[Show abstract][Hide abstract] ABSTRACT: 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.
PLoS ONE 10/2014; 9(10):e108913. DOI:10.1371/journal.pone.0108913 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Perforin-1 (Prf1) is the predominant cytolytic protein secreted by natural killer (NK) cells. For a rapid immune response, resting NK cells contain high Prf1 mRNA concentrations while exhibiting minimal cytotoxicity caused by a blockage of Prf1 protein synthesis, implying that an unknown posttranscriptional regulatory mechanism exists.
We sought to determine whether microRNA-150 (miR-150) posttranscriptionally regulates Prf1 translation in both mouse and human NK cells at rest and at various time points after activation.
Mouse NK cells with a targeted deletion of miR-150 (miR-150(-/-) NK cells), primary human NK cells, and NK92 MI cells were used to investigate the role of miR-150 in NK cells. NK cell cytotoxicity assays and Western blotting proved that activated miR-150(-/-) NK cells expressed upregulated Prf1, augmenting NK cell cytotoxicity. When immunodeficient mice were injected with miR-150(-/-) NK cells, there was a significant reduction in tumor growth and metastasis of B16F10 melanoma.
We report that miR-150 binds to 3' untranslated regions of mouse and human Prf1, posttranscriptionally downregulating its expression. Mouse wild-type NK cells displayed downregulated miR-150 expression in response to IL-15, which led to corresponding repression and induction of Prf1 during rest and after IL-15 activation, respectively.
Our results indicate that miR-150 is a common posttranscriptional regulator for Prf1 in mouse and human NK cells that represses NK cell lytic activity. Thus the therapeutic control of miR-150 in NK cells could enhance NK cell-based immunotherapy against cancer, providing a better clinical outcome.
The Journal of allergy and clinical immunology 03/2014; 134(1). DOI:10.1016/j.jaci.2014.02.018 · 11.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The p53 protein plays a central role in cell cycle arrest and apoptosis in response to diverse stress stimuli. Human ecdysoneless (hEcd) is known for its role in stabilizing the p53 protein level and increasing p53-mediated transcription. Here, we report that thioredoxin interacting protein (TXNIP), a member of the tumor suppressor family, interacts with hEcd and decreases MDM2-mediated p53 ubiquitination, leading to p53 stabilization and an increase in p53 activity. The ectopic overexpression of both TXNIP and Ecd increased actinomycin D-mediated cell death in MCF-7 cells, whereas knockdown of TXNIP and Ecd decreased cell death. These results show that TXNIP is a new regulator of the Ecd-MDM2-p53 loop.
Biochemical and Biophysical Research Communications 07/2013; 438(2). DOI:10.1016/j.bbrc.2013.07.036 · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Reactive oxygen species (ROS) are critical determinants of the fate of hematopoietic stem cells (HSCs) and hematopoiesis. Thioredoxin-interacting protein (TXNIP), which is induced by oxidative stress, is a known regulator of intracellular ROS. Txnip(-/-) old mice exhibited elevated ROS levels in hematopoietic cells and showed a reduction in hematopoietic cell population. Loss of TXNIP led to a dramatic reduction of mouse survival under oxidative stress. TXNIP directly regulated p53 protein by interfering with p53- mouse double minute 2 (MDM2) interactions and increasing p53 transcriptional activity. Txnip(-/-) mice showed downregulation of the antioxidant genes induced by p53. Introduction of TXNIP or p53 into Txnip(-/-) bone marrow cells rescued the HSC frequency and greatly increased survival in mice following oxidative stress. Overall, these data indicate that TXNIP is a regulator of p53 and plays a pivotal role in the maintenance of the hematopoietic cells by regulating intracellular ROS during oxidative stress.
[Show abstract][Hide abstract] ABSTRACT: Vitamin-D3 upregulated protein-1 (VDUP1) is a stress response protein. Pseudomonas aeruginosa (P. aeruginosa) infection is a leading cause of death. Mice infected with live P. aeruginosa exhibit significantly decreased VDUP1 expression. However, the function of VDUP1 during P. aeruginosa-induced mouse bacteremic shock is unknown. To address the function of VDUP1 in P. aeruginosa-infected mice, we constructed a bacteremic shock model wherein both wild-type and VDUP1-deficient mice were infected intra-peritoneally with live P. aeruginosa. We found that VDUP1-deficient mice were more resistant to P. aeruginosa-induced bacteremic shock than wild-type mice, as shown by the increased survival, accelerated bacterial clearance and suppression of cytokine overproduction of the VDUP1-deficient mice. VDUP1 promoted the recruitment of neutrophils into the peritoneal cavities of infected mice. VDUP1 impeded the phagocytosis of non-opsonized P. aeruginosa via phosphatidylinositide 3-kinase (PI3K) pathway in macrophages. P. aeruginosa infection induced the generation of reactive oxygen species (ROS), and the increased production of ROS by the peritoneal cells of VDUP1-deficient mice was advantageous in clearing the bacteria. Overall, VDUP1 aggravates bacteremic shock; thus, VDUP1 can be considered a target molecule for the inhibition of P. aeruginosa-induced bacteremic shock.
[Show abstract][Hide abstract] ABSTRACT: Perforin (Prf1) and granzyme B (GzmB) are essential effector molecules for natural killer (NK)-cell cytotoxicity, but how Prf1 and GzmB expression is regulated during arming of NK cells is poorly defined. We show that human microRNA (miR)-27a* is a negative regulator of NK-cell cytotoxicity by silencing Prf1 and GzmB expression. Human miR-27a* specifically bound to the 3' untranslated regions of Prf1 and GzmB, down-regulating expression in both resting and activated NK cells, and it functioned as a fine-tuner for homeostasis of the net amount of the effector proteins. Consistent with miR-27a* having an inhibitory role, knockdown of miR-27a* in NK cells dramatically increased cytotoxicity in vitro and decreased tumor growth in a human tumor xenograft model. Thus, NK-cell cytotoxicity is regulated, in part, by microRNA, and modulating endogenous miR-27a* levels in NK cells represents a potential immunotherapeutic strategy.
[Show abstract][Hide abstract] ABSTRACT: The IL-22 NKp46(+) innate lymphoid cells, NCR22 cells, are very important for the early host defense against microbial pathogens. We show here that NCR22 cells were differentiated from Lin(-)CD127(+)CD117(+) cells that were derived from hematopoietic precursor cells (HPCs) of mouse bone marrow cells. The combination of low concentrations of IL-23 and IL-15 induced differentiation of NCR22 cells from Lin(-)CD127(+)CD117(+) cells. NCR22 cells expressed a large amount of IL-22 and RORγt, and they had poor cytolytic activity and produced little IFN-γ. Lin(-)CD127(+)CD117(+) cells were very similar to intestinal lamina propria LTi-like cells; both cells dominantly expressed RORγt and IL-22. Meanwhile, Lin(-)CD127(-)CD117(+) cells that were also derived from HPCs did not express RORγt and IL-22, and they developed into conventional NK cells, not into NCR22 cells. These findings revealed that NCR22 cells can be differentiated from Lin(-)CD127(+)CD117(+) cells which are derived from HPCs.
[Show abstract][Hide abstract] ABSTRACT: Natural killer (NK) cells are differentiated from hematopoietic stem cells (HSCs) which are located at the lowest end of an oxygen gradient within the bone marrow (BM). In this report, we investigated whether oxygen tension could affect NK cell differentiation from hematopoietic cells in vitro. We found that hypoxia led to an inhibition of differentiation in NK cells, and increased oxygen supply alleviated this inhibition and restored NK cell differentiation under hypoxic condition. Hypoxia-treated cells demonstrated reduced mRNA expression of transcription factors (TFs) that have important roles in NK cell differentiation, such as EOMES, T-bet, GATA-3 and ETS-1. Moreover, hypoxia-pretreated cells recovered mRNA expression of TFs when the oxygen tension was changed to normoxia. Our findings suggest that oxygen tension modulates in vitro differentiation of NK cells through the regulation of TF expression.
[Show abstract][Hide abstract] ABSTRACT: Natural killer (NK) cells act important roles in innate immunity and adaptive immunity. However, the mechanisms governing NK cell development have not been clearly elucidated. Previous studies have shown that an HMG (high-mobility group) protein, TOX, is important for regulating the differentiation program of developing T cells in mice. In this study, we examined the role of TOX in differentiation of human NK cells. Knockdown of TOX in differentiating cells decreased the NK cell population identified by expression of NK surface markers and receptors. In addition, over-expression of TOX enhanced the differentiation of NK cells which give rise to a population showing effector functions of mature NK cells. Moreover, TOX influenced expression of T-bet (T-box expressed in T cells, also as known as Tbx21) during NK cell development. Overall, these results suggest that TOX is required for IL-15-mediated NK cell differentiation and affected expression of T-bet that plays critical roles in NK differentiation and maturation.
[Show abstract][Hide abstract] ABSTRACT: NK cells are capable of killing virus-infected or tumor cells and producing IFN-gamma. Resting NK cells, however, have only minimal cytolytic activity and secrete a low level of IFN-gamma. The cytokine IL-15 can promote the expression of effector functions by resting NK cells. In this study, we demonstrate that suppressor of cytokine signaling 2 (SOCS2) has a novel role in IL-15-primed human NK cell function. SOCS2 expression was upregulated in NK cells following stimulation with IL-15. During IL-15-mediated NK cell priming, SOCS2 interacted with phosphorylated proline-rich tyrosine kinase 2 (Pyk2) at tyrosine 402 (p-Pyk2(Tyr402)) and induced the proteasome-mediated degradation of p-Pyk2(Tyr402) via ubiquitination. Knockdown of SOCS2 resulted in the accumulation of p-Pyk2(Tyr402) and blocked NK cell effector functions. In addition, NK cell cytolytic activity and IFN-gamma production were inhibited by overexpression of the wild-type of Pyk2 but not by the overexpression of tyrosine 402 mutant of Pyk2. These results suggest that SOCS2 regulates human NK cell effector functions via control of phosphorylated Pyk2 depending on IL-15 existence.
The Journal of Immunology 07/2010; 185(2):917-28. DOI:10.4049/jimmunol.1000784 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: NK cells play crucial roles in innate immunity and adaptive immunity. The detailed mechanisms, however, governing NK cell development remains unclear. In this study, we report that YC-1 significantly enhances NK cell populations differentiated from human umbilical cord blood hematopoietic stem cells (HSCs). NK cells increased by YC-1 display both phenotypic and functional features of fully mature NK (mNK) cells, but YC-1 does not affect the activation of mNK cells. YC-1 did not affect cGMP production and phosphorylation of STAT-5 which is essential for IL-15R signaling. On the other hand, YC-1 increased p38 MAPK phosphorylation during NK cell differentiation. Furthermore, p38 inhibitor SB203580 inhibited the differentiation of NK cells enhanced by YC-1. Taken together, these data suggest that YC-1 enhances NK cell differentiation through the activation of p38 MAPK which is involved in NK cell differentiation.
International immunopharmacology 04/2010; 10(4):481-6. DOI:10.1016/j.intimp.2010.01.009 · 2.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cross-linking of NK activating receptors activates phospholipase-gamma and subsequently induces diacylglycerol and Ca(2+) as second messengers of signal transduction. Previous studies reported that Ras guanyl nucleotide-releasing protein (RasGRP) 1, which is activated by diacylglycerol and Ca(2+), is crucial for TCR-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 RNA interference. 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 phospholipase-gamma activation and NK cell effector functions via the Ras-MAPK pathway.
The Journal of Immunology 11/2009; 183(12):7931-8. DOI:10.4049/jimmunol.0902012 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hematopoietic stem cells (HSCs) are maintained in a quiescent state in bone marrow (BM) niches by intrinsic and extrinsic signals. The mechanisms regulating the quiescence and mobilization of HSCs, however, remain unclear. In this study, we report that the expression of thioredoxin-interacting protein (TXNIP) is decreased during HSC activation. In Txnip(-/-) mice, the long-term reconstituting HSC population is decreased and exhausted, and its capacity to repopulate is rapidly lost. These effects are associated with hyperactive Wnt signaling, an active cell cycle, and reduced p21 expression under conditions of stress. TXNIP deficiency reduced the CXCL12- and osteopontin-mediated interaction between HSCs and the bone marrow, and impaired homing and retention in the osteoblastic niche, resulting in mobilized HSCs. Therefore, we propose that TXNIP is essential for maintaining HSC quiescence and the interaction between HSCs and the BM niche.
The Journal of Immunology 09/2009; 183(4):2495-505. DOI:10.4049/jimmunol.0804221 · 4.92 Impact Factor