 facilitates the development of cardiomyocyte hypertrophy, mitochondrial dysfunction and energy metabolic dysfunction. Strategies targeting inhibition of RIP140 and upregulation of SIRT3 might improve cardiac energy metabolism and suggest therapeutic potential for heart diseases.

Down-Regulated Receptor Interacting Protein 140 Is Involved in Lipopolysaccharide-Preconditioning-Induced Inactivation of Kupffer Cells and Attenuation of Hepatic Ischemia Reperfusion Injury.

BackgroundLipopolysaccharide (LPS) preconditioning is known to attenuate hepatic ischemia/reperfusion injury (I/RI); however, the precise mechanism remains unclear. This study investigated the role of receptor-interacting protein 140 (RIP140) on the protective effect of LPS preconditioning in hepatic I/RI involving Kupffer cells (KCs).MethodsSprague—Dawley rats underwent 70% hepatic ischemia for 90 minutes. LPS (100 μg/kg) was injected intraperitoneally 24 hours before ischemia. Hepatic injury was observed using serum and liver samples. The LPS/NF-κB (nuclear factor-κB) pathway and hepatic RIP140 expression in isolated KCs were investigated.ResultsLPS preconditioning significantly inhibited hepatic RIP140 expression, NF-κB activation, and serum proinflammatory cytokine expression after I/RI, with an observation of remarkably reduced serum enzyme levels and histopathologic scores. Our experiments showed that protection effects could be effectively induced in KCs by LPS preconditioning, but couldn’t when RIP140 was overexpressed in KCs. Conversely, even without LPS preconditioning, protective effects were found in KCs if RIP140 expression was suppressed with siRNA.ConclusionsDown-regulated RIP140 is involved in LPS-induced inactivation of KCs and hepatic I/RI attenuation.