STAT3 Research Articles

Association of the polymorphisms in Th2 chemotaxis-related genes with the development and prognosis of autoimmune thyroid diseases.

The prognosis of autoimmune thyroid disease (AITD) is difficult to predict. Th2 cells suppress the differentiation of Th1 and Th17 cells, which are associated with the prognosis of AITD. However, there are few reports as to whether Th2 chemotaxis-related genes, such as CRTH2 (chemoattractant receptor-homologous molecule expressed on Th2 cells), IL-25, TARC/CCL17 (Thymus and activation regulated chemokine/chemokine ligand 17) or STAT6 (Signal transducer and activator of transcription 6), affect the pathology of and/or susceptibility to AITD. Therefore, in this study, we genotyped functional SNPs in these genes to clarify the association of the genetic differences of genes related to Th2 differentiation and chemotaxis with the development and the prognosis of AITDs. The frequencies of the AA genotype of the CRTH2 rs545659 SNP and the CC genotype and the C allele of the CRTH2 rs634681 SNP were higher in patients with severe HD than in patients with mild HD. The frequency of the CC genotype in the TARC rs223828 SNP was higher in patients with intractable GD than in patients with GD in remission. In conclusion, the CRTH2 rs545659 and rs634681 SNPs were associated with the severity of HD, and the TARC/CCL17 rs223828 SNP was associated with the intractability of GD.

Correlation of IL-6R/STAT3 signaling with invasiveness of pituitary null cell adenomas: A study based on proteomics

Objective To identify the relationship of IL-6R/STAT3 signaling to invasiveness of pituitary null cell adenomas by the study of proteomics. Methods In this study, specimens were collected from 45 cases of pituitary null cell adenomas which were surgical treated at Department of Neurosurgery, Beijing Tiantan Hospital, Capital Medical University. Normal pituitary tissues were collected from 3 body donors who died from traffic accidents. Different proteins between invasive and non-invasive null cell adenomas were determined by nano liquid chromatography tandem mass spectrometry (nano LC-MS/MS) and quantitative proteomics analysis. The signaling pathway related to invasive of null cell adenomas was identified by bioinformatic analysis. The gene expression levels of the signaling pathway was confirmed by qPCR (quantitative polymerase chain reaction) and immunohistochemistry (IHC) in invasive pituitary tumors and non-invasive null cell adenomas. Results A total of 283 differentially expressed proteins (P<0.05) were identified between invasive and non-invasive pituitary null cell adenomas. Among those, 182 and 101 were up-regulated and down-regulated in invasive pituitary null cell adenomas, respectively. The bioinformatic analysis demonstrated that 8 canonical pathways were differentially expressed between normal pituitary glands and pituitary null cell adenomas. IL-6R/STAT3 signaling was related to invasiveness of pituitary null cell adenomas. The IL-6R, JAK2 and STAT3 mRNA expression levels in invasive null cell adenomas (2.2, 2.8 and 4.3, respectively) were significantly higher than those in non-invasive null cell adenomas (1.7, 1.7, 2.9, respectively) based on the results of qPCR (P<0.05). The values of staining intensity of IL-6R, JAK2, STAT3, p-STAT3 and MMP9 (9.2, 8.6, 9.7, 10.5 and 9.8, respectively) were significantly increased in invasive null cell adenomas compared with those in non-invasive null cell adenomas (5.8, 5.3, 5.7, 5.2 and 4.1, respectively) according to the IHC data. Conclusion The IL-6R/STAT3 signaling may play an important role in invasive null cell adenomas by enhancement of MMP9 expression. Key words: Pituitary adenoma; Neoplasm invasiveness; Proteomics; Signal pathways; IL-6R/STAT3

Interleukin?22 promotes proliferation of fibroblast?like synoviocytes from patients with rheumatoid arthritis by inducing STAT3 phosphorylation

To clarify the mechanism by which interleukin?22 (IL?22) promotes the proliferation of fibroblast?like synoviocytes (FLS) from patients with rheumatoid arthritis (RA). FLS were isolated from the synovial tissues of patients with RA and identified by immunohistochemistry for vimentin/CD68. The cells were subcultured and incubated with different concentrations of IL?22 for 24, 48, or 72 h, and their proliferation was examined using MTT assay. After treatment of the cells with IL?22 and AG490, alone or in combination, the expressions of the total and phosphorylated proteins of STAT3, ERK1/2 and P38 were detected with Western blotting. IL?22 significantly increased the proliferation of FLS in a dose?dependent manner (P<0.05). The total protein of STAT3 in the cells showed no significant changes with extended time of IL?22 treatment (P=0.68), but the expression of phosphorylated STAT3 protein increased significantly (P<0.001). The total and phosphorylated proteins of ERK1/2 and P38 underwent no significant changes after IL?22 treatment (P>0.05). A combined treatment with 50 ng/mL IL?22 and 100 µmol/L AG490 resulted in a significant decrease in the proliferation of FLS as compared with IL?22 treatment alone (P<0.01). IL?22 can dose?dependently promote the proliferation of FLS from patients with RA by inducing phosphorylation of STAT3 protein but not through ERK1/2 or P38 signal pathway.

Prevention of Asthma Exacerbation in a Mouse Model by Simultaneous Inhibition of NF-κB and STAT6 Activation Using a Chimeric Decoy Strategy.

Transactivation of inflammatory and immune mediators in asthma is tightly regulated by nuclear factor κB (NF-κB) and signal transducer and activator of transcription 6 (STAT6). Therefore, we investigated the efficacy of simultaneous inhibition of NF-κB and STAT6 using a chimeric decoy strategy to prevent asthma exacerbation. The effects of decoy oligodeoxynucleotides were evaluated using an ovalbumin-induced mouse asthma model. Ovalbumin-sensitized mice received intratracheal administration of decoy oligodeoxynucleotides 3 days before ovalbumin challenge. Fluorescent-dye-labeled decoy oligodeoxynucleotides could be detected in lymphocytes and macrophages in the lung, and activation of NF-κB and STAT6 was inhibited by chimeric decoy oligodeoxynucleotide transfer. Consequently, treatment with chimeric or NF-κB decoy oligodeoxynucleotides protected against methacholine-induced airway hyperresponsiveness, whereas the effect of chimeric decoy oligodeoxynucleotides was significantly greater than that of NF-κB decoy oligodeoxynucleotides. Treatment with chimeric decoy oligodeoxynucleotides suppressed airway inflammation through inhibition of overexpression of interleukin-4 (IL-4), IL-5, and IL-13 and inflammatory infiltrates. Histamine levels in the lung were reduced via suppression of mast cell accumulation. A significant reduction in mucin secretion was observed due to suppression of MUC5AC gene expression. Interestingly, the inhibitory effects on IL-5, IL-13, and histamine secretion were achieved by transfer of chimeric decoy oligodeoxynucleotides only. This novel therapeutic approach could be useful to treat patients with various types of asthma.

IL-13 regulates IL-17C expression by suppressing NF-κB-mediated transcriptional activation in airway epithelial cells

The cytokine interleukin (IL)-17C is highly expressed in epithelial tissues and involved in innate immune responses; however, the regulation of IL-17C expression in the airways remains poorly understood. Here, we show that IL-1β strongly induces both IL-17C mRNA and protein expression in primary normal human bronchial epithelial cells. Conversely, IL-13 significantly reduced the IL-1β-induced IL-17C expression. Attenuation of the nuclear factor (NF)-κB-signaling pathway using an NF-κB-subunit p65-specific small-interfering RNA (siRNA), reduced IL-1β-induced IL-17C expression, demonstrating the importance of NF-κB signaling in IL-17C regulation. The inhibitory effects of IL-13 on IL-17C expression were abolished when the Janus kinase (JAK)/signal transducer and activator of transcription 6 (STAT6)-signaling pathway was impaired, using either the JAK inhibitor ruxolitinib or a STAT6-specific siRNA. Western blot analysis demonstrated that IL-1β promoted both IκB-α phosphorylation and degradation, and p65 nuclear translocation. Although IL-13 induced STAT6 phosphorylation and nuclear translocation, it did not affect the activation of the IL-1β-mediated NF-κB-pathway. Using chromatin immunoprecipitation, we confirmed that IL-1β enhanced p65 binding to regions within the IL-17C promoter that flank putative NF-κB-binding sites (−130/−120 and −157/−147). Interestingly, IL-13 treatment reduced the IL-1β-mediated p65 binding to these regions. These findings demonstrate that NF-κB-mediated transcriptional mechanisms are critically involved in the IL-1β-mediated IL-17C induction, and that IL-13 negatively regulates this induction by suppressing NF-κB-based transcriptional activation.

High STAT4 Expression Indicates Better Disease-free Survival in Patients with Gastric Cancer.

The aim of this study was to investigate the significance of signal transducer and activator of transcription 4 (STAT4) expression and the correlation between STAT4 and interferon gamma (IFN-γ) in patients with gastric cancer. Sixty-two patients who underwent gastrectomy for gastric cancer were enrolled in the study. STAT4 and IFNG mRNA expression was evaluated by quantitative real-time polymerase chain reaction (PCR). Immunohistochemistry was performed to examine CD8+ T-cells, and STAT4 and IFN-γ expression. STAT4 mRNA expression was significantly correlated with IFNG mRNA expression (p<0.05). Regarding disease-free survival, there was a significant difference between the groups with high and low STAT4 expression (5-year disease-free survival: 77.8% and 56.4%, p<0.05). Univariate analysis revealed that tumor differentiation and STAT4 expression were significant factors for tumor recurrence. High expression of STAT4 in gastric cancer predicted a better clinical outcome. STAT4 might be a useful biomarker to identify patients at high risk of recurrence after gastrectomy.

Insight into the molecular recognition mechanism of the coactivator NCoA1 by STAT6

Crucial for immune and anti-inflammatory cellular responses, signal transducer and activator of transcription 6 (STAT6) regulates transcriptional activation in response to interleukin-4 and -13 -induced tyrosine phosphorylation by direct interaction with coactivators. The interaction of STAT6 with nuclear coactivator 1 (NCoA1) is mediated by a short region of the STAT6 transactivation domain that includes the motif LXXLL and interacts with the PAS-B domain of NCoA1. Despite the availability of an X-ray structure of the PAS-B domain/ Leu794-Gly814-STAT6 complex, the mechanistic details of this interaction are still poorly understood. Here, we determine the structure of the NCoA1257–385/STAT6783–814 complex using Nuclear Magnetic Resonance (NMR) and X-ray crystallography. The STAT6783–814 peptide binds with additional N-terminal amino acids to NCoA1257–385, compared to the STAT6794–814 peptide, explaining its higher affinity. Secondary and tertiary structures existing in the free peptide are more highly populated in the complex, suggesting binding by conformational selection.

1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) reduces hepatic injury in concanavalin A-treated mice.

1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG), a chemically synthesized monoacetyldiaglyceride, is one of the constituents in Sika deer antlers and has been known traditionally as having immunomodulatory effects. However, the mechanism by which PLAG controls neutrophil migration, which evokes liver injury in the hepatitis animal model, remains largely unknown. This study was designed to evaluate the immunomodulatory effects of PLAG on cytokine secretion and neutrophil migration in vivo and in vitro. Concanavalin A (Con A) induced leukocyte infiltration in the liver and increased plasma cytokine levels. Pretreatment with PLAG reduced the levels of interleukin (IL)-4, IL-6, IL-10, and CXCL2, but maintained interferon (IFN)-γ levels and modulated neutrophil recruitment toward the liver. Furthermore, the mRNA and protein levels of IL-4 and CXCL2 in liver tissue were also decreased in the Con A-treated mice. Liver histology analyses showed that PLAG reduced Con A-induced hepatic necrosis, which was accompanied by leukocyte infiltration. The in vitro studies revealed that PLAG reduced IL-4 secretion in Con A stimulated T cell and blocked signal transducer and activator of transcription 6 (STAT6) Con A induced hepatocyte. PLAG attenuated IL-4 induced activation of atypical protein kinase C (PKC)/STAT6 in hepatocytes and inhibited neutrophil migration toward the liver tissue through suppression of IL-8/vascular cell adhesion molecule (VCAM) expression. These results suggest that PLAG could mitigate excess neutrophil migration into liver tissue and potentially have a therapeutic effect on immune-mediated liver injury.

Epidermal Growth Factor Receptor Family Inhibition Identifies P38 Mitogen-activated Protein Kinase as a Potential Therapeutic Target in Bladder Cancer

To investigate perturbations in downstream signaling pathway activation and potential resistance mechanisms to epidermal growth factor receptor (EGFR) or human epidermal growth factor receptor 2 (HER2) inhibition in cell line models of bladder cancer. We undertook a structured screening approach by phosphokinase array, followed by validation steps, to detect activated downstream signaling pathway nodes after therapeutic inhibition of EGFR or HER2 in bladder cancer cell lines. Erlotinib treatment of RT112 cells induced phosphorylation of 9 activated phosphoprotein targets (p38 mitogen-activated protein kinase [MAPK] [Thr180/Tyr182], GSK-3α/β [Ser21/9], MEK1/2 [Ser218/222, Ser222/226], Akt (protein kinase B) [Ser473], TOR [target of rapamycin] [Ser2448], Src [Tyr419], p27 [Thr198], p27 [Thr157], and PLCγ-1 [Tyr783]), whereas STAT4 (signal transducer and activator of transcription 4) (Tyr693) phosphorylation was reduced. Of these, p38 MAPK phosphorylation was confirmed to occur in response to inhibition of either EGFR or HER2 signaling through multiple validation steps, including differing bladder cancer cell lines (RT112, UM-UC-3, and T24) and methods of receptor pathway inhibition (erlotinib, lapatinib, and siRNA depletion of EGFR or HER2). Chemical inhibition of p38 MAPK with SB203580 led to inhibition of proliferation in RT112, UM-UC-3, and T24 cell lines (IC50 20.85, 76.78, and 79.12 µM, respectively). Fractional effect analyses indicated a synergistic interaction for inhibition of cell proliferation when combining SB203580 with lapatinib. p38 MAPK is a potential therapeutic target in bladder cancer and this strategy warrants further development in this disease. It may also allow combination therapy strategies to be developed in conjunction with EGFR or HER2 inhibition.

The mechanism of mucin 1 gene by regulated janus protein tyrosine kinase/signal transducer and activators of transcription 3 signaling pathway to inhibit proliferation and promote apoptosis of thyroid cancer cells

Objective To investigate the mechanism of membrane type mucin 1 (MUC1) gene by regulated janus protein tyrosine kinase/signal transducer and activators of transcription 3 (JAK/STAT3) signaling pathway to inhibit proliferation and promote apoptosis of thyroid cancer cells. Methods The expression of MUC1 in thyroid carcinoma and adjacent tissues was detected by Western blotting, MUC1 small interfering RNA (MUC1-siRNA) and negative control (siRNA-NC) were transfected into human thyroid cancer cells SW579, empty cells as the control group; cells were transfected for 48 h, Western blotting was used to detect transfection effect; cell proliferation was detected by cell counting kit-8 (CCK-8); cell apoptosis was detected by flow cytometry; the expression of B cell lymphoma/leukemia-2 associated X protein (bax), B cell lymphoma/leukemia-2 (bcl-2), STAT3, phosphorylated STAT3 (p-STAT3) protein was detected by Western blotting; 15 μmol/L AG490 specific inhibitor of JAK/STAT3 signaling pathway to join cells as the inhibitor group, without inhibitors for the untreated group, cell proliferation and apoptosis in two group was detected by CCK-8 and flow cytometry separately; the expression of bax, bcl-2, STAT3, p-STAT3 protein was detected by Western blotting. Results The expression of MUC1 in thyroid carcinoma tissues (0.434±0.034)was significantly higher than that in paracancerous tissues (0.153±0.018, t=12.651, P=0.000); the cell survival rate [(55.38±7.12)%] and bcl-2 (0.237±0.031), p-STAT3 (0.045±0.013) protein expression in MUC1-siRNA group was significantly lower than the control group [(94.32±5.21)%, 0.425±0.034, 0.122±0.025], cell apoptosis rate [(16.29±1.23)%] and bax (0.248±0.026) protein expression was significantly higher than the control group [(3.06±0.78)%, 0.109±0.019; tSurvival rate=7.645, PSurvival rate=0.002; tbcl-2=7.077, Pbcl-2=0.002; tp-STAT3=4.733, Pp-STAT3=0.009; tApoptosis rate=15.733, PApoptosis rate=0.000; tbax=7.476, Pbax=0.002]. The proliferation and apoptosis of the cells after inhibitor adding were agreed with the results of MUC1-siRNA. Conclusion To silence the expression of MUC1 can significantly inhibit the proliferation of human thyroid carcinoma SW579 cells, and induce cell apoptosis by regulating the ratio of bcl-2/bax, and its mechanism was related to regulation of JAK/STAT3 signaling pathway. Key words: Mucin 1 gene; Thyroid carcinoma; Proliferation; Apoptosis

Expressions and significance of Notch signal pathway and target gene in children with juvenile idiopathic arthritis

Objective To analyze the relationship between Notch signaling pathway and levels of lymphocytes and cytokines in children with juvenile idiopathic arthritis(JIA), and to explore its role in the pathogenesis of JIA. Methods Thirty-five pediatric patients with JIA [males 20 cases, females 15 cases; aged (6.5±4.0) years old, (0.83-15.00 years old)]and 15 healthy children [males 6 cases, females 9 cases; aged (5.0±2.9) years old, (1.0-11.0 years old)]from November 2015 to February 2016 in Guangzhou Women and Children′s Medical Center were included in the study. The JIA group were divided into the systemonset JIA(So-JIA) group (22 cases) and psoriatic JIA(p-JIA) group (13 cases, polyarthritis 7 cases and oligoarthritis 6 cases). The expressions of Notch signaling′s receptor, ligand and target gene mRNA in peripheral blood monouclear cells (PBMC) from the JIA group and the control group were determined by quantitative real-time PCR.The levels of cytokines interleukin (IL) -1β, IL-10, IL-6, IL-17, IL-4 and transforming growth factor-β (TGF-β) were detected by enzyme-linked immunosorbent assay. Results Compared with the healthy control group, the Notch2 receptors (1.6±3.2 vs. 0.4±0.3) expression level, Jagged1 ligand (44.0±79.0 vs. 11.3±1.2) expression levels and the levels of target gene HES1(0.4±0.3 vs. 0.1±0.1) mRNA in the JIA group showed a significant increase, and the differences were all statistically significant (all P<0.05 ). Compared with the healthy control group, the JIA group showed an increased level of IL-1β [(182.22±309.13) ng/L vs. (54.71±20.33) ng/L], IL-10 [(32.99±34.28) ng/L vs. (22.68±4.56) ng/L], IL-6 [(100.48±305.57) ng/L vs. (13.98±2.78) ng/L], IL-17 [(9.11±17.57) ng/L vs. (2.42±0.29) ng/L] and TGF-β [(14.37±9.33) ng/L vs. (5.49±4.49) ng/L], and there were statistically significant differences (all P<0.05) .The expression level of HES1 mRNA was positively correlated with STAT3 mRNA in the So-JIA group (r=0.573, P<0.05). The expression level of HES1 mRNA was positively correlated with CD3+ T(r=0.528), CD19+B(r=0.480), CD3+ CD4+ TH(r=0.457) and CD16+CD56+NK(r=0.598) cell absolute count in the So-JIA group (all P<0.05). The expression level of HES1 mRNA was positively correlated with CD3+ T cell absolute count in the p-JIA group(r=0.577, P<0.05). Conclusion Imbalance between Notch pathway and lymphocytes and cytokines in children with JIA may play an important role in the pathogenesis of JIA. Key words: Juvenile idiopathic arthritis; Notch pathway; Lymphocytes; Cytokines

Targeted Inhibition of the NCOA1/STAT6 Protein–Protein Interaction

The complex formation between transcription factors (TFs) and coactivator proteins is required for transcriptional activity, and thus disruption of aberrantly activated TF/coactivator interactions could be an attractive therapeutic strategy. However, modulation of such protein-protein interactions (PPIs) has proven challenging. Here we report a cell-permeable, proteolytically stable, stapled helical peptide directly targeting nuclear receptor coactivator 1 (NCOA1), a coactivator required for the transcriptional activity of signal transducer and activator of transcription 6 (STAT6). We demonstrate that this stapled peptide disrupts the NCOA1/STAT6 complex, thereby repressing STAT6-mediated transcription. Furthermore, we solved the first crystal structure of a stapled peptide in complex with NCOA1. The stapled peptide therefore represents an invaluable chemical probe for understanding the precise role of the NCOA1/STAT6 interaction and an excellent starting point for the development of a novel class of therapeutic agents.

Therapeutic effect and mechanism of mesenchymal stem cells on acute liver failure

Objective To explore the therapeutic effects of soluble cytokines secreted by mesenchymal stem cells (MSCs) on acute liver failure (ALF). Methods MSCs isolated from Sprague-Dawley rats were determined by FACS analysis. Conditioned medium derived from MSCs (MSCs-CM) was collected and analyzed by a cytokine microarray. SD rats were divided into 3 groups: (1) ALF + dulbecco's modified eagle medium (DMEM) group: 1 ml DMEM was injected into SD rats after D-Gal administration; (2) ALF + MSCs group: 1 ml MSCs (1×106) was injected into SD rats after D-Gal administration; (3) ALF + MSCs-CM group: 1 ml MSCs-CM was injected into SD rats after D-Gal administration. Biochemical indicators, survival rate, histology and inflammatory factors were studied. Exogenous recombinant rat IL-10, anti-rat IL-10 antibody and AG490 (STAT3 signaling pathway inhibitor) were administrated to explore the therapeutic mechanism of MSCs-CM. Results The respective serum biochemical indexes of ALF + DMEM group, ALF + MSCs group, and ALF + MSCs-CM group were: ALT (1 709.8 ± 372.1, 865.5 ± 52.8, 964.7 ± 414.6 U/L), AST (4 234.0 ± 807.3, 2 440.8 ± 511.9, 2 739.8 ± 587.3 U/L), and TBil (79.3 ± 10.9, 43.8 ± 7.0, 61.2 ± 6.7 μg/L). The survival rates of the three groups were 10.0%, 80.0%, and 70.0%, respectively. The levels of inflammatory factors in each group were IFN-γ (69.8 ± 4.7, 46.4 ± 4.3, 54.6 ± 2.4 pg/ml), IL-1β (58.5 ± 7.6, 40.5 ± 6.9, 44.1 ± 6.0 pg/ml), IL-6 (71.9 ± 16.1, 38.4 ± 7.7, 45.3 ± 9.0), and IL-10 (38.3 ± 6.0, 75.4 ± 11.1, 59.6 ± 11.9 pg/ml). Protein microarray results suggested that MSCs-CM expresses a variety of inflammatory-related cytokines, with IL-10 levels being most pronounced. IL-10 (ALT 1 126.9 ± 419.3 U/L, AST 2 370.8 ± 561.2 U/L) alone significantly reduced transaminase levels compared with ALF group (ALT 1 709.8 ± 372.1 U/L, AST 4 234.0 ± 807.3 U/L), while anti-IL-10 antibody (ALT 1 568.5 ± 325.4 U/L, AST 4 043.7 ± 819.0 U/L) neutralized the therapeutic effect of MSCs-CM (ALT 964.7 ± 414.6 U/L, AST 2 739.8 ± 587.3 U/L). IL-10 could significantly increase the level of pSTAT3 in ALF rats (0.93 ± 0.03 vs 0.68 ± 0.01), while STAT3 inhibitor AG490 (0.84 ± 0.04) could decrease the expression of pSTAT3 and reverse the therapeutic effect of IL-10. Conclusion The factors released by MSCs, especially IL-10, have the potential therapeutic effect on ALF, and STAT3 signaling pathway may mediate the anti-inflammation effects of IL-10. Key words: Mesenchymal stem cell; Acute liver failure; Conditioned medium; Immunoregulation; STAT3 signaling pathway

Updates on airway epithelial cells in asthma

Airway epithelial cells, which are the first line of defense against inhaled pathogens and particles, play an important role in initiating airway inflammation, mucous metaplasia and keeping type 2 inflammation in asthma.Mucous metaplasia of airway epithelial cells is one of the important part of airway reconstitution.The mucous overproduction in small airway leads to mucous plug, which is closely related to the high mortality of asthma.Multiple signaling and transcriptional networks influence goblet cell differentiation.These include JAK and the transcription factor STAT6, Notch, EGFR, et al.In this article, the changes of epithelial cells in asthma and its role at the beginning of asthma are described.Furthermore, the progress in airway mucous metaplasia and several related cell signaling pathways were focused on, which may provide some potential target to inhibit the secretion of mucous in asthma. Key words: Airway epithelial cells; Asthma; Mucous metaplasia

Regulatory effect of decitabine on human acute myeloid leukemia cell line HL-60 and its mechanism

Objective To investigate the effect of decitabine (DAC) on human acute myeloid leukemia (AML) cell line HL-60 and the regulating of natural killer (NK) cell activating receptor (NKG2D) ligands (NKG2DL), and to detect the molecular mechanism of JAK-STAT3-SOCS signaling pathway. Methods The effect of DAC on the proliferation of HL-60 was detected by using CCK-8 assay. The cell apoptosis was analyzed by using Annexin-V/PI double standard method. The expressions of receptor NKG2DL including MICA/B and ULBPs in HL-60 cells were detected by using flow cytometry (FCM). The killing activity of NK cells was analyzed by using carboxy fluorescein diacetate succinimidyl ester (CFSE). The expressions of JAK/STAT3 signaling pathway or molecules including STAT3, its upstream kinases JAK1, JAK2 and the negative regulator of STAT3, SOCS-1, SOCS-3 were examined by Western blot. Methylation level of the SOCS-1, SOCS-3 gene after the treatment of DAC was analyzed by using methylation-sensitive high resolution melting (MS-HRM). Results There was an obvious inhibitory effect of DAC on HL-60 cells. The cell viability of HL-60 treated with 0.2, 0.5, and 1.0 μmol/L DAC for 48 h was decreased by (25±11) %, (39±8) % and (50±7) % (P < 0.01) respectively compared with those cells without DAC treatment. The incidence of apoptosis was (24.77±7.50) %, (27.10±4.48) % and (30.53±3.93) % after DAC treatment for 48h respectively, which were higher than that of untreated cells [(3.11±0.50) %] (P < 0.01). DAC induced a significant up-regulation of MICA/B, ULBP-1, ULBP-3 in HL-60 cells, and enhanced the sensitivity of HL-60 cells to NK cytotoxicity. Western blot results showed that a down-regulating expression of STAT3 and JAK1, JAK2 protein was detected, in addition to the phosphor-STAT3 and phosphor-JAKs in HL-60 cells after DAC treatment, but the expressions of SOCS-1 and SOCS-3 protein were increased. HRM results showed that DAC could inhibit the methylation of SOCS-3 gene. Conclusion DAC can inhibit the proliferation of HL-60 cells, upregulate the expression of NKG2DL and enhance the cytotoxicity of NK targeted to HL-60 cells, which might be related to the activity regulation of intracellular JAK-STAT3-SOCS signaling pathway. Key words: Leukemia, myeloid, acute; Cell proliferation; Apoptosis; Molecular mechanisms of action; Decitabine

Renoprotective effect of erythropoietin via modulation of the STAT6/MAPK/NF-κB pathway in ischemia/reperfusion injury after renal transplantation.

Ischemia/reperfusion injury (IRI) commonly occurs in renal transplantation. Erythropoietin (EPO) exerts a protective effect in IRI. To investigate the underlying molecular mechanism, rat models of renal IRI were established and treated with EPO and/or lentivirus-mediated EPO-siRNA, the signal transducer and activator of transcription 6 (STAT6) inhibitor AS1517499, the JNK inhibitor SP600125, the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580, and the nuclear factor (NF)-κB inhibitor lactacystin. Histological examination revealed that EPO protected the kidney from IRI, through decreasing the extent of tissue congestion and inflammatory cell infiltration; however, EPO siRNA did not exert the same protective effect. In addition, the EPO level was inversely associated with renal IRI. EPO downregulated the expression of interferon-γ, interleukin (IL)-4, creatinine and caspase-3, and upregulated the expression of IL-10, thymic stromal lymphopoietin, STAT6, p-JNK and p-p38, while the opposite effects were observed with the administration of EPO-siRNA and the specific respective inhibitors. Further results revealed that MAPK (p-JNK and p-p38) acted upstream of NF-κB, and that NF-κB signaling regulated the expression of caspase-1 and -3, which may be responsible for the cytotoxicity associated with IRI. Taken together, the results of the present study demonstrated that EPO exerted a protective effect in renal IRI via the STAT6/MAPK/NF-κB pathway. This protective effect of EPO may improve reperfusion tolerance in ischemic kidneys and benefit transplant recipients.

High expression of IL-4R enhances proliferation and invasion of hepatocellular carcinoma cells.

In this study, we aimed to investigate the expression and function of interleukin-4 receptor (IL-4R) in hepatocellular carcinoma (HCC). We collected 40 pairs of human HCC and adjacent normal tissue specimens and examined the expression levels of IL-4R. After IL-4R knockdown in HCC cell lines, cell proliferation and invasion ability were examined. Cell cycle and apoptosis were analyzed by flow cytometry. The activity of multiple signaling pathways was examined by Western blot. IL-4R was overexpressed in HCC tumors compared with adjacent normal control tissues and was associated with tumor differentiation status. IL-4R knockdown resulted in enhanced apoptosis, impaired proliferation and reduced invasion of HCC cells. Furthermore, IL-4R knockdown abolished IL-4-induced activation of the Janus Kinase 1 (JAK1)/signal transducer and activator of transcription 6 (STAT6) and JUN N-terminal kinase (JNK)/extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathways. IL-4R plays an important role in regulating HCC cell survival and metastasis, and regulates the activity of the JAK1/STAT6 and JNK/ERK1/2 signaling pathways. We therefore suggest that IL-4/IL-4R may be a new therapeutic target for HCC.

MiR-204/14-3-3ζ axis regulates osteosarcoma cell proliferation through SATA3 pathway.

Hyperproliferation of cells is a major problem is osteosarcoma (OS). So, further elucidation of the molecular mechanisms underlying hyperproliferation of OS is needed. Western blots results showed that 14-3-3ζ protein was upregulated in OS cell lines; 14-3-3ζ knockdown significantly suppressed OS cell proliferation, as well as the protein levels of p-STAT3, c-Myc and Cyclin D1. MicroRNA-204 (miR-204) has been regarded as an essential regulator in cancer carcinogenesis, including OS. Here, we revealed that miR-204 directly targets the 3'UTR of 14-3-3ζ to inhibit its expression, thus to suppress 14-3-3ζ -induced OS cell hyperproliferation. Further, we demonstrated that the STAT3 pathway was involved in miR-204/14-3-3ζ regulation of OS cell proliferation. Our findings provide information about the underlying mechanisms of miR-204/14-3-3ζ in OS cell proliferation through the STAT3 pathway, and suggest miR-204 and 14-3-3ζ as potential therapeutic targets in OS.

Targeting histone-acetyltransferase Tat-interactive protein 60 inhibits intestinal allergy.

The overproduction of IgE plays a critical role in the pathogenesis of allergy; the mechanism is unclear. Histone-acetyltransferase (HAT) activities are required in gene transcription of a large number of molecules in the immune system of the body. This study tests a hypothesis that HAT Tat-interactive protein 60 (Tip60) plays an important role in the initiation of IgE-mediated allergy. The effects of Tip60 on regulating IgE expression were assessed with B cells. An intestinal allergy mouse model was developed to assess the role of Tip60 in the induction of IgE-mediated allergic inflammation. High levels of Tip60 were observed in the peripheral B cells of patients with FA. Tat-interactive protein 60 (Tip60) was required in the expression of IgE and IgG1 in B cells by inducing the chromatin remolding at the gene locus, in which histone acetylation, signal transducer and activator of transcription 6 (STAT6), and nuclear factor-κB at the locus of Iε promoter were markedly increased. Blocking Tip60 significantly attenuated the allergic inflammation in the mouse intestinal mucosa. Tat-interactive protein 60 (Tip60) plays an important role in the induction of IgE in B cells. Blocking Tip60 inhibits the allergic inflammation in the intestine, suggesting Tip60 inhibitor may be a potential anti-allergy drug.

Tim-3 signaling in peripheral NK cells promotes maternal-fetal immune tolerance and alleviates pregnancy loss.

Pregnancy loss occurs in about 15% of clinically recognized pregnancies, and defective maternal-fetal immune tolerance contributes to more than 50% of these events. We found that signaling by the type I membrane protein T cell immunoglobulin and mucin-containing protein 3 (Tim-3) in natural killer (NK) cells had an essential protective role during early pregnancy. Tim-3 on peripheral NK (pNK) cells was transiently increased in abundance during the first trimester of pregnancy, which depended on interleukin-4 (IL-4)-signal transducer and activator of transcription 6 (STAT6) and progesterone signaling. Tim-3+ pNK cells displayed immunosuppressive activities, including the production of anti-inflammatory cytokines and the induction of regulatory T cells (Tregs) in a transforming growth factor-β1 (TGF-β1)-dependent manner. Tim-3 on pNK cells was stimulated by its ligand galectin-9 (Gal-9), leading to signaling by the kinases c-Jun N-terminal kinase (JNK) and AKT. In recurrent miscarriage (RM) patients, Tim-3 abundance on pNK cells was reduced and the immunosuppressive activity of Tim-3+ pNK cells was impaired. Compared to Tim-3+ pNK cells from donors with normal pregnancies, RM patient Tim-3+ pNK cells exhibited changes in DNA accessibility in certain genetic loci, which were reversed by inhibiting accessible chromatin reader proteins. Furthermore, Tim-3+ pNK cells, but not Tim-3- pNK cells, reduced fetal loss in abortion-prone and NK cell-deficient mice. Together, our findings reveal a critical role for Tim-3-Gal-9 signaling-mediated immunoregulation by pNK cells in maternal-fetal immune tolerance and suggest that Tim-3 abundance on pNK cells is a potential biomarker for RM diagnosis.