B-RafV600E inhibitors have been suggested to promote tumor regression with the help of host immunity, but this hypothesis has not been examined directly in detail. In this study, we profiled immunological changes in the tumor microenvironment and tumor infiltrating lymphocytes (TILs) in a B-RafV600E/Pten-driven murine model of melanoma, after administration of the BRafV600E small molecule inhibitor PLX4720. In this model, we found that as tumors developed they gradually acquired immunosuppressive features, including accumulation of regulatory T cells (Tregs) and CD11b+/Gr-1+ myeloid cells and loss of Th1 effector functions on CD4+ TILs, such as CD40L and IFN-γ expression. PLX4720 administration promoted development of a more immune stimulatory microenvironment associated with a relative increase in CD40L and IFNγ expression on intratumoral CD4+ TILs and a reduced accumulation of Tregs and CD11b+/Gr-1+ myeloid cells. Strikingly, CD40L or IFNγ blockade compromised the ability of PLX4720 to inhibit melanoma growth. Supporting this result, agonistic CD40 antibody was sufficient to evoke anti-tumor immunity and suppress tumor growth in tumor-bearing mice. Taken together, our results establish the critical role of immune-related changes with key contributions for CD40L and IFNγ signaling in the anti-tumor responses triggered in vivo by BRafV600E inhibitors.
Braf kinase; melanoma; PLX4720; CD40L; anti-tumor
The establishment of an immunosuppressive tumor microenvironment is a hallmark feature driving cancer cell evasion of immunosurveillance. In a murine melanoma model, we recently demonstrated that decreased intratumoral CD4+ T-cell expression of CD40L and interferon γ (IFNγ) is critical to maintain this immunosuppressive microenvironment. Altered effector functions of tumor-associated CD4+ T cells is essential for B-RafV600E inhibitor-mediated restoration of antitumor immunity.
BRAF; CD40L; IFNγ; T cell; melanoma
The physiological signal activating cytoplasmic accumulation of nuclear receptor interacting protein 140 (RIP140) in adipocytes was unclear. We uncover that endothelin 1 (ET-1) promotes cytoplasmic accumulation of RIP140 in 3T3-L1 adipocytes. We determine ET-1’s signal transduction pathway in adipocytes, which is by activating ETA receptor-PLCβ-nuclear PKCε. Blocking this pathway in 3T3-L1 adipocyte cultures, by treating cells with an ETA antagonist, inhibiting PLCβ, or silencing PKCε, reduces ET-1-stimulated cytoplasmic accumulation of RIP140. In a HFD-fed obese mouse model, administration of a selective ETA antagonist, ambrisentan, effectively dampens cytoplasmic accumulation of RIP140 in the epididymal adipose tissue and reduces HFD-caused adipocyte dysfunctions. Importantly, ambrisentan improves blood glucose control and reduces the severity of hepatic steatosis in HFD-fed mice. This study reports a physiological signal that stimulates nuclear export of RIP140 in adipocytes and provides evidence for a strategy using selective ETA antagonist to treat obesity-induced insulin resistance and, possibly, other metabolic disorders.
RIP140; Endothelin-1; Insulin sensitivity; Cytoplasm; ETA receptor; Ambrisentan
RIP140 (Receptor-interacting protein 140) is highly expressed in mature adipocytes and functions as a co-repressor for gene expression involved in lipid and glucose metabolism. In adipocytes, activated PKCε (Protein kinase C epsilon) phosphorylates nuclear RIP140 which is then subsequently arginine methylated and exported to the cytoplasm. In the cytoplasm, RI140 can elicit additional activities. Here we report a new functional role for cytoplasmic RIP140 in adipocyte in regulating adiponectin secretion. Targeting cytoplasmic RIP140 by knocking down RIP140 itself or its nuclear export trigger, PKCε, promotes the secretion of adiponectin without affecting the production or oligomerization of adiponectin. Consequentially, conditioned media from either RIP140- or PKCε-silenced adipocytes, which contain higher levels of adiponectin, enhance glucose uptake in C2C12 cells and reduce gluconeogenesis in HepG2 cells. Further, these effects can be inhibited by an adiponectin-neutralizing antibody. The effect of cytoplasmic RIP140 in regulating adiponectin secretion is via interacting with AS160, a known RIP140-interacting protein. This study reveals a new functional role for cytoplasmic RIP140 in modulating adiponectin vesicle secretion, and suggests that targeting cytoplasmic RIP140 may be a potentially effective therapeutic strategy to improve adiponectin secretion and possibly to manage metabolic disorders.
RIP140; cytoplasmic; Adiponectin; Insulin sensitivity; Glucose uptake; Gluconeogenesis
Brain tumours that are associated with cerebral aneurysms are rare occurrences, whereas the coexistence of brain tumours and intra-tumoural aneurysms is even rarer. There have been 12 brain tumour cases that have been reported in the literature that describe an aneurysm within a brain tumour, with 4 of these tumours being meningiomas.
A 34-year-old male patient presented with sudden-onset headache, and an inter-hemispheric meningioma with intra-tumoural bleeding was found due to a ruptured embedded anterior cerebral artery aneurysm. The aneurysm was diagnosed incidentally on the third cerebral angiogram, while the initial 2 angiograms were negative. The patient was treated with endovascular aneurysm embolisation that was followed by tumour resection.
This paper is the first case report to describe the coexistence of a meningioma and an aneurysm, which presented with intra-tumoural haemorrhage that was negative on the initial cerebral angiogram. Unlike previous case reports, the aneurysm in this case was located with an anterior cerebral artery distribution.
Meningioma; Haemorrhage; subarachnoid haemorrhage (SAH); aneurysms; computed tomography; Brain magnetic resonance imaging (MRI).
Endotoxin tolerance (ET) triggered by prior exposure to Toll-like receptor (TLR) ligands provides a mechanism to dampen inflammatory cytokines. Receptor-interacting protein 140 (RIP140) interacts with NF-κB to regulate the expression of proinflammatory cytokine genes. We identify lipopolysaccharide (LPS) stimulation of Syk-mediated tyrosine phosphorylation on RIP140 and RelA interaction with RIP140. These events increase recruitment of SOCS1-Rbx1 (SCF) E3 ligase to tyrosine-phosphorylated RIP140, thereby degrading RIP140 to inactivate inflammatory cytokine genes. Macrophages expressing a non-degradable RIP140 were resistant to the establishment of ET for specific genes. The results reveal RelA as an adaptor for SCF ubiquitin ligase to fine-tune NF-κB target genes by targeting co-activator RIP140, and an unexpected role for RIP140 protein degradation in resolving inflammation and ET.
Dental caries is a major public health problem in many countries. Since the last territority-wide dental survey of Hong Kong preschool children was conducted in 2001, a survey to update the information is necessary. This study aimed to describe the dental caries experience of preschool children in Hong Kong and factors affecting their dental caries status.
A stratified random sample of children from seven kindergartens in Hong Kong was surveyed in 2009. Ethical approval from IRB and parental consent was obtained. Clinical examinations of the children were performed by two calibrated examiners using disposable dental mirrors, an intra-oral LED light and ball-ended periodontal probes. A questionnaire to investigate possible explanatory factors for caries status was completed by the children’s parents. Caries experience was recorded using the dmft index. Multifactor-ANOVA was used to study the relationship between dental caries experience, and the background and oral health-related behaviours of the children.
Seven hundred children (53% boys), mean age 5.3 ± 0.7 years were examined. The mean dmft score of the surveyed children was 2.2 and 51% of them had no caries experience (dmft = 0). Most (>95%) of the decayed teeth were untreated. Statistically significant correlations were found between dental caries experience of the children and their oral health-related habits, family income, parental education level and parental dental knowledge.
Early childhood dental caries was prevalent among the preschool children in Hong Kong. Their caries experience was associated with their oral health-related behaviours, socio-economic background, and parental education and dental knowledge.
Dental caries; Oral hygiene; Oral health; Toothbrushing; Preschool children; Hong Kong; China
Recurrent disturbances can have a critical effect on the structure and function of coral reef communities. In this study, long-term changes were examined in the hard coral community at Wanlitung, in southern Taiwan, between 1985 and 2010. In this 26 year interval, the reef has experienced repeated disturbances that include six typhoons and two coral-bleaching events. The frequency of disturbance has meant that species susceptible to disturbance, such as those in the genus Acropora and Montipora have almost disappeared from the reef. Indeed, almost all hard coral species have declined in abundance, with the result that total hard coral cover in 2010 (17.7%) was less than half what it was in 1985 (47.5%). In addition, macro-algal cover has increased from 11.3% in 2003 to 28.5% in 2010. The frequency of disturbance combined with possible chronic influence of a growing human population mean that a diverse reef assemblage is unlikely to persist on this reef into the future.
Receptor-interacting protein 140 (RIP140) is abundantly expressed in mature adipocyte and modulates gene expression involved in lipid and glucose metabolism. Protein kinase C epsilon and protein arginine methyltransferase 1 can sequentially stimulate RIP140 phosphorylation and then methylation, thereby promoting its export to the cytoplasm. Here we report a lipid signal triggering cytoplasmic accumulation of RIP140, and a new functional role for cytoplasmic RIP140 in adipocyte to regulate lipolysis. Increased lipid content, particularly an elevation in diacylglycerol levels, promotes RIP140 cytoplasmic accumulation and increased association with lipid droplets (LDs) by its direct interaction with perilipin. By interacting with RIP140, perilipin more efficiently recruits hormone-sensitive lipase (HSL) to LDs and enhances adipose triglyceride lipase (ATGL) forming complex with CGI-58, an activator of ATGL. Consequentially, HSL can more readily access its substrates, and ATGL is activated, ultimately enhancing lipolysis. In adipocytes, blocking cytoplasmic RIP140 accumulation reduces basal and isoproterenol-stimulated lipolysis and the pro-inflammatory potential of their conditioned media (i.e. activating NF-κB and inflammatory genes in macrophages). These results show that in adipocytes with high lipid contents, RIP140 increasingly accumulates in the cytoplasm and enhances triglyceride catabolism by directly interacting with perilipin. The study suggests that reducing nuclear export of RIP140 might be a useful means of controlling adipocyte lipolysis.
adipocyte; post-translational modification; diacylglyceride; lipid droplet; lipase; lipid; RIP140
Promyelocytic leukemia (Pml) protein is required for Oct4 gene expression and the maintenance of its open chromatin conformation in stem cells. In proliferating stem cells, Pml-nuclear body, along with transcription factors TR2, SF1 and Sp1 and Brg1-dependent chromatin remodeling complex (BRGC), associates with conserved region 1 (CR1) of this promoter to maintain a nucleosome-free region for gene activity. Retinoic acid (RA) rapidly down-regulates Pml, resulting in the replacement of BRGC with Brm-containing remodeling complex (BRMC), disassociation of SF1 and SP1, retaining of TR2, recruitment of RIP140, G9a and HP1γ, and sequential insertion of two nucleosomes on CR1 that progressively displays repressive heterochromatin marks. This study demonstrates a functional role for Pml in maintaining a specific open chromatin conformation of the Oct4 promoter region for its constant expression in stem cells; and illustrates the mechanism underlying RA-induced chromatin remodeling of Oct4 gene in differentiating cells, in which Pml plays a critical role. The study also demonstrates a novel mode of chromatin remodeling which occurs by repositioning and sequentially inserting nucleosomes into a specific region of the gene promoter to compact the chromatin in differentiating cells.
Retinoic acid; P19; Oct4; Pml-NB; TR2; Embryonic stem cells; Chromatin remodeling
Low-income youth experience social-emotional problems linked to chronic stress that are exacerbated by lack of access to care. Drumming is a non-verbal, universal activity that builds upon a collectivistic aspect of diverse cultures and does not bear the stigma of therapy. A pretest-post-test non-equivalent control group design was used to assess the effects of 12 weeks of school counselor-led drumming on social-emotional behavior in two fifth-grade intervention classrooms versus two standard education control classrooms. The weekly intervention integrated rhythmic and group counseling activities to build skills, such as emotion management, focus and listening. The Teacher's Report Form was used to assess each of 101 participants (n = 54 experimental, n = 47 control, 90% Latino, 53.5% female, mean age 10.5 years, range 10–12 years). There was 100% retention. ANOVA testing showed that intervention classrooms improved significantly compared to the control group in broad-band scales (total problems (P < .01), internalizing problems (P < .02)), narrow-band syndrome scales (withdrawn/depression (P < .02), attention problems (P < .01), inattention subscale (P < .001)), Diagnostic and Statistical Manual of Mental Disorders-oriented scales (anxiety problems (P < .01), attention deficit/hyperactivity problems (P < .01), inattention subscale (P < .001), oppositional defiant problems (P < .03)), and other scales (post-traumatic stress problems (P < .01), sluggish cognitive tempo (P < .001)). Participation in group drumming led to significant improvements in multiple domains of social-emotional behavior. This sustainable intervention can foster positive youth development and increase student-counselor interaction. These findings underscore the potential value of the arts as a therapeutic tool.
Receptor Interacting Protein 140 (RIP140), a nuclear receptor corepressor, is important for lipid and glucose metabolism. In adipocytes, RIP140 can be phosphorylated by protein kinase C epsilon (PKCε), followed by arginine methylation, and exported to the cytoplasm. This study demonstrates for the first time a cytoplasmic function for RIP140: to counteract insulin-stimulated glucose transporter 4 (GLUT4) membrane partitioning and glucose uptake in adipocytes. Cytoplasmic RIP140 interacts with the Akt substrate AS160, thereby impeding AS160 phosphorylation by Akt; this in turn reduces GLUT4 trafficking. This signal transduction pathway can be recapitulated in the epididymal adipocytes of diet-induced obese mice: nuclear PKCε is activated, cytoplasmic RIP140 increases, and GLUT4 trafficking and glucose uptake are reduced. The data reveal a new, cytoplasmic, function for RIP140 as a negative regulator of GLUT4 trafficking and glucose uptake, and shed insight into the regulation of basal and insulin-stimulated glucose disposal by a nuclear-initiated counteracting mechanism.
GLUT4; cytoplasmic RIP140; adipocytes; AS160; Akt; post-translational modification
Receptor-interacting protein 140 is a co-regulator for many transcription factors. Previous mass spectrometry studies showed that either phosphorylation or lysine acetylation of RIP140 directly enhanced its trans-repressive activity. In this study, we first identified p300 as a specific lysine acetyltransferase, and extracellular-signal-related kinase 2 (Erk2) as a specific kinase for threonine phosphorylation, of RIP140 in vivo. We further determined two specific acetylated lysine residues (Lys158/Lys287) and phosphorylated threonine residues (Thr202/Thr207) that were critical for its gene repressive activity. We then delineated signal transduction from Erk2-mediated phosphorylation of RIP140 that enhanced its recruiting p300 for subsequent lysine acetylation, and demonstrated the kinetics of activation of this signal transduction pathway in differentiating adipocytes. Finally, the physiological significance of this cell signal transduction pathway was illustrated in rescuing experiments where the defect in fat accumulation of RIP140-null cultures was rescued by re-expressing the wild type RIP140 or its phospho-mimetic mutant, but not its acetylation deficient mutant. These results demonstrate the signal transduction pathway, initiated from Erk2 activation for specific threonine phosphorylation, followed by p300 recruitment for lysine acetylation, which ultimately enhances the gene-repressive activity of RIP140 and its functional role in fat accumulation in differentiated adipoctyes.
Receptor interacting protein 140; post-translational modification; p300; Extracellular-signal-related kinase 2; Acetylation; Phosphorylation; lipid metabolism
TR2 is an orphan nuclear receptor specifically expressed in early embryos (Wei and Hsu, 1994), and a transcription factor for transcriptional regulation of important genes in stem cells including the gate keeper Oct4 (Park et al. 2007). TR2 is known to function as an activator (Wei et al. 2000), or a repressor (Chinpaisal et al., 1998, Gupta et al. 2007). Due to the lack of specific ligands, mechanisms triggering its activator or repressor function have remained puzzling for decades. Recently, we found that all-trans retinoic acid (atRA) triggers the activation of extracellular-signal-regulated kinase 2 (ERK2), which phosphorylates TR2 and stimulates its partitioning to promyelocytic leukemia (PML) nuclear bodies, thereby converting the activator function of TR2 into repression (Gupta et al. 2008; Park et al. 2007). Recruitment of TR2 to PML is a crucial step in the conversion of TR2 from an activator to a repressor. However, it is unclear how phosphorylated TR2 is recruited to PML, an essential step in converting TR2 from an activator to a repressor. In the present study, we use both in vitro and in vivo systems to address the problem of recruiting TR2 to PML nuclear bodies. First, we identify histone deacetylase 3 (HDAC3) as an effector molecule. HDAC3 is known to interact with TR2 (Franco et al. 2001) and this interaction is enhanced by the atRA-stimulated phosphorylation of TR2 at Thr-210 (Gupta et al. 2008). Secondly, in this study, we also find that the carrier function of HDAC3 is independent of its deacetylase activity. Thirdly, we find another novel activity of atRA that stimulates nuclear enrichment of HDAC3 to form nuclear complex with PML, which is ERK2 independent. This is the first report identifying a deacetylase-independent function for HDAC3, which serves as a specific carrier molecule that targets a specifically phosphorylated protein to PML NBs. This is also the first study delineating how protein recruitment to PML nuclear bodies occurs, which can be stimulated by atRA in an ERK2-independent manner. These findings could provide new insights into the development of potential therapeutics and in understanding how orphan nuclear receptor activities can be regulated without ligands.
Receptor interacting protein 140 (RIP140) is a versatile transcriptional co-repressor that plays roles in diverse metabolic processes including fat accumulation in adipocytes. Previously we identified three methylated arginine residues in RIP140, which rendered its export to the cytoplasm; but it was unclear what triggered RIP140 arginine methylation.
In this study, we determined the activated PKCε as the specific trigger for RIP140 arginine methylation and its subsequent export. We identified two PKCε–phosphorylated residues of RIP140, Ser-102 and Ser-1003, which synergistically stimulated direct binding of RIP140 by 14-3-3 that recruited protein arginine methyl transferase 1 to methylate RIP140. The methylated RIP140 then preferentially recruited exportin 1 for nuclear export. As a result, the nuclear gene-repressive activity of RIP140 was reduced. In RIP140 null adipocyte cultures, the defect in fat accumulation was effectively rescued by the phosphoylation-deficient mutant RIP140 that resided predominantly in the nucleus, but less so by the phospho-mimetic RIP140 that was exported to the cytoplasm.
This study uncovers a novel means, via a cascade of protein modifications, to inactivate, or suppress, the nuclear action of an important transcription coregulator RIP140, and delineates the first specific phosphorylation-arginine methylation cascade that could alter protein subcellular distribution and biological activity.
Earlier studies have shown that the C-terminal half of helix 6 (H6) of the influenza A virus matrix protein (M1) containing the YRKL sequence is involved in virus budding (E. K.-W. Hui, S. Barman, T. Y. Yang, and D. P. Nayak, J. Virol. 77:7078-7092, 2003). In this report, we show that the YRKL sequence is the L domain motif of influenza virus. Like other L domains, YRKL can be inserted at different locations on the mutant M1 protein and can restore virus budding in a position-independent manner. Although YRKL is a part of the nuclear localization signal (NLS), the function of YRKL was independent of the NLS activity and the NLS function of M1 was not required for influenza virus replication. Some mutations in YRKL and the adjacent region caused a reduction in the virus titer by blocking virus release, and some affected virus morphology, producing elongated particles. Coimmunoprecipitation and Western blotting analyses showed that VPS28, a component of the ESCRT-I complex, and Cdc42, a member of the Rho family GTP-binding proteins, interacted with the M1 protein via the YRKL motif. In addition, depletion of VPS28 and Cdc42 by small interfering RNA resulted in reduction of influenza virus production. Moreover, overexpression of dominant-negative Cdc42 inhibited influenza virus replication, whereas a constitutively active Cdc42 mutant enhanced virus production in infected cells. These results indicated that VPS28, a component of ESCRT-I, and Cdc42, a small G protein, are associated with the M1 protein and involved in the influenza virus life cycle.