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Guo Chen, JiHong Tang, Zhenhua Ni, Qingge Chen, Zhao Li, Wei Yang, Jia ...
2015 Volume 38 Issue 4 Pages
507-513
Published: April 01, 2015
Released on J-STAGE: April 01, 2015
Advance online publication: February 04, 2015
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Resveratrol, a natural polyphenolic compound known for its antioxidative and antiinflammatory effects, exerts antiasthmatic effects, although the mechanism underlying these effects remains elusive. The phosphatase and tensin homology deleted on chromosome ten gene (PTEN) is involved in the pathogenesis of asthma, and PTEN overexpression in asthmatic mice improved asthma symptoms. To investigate whether the antiasthmatic mechanisms of resveratrol correlated with the upregulation of PTEN expression, an ovalbumin (OVA)-induced murine asthma model was used to determine the effectiveness of resveratrol treatment. PTEN mRNA and protein expression was assessed with real-time polymerase chain reaction (PCR) and immunochemistry. To determine whether airway remodeling occurred, the inner airway wall, mucous layer, and smooth muscle areas were each determined using an image analysis system. The lung epithelial cell line 16HBE was used to study the regulation of PTEN expression levels by resveratrol
in vitro. Our data demonstrated that resveratrol inhibited OVA-induced airway inflammation and airway remodeling in asthmatic mice. PTEN expression was decreased in the murine asthma model, although the expression of PTEN was restored following treatment with resveratrol. Correlation efficiency analysis showed that PTEN expression was associated with the degree of airway remodeling. Further
in vitro studies demonstrated that the inhibition of Sirtuin 1 (SIRT1) activity by a SIRT1 inhibitor and RNA interference decreased PTEN protein expression, while resveratrol attenuated the decreases in PTEN expression induced by the SIRT1 inhibitor. These data suggest the mechanism of the antiasthmatic effects of resveratrol in an OVA-induced murine asthma model, which resulted in the upregulation of PTEN
via SIRT1 activation.
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Yoshitaka Kihira, Ariunzaya Burentogtokh, Mari Itoh, Yuki Izawa-Ishiza ...
2015 Volume 38 Issue 4 Pages
514-521
Published: April 01, 2015
Released on J-STAGE: April 01, 2015
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Glucagon-like peptide-1 (GLP-1), an incretin hormone, is secreted from L cells located in the intestinal epithelium. It is known that intestinal oxygen tension is decreased postprandially. In addition, we found that the expression of hypoxia-inducible factor-1α (HIF-1α), which accumulates in cells under hypoxic conditions, was significantly increased in the colons of mice with food intake, indicating that the oxygen concentration is likely reduced in the colon after eating. Therefore, we hypothesized that GLP-1 secretion is affected by oxygen tension. We found that forskolin-stimulated GLP-1 secretion from GLUTag cells, a model of intestinal L cells, is suppressed in hypoxia (1% O
2). Forskolin-stimulated elevations of preproglucagon (ppGCG) and proprotein convertase 1/3 (PC1/3) mRNA expression were decreased under hypoxic conditions. The finding that H89, a protein kinase A (PKA) inhibitor, inhibited the forskolin-stimulated increase of ppGCG and PC1/3 indicated that the cAMP-PKA pathway is involved in the hypoxia-induced suppression of the genes. Hypoxia decreased hexokinase 2 mRNA and protein expression and increased lactate dehydrogenase A mRNA and protein expression. Concomitantly, lactate production was increased and ATP production was decreased. Together, the results indicate that hypoxia decreases glucose utilization for ATP production, which probably causes a decrease in cAMP production and in subsequent GLP-1 production. Our findings suggest that the postprandial decrease in oxygen tension in the intestine attenuates GLP-1 secretion.
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Kuan Ming Chiu, Chia Chan Wu, Ming Jiuh Wang, Ming Yi Lee, Su Jane Wan ...
2015 Volume 38 Issue 4 Pages
522-530
Published: April 01, 2015
Released on J-STAGE: April 01, 2015
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The excessive release of glutamate is a critical element in the neuropathology of epilepsy, and bupivacaine, a local anesthetic agent, has been shown to inhibit the release of glutamate in rat cerebrocortical nerve terminals. This study investigated whether bupivacaine produces antiseizure and antiexcitotoxic effects using a kainic acid (KA) rat model, an animal model used for temporal lobe epilepsy, and excitotoxic neurodegeneration experiments. The results showed that administering bupivacaine (0.4 mg/kg or 2 mg/kg) intraperitoneally to rats 30 min before intraperitoneal injection of KA (15 mg/kg) increased seizure latency and reduced the seizure score. In addition, bupivacaine attenuated KA-induced hippocampal neuronal cell death, and this protective effect was accompanied by the inhibition of microglial activation and production of proinflammatory cytokines such as interleukin (IL)-1β, IL-6, and tumor necrosis factor-α in the hippocampus. Moreover, bupivacaine shortened the latency of escaping onto the platform in the Morris water maze learning performance test. Collectively, these data suggest that bupivacaine has therapeutic potential for treating epilepsy.
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Qing-Quan Lian, Pei-Pei Pan, Jun-Wei Li, Han Lin, Guo-Xin Hu, Ming-Zha ...
2015 Volume 38 Issue 4 Pages
531-535
Published: April 01, 2015
Released on J-STAGE: April 01, 2015
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The microsomal CYP2C9 alleles involved in the biotransformation of propofol, a widely used anesthetic agent, were investigated
in vitro. To examine the enzymatic activity of the CYP2C9 alleles, kinetic parameters for propofol 4-hydroxylation were determined in recombinant human P450s CYP2C9 microsomes from
Sf21 insects cells carrying CYP2C9*1 and other variants. Some of the variants showed decreased enzyme activity compared with the wild type, as previously reported. Two variants (CYP2C9*36 and *56) were found substantially to increase intrinsic clearance relative to the wild type variant. Most variants significantly (
p<0.05) decreased intrinsic clearance of propofol compared with the wild type, except *11, *47, and *54. This study is the first to report these rare alleles for propofol metabolism, providing fundamental data for further clinical studies on CYP2C9 alleles for propofol metabolism
in vivo.
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Yasumichi Inoue, Kenji Abe, Kikuo Onozaki, Hidetoshi Hayashi
2015 Volume 38 Issue 4 Pages
536-544
Published: April 01, 2015
Released on J-STAGE: April 01, 2015
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We have previously reported that transforming growth factor-β (TGF-β) down-regulates interferon-γ (IFN-γ) production in an interleukin-18 (IL-18) treated mouse natural killer (NK) cell line, LNK5E6. In LNK5E6 cells, TGF-β exhibited no inhibition of the IL-18-induced transcription of
IFN-γ, but did stimulate the degradation of
IFN-γ mRNA induced by IL-18. In the present study, we investigated the mechanism of the down-regulatory effects of TGF-β on
IFN-γ mRNA expression in a human myelomonocytic cell line, KG-1, which produces IFN-γ in response to IL-18 alone. Interestingly, IL-18 induced the production of the IFN-γ through the stabilization of
IFN-γ mRNA, but not the enhanced transcription of
IFN-γ gene. The stability of
IFN-γ mRNA was regulated by mRNA destabilizing elements in the 3′untranslated region (UTR) of
IFN-γ mRNA, especially adenylate-uridylate (AU)-rich elements (AREs) in the 5′ half of 3′UTR. Tristetraprolin (TTP), one of the ARE-binding proteins, destabilizes
IFN-γ mRNA, and IL-18 repressed the expression of
TTP mRNA. Moreover, TGF-β repressed the IL-18-induced expression of
IFN-γ mRNA through the induction of
TTP mRNA to destabilize
IFN-γ mRNA. Our data is the first to reveal that the crosstalk between IL-18 and TGF-β through the expression of TTP regulates the production of IFN-γ.
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Tomoya Narawa, Toshiaki Yano, Tomoo Itoh
2015 Volume 38 Issue 4 Pages
545-551
Published: April 01, 2015
Released on J-STAGE: April 01, 2015
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The stereoselective transport of methotrexate (L-amethopterin, L-MTX) and its enantiomer (D-amethopterin, D-MTX) by the rat proton-coupled folate transporter (rPCFT) were examined using rPCFT-expressing HEK293 cells. The initial rate of uptake of [
3H]-L-MTX by the rPCFT followed Michaelis–Menten kinetics, with a
Km value of 2.1 µM. Dixon plots revealed that the uptake of L-MTX by the rPCFT was inhibited in a competitive manner by unlabeled L-MTX and D-MTX, with
Ki values of approximately 1.3 and 150 µM, respectively. The initial rate of uptake of D-MTX by the rPCFT also followed Michaelis–Menten kinetics, with a
Km value of 190 µM. The results of the current study demonstrate that the different enantiomers of MTX are transported in a highly stereoselective manner by the rPCFT, with the uptake clearance of L-MTX being approximately 46-fold greater than that of D-MTX. The observed stereoselectivity of the rPCFT was found to be comparable with that of the human PCFT.
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Seong-Il Kang, Hye-Sun Shin, Se-Jae Kim
2015 Volume 38 Issue 4 Pages
552-558
Published: April 01, 2015
Released on J-STAGE: April 01, 2015
Advance online publication: January 24, 2015
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Sinensetin is a rare polymethoxylated flavone (PMF) found in certain citrus fruits. In this study, we investigated the effects of sinensetin on lipid metabolism in 3T3-L1 cells. Sinensetin promoted adipogenesis in 3T3-L1 preadipocytes growing in incomplete differentiation medium, which did not contain 3-isobutyl-1-methylxanthine. Sinensetin up-regulated expression of the adipogenic transcription factors peroxisome proliferator-activated receptor γ, CCAAT/enhancer-binding protein (C/EBP) α, and sterol regulatory element-binding protein 1c. It also potentiated expression of C/EBPβ and activation of cAMP-responsive element-binding protein. Sinensetin enhanced activation of protein kinase A and increased intracellular cAMP levels in 3T3-L1 preadipocytes. In mature 3T3-L1 adipocytes, sinensetin stimulated lipolysis
via a cAMP pathway. Taken together, these results suggest that sinensetin enhances adipogenesis and lipolysis by increasing cAMP levels in adipocytes.
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Takanori Kanazawa, Mamiko Yamazaki, Tsunehiko Fukuda, Yuuki Takashima, ...
2015 Volume 38 Issue 4 Pages
559-565
Published: April 01, 2015
Released on J-STAGE: April 01, 2015
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To develop a versatile nuclear-targeted gene vector, nuclear localization signal (NLS) oligopeptides combining cysteine (C), histidine (H), and stearic acid (STR) were investigated in this study. The original SV40 sequence (
SV40: Pro-Lys-Lys-Lys-Arg-Lys-Val) was selected as the NLS sequence, and physical characterizations of various NLS-based oligopeptides (C
SV40C, STR-C
SV40C, and STR-CH
2SV40H
2C), including mean diameter, zeta-potential, complex condensation, and decondensation, were evaluated. In addition, cellular and nuclear uptake of plasmid DNA (pDNA) and gene expression in COS7 and dendritic cells (JAWS II) were determined. As a result, C and STR enhanced formation of a smaller and more stable nano-complex with pDNA based on ionic interactions, the disulfide linkage and hydrophobic interactions. STR-C
SV40C and STR-CH
2SV40H
2C had significantly higher cellular uptake ability and transfection efficiency than
SV40 and C
SV40C. In particular, STR-CH
2SV40H
2C had higher nuclear uptake and gene expression efficiency than STR-C
SV40C. Furthermore, STR-CH
2SV40H
2C could deliver pDNA to the nuclei and had high gene expression efficiency in dendritic cells. Our results indicate that STR-CH
2SV40H
2C is a promising gene delivery system in non- or slow-dividing cells.
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Teruaki Akao, Yugi Oyanagi, Satoshi Shiotsuki, Yoko Ishii, Masakiyo Sa ...
2015 Volume 38 Issue 4 Pages
566-570
Published: April 01, 2015
Released on J-STAGE: April 01, 2015
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Ulcerative colitis induced by dextran sulfate sodium (DSS) is one of the most widely used experimental animal models. However, the mechanism responsible for the pathogenesis of the colitis is still unclear. The aim of the present study was to clarify the events occurring after administration of DSS to rats focusing on the relationship between the intestinal bacterial metabolism of DSS and the intestinal mucosal lesions in the model. Within 2 d after DSS administration, severe injury of the cecal mucosa was evident, together with bloody feces and blood in the cecum. However, these lesions were repressed by administration of antibiotics. On the other hand, DSS was found to be metabolized under anaerobic conditions upon incubation with cecal content
in vitro, first being desulfated and then undergoing carbohydrate moiety degradation. However, no such metabolic process occurred when cecal content from rats that had been administered antibiotics was employed. These results indicate that the initial step of DSS-induced ulcerative colitis is lesioning of the cecal mucosa, which is related to metabolism of DSS by intestinal bacteria.
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Komsak Pintha, Supachai Yodkeeree, Pornngarm Limtrakul
2015 Volume 38 Issue 4 Pages
571-581
Published: April 01, 2015
Released on J-STAGE: April 01, 2015
Advance online publication: January 20, 2015
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Supplementary material
Proanthocyanidin is one of the main active compounds found in red jasmine rice. We previously reported that red rice extract could reduce cancer cell invasion. However, the direct effect of proanthocyanidin from red rice on the invasion of cancer cells and the exact molecular mechanism remained unclear. Here, we report for the first time that proanthocyanidin-rich fraction from red rice (PRFR) reduced the migration and invasion of MDA-MB-231 human breast cancer cells. The types of proanthocyanidin in PRFR were identified as procyanidins and prodelphinidins by acid hydrolysis. For cancer cell invasion, degradation of the extracellular matrix (ECM) is required. Treatment of the cells with PRFR reduced the expression of ECM degradation-associated proteins, including matrix metalloproteinase-9 (MMP-9), membrane type-1 matrix metalloproteinase, urokinase plasminogen activator, urokinase plasminogen activator receptor and plasminogen activator-1. Moreover, PRFR also reduced the activity of collagenase and MMP-9. Furthermore, PRFR significantly suppressed the expression of intercellular adhesion molecule-1 and interleukin-6. We also found that PRFR reduced the DNA-binding activity of nuclear factor kappa B (NF-κB), which is the expressed mediator of ECM degradation-associated proteins. These results suggest that proanthocyanidin from red rice mediates MDA-MB-231 breast cancer cell invasion by altering the expression of the invasion-associated proteins, possibly by targeting NF-κB activity.
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Hiroki Ohya, Yoshihiko Shibayama, Jiro Ogura, Katsuya Narumi, Masaki K ...
2015 Volume 38 Issue 4 Pages
582-586
Published: April 01, 2015
Released on J-STAGE: April 01, 2015
Advance online publication: February 17, 2015
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Regorafenib is a small molecule inhibitor of tyrosine kinases, and has been shown to improve the outcomes of patients with advanced colorectal cancer and advanced gastrointestinal stromal tumors. The transport profiles of regorafenib by various transporters were evaluated. HEK293/organic anion transporting polypeptide 1B1 (OATP1B1) cells exhibited increased drug sensitivity to regorafenib. Regorafenib inhibited the uptake of
3H-estrone sulfate by HEK293/OATP1B1 cells in a dose-dependent manner, but did not affect its elimination by P-glycoproteins. The concentration of regorafenib was significantly lower in LLC-PK1/multidrug resistance protein 2 (MRP2) cells than in LLC-PK1 cells treated with the MRP2 inhibitor, MK571. MK571 abolished the inhibitory effects of regorafenib on intracellular accumulation in LLC-PK1/MRP2 cells. The uptake of regorafenib was significantly higher in HEK293/OATP1B1 cells than in OATP1B1-mock cells. Transport kinetics values were estimated to be
Km=15.9 µM and
Vmax=1.24 nmol/mg/min. No significant difference was observed in regorafenib concentrations between HEK293/OATP1B3 and OATP1B3-mock cells. These results indicated that regorafenib is a substrate for MRP2 and OATP1B1, and also suggest that the substrate preference of regorafenib may implicate the pharmacokinetic profiles of regorafenib.
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Maiko Machida, Yuki Miyamura, Takuji Machida, Kinuko Koyama, Kenji Iiz ...
2015 Volume 38 Issue 4 Pages
587-593
Published: April 01, 2015
Released on J-STAGE: April 01, 2015
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We investigated the effects of olanzapine on cisplatin-induced pica (the consumption of non-nutrient materials such as kaolin) and glucose homeostasis in rats to clarify the effects of olanzapine when used as an anti-emetic drug. Rats were injected intraperitoneally (i.p.) with either 5 mg/kg cisplatin or saline. Additionally, 2 or 10 mg/kg olanzapine were administered i.p. to the rats 10 min before the administration of cisplatin and subsequently administered every 24 h for 3 d. Kaolin and food intake was measured using an automatic monitoring apparatus. Plasma glucose levels were measured by an enzyme electrode method. The plasma levels of insulin and intact proinsulin were measured by enzyme-linked immunosorbent assay (ELISA). The proinsulin-to-insulin (P/I) ratio was calculated. Cisplatin significantly increased kaolin intake, but decreased food intake and body weight up to 72 h. Olanzapine had no effect on these parameters. Neither olanzapine nor cisplatin alone had a significant effect on the plasma levels of glucose, insulin, or proinsulin. However, a combination of olanzapine and cisplatin significantly decreased plasma insulin levels, but increased plasma intact proinsulin levels and the P/I ratio. Our results suggest that an additive deterioration of insulin-secreting beta-cell function and disturbance of glucose homeostasis should be considered during treatment of patients with olanzapine for cisplatin-induced nausea and vomiting.
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Shigeyuki Ohta, Sayaka Sakaguchi, Yuki Kobayashi, Norikazu Mizuno, Ken ...
2015 Volume 38 Issue 4 Pages
594-600
Published: April 01, 2015
Released on J-STAGE: April 01, 2015
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GPR56 is a member of the adhesion G protein-coupled receptor (GPCR) and is highly expressed in parts of tumor cells. The involvement of GPR56 in tumorigenesis has been reported. We generated agonistic monoclonal antibodies against human GPR56 and analyzed the action and signaling pathway of GPR56. The antibodies inhibited cell migration through the Gq and Rho pathway in human glioma U87-MG cells. Co-immunoprecipitation analysis indicated that the interaction between the GPR56 extracellular domain and transmembrane domain was potentiated by agonistic antibodies. These results demonstrated that functional antibodies are invaluable tools for GPCR research and should open a new avenue for therapeutic treatment of tumors.
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Noriaki Nagai, Yoshimasa Ito, Tadatoshi Tanino
2015 Volume 38 Issue 4 Pages
601-610
Published: April 01, 2015
Released on J-STAGE: April 01, 2015
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The accumulation of amyloid β
1–42 peptide (Aβ
1–42) in retina is implicated in the development of retinal ganglion cell apoptosis and diabetic retinopathy. In this study we demonstrate that spontaneous diabetes mellitus Otsuka Long-Evans Tokushima Fatty (OLETF) rats can be used as an animal model in studies to identify the expression of Aβ in diabetic retinas. In addition, we investigated the relation between glucose level and Aβ production in the retinas of OLETF rats. In the retinas of Long-Evans Tokushima Otsuka (LETO) rats used as normal controls and OLETF rats, no expression of neprilysin (NEP), which degrades Aβ, was detected, and the expression levels of genes associated with Aβ production (amyloid precursor protein, β site APP cleaving enzyme, and presenilin) and Aβ
1–42 levels in the retinas of 60-week-old OLETF rats with diabetes mellitus were significantly higher than in 60-week-old LETO rat retinas. Furthermore, the increase in the expression levels of genes associated with Aβ production was enhanced by administration of glucose (3.0 g/kg; OGT test), and close relations between the retinal Aβ
1–42 level and plasma blood glucose and HbA1c were observed. In conclusion, we have found that Aβ accumulates easily in the retinas of LETO and OLETF rats due to the absence of NEP. In addition, we determined that the accumulation of Aβ
1–42 in the retinas of OLETF rats is promoted by high plasma glucose levels. Therefore OLETF rats may be a suitable model for studies to identify the expression of Aβ in diabetic retinas.
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Kaoru Suzuki, Fuminori Otsuka, Hirotomo Yamada, Shinji Koizumi
2015 Volume 38 Issue 4 Pages
611-617
Published: April 01, 2015
Released on J-STAGE: April 01, 2015
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Metal responsive element (MRE)-binding transcription factor-1 (MTF-1) is a zinc finger (ZF) transcription factor that plays a key role in heavy metal homeostasis by regulating relevant genes in response to metals. MTF-1 is known to be activated by heavy metals such as Zn and Cd, but the mechanism of activation remains unclear. In the present study, Cys and His residues of human MTF-1 (hMTF-1), some of which may be involved in interaction with metals or with each other, were screened for their contribution to Zn-dependent transcription. To avoid poor induction ratios of previous transfection assays, we re-examined experimental conditions to establish an assay able to correctly detect Zn-responsive transcription. Using this assay, a series of Cys and/or His substitution mutants were analyzed over the entire hMTF-1 molecule. In five out of the six ZFs (ZF1 to ZF5), Cys mutations that disrupt the ZF structure abolished response to Zn. Of these, ZF5 was shown for the first time to be essential for Zn-responsive transcription, despite it being unnecessary for Zn-induced DNA binding. These results indicate that Zn activation of hMTF-1 involves an additional process besides induction of DNA binding activity. Our assay also confirmed the importance of Cys in the acidic activation domain, as well as those in the C-terminal Cys cluster, implicated in transcription in other studies. The identified Cys residues might contribute to metal response of hMTF-1 through direct metal binding and/or intramolecular interactions, analysis of which will be helpful in understanding the mechanism of metal response.
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Chiharu Miyajima, Yasumichi Inoue, Hidetoshi Hayashi
2015 Volume 38 Issue 4 Pages
618-624
Published: April 01, 2015
Released on J-STAGE: April 01, 2015
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Tribbles 1 (TRB1) is one of the mammalian orthologs of
Drosophila Tribbles, which regulates development and cell proliferation. TRB1 is suggested to act as a scaffold protein in signaling pathways for important cellular processes. TRB1 has also been identified as a myeloid oncogenic driver and mediates leukemogenesis through the mitogen-activated protein extracellular kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway and CCAAT/enhancer binding protein (C/EBP) transcriptional factors. However, the physiological roles of TRB1 in solid tumors have not been clarified. Here, we show that TRB1 interacts with p53 and suppresses its tumor suppressor activity. TRB1 knockdown enhances transcriptional activity of p53 and decreases cell viability. Interestingly, TRB1 enhances histone deacety lase 1 (HDAC1)-mediated p53 deacetylation and decreases DNA binding of p53. These results suggest that TRB1 is involved in the proliferation of tumor cells by inhibiting the activities of tumor suppressor p53 in solid tumors.
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