Placental growth factor promotes epithelial-mesenchymal transition-like changes in ARPE-19 cells under hypoxia.

Purpose: To investigate the role of placental growth factor (PGF) in the epithelial-mesenchymal transition (EMT) of ARPE-19 cells under hypoxia, and whether the NF-κB signaling pathway is involved in this process. Methods: ARPE-19 cells were treated in five groups: a control group, hypoxia group, PGF group, hypoxia+PGF group, and NF-κB-blocked group. A chemical hypoxia model was established in the ARPE-19 cells by adding CoCl2 to the culture medium. The morphological changes after treatment were observed. The proliferation rates were measured with 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The migration abilities were measured with scratch assay. The EMT biomarkers were measured with quantitative real-time PCR (qRT-PCR), western blotting, and immunofluorescence. The relative protein expression of components of the NF-κB signaling pathway was measured with western blotting and immunofluorescence. Results: Cells treated with PGF under hypoxia exhibited morphological changes consistent with the transition from an epithelial to a mesenchymal phenotype. In the ARPE-19 cells, exogenous PGF under hypoxia increased the proliferation rate compared to the rate under hypoxia alone (p<0.05) and increased the migration rate (p<0.05). Treatment of hypoxia-exposed cells with PGF caused decreased expression of the epithelial biomarkers E-cadherin and ZO-1 (both p<0.05) and increased expression of the mesenchymal marker α-SMA (p<0.05) by enhancing the phosphorylation of NF-κB p65 of the total protein, promoting the translocation of p65 to the nucleus, and inducing the degradation of IκB-α (a negative regulator of the NF-κB pathway) in the ARPE-19 cells. Additionally, the effect of PGF-induced EMT in the ARPE-19 cells under hypoxia was counteracted with BAY 11-7082 (a selective NF-κB inhibitor). Conclusions: Exogenous PGF promotes EMT-like changes in ARPE-19 cells under hypoxia by activating the NF-κB signaling pathway. The study results suggest that PGF may play a role in scar formation in neovascular age-related macular degeneration (AMD) and that the inhibition of PGF may be a promising target for the prevention and treatment of AMD.

Altered expression of histone deacetylases, inflammatory cytokines and contractile-associated factors in uterine myometrium of Long Evans rats gestationally exposed to benzo[a]pyrene.

Etiology of preterm birth (PTB) is multifactorial; therefore, decreasing the incidence of PTB is a major challenge in the field of obstetrics. Epidemiological studies have reported an association between toxicants and PTB. However, there are no studies on the role of benzo[a]pyrene (BaP), an environmental toxicant, in the incidence of PTB. We first assessed the effects of BaP (150 and 300 µg kg(-1) body weight) dosed via gavage from day 14 to 17 of pregnancy on gestation length in Long Evans rats. We further assessed the histopathology of the uterus, expression of inflammatory cytokines, contractile-associated factors, histone deacetylases (HDACs) and NFқB-p65 in myometrium collected on day 22 postpartum versus vehicle-treated controls. In our study, rats exposed to BaP delivered prematurely (P < 0.05) compared to control. Hematoxylin and eosin staining of uterus showed squamous metaplasia, glandular and stromal hyperplasia in BaP-exposed rats versus control. The concentrations of BaP metabolites measured by high-pressure liquid chromatography were higher in uterine myometrium of BaP-exposed rats while they were undetectable in controls. Quantitative real-time polymerase chain reaction showed significant increases in mRNA expression of interleukin-1ß and -8, tumor necrosis factor-α, connexin 43, cyclo-oxygenase-2 and prostaglandin F2α receptor as compared to controls (P < 0.05). Western blot analysis revealed that BaP exposure caused decreases in class I HDACs 1 and 3 and increases in class II HDAC 5, cyclo-oxygenase-2 and nuclear translocation of NFκB-p65 relative to controls. Our results suggest that gestational exposure to BaP increases incidence of PTB through epigenetic changes that causes increases in the expression of contractile-associated factors through the NFκB pathway. Copyright © 2015 John Wiley & Sons, Ltd.

Promoted Interaction of Nuclear Factor-κB With Demethylated Purinergic P2X3 Receptor Gene Contributes to Neuropathic Pain in Rats With Diabetes.

Painful diabetic neuropathy is a common complication of diabetes produced by mechanisms that as yet are incompletely defined. The aim of this study was to investigate the roles of nuclear factor-κB (NF-κB) in the regulation of purinergic receptor P2X ligand-gated ion channel 3 (P2X3R) plasticity in dorsal root ganglion (DRG) neurons of rats with painful diabetes. Here, we showed that hindpaw pain hypersensitivity in streptozocin-induced diabetic rats was attenuated by treatment with purinergic receptor antagonist suramin or A-317491. The expression and function of P2X3Rs was markedly enhanced in hindpaw-innervated DRG neurons in diabetic rats. The CpG (cytosine guanine dinucleotide) island in the p2x3r gene promoter region was significantly demethylated, and the expression of DNA methyltransferase 3b was remarkably downregulated in DRGs in diabetic rats. The binding ability of p65 (an active form of NF-κB) with the p2x3r gene promoter region and p65 expression were enhanced significantly in diabetes. The inhibition of p65 signaling using the NF-κB inhibitor pyrrolidine dithiocarbamate or recombinant lentiviral vectors designated as lentiviral vector-p65 small interfering RNA remarkably suppressed P2X3R activities and attenuated diabetic pain hypersensitivity. Insulin treatment significantly attenuated pain hypersensitivity and suppressed the expression of p65 and P2X3Rs. Our findings suggest that the p2x3r gene promoter DNA demethylation and enhanced interaction with p65 contributes to P2X3R sensitization and diabetic pain hypersensitivity.

Single administration of ultra-low-dose lipopolysaccharide in rat early pregnancy induces TLR4 activation in the placenta contributing to preeclampsia.

Balanced immune responses are essential for the maintenance of successful pregnancy. Aberrant responses of immune system during pregnancy increase the risk of preeclampsia. Toll-like receptor 4 (TLR4) plays a crucial role in the activation of immune system at the maternal-fetal interface. This study aimed to generate a rat model of preeclampsia by lipopolysaccharide (LPS, a TLR4 agonist) administration on gestational day (GD) 5 as rats are subjected to placentation immediately after implantation between GDs 4 and 5, and to assess the contribution of TLR4 signaling to the development of preeclampsia. Single administration of 0.5 µg/kg LPS significantly increased blood pressure of pregnant rats since GD 6 (systolic blood pressure, 124.89 ± 1.79 mmHg versus 119.02 ± 1.80 mmHg, P < 0.05) and urinary protein level since GD 9 (2.02 ± 0.29 mg versus 1.11 ± 0.18 mg, P < 0.01), but barely affected blood pressure or proteinuria of virgin rats compared with those of saline-treated pregnant rats. This was accompanied with adverse pregnancy outcomes including fetal growth restriction. The expression of TLR4 and NF-κB p65 were both increased in the placenta but not the kidney from LPS-treated pregnant rats, with deficient trophoblast invasion and spiral artery remodeling. Furthermore, the levels of inflammatory cytokines were elevated systemically and locally in the placenta from pregnant rats treated with LPS. TLR4 signaling in the placenta was activated, to which that in the placenta of humans with preeclampsia changed similarly. In conclusion, LPS administration to pregnant rats in early pregnancy could elicit TLR4-mediated immune response at the maternal-fetal interface contributing to poor early placentation that may culminate in the preeclampsia-like syndrome.

The Extracellular IFI16 Protein Propagates Inflammation in Endothelial Cells Via p38 MAPK and NF-κB p65 Activation.

The nuclear interferon-inducible-16 (IFI16) protein acts as DNA sensor in inflammasome signaling and as viral restriction factor. Following Herpesvirus infection or UV-B treatment, IFI16 delocalizes from the nucleus to the cytoplasm and is eventually released into the extracellular milieu. Recently, our group has demonstrated the occurrence of IFI16 in sera of systemic-autoimmune patients that hampers biological activity of endothelia through high-affinity membrane binding. As a continuation, we studied the activity of endotoxin-free recombinant IFI16 (rIFI16) protein on primary endothelial cells. rIFI16 caused dose/time-dependent upregulation of IL-6, IL-8, CCL2, CCL5, CCL20, ICAM1, VCAM1, and TLR4, while secretion of IL-6 and IL-8 was amplified with lipopolysaccharide synergy. Overall, cytokine secretion was completely inhibited in MyD88-silenced cells and partially by TLR4-neutralizing antibodies. By screening downstream signaling pathways, we found that IFI16 activates p38, p44/42 MAP kinases, and NF-kB. In particular, activation of p38 is an early event required for subsequent p44/42 MAP kinases activity and cytokine induction indicating a key role of this kinase in IFI16 signaling. Altogether, our data conclude that extracellular IFI16 protein alone or by synergy with lipopolysaccharide acts like Damage-associated molecular patterns propagating "Danger Signal" through MyD88-dependent TLR-pathway.

Oxytocin activates NF-κB-mediated inflammatory pathways in human gestational tissues.

Human labour, both at term and preterm, is preceded by NF-κB-mediated inflammatory activation within the uterus, leading to myometrial activation, fetal membrane remodelling and cervical ripening. The stimuli triggering inflammatory activation in normal human parturition are not fully understood. We show that the neurohypophyseal peptide, oxytocin (OT), activates NF-κB and stimulates downstream inflammatory pathways in human gestational tissues. OT stimulation (1 pM-100 nM) specifically via its receptor (OTR) in human myometrial and amnion primary cells led to MAPK and NF-κB activation within 15 min and maximal p65-subunit nuclear translocation within 30 min. Both in human myometrium and amnion, OT-induced activation of the canonical NF-κB pathway upregulated key inflammatory labour-associated genes including IL-8, CCL5, IL-6 and COX-2. IKKß inhibition (TPCA1; 10 µM) suppressed OT-induced NF-κB-p65 phosphorylation, whereas p65-siRNA knockdown reduced basal and OT-induced COX-2 levels in myometrium and amnion. In both gestational tissues, MEK1/2 (U0126; 10 µM) or p38 inhibition (SB203580; 10 µM) suppressed OT-induced COX-2 expression, but OT-induced p65-phosphorylation was only inhibited in amnion, suggesting OT activation of NF-κB in amnion is MAPK-dependent. Our data provide new insight into the OT/OTR system in human parturition and suggest that its therapeutic modulation could be a strategy for regulating both contractile and inflammatory pathways in the clinical context of term/preterm labour.

Disturbed flow enhances inflammatory signaling and atherogenesis by increasing thioredoxin-1 level in endothelial cell nuclei.

BACKGROUND: Oxidative stress occurs with disturbed blood flow, inflammation and cardiovascular disease (CVD), yet free-radical scavenging antioxidants have shown limited benefit in human CVD. Thioredoxin-1 (Trx1) is a thiol antioxidant protecting against non-radical oxidants by controlling protein thiol/disulfide status; Trx1 translocates from cytoplasm to cell nuclei due to stress signaling, facilitates DNA binding of transcription factors, e.g., NF-κB, and potentiates inflammatory signaling. Whether increased nuclear Trx1 contributes to proatherogenic signaling is unknown. METHODOLOGY/PRINCIPAL FINDINGS: In vitro and in vivo atherogenic models were used to test for nuclear translocation of Trx1 and associated proinflammatory signaling. Disturbed flow by oscillatory shear stress stimulated Trx1 nuclear translocation in endothelial cells. Elevation of nuclear Trx1 in endothelial cells and transgenic (Tg) mice potentiated disturbed flow-stimulated proinflammatory signaling including NF-κB activation and increased expression of cell adhesion molecules and cytokines. Tg mice with increased nuclear Trx1 had increased carotid wall thickening due to disturbed flow but no significant differences in serum lipids or weight gain compared to wild type mice. Redox proteomics data of carotid arteries showed that disturbed flow stimulated protein thiol oxidation, and oxidation was higher in Tg mice than wild type mice. CONCLUSIONS/SIGNIFICANCE: Translocation of Trx1 from cytoplasm to cell nuclei plays an important role in disturbed flow-stimulated proatherogenesis with greater cytoplasmic protein oxidation and an enhanced nuclear transcription factor activity. The results suggest that pharmacologic interventions to inhibit nuclear translocation of Trx1 may provide a new approach to prevent inflammatory diseases or progression.

Valproic acid inhibits the proliferation of SHSY5Y neuroblastoma cancer cells by downregulating URG4/URGCP and CCND1 gene expression.

Valproic acid (VPA), used for the treatment of epilepsy and bipolar disorder, regulates several signaling pathways in brain cells. The up-regulated gene 4 (URG4/URGCP) is a novel gene located on 7p13. URG4/URGCP stimulates cyclin D1 (CCND1) mRNA expression, and URG4/URGCP silencing diminishes CCND1 mRNA expression in HepG2 cells. This study was performed to investigate the anti-cancer mechanism of action of VPA by analyzing the expression of novel gene URG4/URGCP, CCND1, p21, p53, p65 (RelA), Bax, and Bcl-2 in SHSY5Y neuroblastoma (NB) cancer cells. Cytotoxic effects of VPA in SHSY5Y were noticed in time and dose dependent manner with the IC50 doses within the range of 0.5-10 mM. IC50 doses in the SHSY5Y were detected as 7.5 mM. Expression profiles were determined by semi quantitative RT-PCR and URG4/URGCP protein change by western blot analysis. Our results suggest that VPA induces cell cycle arrest in SHSY5Y due to the decrease in URG4/URGCP, CCND1 gene expression and the increase in p65. To conclude, VPA may be a prospective agent for the treatment of NB as a single agent or in combination with other drugs. Thus, more studies should be designed to find a safe dose with the best effects of VPA.

MicroRNAs regulate human adipocyte lipolysis: effects of miR-145 are linked to TNF-α.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression and have multiple effects in various tissues including adipose inflammation, a condition characterized by increased local release of the pro-lipolytic cytokine tumor necrosis factor-alpha (TNF-α). Whether miRNAs regulate adipocyte lipolysis is unknown. We set out to determine whether miRNAs affect adipocyte lipolysis in human fat cells. To this end, eleven miRNAs known to be present in human adipose tissue were over-expressed in human in vitro differentiated adipocytes followed by assessments of TNF-α and glycerol levels in conditioned media after 48 h. Three miRNAs (miR-145, -26a and let-7d) modulated both parameters in parallel. However, while miR-26a and let-7d decreased, miR-145 increased both glycerol release and TNF-α secretion. Further studies were focused therefore on miR-145 since this was the only stimulator of lipolysis and TNF-α secretion. Time-course analysis demonstrated that miR-145 over-expression up-regulated TNF-α expression/secretion followed by increased glycerol release. Increase in TNF-α production by miR-145 was mediated via activation of p65, a member of the NF-κB complex. In addition, miR-145 down-regulated the expression of the protease ADAM17, resulting in an increased fraction of membrane bound TNF-α, which is the more biologically active form of TNF-α. MiR-145 overexpression also increased the phosphorylation of activating serine residues in hormone sensitive lipase and decreased the mRNA expression of phosphodiesterase 3B, effects which are also observed upon TNF-α treatment in human adipocytes. We conclude that miR-145 regulates adipocyte lipolysis via multiple mechanisms involving increased production and processing of TNF-α in fat cells.

Hydrogen sulfide suppresses high glucose-induced expression of intercellular adhesion molecule-1 in endothelial cells.

Hydrogen sulfide (H2S) is a newly identified endogenous gasotransmitter that has been implicated in the pathophysiology of several biologic systems. However, the role of H2S in the pathogenesis of diabetic vascular injury remains unclear. The aims of this study were to determine the effect of H2S on the high glucose (HG)-induced expression of intercellular adhesion molecule-1 (ICAM-1) in human umbilical vein endothelial cells and to explore the possible underlying mechanisms. Human umbilical vein endothelial cells were exposed either to a normal concentration of D-glucose (5.5 mmol/L) or to HG (16.7 mmol/L) in the absence or presence of NaHS for the indicated periods. The ICAM-1 protein and messenger RNA (mRNA) levels were analyzed by Western blotting and real-time reverse transcriptase-polymerase chain reaction, respectively. Exposure to HG for 48 or 72 hours significantly increased ICAM-1 expression at both the protein and mRNA levels, and these increases correlated with increases in both the production of intracellular reactive oxygen species and the activation of nuclear factor-κB. Pretreatment with NaHS inhibited HG-induced ICAM-1 expression at both the protein and mRNA levels and resulted in a reduction in the intracellular reactive oxygen species level and the suppression of nuclear factor-κB activity. NaHS also inhibited tumor necrosis factor-α-induced ICAM-1 protein expression, which was similar to the effect of antioxidant N-acetyl-L-cysteine. These findings indicate that H2S might protect against HG-induced vascular damage by down-regulating ICAM-1 expression in endothelial cells.

Lipid metabolism enzyme 5-LOX and its metabolite LTB4 are capable of activating transcription factor NF-κB in hepatoma cells.

The issue that lipid metabolism enzyme and its metabolites regulate transcription factors in cancer cell is not fully understood. In this study, we first report that the lipid metabolism enzyme 5-Lipoxygenase (5-LOX) and its metabolite leukotriene B4 (LTB4) are capable of activating nuclear factor-κB (NF-κB) in hepatoma cells. We found that the treatment of MK886 (an inhibitor of 5-LOX) or knockdown of 5-LOX was able to downregulate the expression of NF-κB p65 at the mRNA level and decreased the phosphorylation level of inhibitor κBα (IκBα) in the cytoplasm of hepatoma HepG2 or H7402 cells, which resulted in the decrease of the level of nuclear NF-κB p65. These were confirmed by immunofluorescence staining in HepG2 cell. Moreover, the above treatments were able to decrease the transcriptional activity of NF-κB in the cells. The LTB4, one of metabolites of 5-LOX, is responsible for 5-LOX-activated NF-κB in a dose-dependent manner. Thus, we conclude that the lipid metabolism enzyme 5-LOX and its metabolite LTB4 are capable of activating transcription factor NF-κB in hepatoma cells. Our finding provides new insight into the significance of lipid metabolism in activation of transcription factors in cancer.

Quantifying nuclear p65 as a parameter for NF-κB activation: Correlation between ImageStream cytometry, microscopy, and Western blot.

The nuclear factor kappa B (NF-κB) pathway, which regulates many cellular processes including proliferation, apoptosis, and survival, has emerged as an important therapeutic target in cancer. Activation of the NF-κB transcription factor is associated with nuclear translocation of the p65 component of the complex. Conventional methods employed to determine nuclear translocation of NF-κB either lack statistical robustness (microscopy) or the ability to discern heterogeneity within the sampled populations (Western blotting and Gel Shift assays). The ImageStream platform combines the high image content information of microscopy with the high throughput and multiparameter analysis of flow cytometry which overcomes the aforementioned limitations of conventional assays. It is demonstrated that ImageStream assessment of receptor-mediated (TNFα) and drug (Daunorubicin, DNR)-induced NF-κB translocation in leukemic cell lines correlates well with microscopy analysis and Western blot analysis. It is further demonstrated that ImageStream cytometry enables quantitative assessment of p65 translocation in immunophenotypically defined subpopulations; and that this assessment is highly reproducible. It is also demonstrated that, quantitatively, the DNR-induced nuclear translocation of NF-κB correlates well with a biological response (apoptosis). We conclude that the ImageStream has the potential to be a powerful tool to evaluate NF-κB /p65 activity as a determinant of response to therapies designed to target aberrant NF-κB signaling activities.

Ceftiofur impairs pro-inflammatory cytokine secretion through the inhibition of the activation of NF-kappaB and MAPK.

Ceftiofur is a new broad-spectrum, third-generation cephalosporin antibiotic for veterinary use. Immunopharmacological studies can provide new information on the immunomodulatory activities of some drugs, including their effect on cytokine productions. For this reason, we investigated the effect of ceftiofur on cytokine productions in vitro. We found that ceftiofur can downregulate tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and interleukin-6 (IL-6), but did not affect interleukin-10 (IL-10) production. We further investigated signal transduction mechanisms to determine how ceftiofur affects. RAW 264.7 cells were pretreated with 1, 5, or 10 mg/L of ceftiofur 1 h prior to treatment with 1 mg/L of LPS. Thirty minutes later, cells were harvested and mitogen activated protein kinases (MAPKs) activation was measured by Western blot. Alternatively, cells were fixed and nuclear factor-kappaB (NF-kappaB) activation was measured using immunocytochemical analysis. Signal transduction studies showed that ceftiofur significantly inhibited extracellular signal-regulated kinase (ERK), p38, and c-jun NH(2)-terminal kinase (JNK) phosphorylation protein expression. Ceftiofur also inhibited p65-NF-kappaB translocation into the nucleus. Therefore, ceftiofur may inhibit LPS-induced production of inflammatory cytokines by blocking NF-kappaB and MAPKs signaling in RAW264.7 cells.

Rhinovirus replication in human macrophages induces NF-kappaB-dependent tumor necrosis factor alpha production.

Rhinoviruses (RV) are the major cause of acute exacerbations of asthma and chronic obstructive pulmonary disease (COPD). Rhinoviruses have been shown to activate macrophages, but rhinovirus replication in macrophages has not been reported. Tumor necrosis factor alpha (TNF-alpha) is implicated in the pathogenesis of acute exacerbations, but its cellular source and mechanisms of induction by virus infection are unclear. We hypothesized that rhinovirus replication in human macrophages causes activation and nuclear translocation of NF-kappaB, leading to TNF-alpha production. Using macrophages derived from the human monocytic cell line THP-1 and from primary human monocytes, we demonstrated that rhinovirus replication was productive in THP-1 macrophages, leading to release of infectious virus into supernatants, but was limited in monocyte-derived macrophages, likely due to type I interferon production, which was robust in monocyte-derived but deficient in THP-1-derived macrophages. Similar to bronchial epithelial cells, only small numbers of cells supported complete virus replication. We demonstrated RV-induced activation of NF-kappaB and colocalization of p65/NF-kappaB nuclear translocation with virus replication in both macrophage types. The infection induced TNF-alpha release in a time- and dose-dependent, RV serotype- and receptor-independent manner and was largely (THP-1 derived) or completely (monocyte derived) dependent upon virus replication. Finally, we established the requirement for NF-kappaB but not p38 mitogen-activated protein kinase in induction of TNF-alpha. These data suggest RV infection of macrophages may be an important source of proinflammatory cytokines implicated in the pathogenesis of exacerbations of asthma and COPD. They also confirm inhibition of NF-kappaB as a promising target for development of new therapeutic intervention strategies.