HSV-2 induces TLRs and NF-κB-dependent cytokines in cervical epithelial cells

Herpes simplex virus type 2 (HSV-2) is one of the most common sexually transmitted pathogens worldwide. The host immune response induced by viral infection is cell-type specific. Little is known about the innate immune response to this virus in its natural host cells. In this study, we established an in vitro HSV-2 infection model with human cervical epithelial (HCE) cells. The viral infection was sufficient to induce expression of Toll-like receptors (TLRs), and Western blot and reporter assays suggest that HSV-2 infection leads to dramatic activation of the NF-κB signaling pathway. More importantly, our data provide direct evidence that the activation of NF-κB is required for the production of both IL-6 and IFN-β induced by HSV-2 in HCE cells. Taken together, our results suggest the potential contributions of TLRs and a critical role of NF-κB in the innate immune response to HSV-2 in HCE cells.     

Inhibition of Aurora-A kinase induces cell cycle arrest in epithelial ovarian cancer stem cells by affecting NFκB pathway

Recurrent ovarian cancer is resistant to conventional chemotherapy. A sub-population of ovarian cancer cells, the epithelial ovarian cancer stem cells (EOC stem cells) have stemness properties, constitutive NFκB activity, and represent the chemoresistant population. Currently, there is no effective treatment that targets these cells. Aurora-A kinase (Aurora-A) is associated with tumor initiation and progression and is overexpressed in numerous malignancies. The aim of this study is to determine the effect of Aurora-A inhibition in EOC stem cells. EOC stem cells were treated with the Aurora-A inhibitor, MK-5108. Cell growth was monitored by Incucyte real-time imaging system, cell viability was measured using the Celltiter 96 assay and cytokine levels were quantified using xMAP technology. The intracellular changes associated with MK-5108 treatment are: (1) polyploidy and cell cycle arrest; (2) inhibition of NFκB activity; (3) decreased cytokine production; and (4) nuclear accumulation of IκBα. Thus, inhibition of Aurora-A decreases cell proliferation in the EOC stem cells by inducing cell cycle arrest and affecting the NFκB pathway. As EOC stem cells represent a source of recurrence and chemoresistance, these results suggest that Aurora-A inhibition may effectively target the cancer stem cell population in ovarian cancer.Key words: ovarian cancer stem cells, aurora-A kinase, cell cycle arrest, nuclear factor kappaB     

Bacterial neuraminidase increases IL-8 production in lung epithelial cells via NF-κB-dependent pathway

Bacterial neuraminidase, a sialic acid-degrading enzyme, is one of the virulent factors produced in pathogenic bacteria like as other bacterial components. However little is known about whether bacterial neuraminidase can initiate or modify a cellular response, such as cytokine production, in epithelial cells at infection and inflammation. We demonstrate here that bacterial neuraminidase, but not heat-inactivated neuraminidase, up-regulates expression of interleukin-8 (IL-8) mRNA and protein in lung epithelial A549 and NCI-H292 cells. We also show that bacterial neuraminidase significantly up-regulates IL-8 promoter activity as well as nuclear factor-kappaB (NF-κB) reporter activity. Moreover, inhibition of NF-κB signaling suppressed IL-8 mRNA expression induced by bacterial neuraminidase. Taken together, desialylation-induced IL-8 production in lung epithelial cells may play an important role in infection-associated inflammatory events.     

Vibrio vulnificus AphB is involved in interleukin-8 production via an NF-κB-dependent pathway in human intestinal epithelial cells

We previously reported that the aphB gene mutant of Vibrio vulnificus had significantly impaired motility and adherence to host cells. In this study, we investigated the role of V. vulnificus AphB on the production of interleukin-8 (IL-8), a proinflammatory cytokine, as well as its underlying mechanism in human intestinal epithelial INT-407 cells. The aphB gene mutation significantly reduced the ability of V. vulnificus to stimulate IL-8 production and IL-8 gene promoter activation in INT-407 cells. The V. vulnificusaphB mutant also induced lower levels of NF-κB DNA binding activity and NF-κB minimal promoter activation than did the wild-type of V. vulnificus. Importantly, the observed reductions in IL-8 production, IL-8 gene promoter activation and NF-κB DNA binding activity were significantly restored by complementing the aphB gene into the V. vulnificusaphB mutant. These results indicate that V. vulnificus AphB is involved in the IL-8 production via an NF-κB dependent pathway in human intestinal epithelial cells.     

Id-1 promotes migration and invasion of non-small cell lung cancer cells through activating NF-κB signaling pathway

Background

Numerous studies have shown that Id-1 (Inhibitor of differentiation 1) is upregulated in several cancers and associated with tumor malignant characters. However, the clinical significance and biological role of Id-1 in non-small cell lung cancer (NSCLC) remains unclear.

Methods

We used RT-PCR, Western blot and Immunohistochemistry to measure Id-1 expression in NSCLC tissues and matched adjacent noncancerous tissues. The expression pattern of Id-1 in NSCLC tissues was determined by scoring system of immunohistochemical analysis. The Kaplan-Meier method was used to calculate the survival curve, and log-rank test to determine statistical significance. The Id-1 gene was overexpressed or downreuglated with Lentiviral vectors in NSCLC cells. And, the migration ability of NSCLC cells was tested in a Transwell Boyden Chamber.

Results

We found that Id-1 is generally expressed higher in NSCLC tissues compared with matched adjacent noncancerous tissues. We also found that high Id-1 expression in tumor tissues is significantly correlated with tumor progression and poor survival in NSCLC patients. Furthermore, our experimental data revealed that knockdown of Id-1 significantly suppressed the proliferation, migration and invasion of NSCLC cells, whereas ectopic expression of Id-1 promoted the malignant phenotype of NSCLC cells. Mechanistic study showed that NF-κB signaling pathway contributed to the effects of Id-1 in NSCLC cells. Moreover, blocking the NF-κB pathway significantly inhibited the tumor-promoting actions of Id-1 in NSCLC cells.

Conclusions

We identified a tumorigenic role of Id-1 in NSCLC and provided a novel therapeutic target for NSCLC patients.

     

MiR-499 induces cardiac differentiation of rat mesenchymal stem cells through wnt/β-catenin signaling pathway

ObjectiveTo test the hypothesis that over-expressing miR-499 in rat bone marrow-derived mesenchymal stem cells (BM-MSCs) induces them to differentiate into cardiomyocyte-like cells through the wnt/β-catenin signaling pathway.MethodsRat BM-MSCs were infected with lentiviral vectors bearing miR-499. The expression of cardiac-specific markers, NKx2.5, GATA4, MEF2C, and cTnI in these cells were examined by rtPCR or Western blot analysis and the activity of the wnt/β-catenin signaling pathway was evaluated by measuring the phosphorylation status of β-catenin.ResultsOver-expression of miR-499 in rat BM-MSCs increased the expression of cardiac-specific genes, such as NKx2.5, GATA4, MEF2C, and cTnI and decreased the ratio of phosphorylated/dephosphorylated β-catenin in the wnt/β-catenin signaling pathway, thus activating the pathway. Knocking down the expression of Dvl, an adaptor molecule in the wnt/β-catenin signaling, partially blocked the role of the miR-499 and decreased those cardiac-specific genes.ConclusionOver-expression of miR-499 in rat BM-MSCs induces them toward cardiac differentiation through the activating the wnt/β-catenin signal pathway.     

Effect of Helicobacter pylori’s vacuolating cytotoxin on the autophagy pathway in gastric epithelial cells

Host cell responses to Helicobacter pylori infection are complex and incompletely understood. Here, we report that autophagy is induced within human-derived gastric epithelial cells (AGS) cells in response to H. pylori infection. These autophagosomes were distinct and different from the large vacuoles induced during H. pylori infection. Autophagosomes were detected by transmission electron microscopy, conversion of LC3-I to LC3-II, GFP-LC3 recruitment to autophagosomes, and depended on Atg5 and Atg12. The induction of autophagy depended on the vacuolating cytotoxin (VacA) and, moreover, VacA was sufficient to induce autophagosome formation. The channel forming activity of VacA was necessary for inducing autophagy. Intracellular VacA partially co-localized with GFP-LC3, indicating that the toxin associates with autophagosomes. The inhibition of autophagy increased the stability of intracellular VacA, which in turn resulted in enhanced toxin-mediated cellular vacuolation. These findings suggest that the induction of autophagy by VacA may represent a host mechanism to limit toxin-induced cellular damage.     

Inhibition of Aurora-A kinase induces cell cycle arrest in epithelial ovarian cancer stem cells by affecting NFĸB pathway

Recurrent ovarian cancer is resistant to conventional chemotherapy. A sub-population of ovarian cancer cells, the epithelial ovarian cancer stem cells (EOC stem cells) have stemness properties, constitutive NFκB activity, and represent the chemoresistant population. Currently, there is no effective treatment that targets these cells. Aurora-A kinase (Aurora-A) is associated with tumor initiation and progression and is overexpressed in numerous malignancies. The aim of this study is to determine the effect of Aurora-A inhibition in EOC stem cells. EOC stem cells were treated with the Aurora-A inhibitor, MK-5108. Cell growth was monitored by Incucyte real-time imaging system, cell viability was measured using the Celltiter 96 assay and cytokine levels were quantified using xMAP technology. The intracellular changes associated with MK-5108 treatment are: (1) polyploidy and cell cycle arrest; (2) inhibition of NFκB activity; (3) decreased cytokine production; and (4) nuclear accumulation of IκBα. Thus, inhibition of Aurora-A decreases cell proliferation in the EOC stem cells by inducing cell cycle arrest and affecting the NFκB pathway. As EOC stem cells represent a source of recurrence and chemoresistance, these results suggest that Aurora-A inhibition may effectively target the cancer stem cell population in ovarian cancer.     

Trophinin-mediated cell adhesion induces apoptosis of human endometrial epithelial cells through PKC-δ

Trophinin is an intrinsic membrane protein expressed in trophectoderm cells of embryos and in uterine epithelial cells. Trophinin potentially mediates apical cell adhesion at human embryo implantation sites through trophinin-trophinin binding in these two cell types. Trophinin-mediated cell adhesion activates trophectoderm cells for invasion, whereas the effect of adhesion on maternal side is not known. We show that addition of GWRQ peptide, a previously established peptide that mimics trophinin-mediated cell adhesion, to human endometrial epithelial cells expressing trophinin induces their apoptosis. FAS involvement was excluded, as GWRQ did not bind to FAS, and FAS knockdown did not alter GWRQ-induced apoptosis. Immunoblotting analyses of protein kinases revealed an elevation of PKC-δ protein in GWRQ-bound endometrial epithelial cells. In the absence of GWRQ, PKC-δ associated with trophinin and remained cytoplasmic, but after GWRQ binding to the trophinin extracellular domain, PKC-δ became tyrosine phosphorylated, dissociated from trophinin and entered the nucleus. In PKC-δ knockdown endometrial cells, GWRQ did not induce apoptosis. These results suggest that trophinin-mediated cell adhesion functions as a molecular switch to induce apoptosis through the PKC-δ pathway in endometrial epithelial cells. Thus, trophinin-mediated induction of apoptosis of endometrial epithelial cells, which function as a barrier to embryo invasion, allows trophoblast invasion of maternal tissue and embryo implantation in humans.Key words: blastocyst, embryo implantation, apoptosis, cell adhesion, signal transduction     

BMAL1 regulates balance of osteogenic–osteoclastic function of bone marrow mesenchymal stem cells in type 2 diabetes mellitus through the NF-κB pathway

In bone marrow mesenchymal stem cell (BMSCs), type 2 diabetes mellitus (T2DM) induces metabolic and functional disorders, leading to imbalanced bone resorption and formation and bone loss. Brain and muscle ARNT-like protein 1 (BMAL1) is involved in regulating T2DM-related suppression of BMSCs osteogenesis and bone formation. However, the relationship between BMAL1 and bone remodelling, especially bone resorption in T2DM, is unclear. We investigated the antergic role played by BMAL1 in T2DM-prompted imbalance in BMSCs osteogenic–osteoclastic function. BMAL1 was inhibited and the receptor activator of nuclear factor-κB ligand/osteoprotegerin (RANKL/OPG) ratio was increased in diabetic BMSCs. Inhibitor κB (IκB) expression was decreased, whereas phosphorylated-p65 (p-p65), caspase-3, and p-IκB expression were increased in diabetic BMSCs. BMAL1 overexpression recovered the osteogenesis ability and suppressed osteoclastic induction capability of BMSCs to improve bone metabolism and function, which was partially due to NF-κB pathway activity inhibition. Our results provide evidence about the role of BMAL1 in T2DM-prompted BMSCs differentiation dysfunction, i.e. partially decreasing NF-κB pathway expression. In T2DM, it might be possible to use overexpressed BMAL1 to re-establish the homeostasis of bone metabolism.     

microRNA-95 knockdown inhibits epithelial–mesenchymal transition and cancer stem cell phenotype in gastric cancer cells through MAPK pathway by upregulating DUSP5

This study investigated the role of microRNA-95 (miR-95) in gastric cancer (GC) and to elucidate the underlying mechanism. Initially, bioinformatic prediction was used to predict the differentially expressed genes and related miRNAs in GC. miR-95 and DUSP5 expression was altered in GC cell line (MGC803) to evaluate their respective effects on the epithelial–mesenchymal transition (EMT) process, cellular processes (cell proliferation, migration, invasion, cell cycle, and apoptosis), cancer stem cell (CSC) phenotype, as well as tumor growth ability. It was further predicted in bioinformatic prediction and verified in GC tissue and cell line experiments that miR-95 was highly expressed in GC. miR-95 negatively regulated DUSP5, which resulted in the MAPK pathway activation. Inhibited miR-95 or overexpressed DUSP5 was observed to inhibit the levels of CSC markers (CD133, CD44, ALDH1, and Lgr5), highlighting the inhibitory role in the CSC phenotype. More important, evidence was obtained demonstrating that miR-95 knockdown or DUSP5 upregulation exerted an inhibitory effect on the EMT process, cellular processes, and tumor growth. Together these results, miR-95 knockdown inhibited GC development via DUSP5-dependent MAPK pathway.     

Tumor necrosis factor-alpha promotes survival in methotrexate-exposed macrophages by an NF-κB-dependent pathway

Introduction  

Methotrexate (MTX) induces macrophage apoptosis in vitro, but there is not much evidence for increased synovial macrophage apoptosis in MTX-treated patients. Macrophage apoptosis is reported, however, during clinical response to anti-tumor necrosis factor-alpha (TNF-α) treatments. This implies that TNF-α promotes macrophage survival and suggests that TNF-α may protect against MTX-induced apoptosis. We, therefore, investigated this proposal and the macrophage signaling pathways underlying it.     

Roles of Wnt/β-catenin signaling in epithelial differentiation of mesenchymal stem cells

Bone marrow-derived mesenchymal stem cells (MSCs) have been demonstrated to be able to differentiate into epithelial lineage, but the precise mechanisms controlling this process are unclear. Our aim is to explore the roles of Wnt/β-catenin in the epithelial differentiation of MSCs. Using indirect co-culture of rat MSCs with rat airway epithelial cells (RTE), MSCs expressed several airway epithelial markers (cytokeratin 18, tight junction protein occudin, cystic fibrosis transmembrance regulator). The protein levels of some important members in Wnt/β-catenin signaling were determined, suggested down-regulation of Wnt/β-catenin with epithelial differentiation of MSCs. Furthermore, Wnt3α can inhibit the epithelial differentiation of MSCs. A loss of β-catenin induced by Dickkopf-1 can enhance MSCs differentiation into epithelial cells. Lithium chloride transiently activated β-catenin expression and subsequently decreased β-catenin level and at last inhibited MSCs to differentiate into airway epithelium. Taken together, our study indicated that RTE cells can trigger epithelial differentiation of MSCs. Blocking Wnt/β-catenin signaling may promote MSCs to differentiate towards airway epithelial cells.