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1.  Integrated mRNA-MicroRNA Profiling of Human NK Cell Differentiation Identifies MiR-583 as a Negative Regulator of IL2Rγ Expression 
PLoS ONE  2014;9(10):e108913.
Natural killer (NK) cells are innate immune effector cells that protect against cancer and some viral infections. Until recently, most studies have investigated the molecular signatures of human or mouse NK cells to identify genes that are specifically expressed during NK cell development. However, the mechanism regulating NK cell development remains unclear. Here, we report a regulatory network of potential interactions during in vitro differentiation of human NK cells, identified using genome-wide mRNA and miRNA databases through hierarchical clustering analysis, gene ontology analysis and a miRNA target prediction program. The microRNA (miR)-583, which demonstrated the largest ratio change in mature NK cells, was highly correlated with IL2 receptor gamma (IL2Rγ) expression. The overexpression of miR-583 had an inhibitory effect on NK cell differentiation. In a reporter assay, the suppressive effect of miR-583 was ablated by mutating the putative miR-583 binding site of the IL2Rγ 3′ UTR. Therefore, we show that miR-583 acts as a negative regulator of NK cell differentiation by silencing IL2Rγ. Additionally, we provide a comprehensive database of genome-wide mRNA and miRNA expression during human NK cell differentiation, offering a better understanding of basic human NK cell biology for the application of human NK cells in immunotherapy.
PMCID: PMC4196775  PMID: 25313504
2.  The EF-hand calcium-binding protein tescalcin is a potential oncotarget in colorectal cancer 
Oncotarget  2014;5(8):2149-2160.
Tescalcin (TESC) is an EF-hand calcium binding protein that is differentially expressed in several tissues, however it is not reported that the expression and functional roles of TESC in colorectal cancer. Levels of messenger RNA (mRNA) and protein expression of TESC in colorectal cancer tissues were assessed using RT-PCR, real time PCR, immunohistochemistry, and clinicopathologic analyses. Quantitative analysis of TESC levels in serum specimens was performed using sandwich ELISA. Colorectal cancer cells transfected with TESC small interfering RNA and short hairpin RNA were examined in cell proliferation assays, phospho-MAPK array, and mouse xenograft models. Here we demonstrated that TESC is overexpressed in colorectal cancer (CRC), but was not expressed in normal mucosa and premalignant dysplastic lesions. Furthermore, serum TESC levels were elevated in patients with CRC. Knockdown of TESC inhibited the Akt-dependent NF-κB pathway and decreased cell survival in vitro. Depletion of TESC reduced tumor growth in a CRC xenograft model. Thus, TESC is a potential diagnostic marker and oncotarget in colorectal cancer.
PMCID: PMC4039152  PMID: 24811141
Tescalcin; colorectal cancer; cell growth; tumor growth, NF-κB
3.  TXNIP Deficiency Exacerbates Endotoxic Shock via the Induction of Excessive Nitric Oxide Synthesis 
PLoS Pathogens  2013;9(10):e1003646.
Thioredoxin-interacting protein (TXNIP) has multiple functions, including tumor suppression and involvement in cell proliferation and apoptosis. However, its role in the inflammatory process remains unclear. In this report, we demonstrate that Txnip−/− mice are significantly more susceptible to lipopolysaccharide (LPS)-induced endotoxic shock. In response to LPS, Txnip−/− macrophages produced significantly higher levels of nitric oxide (NO) and inducible nitric oxide synthase (iNOS), and an iNOS inhibitor rescued Txnip−/− mice from endotoxic shock-induced death, demonstrating that NO is a major factor in TXNIP-mediated endotoxic shock. This susceptibility phenotype of Txnip−/− mice occurred despite reduced IL-1β secretion due to increased S-nitrosylation of NLRP3 compared to wild-type controls. Taken together, these data demonstrate that TXNIP is a novel molecule that links NO synthesis and NLRP3 inflammasome activation during endotoxic shock.
Author Summary
TXNIP has many biological functions, including the inhibition of tumor growth, suppression of hepatocarcinogenesis, and regulation of glucose metabolism and reactive oxygen species (ROS) generation in different cell types. However, little is known about its role in the inflammatory process. In this study, our results demonstrate that TXNIP plays a critical role in the control of lethal endotoxin-induced shock by controlling NO production in innate immune cells via the regulation of iNOS expression. This regulation is mediated through changes in the activation and translocation of NF-κB that affect the NF-κB/iNOS pathway. In addition, excessive NO reduces the production of IL-1β via S-nitrosylation of the NLRP3 inflammasome. Subsequently, the survival of Txnip−/− mice is significantly decreased due to hypothermia and hypoglycemia. Overall, these results suggest that TXNIP is a novel therapeutic target for the treatment of inflammatory diseases.
PMCID: PMC3789754  PMID: 24098117
4.  IL-15-Induced IL-10 Increases the Cytolytic Activity of Human Natural Killer Cells 
Molecules and Cells  2011;32(3):265-272.
Interleukin 10 (IL-10) is a multifunctional cytokine that regulates diverse functions of immune cells. Natural killer (NK) cells express the IL-10 and IL-10 receptor, but little is known about the function of IL-10 on NK cell activation. In this study, we show the expression and role of IL-10 in human NK cells. Among the cytokines tested, IL-15 was the most potent inducer of IL-10, with a maximal peak expression at 5 h after treatment. Furthermore, IL-10 receptor was shown to be expressed in NK cells. IL-10 alone had a significant effect on NK cytotoxicity which additively increased NK cell cytotoxicity in the presence of IL-15. Neutralizing IL-10 with anti-IL-10 antibody suppressed the inductive effect of IL-10 on NK cell cytotoxicity; however, IL- 10 had no effect on IFN-γ or TNF-α production or NK cell activatory receptor expression. STAT signals are implicated as a key mediator of IL-10/IL-15 cytotoxicity response. Thus, the effect of IL-10 on NK cells is particularly interesting with regard to the STAT3 signal that was enhanced by IL-10 or IL-15.
PMCID: PMC3887627  PMID: 21809216
cytotoxicity; IL-10; IL-15; natural killer cell
5.  A 12-week after-school physical activity programme improves endothelial cell function in overweight and obese children: a randomised controlled study 
BMC Pediatrics  2012;12:111.
Endothelial dysfunction is associated with childhood obesity and is closely linked to the amount and function of endothelial progenitor cells. However, it remains unclear whether endothelial progenitor cells increase with after-school exercise in overweight and obese children. The purpose of this study was to investigate the effects of an after-school exercise programme on endothelial cell function in overweight and obese children.
A total of 29 overweight/obese children (12.2 ± 0.1 years) were randomly divided into control (i.e. no after-school exercise, n = 14) and after-school exercise (n = 15) groups. The 12-week after-school exercise intervention consisted of 3 days of combined aerobic and resistance exercise per week. Each 80-minute exercise programme included 10 minutes of warm-up and 10 minutes of cool-down after school. CD34+ (a cell surface marker on hematopoietic stem cells), CD133+ (a cell surface marker on hematopoietic progenitor cells) and CD34+/CD133+ (considered as endothelial progenitor cells) were measured at baseline and after 12 weeks using flow cytometry.
Increased percentages of CD34+, CD133+ and CD34+/CD133+ cells were observed in the after-school exercise group (p = 0.018; p = 0.001; p = 0.002, respectively) compared with the control group. Carotid intima-media thickness decreased after 12 weeks in the after-school exercise group (p = 0.020) compared with the control group.
This study provides preliminary evidence that a combined after-school exercise programme may represent an effective intervention strategy for improving vascular repair and endothelial cell function, leading to improved cardiovascular health in overweight and obese children.
Trial registration
Current Controlled Trials ISRCTN19037201
PMCID: PMC3447644  PMID: 22849607
After-school exercise programme; Carotid intima-media thickness; Endothelial cell function; Endothelial progenitor cells; Overweight/obese children
6.  RasGRP1 is required for human NK cell function1 
Cross-linking of NK activating receptors activates PLC-γ and subsequently induces DAG and Ca2+ as second messengers of signal transduction. Previous studies reported that Ras guanyl nucleotide-releasing protein (RasGRP) 1, which is activated by DAG and Ca2+, is crucial for T cell receptor-mediated Ras-ERK activation. We now report that RasGRP1, which can also be detected in human NK cells, plays an essential role in NK cell effector functions. To examine the role of RasGRP1 in NK cell functions, the expression of RasGRP1 was suppressed using RNAi. Knockdown of RasGRP1 significantly blocked ITAM-dependent cytokine production as well as NK cytotoxicity. Biochemically, RasGRP1-knockdown NK cells showed markedly decreased ability to activate Ras, ERK and JNK. Activation of the Ras-MAPK pathway was independently shown to be indispensable for NK cell effector functions via the use of specific pharmacological inhibitors. Our results reveal that RasGRP1 is required for the activation of the Ras-MAPK pathway leading to NK cell effector functions. Moreover, our data suggest that RasGRP1 might act as an important bridge between PLC-γ activation and NK cell effector functions via the Ras-MAPK pathway.
PMCID: PMC2896689  PMID: 19933860
7.  Pseudomonas aeruginosa Eliminates Natural Killer Cells via Phagocytosis-Induced Apoptosis 
PLoS Pathogens  2009;5(8):e1000561.
Pseudomonas aeruginosa (PA) is an opportunistic pathogen that causes the relapse of illness in immunocompromised patients, leading to prolonged hospitalization, increased medical expense, and death. In this report, we show that PA invades natural killer (NK) cells and induces phagocytosis-induced cell death (PICD) of lymphocytes. In vivo tumor metastasis was augmented by PA infection, with a significant reduction in NK cell number. Adoptive transfer of NK cells mitigated PA-induced metastasis. Internalization of PA into NK cells was observed by transmission electron microscopy. In addition, PA invaded NK cells via phosphoinositide 3-kinase (PI3K) activation, and the phagocytic event led to caspase 9-dependent apoptosis of NK cells. PA-mediated NK cell apoptosis was dependent on activation of mitogen-activated protein (MAP) kinase and the generation of reactive oxygen species (ROS). These data suggest that the phagocytosis of PA by NK cells is a critical event that affects the relapse of diseases in immunocompromised patients, such as those with cancer, and provides important insights into the interactions between PA and NK cells.
Author Summary
Phagocytic leukocytes, including neutrophils and macrophages, are critical for innate immunity against invading bacteria. Binding and internalization of bacteria by these immune cells stimulates a variety of anti-microbial activities. Although the immune cells are specialized for elimination of bacteria, cellular apoptosis by bacterial phagocytosis has emerged as an important mechanism of pathogenesis. NK cells are non-phagocytic lymphocytes that are responsible for innate immunity via elimination of virus or bacteria-infected cells, as well as transformed cells. We found that PA invades NK cells and that this phagocytic event results in the generation of ROS within the NK cells, leading to apoptosis. The elimination of NK cells, at least in part, may be responsible for the relapse in PA-infected cancer patients. Based on these findings, studies on the interactions between bacterial determinants and host receptors should provide further insight into the mechanisms of bacterial pathogenesis.
PMCID: PMC2726936  PMID: 19714221
8.  Hypoxia-induced IL-18 Increases Hypoxia-inducible Factor-1α Expression through a Rac1-dependent NF-κB Pathway 
Molecular Biology of the Cell  2008;19(2):433-444.
Interleukin-18 (IL-18) plays pivotal roles in linking inflammatory immune responses and tumor progression and metastasis, yet the manner in which this occurs remains to be sufficiently clarified. Here we report that hypoxia induces the transcription and secretion of IL-18, which subsequently induces the expression of hypoxia-inducible factor-1α (HIF-1α). Mechanistically, IL-18 induces HIF-1α through the activity of the GTPase Rac1, which inducibly associates with the IL-18 receptor β (IL-18Rβ) subunit, via a PI3K-AKT-NF-κB–dependent pathway. Importantly, the knockdown of the IL-18Rβ subunit inhibited IL-18–driven tumor cell metastasis. Collectively, these findings demonstrate a feed-forward pathway in HIF-1α–mediated tumor progression, in which the induction of IL-18 by hypoxia or inflammatory cells augments the expression of both HIF-1α and tumor cell metastasis.
PMCID: PMC2230593  PMID: 18003981

Results 1-8 (8)