The mutation of BCOR is highly recurrent and oncogenic in mature T-cell lymphoma.

BACKGROUND: BCOR acts as a corepressor of BCL6, a potent oncogenic protein in cancers of the lymphoid lineage. We have found the recurrent somatic mutation of BCOR occurred in mature T-cell lymphoma (TCL). The role of BCOR mutation in lymphoid malignancies is unknown. METHODS: Lymphoma patient samples were analyzed to identify missense mutations in BCOR using Sanger sequencing. Transfection, RNA interference, immunoprecipitation, western blotting, cell proliferation, cytokine assays and quantitative real-time PCR were employed to determine the functional relevance of the novel K607E mutation in BCOR. The significant transcriptional changes were analyzed by performing DNA microarray profiling in cells expressing BCOR K607E mutant. RESULTS: One hundred thirty-seven lymphoma patient samples were analyzed to identify K607E mutation of the BCOR gene. The BCOR K607E mutation was identified in 15 of 47 NK/T cell lymphoma cases (31.9%), 2 of 18 angioimmunoblastic T-cell lymphoma cases (11.1%), 10 of 30 peripheral T-cell lymphoma, not otherwise specified cases (33.3%), and 13 of 42 diffuse large B-cell lymphoma cases (30.9%). Molecular analysis of BCOR K607E mutation revealed that compared to the wild-type BCOR, the mutant BCOR bound to the BCL6, PCGF1, and RING1B proteins with lesser affinity. Ectopic expression of BCOR K607E mutant significantly enhanced cell proliferation, AKT phosphorylation and the expression of interleukin-2 (IL-2) with up-regulated expression of HOX and S100 protein genes in T cells. BCOR silencing also significantly enhanced cell proliferation, AKT phosphorylation, and IL-2 production. CONCLUSIONS: Functional analyses indicated that K607E mutation of BCOR is oncogenic in nature and can serve as a genetic marker of T-cell lymphoma.

Inflammatory mediators ATP and S100A12 activate the NLRP3 inflammasome to induce MUC5AC production in airway epithelial cells.

Danger-associated molecular patterns (DAMPs) play a proinflammatory role in the pathogenesis of airway obstructive diseases such as severe asthma and chronic obstructive pulmonary disease. The NLRP3 inflammasome is a cytosolic multiprotein platform that activates the caspase-1 pathway in response to inflammatory stimuli such as DAMPs. ATP and S100 proteins are newly identified DAMPs that accumulate in inflamed airways. We previously demonstrated that S100A8, S100A9, and S100A12 induce production and secretion of MUC5AC, a major mucin in the conducting airway mucosa. The purpose of this study was to determine the involvement of NLRP3 inflammasome in, and the contribution of ATP to, S100 protein-induced MUC5AC production by NCI-H292 mucoepidermoid carcinoma cells. Stimulation with either S100A12 or ATP led to MUC5AC production at comparable levels. Simultaneous treatment with both stimuli resulted in additive increases in NLRP3, active caspase-1, IL-1ß, NLRP3/caspase-1 colocalization, and MUC5AC. NLRP3 siRNA or inhibitors of NF-κB, NLRP3 inflammasome oligomerization, or caspase-1 nearly completely inhibited ATP- and S100A12-mediated MUC5AC production. Furthermore, S100A12-as well as ATP-mediated MUC5AC production was almost equally blunted by both nonspecific and specific antagonists of the purinergic receptor P2X7, a principal receptor mediating NLRP3 inflammasome activation by ATP. Thus, these two danger signals contribute to MUC5AC production in airway epithelial cells through overlapping signaling pathways for NLRP3 inflammasome activation.

GPNMB promotes proliferation of developing eosinophils.

Glycoprotein non-metastatic melanoma protein B (GPNMB) is a type I transmembrane protein that is expressed in a wide variety of cell types, including haematopoietic lineages. We previously demonstrated that GPNMB is one of the most highly expressed genes at an early and intermediate stage of eosinophil development. We herein examined GPNMB expression and its possible functional effect using cord blood (CB) CD34+ haematopoietic stem cells differentiating toward eosinophils during a 24-day culture period. Western blot and confocal microscopy analyses showed that GPNMB reached its highest levels at day 12 with most GPNMB-positive cells also expressing major basic protein 1 (MBP1), an eosinophil granule protein. GPNMB declined thereafter, but was still present at an appreciable level at day 24, the time when CB eosinophils most abundantly expressed MBP1 and were thus considered fully differentiated. When the developing CB cells were cultured in the presence of a blocking anti-GPNMB antibody, cell proliferation was significantly reduced. In agreement, ectopic expression of GPNMB in heterologous cells resulted in a significant increase in cell proliferation, while small interfering RNA of GPNMB inhibited the GPNMB-mediated proliferation. Thus, GPNMB is expressed in a temporal manner during eosinophil development and delivers a proliferative signal upon activation.

S100A8, S100A9 and S100A12 activate airway epithelial cells to produce MUC5AC via extracellular signal-regulated kinase and nuclear factor-κB pathways.

Airway mucus hyperproduction is a common feature of chronic airway diseases such as severe asthma, chronic obstructive pulmonary disease and cystic fibrosis, which are closely associated with neutrophilic airway inflammation. S100A8, S100A9 and S100A12 are highly abundant proteins released by neutrophils and have been identified as important biomarkers in many inflammatory diseases. Herein, we report a new role for S100A8, S100A9 and S100A12 for producing MUC5AC, a major mucin protein in the respiratory tract. All three S100 proteins induced MUC5AC mRNA and the protein in normal human bronchial epithelial cells as well as NCI-H292 lung carcinoma cells in a dose-dependent manner. A Toll-like receptor 4 (TLR4) inhibitor almost completely abolished MUC5AC expression by all three S100 proteins, while neutralization of the receptor for advanced glycation end-products (RAGE) inhibited only S100A12-mediated production of MUC5AC. The S100 protein-mediated production of MUC5AC was inhibited by the pharmacological agents that block prominent signalling molecules for MUC5AC expression, such as mitogen-activated protein kinases, nuclear factor-κB (NF-κB) and epidermal growth factor receptor. S100A8, S100A9 and S100A12 equally elicited both phosphorylation of extracellular signal-regulated kinase (ERK) and nuclear translocation of NF-κB/degradation of cytosolic IκB with similar kinetics through TLR4. In contrast, S100A12 preferentially activated the ERK pathway rather than the NF-κB pathway through RAGE. Collectively, these data reveal the capacity of these three S100 proteins to induce MUC5AC production in airway epithelial cells, suggesting that they all serve as key mediators linking neutrophil-dominant airway inflammation to mucin hyperproduction.

Use of surface-enhanced Raman scattering to quantify EGFR markers uninhibited by cetuximab antibodies.

Epidermal growth factor receptor (EGFR) has been recognized as an important prognostic marker expressed in cancer cells because its activation is associated with key features of cancer including tumor growth, survival, angiogenesis, and metastasis. Cetuximab is the first monoclonal antibody drug that targets EGFR overexpressed in cancer cells. It easily binds to EGFR, thereby down-regulating the receptor, blocking EGFR-mediated tyrosine kinase activity, and inhibiting cellular proliferation. Thus, EGFR-cetuximab binding can be quantified to monitor receptor status and the prognosis of cancer therapy. In this work, we report using SERS imaging to assess the inhibitory effect of cetuximab on EGFR expressed on cancer cells. From SERS mapping images using silica-encapsulated gold nanotags, the localized spatial distribution of EGFR that was not inhibited by cetuximab could be determined. Furthermore, EGFR expression could be accurately quantified through the statistical analysis of surface-enhanced Raman scattering (SERS) spectral data. Our experimental data demonstrate the feasibility of SERS imaging to improve the prognostic efficacy of cetuximab treatment.

Epithelial-to-mesenchymal transition of human lung alveolar epithelial cells in a microfluidic gradient device.

Epithelial-to-mesenchymal transition (EMT), a process in which epithelial cells undergo phenotypic transitions to fibrotic cells, is induced by stimulants including transforming growth factor-beta1 (TGF-ß1). In the present study, we developed a microfluidic gradient device to reproduce EMT in A549 human lung alveolar epithelial cells in response to TGF-ß1 gradients. The device was directly mounted on the cells that had grown in cell culture plates and produced a stable concentration gradient of TGF-ß1 with negligible shear stress, thereby providing a favorable environment for the anchorage-dependent cells. A549 cells elongated with the characteristic spindle-shaped morphological changes with upregulation of alpha-smooth muscle actin, a mesenchyme marker, in a gradient-dependent manner, suggestive of EMT progression. We observed that at higher TGF-ß1 concentrations ranging from 5 to 10 ng/mL, the cultures in the microfluidic device allowed to quantitatively pick up subtle differences in the EMT cellular response as compared with plate cultures. These results suggest that the microfluidic gradient device would accurately determine the optimal concentrations of TGF-ß1, given that epithelial cells of different tissue origins greatly vary their responses to TGF-ß1. Therefore, this microfluidic device could be a powerful tool to monitor EMT induced by a variety of environmental stresses including cigarette smoke with high sensitivity.

MUC5AC expression through bidirectional communication of Notch and epidermal growth factor receptor pathways.

Hyperproduction of goblet cells and mucin in the airway epithelium is an important feature of airway inflammatory diseases. We investigated the involvement of Notch signaling in MUC5AC expression in NCI-H292 cells, a human lung carcinoma cell line. Epidermal growth factor (EGF) stimulated generation of the Notch intracellular domain (NICD) in a RBP-Jκ-dependent manner. Treatment with γ-secretase inhibitors L-685,458 or DAPT or introduction of small interfering RNA directed against Notch1 reduced EGF-induced MUC5AC expression. The inhibitory effect of L-685,458 on EGF-induced MUC5AC mRNA and protein expression was also observed in primary human bronchial epithelial cells. Blockage of Notch signaling with L-685,458 or Notch siRNA resulted in a decrease in EGF-induced phosphorylation of ERK. These results suggested that ERK activation is necessary for the regulation of EGF receptor (EGFR)-mediated MUC5AC expression by Notch signaling. Conversely, forced expression of NICD induced both EGFR and ERK phosphorylation with MUC5AC expression even in the absence of EGF. Treatment of the NICD-expressing cells with EGF further augmented ERK phosphorylation in an additive manner. The ERK phosphorylation induced by exogenous NICD was inhibited by treatment with an Ab that antagonizes EGFR activity as well as by inhibitors of EGFR and ERK, implying that Notch signaling induces MUC5AC expression by activating the EGFR pathway. Collectively, these results suggest that MUC5AC expression is regulated by a bidirectional circuit between Notch and EGFR signaling pathways.

Mucin (MUC5AC) expression by lung epithelial cells cultured in a microfluidic gradient device.

We have developed a microfluidic gradient device for controlling mucin gene expression of NCI-H292 epithelial cells derived from lung tissues. We hypothesized that gradient profiles would control mucin gene expression of lung epithelial cells. However, it was not possible to generate various stable gradient profiles using conventional culture methods. To address this limitation, we used a microfluidic gradient device to create various gradient profiles (i.e. non-linear, linear, and flat) in a temporal and spatial manner. NCI-H292 lung epithelial cells were exposed to concentration gradients of epidermal growth factor in a microfluidic gradient device with continuous medium perfusion. We demonstrated an effect of gradient profiles on mucin expression of lung epithelial cells cultured in the microfluidic gradient device. It was revealed that NCI-H292 lung epithelial cells exposed to the flat gradient profile of the epidermal growth factor exhibited high expression of mucin as compared with cells exposed to non-linear and linear gradient profiles. Therefore, this microfluidic gradient device could be a potentially useful tool for regulating the mucin expression of lung epithelial cells exposed to chemokine gradient profiles.

The crucial role of GATA-1 in CCR3 gene transcription: modulated balance by multiple GATA elements in the CCR3 regulatory region.

GATA-1, a zinc finger-containing transcription factor, regulates not only the differentiation of eosinophils but also the expression of many eosinophil-specific genes. In the current study, we dissected CCR3 gene expression at the molecular level using several cell types that express varying levels of GATA-1 and CCR3. Chromatin immunoprecipitation analysis revealed that GATA-1 preferentially bound to sequences in both exon 1 and its proximal intron 1. A reporter plasmid assay showed that constructs harboring exon 1 and/or intron 1 sequences retained transactivation activity, which was essentially proportional to cellular levels of endogenous GATA-1. Introduction of a dominant-negative GATA-1 or small interfering RNA of GATA-1 resulted in a decrease in transcription activity of the CCR3 reporter. Both point mutation and EMSA analyses demonstrated that although GATA-1 bound to virtually all seven putative GATA elements present in exon 1-intron 1, the first GATA site in exon 1 exhibited the highest binding affinity for GATA-1 and was solely responsible for GATA-1-mediated transactivation. The fourth and fifth GATA sites in exon 1, which were postulated previously to be a canonical double-GATA site for GATA-1-mediated transcription of eosinophil-specific genes, appeared to play an inhibitory role in transactivation, albeit with a high affinity for GATA-1. Furthermore, mutation of the seventh GATA site (present in intron 1) increased transcription, suggesting an inhibitory role. These data suggest that GATA-1 controls CCR3 transcription by interacting dynamically with the multiple GATA sites in the regulatory region of the CCR3 gene.

Effect of single nucleotide polymorphisms within the interleukin-4 promoter on aspirin intolerance in asthmatics and interleukin-4 promoter activity.

OBJECTIVE: Aspirin affects interleukin-4 (IL-4) synthesis; however, the genetic role of IL-4 has not been evaluated in asthmatics with aspirin hypersensitivity. The objective of the study was to examine the influence of single nucleotide polymorphisms (SNPs) in IL-4 gene on aspirin hypersensitivity in asthmatics at the genetic and molecular levels. METHODS: Aspirin-intolerant (AIA, n=103) and aspirin-tolerant asthmatics (n=270) were genotyped and functional promoter assays were performed. RESULTS: Of 15 SNPs tested, seven (-589T>C (rs2243250) in promoter, -33T>C (rs2070874) in the 5'-untranslated region, +4047A>G (rs2243266), +4144C>G (rs2243267), +4221C>A (rs2243268), +4367G>A (rs2243270), and +5090A>G (rs2243274) in introns) were significantly associated with AIA risk. The frequency of the rare allele (C) of -589T>C was higher in the AIA group than in the aspirin-tolerant asthmatic group (P=0.016), and a gene dose-dependent decline in forced expiratory volume in 1 s was noted after an aspirin challenge (P=0.0009). Aspirin unregulated IL-4 mRNA production in Jurkat T and K562 leukemia cells. A reporter plasmid assay revealed that aspirin augmented IL-4 promoter transactivation with the -589T>C C and -33T>C C alleles, compared with that bearing the -589T>C T and -33T>C T alleles. Further, electrophoretic mobility shift assay showed the formation of nuclear complexes with -33T>C and -589T>C allele-containing probes; this was augmented by aspirin. The complexes formed with the -33T>C and -589T>C probes were shifted by treatment with anti-CCAAT-enhancer-binding proteins ß and anti-nuclear factor of activated T-cells antibodies, respectively, indicating the inclusion of these transcription factors. CONCLUSION: Aspirin may regulate IL4 expression in an allele-specific manner by altering the availability of transcription factors to the key regulatory elements in the IL4 promoter, leading to aspirin hypersensitivity.

Gamma-secretase inhibitor reduces allergic pulmonary inflammation by modulating Th1 and Th2 responses.

RATIONALE: Gamma-secretase inhibitor (GSI) has been used to effectively block Notch signaling, which is implicated in the differentiation and functional regulation of T helper (Th) effector cells. In asthma, a subset of CD4(+) T cells is believed to initiate and perpetuate the disease. OBJECTIVES: The aim of this study was to evaluate the therapeutic potential of GSI against allergic asthma. METHODS: GSI was administered to an ovalbumin-sensitized mouse via an intranasal route at the time of ovalbumin challenge. MEASUREMENTS AND MAIN RESULTS: The administration of GSI inhibits asthma phenotypes, including eosinophilic airway inflammation, goblet cell metaplasia, methacholine-induced airway hyperresponsiveness, and serum IgE production. GSI treatment of bronchoalveolar lavage cells stimulated via TCR or non-TCR pathways led to a decrease in Th2 cytokine production with a concomitant increase in Th1 cytokine secretion. Expression of Hes-1, a target of Notch signaling, was down-regulated in conjunction with a reduction of Notch intracellular domain and GATA-3 levels after GSI treatment of bronchoalveolar lavage cells. GSI treatment resulted in an inhibition of NF-kappaB activation, and combined treatment with GSI and an NF-kappaB inhibitor augmented IFN-gamma production in a synergistic manner. CONCLUSIONS: These data suggest that GSI directly regulates Th1 and Th2 responses in allergic pulmonary inflammation through a Notch signaling-dependent pathway and that GSI is of high therapeutic value for treating asthma by inhibiting airway inflammatory responses.

Regulation of functional phenotypes of cord blood derived eosinophils by gamma-secretase inhibitor.

Eosinophils develop from stem cells in the bone marrow under the influence of hematopoietic cytokines, particularly IL-5. Previously, we have demonstrated that blockage of Notch signaling by a gamma-secretase inhibitor (GSI) promotes the differentiation of umbilical cord blood (UCB)-derived eosinophils. These highly major basic protein (MBP)-positive eosinophils cultured in the presence of the inhibitor lack the migratory response to eotaxin, although their CCR3 levels are similar to those of eosinophils cultured without the inhibitor. We investigated the mechanism underlying the differential responses of differentiating eosinophils and their functionalities in response to eosinophil-active cytokines in the presence and absence of GSI. UCB cells cultured for 4 weeks with hematopoietic cytokines in the presence or absence of GSI were monitored for extracellular signal-regulated kinase (ERK) phosphorylation, MBP expression, and functionality. Eosinophil differentiation from UCB cells was accompanied by activation of the ERK1/2 pathway during the 4-week culture period. In particular, strong ERK1/2 phosphorylation was observed in eosinophils during the final stage of culture when GSI was present. Consistent with this finding, ERK inhibition nullified the effect of GSI on eosinophil differentiation. Eosinophils cultured with GSI resembled airway eosinophils rather than peripheral blood eosinophils based on reduced IL-5Ralpha expression, blunted eosinophil cationic protein (ECP) degranulation, and decreased IL-13 and granulocyte macrophage-colony-stimulating factor production. These results suggest that Notch signaling regulates the terminal differentiation and subsequent effector phenotypes of eosinophils, partly through modulation of the ERK pathway. GSI has therapeutic potential for eosinophilic inflammatory diseases, such as asthma.