Kang HB, Kim YE, Kwon HJ, Sok DE, Lee Y.. Enhancement of NF-kappaB expression and activity upon differentiation of human embryonic stem cell line SNUhES3. Stem Cells Dev 16: 615-623
ABSTRACT NF-kappaB is involved in many biological processes including proliferation, survival, and differentiation. Because human embryonic stem (ES) cells have the potential to differentiate to various lineages, understanding mechanisms involved in stemness and lineage differentiation is an important issue. We investigated expression of NF-kappaB in the human ES cell lines SNUhES3 and MizhES4 and found that expression of NF-kappaB mRNA and protein in these two cell lines was significantly lower compared to those of other adult cell lines. However, when SNUhES3 cells were induced to differentiate by retinoic acid, expression levels of NF-kappaB significantly increased compared to undifferentiated SNUhES3 cells. As the components of tumor necrosis factor-alpha (TNF-alpha) signaling are expressed comparably in undifferentiated and differentiated SNUhES3 cells, we examined the responsiveness of SNUhES3 cells to treatment with TNF-alpha, an agonist of NF-kappaB signaling. Nuclear localization of NF-kappaB in response to TNF-alpha was evident in differentiated, but not undifferentiated, SNUhES3 cells. In agreement with this observation, induction of interleukin-8 (IL-8) in response to TNF-alpha was seen only in differentiated SNUhES3 cells. On the basis of an IkappaB kinase (IKK) inhibitor study, expression of IL-8 induced by TNF-alpha was dependent on NF-kappaB activity. Taken together, our results suggest that expression and activity of NF-kappaB is comparatively low in undifferentiated human ES cells, but increases during differentiation of the ES cells.
- SourceAvailable from: Rodney P. Dekoter
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- "Results are of three experiments, each conducted in duplicate. determine the relative amount of immunoprecipitated DNA from the PAD4 promoter region, the IL8 promoter region with which p50 and p65 are known to interact with as a positive control, and the negative control C4ORF11 (Fig. 5; Kang et al., 2007). ChIP analysis confirmed that p65 was highly enriched at the IL8 promoter but not significantly enriched at the PAD4 promoter upon TNF-α stimulation (Fig. 5A, B). "
ABSTRACT: High titers of anti-citrullinated protein/peptide antibodies (ACPAs) have been detected in sera of rheumatoid arthritis (RA) patients, implicating citrullinating enzymes in the pathogenesis of RA. Peptidylarginine deiminase type IV (PAD4) is a member of the PAD family of citrullinating enzymes and has been linked to RA. Therefore, our aim was to determine how transcription of PAD4 is regulated in the human myeloid lineage. We located the PAD4 transcription start site and promoter and phylogenetic comparisons of the area identified a 200bp conserved region. Bioinformatics analysis predicted the presence of a NF-κB binding site and we tested this via luciferase assays. Intriguingly, mutation of the predicted NF-κB site significantly increased biological activity. We used RT-qPCR to quantify PAD4 expression in HL-60 cells treated with TNF-α to activate the canonical NF-κB pathway and found that PAD4 mRNA was reduced in response to TNF-α treatment. Finally, we used chromatin immunoprecipitation (ChIP) to determine NF-κB enrichment at the PAD4 promoter and the p50 subunit of NF-κB was more highly enriched than p65 at the PAD4 promoter. These results suggest that the p50 subunit of NF-κB may play a role in the repression of PAD4 transcription during inflammation.Gene 10/2013; 533(1). DOI:10.1016/j.gene.2013.09.108 · 2.14 Impact Factor
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- "IL-6 and IL-8 are cytokines involved in infection and immunity, inflammation, autoimmune diseases and cancer  . Both cytokines are transcribed by the nuclear factor k B (NFkB) complex in response to bacterial lipopolysaccharides (LPS) and tumor necrosis factor-alpha (TNF-alpha)  . In a recent report IL-6 and IL-8 over expression in the epithelial lung cancer cell line H460a was identified as driver of an in vivo resistance "
ABSTRACT: We previously identified the tumor suppressor INT6/eIF3e as a novel down regulator of HIF2α. Small interfering RNA targeting Int6 (siRNA-Int6) in HeLa cells led to normoxic stabilization of HIF2α, with concomitant transcription of angiogenic factors, including angiopoietin, basic fibroblast growth factor, and vascular endothelial growth factor. Here we used HIF2α normoxic up-regulation via Int6 silencing to investigate the role of HIF2α in endothelial cells. As a result Int6 silencing in human umbilical vein endothelial cells (HUVECs) and in human aortic endothelial cells (HAECs) led to robust enhanced cord formation and the medium supernatant of Int6 silenced HUVECs enhanced migration of untreated HUVECs, indicating a HIF2α triggered secretory signaling. Within the responsible genes were the cytokines interleukin-6 (IL-6) and IL-8 and not unlike in HeLa cells vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF) and epidermal growth factor (EGF). In addition application of IL-6 and IL-8 antibodies to the medium of Int6 silenced HUVECs could reverse the enhanced migration effect and also abrogated their tube formation. Finally, a CHIP assay analysis confirmed hypoxia-responsive elements (HREs) in the IL-6 and IL-8 promoters. Our results demonstrate that expression of both IL-6 and IL-8 is regulated by HIF2α and we suggest that IL-6 and IL-8 are HIF2α controlled cytokines for angiogenesis particularly in endothelial cells.Cytokine 03/2013; 62(1). DOI:10.1016/j.cyto.2013.01.021 · 2.66 Impact Factor
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- "It has been shown that expression of NFκB significantly increased following differentiation of embryonic stem (ES) cells with retinoic acid. Additionally, nuclear localization of NFκB in response to TNFα, an agonist of NFκB signaling, was evident in differentiated, but not in undifferentiated ES cells . Similar results have been obtained by other authors . "
ABSTRACT: Cancer initiating cells have been described to be the only cell population with tumorigenic capacity in glioblastoma multiforme, one of the most aggressive and untreatable cancers. Recent work from our group described that NFκB pathway was activated in glioblastoma initiating cells undergoing differentiation, and that blockade of this activation promoted senescence of differentiating cells. NFκB activation in cancer may be the result of either exposure to proinflammatory stimuli in the tumor microenvironment or upregulation of the signaling pathway by upstream regulators. Appropriate control of NFκB activity, which can be achieved by gene modification or pharmacological strategies, would provide a potential approach for the management of NFκB related tumors, including glioblastoma. Here, we summarize the current knowledge of the relevance of NFκB in cancer and its possible role as a target of therapeutic intervention..Oncotarget 09/2011; 2(8):646-53. DOI:10.18632/oncotarget.322 · 6.36 Impact Factor