The UVB-Stimulated Expression of Transglutaminase 1 Is Mediated Predominantly via the NFκB Signaling Pathway: New Evidence of Its Significant Attenuation through the Specific Interruption of the p38/MSK1/NFκBp65 Ser276 Axis

The influence of ultraviolet B (UVB) radiation on transglutaminase 1 (TGase 1), a major factor that regulates skin keratinization, has not been sufficiently characterized especially at the gene or protein level. Thus, we determined whether UVB affects the expression of TGase 1 in human keratinocytes and clarified the intracellular stress signaling mechanism(s) involved. Exposure of human keratinocytes to UVB significantly up-regulated the expression of TGase 1 at the gene and protein levels. Treatment with inhibitors of p38, MEK, JNK or NFκB significantly abolished the UVB-stimulated protein expression of TGase 1. Treatment with astaxanthin immediately after UVB irradiation did not attenuate the increased phosphorylation of Ser536/Ser468NFκBp65, c-Jun, ATK-2 and CK2, and did not abrogate the increased or diminished protein levels of c-Jun/c-Fos or I-κBα, respectively. However, the same treatment with astaxanthin significantly abolished the UVB-stimulated expression of TGase 1 protein, which was accompanied by the attenuated phosphorylation of Thr565/Ser376/Ser360MSK1, Ser276NFκBp65 and Ser133CREB. The MSK1 inhibitor H89 significantly down-regulated the increased protein expression of TGase 1 in UVB-exposed human keratinocytes, which was accompanied by an abrogating effect on the increased phosphorylation of Ser276NFκBp65 and Ser133CREB but not Thr565/Ser376/Ser360MSK1. Transfection of human keratinocytes with MSK1 siRNA suppressed the UVB-stimulated protein expression of TGase 1. These findings suggest that the UVB-stimulated expression of TGase 1 is mediated predominantly via the NFκB pathway and can be attenuated through a specific interruption of the p38/MSK1/NFκBp65Ser276 axis.     

Exploring genome-wide DNA methylation profiles altered in hepatocellular carcinoma using Infinium HumanMethylation 450 BeadChips

Hepatocellular carcinoma (HCC) incidence has increased in the US and also has one of the fastest growing death rates of any cancer. The purpose of the current study was to discover novel genome-wide aberrant DNA methylation patterns in HCC tumors that are predominantly HCV-related. Infinium HumanMethylation 450K BeadChip arrays were used to examine genome-wide DNA methylation profiles in 66 pairs of HCC tumor and adjacent non-tumor tissues. After Bonferroni adjustment, a total of 130,512 CpG sites significantly differed in methylation level in tumor compared with non-tumor tissues, with 28,017 CpG sites hypermethylated and 102,495 hypomethylated in tumor tissues. Absolute tumor/non-tumor methylation differences ≥ 20% were found in 24.9% of the hypermethylated and 43.1% of the hypomethylated CpG sites; almost 10,000 CpG sites have ≥ 30% DNA methylation differences. Most (60.1%) significantly hypermethylated CpG sites are located in CpG islands, with 21.6% in CpG shores and 3.6% in shelves. In contrast, only a small proportion (8.2%) of significantly hypomethylated CpG sites are situated in islands, while most are found in open sea (60.2%), shore (17.3%) or shelf (14.3%) regions. A total of 2,568 significant CpG sites (2,441 hypermethylated and 127 hypomethylated) covering 589 genes are located within 684 differentially methylated regions defined as regions with at least two significant CpG sites displaying > 20% methylation differences in the same direction within 250-bp. The top 500 significant CpG sites can significantly distinguish HCC tumor from adjacent tissues with one misclassification. Within adjacent non-tumor tissues, we also identified 75 CpG sites significantly associated with gender, 228 with HCV infection, 17,207 with cirrhosis, and 56 with both HCV infection and cirrhosis after multiple comparisons adjustment. Aberrant DNA methylation profiles across the genome were identified in tumor tissues from US HCC cases that are predominantly related to HCV infection. These results demonstrate the significance of aberrant DNA methylation in HCC tumorigenesis.     

WAVE3-NFκB Interplay Is Essential for the Survival and Invasion of Cancer Cells

The WAVE3 cytoskeletal protein promotes cancer invasion and metastasis. We have shown that the WAVE3-mediated activation of cancer cell invasion is due, in part, to its regulation of expression and activity of key metalloproteinases (MMPs), including MMP9, which is centrally involved in invadopodia-mediated degradation of the extracellular matrix (ECM). MMP9 is also a major NFκB target gene, suggesting a potential linkage of WAVE3 to this pathway, which we sought to investigate. Mechanistically, we found that loss of WAVE3 in cancer cells leads to inhibition of NFκB signaling as a result of a decrease in the nuclear translocation of NFκB and therefore loss of activation of NFκB target genes. Conversely, overexpression of WAVE3 was sufficient to enhance NFκB activity. Both pharmacologic and genetic manipulations of NFκB effector molecules show that the biological consequence of loss of WAVE3 function in the NFκB pathway result the inhibition of invadopodia formation and ECM degradation by cancer cells, and these changes are a consequence of decreased MMP9 expression and activity. Loss of WAVE3 also sensitized cancer cells to apoptosis and cell death driven by TNFα, through the inhibition of the AKT pro-survival pathway. Our results identify a novel function of WAVE3 in NFκB signaling, where its activity is essential for the regulation of invadopodia and ECM degradation. Therefore, targeted therapeutic inhibition of WAVE3 will sensitize cancer cells to apoptosis and cell death, and suppress cancer invasion and metastasis.     

A combination of Lox-1 and Nox1 regulates TLR9-mediated foam cell formation

The formation of foam cells is the hallmark of early atherosclerotic lesions, and the uptake of modified low-density lipoprotein (LDL) by macrophage scavenger receptors is thought to be a key process in their formation. In this study, we examined the role of lectin-like oxLDL receptor-1 (Lox-1) and NADPH oxidase 1 (Nox1) in toll-like receptor 9 (TLR9)-mediated foam cell formation. TLR9 activation of Raw264.7 cells or mouse primary peritoneal macrophages by CpG ODN treatment enhanced Lox-1 gene and protein expression. In addition, CpG ODN-induced Nox1 mRNA expression, which in turn increased foam cell formation. The inhibition of CpG ODN-induced reactive oxygen species (ROS) generation by treatment with antioxidants, as well as with knockdown of Nox1 using siRNA, suppressed the formation of foam cells. The induction of Lox-1 and Nox1 by CpG ODN was regulated via the TLR9-p38 MAPK signaling pathway. CpG ODN also increased NFκB activity, and a potent inhibitor of NFκB that significantly blocked CpG-induced Nox1 expression, suggesting that Nox1 regulation is mediated through an NFκB-dependent mechanism. Taken together, these results suggest that a combination of Lox-1 and Nox1 plays a key role in the TLR9-mediated formation of foam cells via the p38 MAPK pathway.     

The Nexus between VEGF and NFκB Orchestrates a Hypoxia-Independent Neovasculogenesis

Nuclear Factor-Kappa B [NFκB] activation triggers the elevation of various pro-angiogenic factors that contribute to the development and progression of diabetic vasculopathies. It has been demonstrated that vascular endothelial growth factor [VEGF] activates NFκB signaling pathway. Under the ischemic microenvironments, hypoxia-inducible factor-1 [HIF-1] upregulates the expression of several proangiogenic mediators, which play crucial roles in ocular pathologies. Whereas YC-1, a soluble guanylyl cyclase [sGC] agonist, inhibits HIF-1 and NFκB signaling pathways in various cell and animal models. Throughout this investigation, we examined the molecular link between VEGF and NFκB under a hypoxia-independent microenvironment in human retinal microvascular endothelial cells [hRMVECs]. Our data indicate that VEGF promoted retinal neovasculogenesis via NFκB activation, enhancement of its DNA-binding activity, and upregulating NFκB/p65, SDF-1, CXCR4, FAK, αVβ3, α5β1, EPO, ET-1, and MMP-9 expression. Conversely, YC-1 impaired the activation of NFκB and its downstream signaling pathways, via attenuating IκB kinase phosphorylation, degradation and activation, and thus suppressing p65 phosphorylation, nuclear translocation, and inhibiting NFκB-DNA binding activity. We report for the first time that the nexus between VEGF and NFκB is implicated in coordinating a scheme that upregulates several pro-angiogenic molecules, which promotes retinal neovasculogenesis. Our data may suggest the potential use of YC-1 to attenuate the deleterious effects that are associated with hypoxia/ischemia-independent retinal vasculopathies.     

Bindarit: An anti-inflammatory small molecule that modulates the NFκB pathway

The activation of nuclear factor (NF)κB pathway and its transducing signaling cascade has been associated with the pathogenesis of many inflammatory diseases. The central role that IκBα and p65 phosphorylation play in regulating NFκB signalling in response to inflammatory stimuli made these proteins attractive targets for therapeutic strategies. Although several chemical classes of NFκB inhibitors have been identified, it is only for a few of those that a safety assessment based on a comprehensive understanding of their pharmacologic mechanism of action has been reported. Here, we describe the specific anti-inflammatory effect of bindarit, an indazolic derivative that has been proven to have anti-inflammatory activity in a variety of models of inflammatory diseases, including lupus nephritis, arthritis and pancreatitis. The therapeutic effects of bindarit have been associated with its ability to selectively interfere with monocyte recruitment and the “early inflammatory response,” although its specific molecular mechanisms have remained ill-defined. For this purpose, we investigated the effect of bindarit on the LPS-induced production of inflammatory cytokines (MCP-1 and MCPs, IL-12β/p40, IL-6 and IL-8/KC) in both a mouse leukaemic monocyte-macrophage cell line and bone marrow-derived macrophages (BMDM). Bindarit inhibits the LPS-induced MCP-1 and IL-12β/p40 expression without affecting other analyzed cytokines. The effect of bindarit is mediated by the downregulation of the classical NFκB pathway, involving a reduction of IκBα and p65 phosphorylation, a reduced activation of NFκB dimers and a subsequently reduced nuclear translocation and DNA binding. Bindarit showed a specific inhibitory effect on the p65 and p65/p50 induced MCP-1 promoter activation, with no effect on other tested activated promoters. We conclude that bindarit acts on a specific subpopulation of NFκB isoforms and selects its targets wihtin the whole NFκB inflammatory pathway. These findings pave the way for future applications of bindarit as modulator of the inflammatory response.Key words: inflammation, NFκB, MCP-1, IL-12β/p40, macrophages, lipopolysaccharide, bindarit     

TWEAK Activates the Non-Canonical NFκB Pathway in Murine Renal Tubular Cells: Modulation of CCL21

TWEAK is a member of the TNF superfamily of cytokines that contribute to kidney tubulointerstitial injury. It has previously been reported that TWEAK induces transient nuclear translocation of RelA and expression of RelA-dependent cytokines in renal tubular cells. Additionally, TWEAK induced long-lasting NFκB activation suggestive of engagement of the non-canonical NFκB pathway. We now explore TWEAK-induced activation of NFκB2 and RelB, as well as expression of CCL21, a T-cell chemotactic factor, in cultured murine tubular epithelial cells and in healthy kidneys in vivo. In cultured tubular cells, TWEAK and TNFα activated different DNA-binding NFκB complexes. TWEAK-induced sustained NFκB activation was associated with NFκB2 p100 processing to p52 via proteasome and nuclear translocation and DNA-binding of p52 and RelB. TWEAK, but not TNFα used as control), induced a delayed increase in CCL21a mRNA (3.5±1.22-fold over control) and CCL21 protein (2.5±0.8-fold over control), which was prevented by inhibition of the proteasome, or siRNA targeting of NIK or RelB, but not by RelA inhibition with parthenolide. A second NFκB2-dependent chemokine, CCL19, was upregulates by TWEAK, but not by TNFα. However, both cytokines promoted chemokine RANTES expression (3-fold mRNA at 24 h). In vivo, TWEAK induced nuclear NFκB2 and RelB translocation and CCL21a mRNA (1.5±0.3-fold over control) and CCL21 protein (1.6±0.5-fold over control) expression in normal kidney. Increased tubular nuclear RelB and tubular CCL21 expression in acute kidney injury were decreased by neutralization (2±0.9 vs 1.3±0.6-fold over healthy control) or deficiency of TWEAK (2±0.9 vs 0.8±0.6-fold over healthy control). Moreover, anti-TWEAK treatment prevented the recruitment of T cells to the kidney in this model (4.1±1.4 vs 1.8±1-fold over healthy control). Our results thus identify TWEAK as a regulator of non-canonical NFκB activation and CCL21 expression in tubular cells thus promoting lymphocyte recruitment to the kidney during acute injury.     

基于SNP共表达网络肝癌分子分型及预后分析

基于SNP突变数据与mRNA表达谱关联分析,构建一种肝癌分子分型方法并对比不同分型预后的差异,并对不同分型肝癌的发生发展机制进一步研究。首先通过TCGA数据库收集359例肝细胞癌患者的SNP突变数据和mRNA表达数据,采用Wilcoxon秩和检验,筛选突变后差异表达基因,并通过生物信息学工具String和Cytoscape构建差异表达基因的蛋白互作网络,筛选连接度最高的10个Hub基因。利用Consensus Cluster Plus软件包,基于Hub基因mRNA表达水平构建NMF分子分型模型,再结合生存数据评估各分型患者的预后。最后利用加权基因共表达网络分析(WGCNA),识别与肝癌分子分型相关的模块,并针对关键模块的基因进行通路富集,从而对不同分型肝癌的基因表达谱进行比较。结果：NMF模型将肝癌分为高危、低危2个分型,其中CDKN2A和FOXO1基因对分型贡献度高。生存分析显示低危组患者的生存情况显著优于高危组,高危组富集多个与肿瘤细胞侵蚀、转移、复发过程相关的信号通路,低危组则与细胞周期和胰液分泌相关。本研究在无先验性信息的前提下,基于突变后显著差异表达的Hub基因表达水平构建的...     

The IKK Inhibitor Bay 11-7082 Induces Cell Death Independent from Inhibition of Activation of NFκB Transcription Factors

Multiple myeloma (MM) displays an NFκB activity-related gene expression signature and about 20% of primary MM samples harbor genetic alterations conducive to intrinsic NFκB signaling activation. The relevance of blocking the classical versus the alternative NFκB signaling pathway and the molecular execution mechanisms involved, however, are still poorly understood. Here, we comparatively tested NFκB activity abrogation through TPCA-1 (an IKK2 inhibitor), BAY 11-7082 (an IKK inhibitor poorly selective for IKK1 and IKK2), and MLN4924 (an NEDD8 activating enzyme (NAE)-inhibitor), and analyzed their anti-MM activity. Whereas TPCA-1 interfered selectively with activation of the classical NFκB pathway, the other two compounds inhibited classical and alternative NFκB signaling without significant discrimination. Noteworthy, whereas TPCA-1 and MLN4924 elicited rather mild anti-MM effects with slight to moderate cell death induction after 1 day BAY 11-7082 was uniformly highly toxic to MM cell lines and primary MM cells. Treatment with BAY 11-7082 induced rapid cell swelling and its initial effects were blocked by necrostatin-1 or the ROS scavenger BHA, but a lasting protective effect was not achieved even with additional blockade of caspases. Because MLN4924 inhibits the alternative NFκB pathway downstream of IKK1 at the level of p100 processing, the quite discordant effects between MLN4924 and BAY 11-7082 must thus be due to blockade of IKK1-mediated NFκB-independent necrosis-inhibitory functions or represent an off-target effect of BAY 11-7082. In accordance with the latter, we further observed that concomitant knockdown of IKK1 and IKK2 did not have any major short-term adverse effect on the viability of MM cells.     

Dimethyl Fumarate Inhibits the Nuclear Factor κB Pathway in Breast Cancer Cells by Covalent Modification of p65 Protein

In breast tumors, activation of the nuclear factor κB (NFκB) pathway promotes survival, migration, invasion, angiogenesis, stem cell-like properties, and resistance to therapy—all phenotypes of aggressive disease where therapy options remain limited. Adding an anti-inflammatory/anti-NFκB agent to breast cancer treatment would be beneficial, but no such drug is approved as either a monotherapy or adjuvant therapy. To address this need, we examined whether dimethyl fumarate (DMF), an anti-inflammatory drug already in clinical use for multiple sclerosis, can inhibit the NFκB pathway. We found that DMF effectively blocks NFκB activity in multiple breast cancer cell lines and abrogates NFκB-dependent mammosphere formation, indicating that DMF has anti-cancer stem cell properties. In addition, DMF inhibits cell proliferation and significantly impairs xenograft tumor growth. Mechanistically, DMF prevents p65 nuclear translocation and attenuates its DNA binding activity but has no effect on upstream proteins in the NFκB pathway. Dimethyl succinate, the inactive analog of DMF that lacks the electrophilic double bond of fumarate, is unable to inhibit NFκB activity. Also, the cell-permeable thiol N-acetyl l-cysteine, reverses DMF inhibition of the NFκB pathway, supporting the notion that the electrophile, DMF, acts via covalent modification. To determine whether DMF interacts directly with p65, we synthesized and used a novel chemical probe of DMF by incorporating an alkyne functionality and found that DMF covalently modifies p65, with cysteine 38 being essential for the activity of DMF. These results establish DMF as an NFκB inhibitor with anti-tumor activity that may add therapeutic value in the treatment of aggressive breast cancers.     

TRAF2 recruitment via T61 in CD30 drives NFκB activation and enhances hESC survival and proliferation

CD30 (TNFRSF8), a tumor necrosis factor receptor family protein, and CD30 variant (CD30v), a ligand-independent form encoding only the cytoplasmic signaling domain, are concurrently overexpressed in transformed human embryonic stem cells (hESCs) or hESCs cultured in the presence of ascorbate. CD30 and CD30v are believed to increase hESC survival and proliferation through NFκB activation, but how this occurs is largely unknown. Here we demonstrate that hESCs that endogenously express CD30v and hESCs that artificially overexpress CD30v exhibit increased ERK phosphorylation levels, activation of the canonical NFκB pathway, down-regulation of the noncanonical NFκB pathway, and reduced expression of the full-length CD30 protein. We further find that CD30v, surprisingly, resides predominantly in the nucleus of hESC. We demonstrate that alanine substitution of a single threonine residue at position 61 (T61) in CD30v abrogates CD30v-mediated NFκB activation, CD30v-mediated resistance to apoptosis, and CD30v-enhanced proliferation, as well as restores normal G₂/M-checkpoint arrest upon H₂O₂ treatment while maintaining its unexpected subcellular distribution. Using an affinity purification strategy and LC-MS, we identified TRAF2 as the predominant protein that interacts with WT CD30v but not the T61A-mutant form in hESCs. The identification of Thr-61 as a critical residue for TRAF2 recruitment and canonical NFκB signaling by CD30v reveals the substantial contribution that this molecule makes to overall NFκB activity, cell cycle changes, and survival in hESCs.     

Aberrant CpG methylation of the TFAP2A gene constitutes a mechanism for loss of TFAP2A expression in human metastatic melanoma

Metastatic melanoma is a deadly treatment-resistant form of skin cancer whose global incidence is on the rise. During melanocyte transformation and melanoma progression the expression profile of many genes changes. Among these, a gene implicated in several steps of melanocyte development, TFAP2A, is frequently silenced; however, the molecular mechanism of TFAP2A silencing in human melanoma remains unknown. In this study, we measured TFAP2A mRNA expression in primary human melanocytes compared to 11 human melanoma samples by quantitative real-time RT-PCR. In addition, we assessed CpG DNA methylation of the TFAP2A promoter in these samples using bisulfite sequencing. Compared to primary melanocytes, which showed high TFAP2A mRNA expression and no promoter methylation, human melanoma samples showed decreased TFAP2A mRNA expression and increased promoter methylation. We further show that increased CpG methylation correlates with decreased TFAP2A mRNA expression. Using The Cancer Genome Atlas, we further identified TFAP2A as a gene displaying among the most decreased expression in stage 4 melanomas vs. non-stage 4 melanomas, and whose CpG methylation was frequently associated with lack of mRNA expression. Based on our data, we conclude that TFAP2A expression in human melanomas can be silenced by aberrant CpG methylation of the TFAP2A promoter. We have identified aberrant CpG DNA methylation as an epigenetic mark associated with TFAP2A silencing in human melanoma that could have significant implications for the therapy of human melanoma using epigenetic modifying drugs.     

Aberrant CpG methylation of the TFAP2A gene constitutes a mechanism for loss of TFAP2A expression in human metastatic melanoma

Metastatic melanoma is a deadly treatment-resistant form of skin cancer whose global incidence is on the rise. During melanocyte transformation and melanoma progression the expression profile of many genes changes. Among these, a gene implicated in several steps of melanocyte development, TFAP2A, is frequently silenced; however, the molecular mechanism of TFAP2A silencing in human melanoma remains unknown. In this study, we measured TFAP2A mRNA expression in primary human melanocytes compared to 11 human melanoma samples by quantitative real-time RT-PCR. In addition, we assessed CpG DNA methylation of the TFAP2A promoter in these samples using bisulfite sequencing. Compared to primary melanocytes, which showed high TFAP2A mRNA expression and no promoter methylation, human melanoma samples showed decreased TFAP2A mRNA expression and increased promoter methylation. We further show that increased CpG methylation correlates with decreased TFAP2A mRNA expression. Using The Cancer Genome Atlas, we further identified TFAP2A as a gene displaying among the most decreased expression in stage 4 melanomas vs. non-stage 4 melanomas, and whose CpG methylation was frequently associated with lack of mRNA expression. Based on our data, we conclude that TFAP2A expression in human melanomas can be silenced by aberrant CpG methylation of the TFAP2A promoter. We have identified aberrant CpG DNA methylation as an epigenetic mark associated with TFAP2A silencing in human melanoma that could have significant implications for the therapy of human melanoma using epigenetic modifying drugs.