TNFα-induced Gene Research Articles

TNFα increases the degradation of pyruvate dehydrogenase kinase 4 by the Lon protease to support proinflammatory genes

The endothelium is a major target of the proinflammatory cytokine, tumor necrosis factor alpha (TNFα). Exposure of endothelial cells (EC) to proinflammatory stimuli leads to an increase in mitochondrial metabolism; however, the function and regulation of elevated mitochondrial metabolism in EC in response to proinflammatory cytokines remain unclear. Studies using high-resolution metabolomics and 13C-glucose and 13C-glutamine labeling flux techniques showed that pyruvate dehydrogenase activity (PDH) and oxidative tricarboxylic acid cycle (TCA) flux are elevated in human umbilical vein ECs in response to overnight (16 h) treatment with TNFα (10 ng/mL). Mechanistic studies indicated that TNFα mediated these metabolic changes via mitochondrial-specific protein degradation of pyruvate dehydrogenase kinase 4 (PDK4, inhibitor of PDH) by the Lon protease via an NF-κB-dependent mechanism. Using RNA sequencing following siRNA-mediated knockdown of the catalytically active subunit of PDH, PDHE1α (PDHA1 gene), we show that PDH flux controls the transcription of approximately one-third of the genes that are up-regulated by TNFα stimulation. Notably, TNFα-induced PDH flux regulates a unique signature of proinflammatory mediators (cytokines and chemokines) but not inducible adhesion molecules. Metabolomics and ChIP sequencing for acetylated modification on lysine 27 of histone 3 (H3K27ac) showed that TNFα-induced PDH flux promotes histone acetylation of specific gene loci via citrate accumulation and ATP-citrate lyase-mediated generation of acetyl CoA. Together, these results uncover a mechanism by which TNFα signaling increases oxidative TCA flux of glucose to support TNFα-induced gene transcription through extramitochondrial acetyl CoA generation and histone acetylation.

Late phase endothelial cell inflammation is characterized by interferon response genes and driven by JAK/STAT, not NFκB

Chronic vascular inflammation underlies many diseases, including atherosclerosis, autoimmune vasculitides and transplant rejection. The resolution of inflammation is critical for proper healing and restoration of homeostasis, but the timing and signaling mechanisms involved in the return to a non-inflamed state are not well understood. Pro-adhesive gene expression, phenotype and secretome of human endothelial cells was measured in primary human aortic endothelium under chronic TNFα stimulation, and after short-term TNFα priming followed by withdrawal. The effects of NFκB, MAPK and JAK1/2 inhibitors on TNFα-induced gene expression were tested. The majority of inducible TNFα effectors, such as E-selectin, VCAM-1 and most chemokines, required continuous exposure for reinforcement of the altered phenotype, and were NFκB dependent. However, 3 h priming with TNFα induced late phase STAT activation and interferon response genes after 18 h, as well as enhanced ICAM-1, BST2 and CXCR3 ligand expression. Chronic activation was autonomous of continuous TNFα, and could be blocked by the JAK1/2 inhibitor ruxolitinib. The results demonstrate that NFκB is not a significant driver of the later phase of endothelial cell activation by TNFα, but that sustained inflammation is JAK1/2-dependent and characterized by adaptive chemokines.

Human Lung Fibroblasts Exhibit Induced Inflammation Memory via Increased IL6 Gene Expression and Release.

Fibroblasts of different origins are known to possess stromal memory after inflammatory episodes. However, there are no studies exploring human lung fibroblast memory which may predict a subsequent inflammatory response in chronic respiratory diseases and COVID-19. MRC-5 and HF19 human lung fibroblast cell lines were treated using different primary and secondary stimulus combinations: TNFα–WD–TNFα, Poly (I:C)–WD–TNFα, TNFα–WD–Poly (I:C), or LPS–WD–TNFα with a 24-h rest period (withdrawal period; WD) between the two 24-h stimulations. TLR3 and NF-κB inhibitors were used to determine pathways involved. The effect of SARS-Cov-2 spike protein to inflammatory response of lung fibroblasts was also investigated. mRNA expressions of genes and IL6 release were measured using qRT-PCR and ELISA, respectively. Statistical significance was determined by using one- or two-way ANOVA, followed by Bonferroni’s post hoc analysis for comparison of multiple groups. Preexposure with Poly (I:C) significantly increased TNFα-induced IL6 gene expression and IL6 release in both cell lines, while it affected neither gene expressions of IL1B, IL2, IL8, and MMP8 nor fibrosis-related genes: ACTA2, COL1A1, POSTN, and TGFB1. Inhibition of TLR3 or NF-κB during primary stimulation significantly downregulated IL6 release. Simultaneous treatment of MRC-5 cells with SARS-CoV-2 spike protein further increased TNFα-induced IL6 release; however, preexposure to Poly (I:C) did not affect it. Human lung fibroblasts are capable of retaining inflammatory memory and showed an augmented response upon secondary exposure. These results may contribute to the possibility of training human lung fibroblasts to respond suitably on inflammatory episodes after viral infection.

Abstract 203: Synergism Of TNFα And IFNγ Elicits Transcriptional Cooperativity At The CXCL9,-10,-11 Locus In Human Endothelial Cells And Is Ultrasensitive To BRD4 And CBP/P300 Inhibition.

Background/Rationale: Combinatorial signaling by proinflammatory cytokines drives the pathogenesis of many cardiovascular diseases ranging from acute SARS-COV-2 vascular complications to chronic atherosclerosis. TNFα and IFNγ synergistically induce genes that amplify inflammatory cascades, causing tissue damage and high mortality in humans. Characterization of transcriptional mechanisms governing synergistic gene induction holds the promise of revealing novel therapeutic targets while also improving our understanding of how inflammatory states are propagated. Objective: To delineate unique mechanisms of synergistic gene transcription in human aortic endothelial cells (HAECs) exposed to high levels of both TNFα and IFNγ. Methods & Result: Using RNA-sequencing, we defined the dynamic transcriptome in HAECs stimulated with TNFα, IFNγ, or combination for one hour. We devised a novel statistical approach that generates a positive cooperativity score (PCS) for each gene in order to define the TNFα/IFNγ synergy signature in HAECs. This approach revealed the chemokines CXCL9, -10, -11 as highly synergistic in their expression (PCS=61.9, 18.3, 8.9; log 2 [Combination/No Treatment]=8.1, 2.1, 3.1 respectively), in contrast to SELE , a TNFα-induced gene (PCS=0.4; log 2 [Combination/No Treatment]=6.6). Furthermore, pathway analysis revealed strong enrichment for chemotaxis programs among the synergy gene set. To deconvolute transcriptional coactivator dependencies at these exemplary genes, we conducted a targeted mini-screen of transcriptional inhibitors along with biochemical modelling of inhibitor dose-response curves. We discover that CXCL9, -10, -11 induction is sensitively disrupted by inhibition of BRD4 and CBP/p300 (hill coefficient (HC) > 2) coactivators as compared to SELE (HC<2). Conclusions: These results provide definitive evidence of acute transcriptional synergy at the chemokine genes CXCL9, -10, -11 elicited by TNFα/IFNγ activity in HAECs. As these chemokines are associated with poor outcomes in sepsis, these results also implicate targeted inhibition of transcriptional coactivators as a potential therapeutic strategy for inflammatory cardiovascular diseases.

The HSP90 inhibitor RGRN‐305 exhibits strong immunomodulatory effects in human keratinocytes

Keratinocytes are the key cellular target for IL-17A-mediated effects in psoriasis and HSP90 is important for IL-17A-mediated signalling. RGRN-305 is a novel HSP90 inhibitor reported to reduce psoriatic phenotypes in preclinical animal models. The aim of this study was to investigate the effect of RGRN-305 on a psoriasis-like inflammatory response in human keratinocytes in vitro. Using RT-qPCR, we demonstrated a significantly increased expression of the HSP90 isoforms HSP90AB1, HSP90B1 and TRAP1 in lesional compared with non-lesional psoriatic skin. In a psoriasis-like setting where keratinocytes were stimulated with TNFα and/or IL-17A, we analysed the mRNA expression using the NanoString nCounter technology and demonstrated that the HSP90 inhibitor RGRN-305 significantly reduced the IL-17A- and TNFα-induced gene expression of a number of proinflammatory genes, including the psoriasis-associated genes CCL20, NFKBIZ, IL36G and IL23A. In agreement with the mRNA data, the protein level of CCL20, IκBζ and IL-36γ were inhibited by RGRN-305 as demonstrated by western blotting and ELISA. Interestingly, when keratinocytes were stimulated with a TLR3 agonist, RGRN-305 also demonstrated potent immunomodulatory effects, significantly inhibiting poly(I:C)-induced expression of the proinflammatory genes TNFα, IL1B, IL6 and IL23A. Taken together, our data support a role for HSP90 not only in the pathogenesis of psoriasis, but also in broader immune responses. Therefore, HSP90 provides an attractive target for the treatment of psoriasis and other diseases where the innate immune system plays an important role.

Transcription factor EGR-1 transactivates the MMP1 gene promoter in response to TNFα in HaCaT keratinocytes

Matrix metalloproteinase 1 (MMP-1), a calcium-dependent zinc- containing collagenase, is involved in the initial degradation of native fibrillar collagen. Tissue necrosis factor-alpha (TNFα) is a pro-inflammatory cytokine that is rapidly produced by dermal fibroblasts, monocytes/macrophages, and keratinocytes and regulates inflammation and damaged-tissue remodeling. MMP-1 is induced by TNFα and plays a critical role in tissue remodeling and skin aging processes. However, the regulation of the MMP1 gene by TNFα is not fully understood. We aimed to find additional cis-acting elements involved in the regulation of TNFα-induced MMP1 gene transcription in addition to the nuclear factor-kappa B (NF-kB) and activator protein 1 (AP1) sites. Assessments of the 5’-regulatory region of the MMP1 gene, using a series of deletion constructs, revealed the requirement of the early growth response protein 1 (EGR-1)-binding sequence (EBS) in the proximal region for proper transcription by TNFα. Ectopic expression of EGR-1, a zinc-finger transcription factor that binds to G-C rich sequences, stimulated MMP1 promoter activity. The silencing of EGR-1 by RNA interference reduced TNFα-induced MMP-1 expression. EGR-1 directly binds to the proximal region and transactivates the MMP1 gene promoter. Mutation of the EBS within the MMP1 promoter abolished EGR-1-mediated MMP-1 promoter activation. These data suggest that EGR-1 is required for TNFα-induced MMP1 transcriptional activation. In addition, we found that all three MAPKs, ERK1/2, JNK, and p38 kinase, mediate TNFα-induced MMP-1 expression via EGR-1 upregulation. These results suggest that EGR-1 may represent a good target for the development of pharmaceutical agents to reduce inflammation-induced MMP-1 expression.

The CSF-1-receptor inhibitor, JNJ-40346527 (PRV-6527), reduced inflammatory macrophage recruitment to the intestinal mucosa and suppressed murine T cell mediated colitis.

Background & aimsOriginally believed to be primarily a disorder of T-cell signaling, evidence shows that macrophage-lineage cells also contribute to the pathogenesis of Crohn’s disease (CD). Colony stimulating factor-1 (CSF-1) is a key regulator of the macrophage lineage, but its role in CD has not been well established. We examined transcriptional data from CD mucosa for evidence of CSF-1 pathway activation and tested JNJ-40346527 (PRV-6527), a small molecule inhibitor of CSF-1 receptor kinase (CSF-1R), for its ability to inhibit disease indices in murine colitis.MethodsA CSF-1 pathway gene set was created from microarray data of human whole blood cultured ex vivo with CSF-1 and compared to a TNFα-induced gene set generated from epithelial-lineage cells. Gene set variation analysis was performed using existing Crohn’s mucosa microarray data comparing patients who either responded or failed to respond to anti-TNFα therapy. Commencing day 14 or day 21, mice with T-cell transfer colitis were treated with vehicle or JNJ-40346527 until study termination (day 42). Endpoints included colon weight/length ratios and histopathology scores, and macrophage and T cells were assessed by immunohistochemistry. Mucosal gene expression was investigated using RNAseq.ResultsBoth the CSF-1 and the TNFα gene sets were enriched in the colonic mucosal transcriptomes of Crohn’s disease and in mouse colitis, and expression of both gene sets was highest in patients who did not respond to anti-TNFα therapy. In these patients neither set was reduced by therapy. In the mouse model, JNJ-40346527 inhibited the increase in colon weight/length ratio by ∼50%, reduced histological disease scores by ∼60%, and reduced F4/80+ mononuclear cell and CD3+ lymphocyte numbers. RNAseq analysis confirmed the CSF-1 gene set was sharply reduced in treated mice, as were gene sets enriched in “M1” inflammatory and “M0” resident macrophages and in activated T cells.ConclusionsCSF-1 biology is activated in Crohn’s disease and in murine T cell transfer colitis. Inhibition of CSF-1R by JNJ-40346527 was associated with attenuated clinical disease scores and reduced inflammatory gene expression in mice. These data provide rationale for testing JNJ-40346527 (PRV-6527) in human inflammatory bowel disease.

Chemotherapeutic Drugs Inhibiting Topoisomerase 1 Activity Impede Cytokine-Induced and NF-κB p65-Regulated Gene Expression.

Inhibitors of DNA topoisomerase I (TOP1), an enzyme relieving torsional stress of DNA by generating transient single-strand breaks, are clinically used to treat ovarian, small cell lung and cervical cancer. As torsional stress is generated during transcription by progression of RNA polymerase II through the transcribed gene, we tested the effects of camptothecin and of the approved TOP1 inhibitors Topotecan and SN-38 on TNFα-induced gene expression. RNA-seq experiments showed that inhibition of TOP1 but not of TOP2 activity suppressed the vast majority of TNFα-triggered genes. The TOP1 effects were fully reversible and preferentially affected long genes. TNFα stimulation led to inducible recruitment of TOP1 to the gene body of IL8, where its inhibition by camptothecin reduced transcription elongation and also led to altered histone H3 acetylation. Together, these data show that TOP1 inhibitors potently suppress expression of proinflammatory cytokines, a feature that may contribute to the increased infection risk occurring in tumor patients treated with these agents. On the other hand, TOP1 inhibitors could also be considered as a therapeutic option in order to interfere with exaggerated cytokine expression seen in several inflammatory diseases.

Skeletal Muscle-specific PGC-1\u03b1 Overexpression Suppresses Atherosclerosis in Apolipoprotein E-Knockout Mice

Endurance exercise training prevents atherosclerosis. Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) increases myokine secretion from the skeletal muscle, and these myokines have been shown to affect the function of multiple organs. Since endurance exercise training increases PGC-1α expression in skeletal muscles, we investigated whether skeletal muscle-specific PGC-1α overexpression suppresses atherosclerosis. Apolipoprotein E-knockout (ApoE-KO)/PGC-1α mice, which overexpress PGC-1α in the skeletal muscle of ApoE-KO mice, were sacrificed, and the atherosclerotic plaque area, spontaneous activity, plasma lipid profile, and aortic gene expression were measured. Immunohistochemical analyses were also performed. The atherosclerotic lesions in ApoE-KO/PGC-1α mice were 40% smaller than those in ApoE-KO mice, concomitant with the reduction in vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemoattractant protein-1 (MCP-1) mRNA and protein levels in the aorta. Spontaneous activity and plasma lipid profiles were not changed by the overexpression of PGC-1α in the skeletal muscle. In human umbilical vein endothelial cells, Irisin and β-aminoisobutyric acid (BAIBA), PGC-1α-dependent myokines, inhibited the tumor necrosis factor α-induced VCAM-1 gene and protein expression. BAIBA also inhibited TNFα-induced MCP-1 gene expression. These results showed that the skeletal muscle-specific overexpression of PGC-1α suppresses atherosclerosis and that PGC-1α-dependent myokines may be involved in the preventive effects observed.

Inhibition of TNFα-induced interleukin-6 gene expression by barley (Hordeum vulgare) ethanol extract in BV-2 microglia.

Inflammation in the central nervous system is closely associated with pathological neurodegenerative diseases as well as psychiatric disorders. Prolonged activation of microglia can produce many inflammatory mediators, which may result in pathological neurotoxic side effects. Interleukin (IL)-6 serves as a hallmark of the injured brain. Whole grains are known to contain many bioactive components. However, little information is available about anti-neuroinflammatory effects of grains in the CNS. This study aims to investigate the effect of Hordeum vulgare ethanol extract (HVE) on the suppression of IL-6 expression in BV2 microglia. Inhibitory effects of HVE on IL-6 expression were analyzed by immunoblot anaysis, immunofluoresce microscopic analysis, reverse transcription-polymerase chain reaction, and luciferase promoter reporter assay. HVE inhibited TNFα-induced phosphorylation of IKKα/β, IκB, and p65/RelA NF-κB. TNFα-induced IL-6 mRNA expression and promoter activity were reduced by HVE. Point mutation of NF-κB-binding site within the IL-6 gene promoter abolished TNFα-induced reporter activity, whereas exogenous expression of p65 NF-κB enhanced IL-6 promoter activity. NF-κB-binding site within the IL-6 promoter region is a HVE target element involved in the inhibition of TNFα-induced IL-6 gene transcription. HVE inhibits TNFα-induced IL-6 expression via suppression of NF-κB signaling in BV2 microglial cells.

Loss of DBC1 (CCAR2) affects TNFα-induced lipolysis and Glut4 gene expression in murine adipocytes.

STAT5A (signal transducer and activator of transcription 5A) is a transcription factor that plays a role in adipocyte development and function. In this study, we report DBC1 (deleted in breast cancer 1; also known as CCAR2) as a novel STAT5A-interacting protein. DBC1 has been primarily studied in tumor cells, but there is evidence that loss of this protein may promote metabolic health in mice. Currently, the functions of DBC1 in mature adipocytes are largely unknown. Using immunoprecipitation and immunoblotting techniques, we confirmed that there is an association between endogenous STAT5A and DBC1 proteins under physiological conditions in the adipocyte nucleus that is not dependent upon STAT5A tyrosine phosphorylation. We used siRNA to knockdown DBC1 in 3T3-L1 adipocytes to determine the impact on STAT5A activity, adipocyte gene expression, and TNFα (tumor necrosis factor α)-regulated lipolysis. The loss of DBC1 did not affect the expression of several STAT5A target genes including Socs3, Cish, Bcl6, Socs2, and Igf1 However, we did observe decreased levels of TNFα-induced glycerol and free fatty acids released from adipocytes with reduced DBC1 expression. In addition, DBC1-knockdown adipocytes had increased Glut4 expression. In summary, DBC1 can associate with STAT5A in adipocyte nucleus, but it does not appear to impact regulation of STAT5A target genes. Loss of adipocyte DBC1 modestly increases Glut4 gene expression and reduces TNFα-induced lipolysis. These observations are consistent with in vivo observations that show loss of DBC1 promotes metabolic health in mice.

A novel regulatory function of CDKN1A/p21 in TNFα-induced matrix metalloproteinase 9-dependent migration and invasion of triple-negative breast cancer cells

Metastasis is the leading cause of mortality in patients with highly invasive cancers and, as such, is a major problem for medicine. It has been increasingly recognized that cancer-related inflammation plays an important role in promoting invasion and the metastatic process in which cell motility and upregulation of proteolytic enzymes are crucial events. TNFα is a proinflammatory cytokine known to stimulate synthesis of MMP9, a zinc- and calcium-dependent endopeptidase contributing to the regulation of ECM remodeling and cell signaling. However, the precise molecular mechanism of TNFα-induced MMP9 gene expression in cancers is still not fully understood. This study shows that TNFα-induced cell migration and invasion involve ERK1/2-dependent up-regulation of CDKN1A/p21 expression in highly aggressive breast cancer cells and that CDKN1A/p21 plays an important regulatory role in TNFα-induced MMP9 gene expression, indicating an unknown function of CDKN1A/p21 as a regulator of proteolytic activity in cancer cells.

The Lactobacillus rhamnosus R0011 secretome attenuates TNFα-induced pro-inflammatory gene expression in human HT-29 intestinal epithelial cells

Abstract Probiotic lactic acid bacteria have been associated with a wide array of host-immune modulatory effects including modulation of pro-inflammatory gene expression. However, many questions remain about the exact cellular mechanisms through which these bacteria modulate host cell responses following pro-inflammatory challenge. The aim of this study was to elucidate the effects of the Lactobacillus rhamnosus R0011 secretome on TNFα-induced pro-inflammatory gene expression. Previous analysis has shown that the <10kDa fraction of the Lr R0011 secretome attenuates TNFα-induced IL-8 production from HT-29 intestinal epithelial cells (IECs). Whole-genome wide microarray analysis was used to further interrogate the impact of the <10kDa fraction of the Lr R0011 secretome on TNFα-induced gene expression. Contact with the <10kDa secretome fraction alone induced minimal changes in global gene expression by HT-29 IECs, with no increases in pro-inflammatory gene expression. However, the <10kDa fraction of the Lr R0011 secretome attenuated TNFα-induced expression of the pro-inflammatory mediators CCL10, CXCL1, CXCL10, CXCL11, IL-1β, IL-8, IL-17C, IL-23A, IL-32, and PTGS2, when compared to HT-29 IEC treated with TNFα alone. Co-challenge of HT-29 IECs with the <10kDa fraction of the Lr R0011 secretome and TNFα also resulted in an increase in the expression of dual specificity phosphatase 1 (DUSP1), a key regulator of the MAPK pathway, and of activating transcription factor 3 (ATF3), a negative regulator of innate immunity, relative to HT-29 IECs treated with TNFα alone. This provides insight into mechanisms through which these bacteria may influence innate immune activity at the IEC level through soluble mediators.

Modulation of the TNFα-induced gene expression profile of intestinal epithelial cells by soy fermented with lactic acid bacteria

The effect of soy ferments on gene expression induced by the proinflammatory cytokine tumour necrosis factor α (TNFα) at the intestinal epithelial cell (IEC) level was evaluated. Microarray-based transcriptional analysis revealed that soy fermented with Lactobacillus helveticus R0052 and Streptococcus thermophilus R0083 (SF-Lh) down-regulated 33 of 40 proinflammatory genes up-regulated by TNFα in HT-29 IEC, attenuating expression of genes encoding several proinflammatory cytokines and cell adhesion molecules. TNFα-mediated activation of gene expression associated with the NF-κB pathway was also repressed, as was Interleukin-8 (IL-8) production. In contrast, soy fermented with Bifidobacterium longum R0175 and S. thermophilus R0083 (SF-Bl) up-regulated expression of three proinflammatory genes induced by TNFα. Increased intracellular levels of hydrogen peroxide (H2O2) were detected in HT-29 IEC following incubation with SF-Lh. These results indicate that SF-Lh has immunomodulatory activity through reduction of proinflammatory gene expression at the IEC level.

The TrxG Complex Mediates Cytokine Induced De Novo Enhancer Formation in Islets.

To better understand how β-cells respond to proinflammatory cytokines we mapped the locations of histone 3 lysine 4 monomethylation (H3K4me1), a post-translational histone modification enriched at active and poised cis-regulatory regions, in IFNγ, Il-1β, and TNFα treated pancreatic islets. We identified 96,721 putative cis-regulatory loci, of which 3,590 were generated de novo, 3,204 had increased H3K4me1, and 5,354 had decreased H3K4me1 in IFNγ, Il-1β, and TNFα exposed islets. Roughly 10% of the de novo and increased regions were enriched for the repressive histone modification histone 3 lysine 27 trimethylation (H3K27me3) in untreated cells, and these were frequently associated with chemokine genes. We show that IFNγ, Il-1β, and TNFα exposure overcomes this repression and induces chemokine gene activation in as little as three hours, and that this expression persists for days in absence of continued IFNγ, Il-1β, and TNFα exposure. We implicate trithorax group (TrxG) complexes as likely players in the conversion of these repressed loci to an active state. To block the activity of these complexes, we suppressed Wdr5, a core component of the TrxG complexes, and used the H3K27me3 demethylase inhibitor GSK-J4. We show that GSK-J4 is particularly effective in blunting IFNγ, Il-1β, and TNFα-induced chemokine gene expression in β-cells; however, it induced significant islet-cell apoptosis and β-cell dysfunction. Wdr5 suppression also reduced IFNγ, Il-1β, and TNFα induced chemokine gene expression in β-cells without affecting islet-cell survival or β-cell function after 48hrs, but did begin to increase islet-cell apoptosis and β-cell dysfunction after four days of treatment. Taken together these data suggest that the TrxG complex is potentially a viable target for preventing cytokine induced chemokine gene expression in β-cells.

Abstract 4042: Kruppel-like factor 5 promotes breast cancer proliferation, migration and invasion partially through upregulating the transcription of TNFα-induced protein 2

Abstract The Krupple-like factor 5 (KLF5) transcription factor is highly expressed in high-grade and basal-like TNBC breast cancers. Our previous studies reported that KLF5 promotes breast cancer cell proliferation and survival through target genes, such as FGF-BP and mPGES1. However, the mechanism by which KLF5 promotes cell migration and invasion is still not completely understood. In this study, we demonstrated that TNFAIP2, a TNFα-induced gene, is a KLF5 direct downstream target gene. The expression of the TNFAIP2 is highly correlated with the expression of the KLF5 protein in breast epithelial cell lines at both mRNA and protein levels. Manipulation of KLF5 expression positively alters TNFAIP2 mRNA and protein expression levels in multiple breast cell lines. KLF5 binds to TNFAIP2 gene promoter and activates its transcription. Functionally, TNFAIP2 promotes cell proliferation and xenograft growth in NOD-SCID mice. Importantly, knockdown of either KLF5 or TNFAIP2 significantly reduces cancer cell migration and invasion. Overexpression of TNFAIP2 rescues KLF5 knockdown-induced migration suppression in HCC1937 cells. TNFAIP2 interacts with two small GTPase Cdc42 and Rac1, increases their activities, and changes actin skeleton and cell morphyology. Finally, KLF5 and TNFAIP2 promote cell migration and invasion through activating Cdc42 and Rac1. These findings collectively suggest that TNFAIP2 is a KLF5 direct target gene and both KLF5 and TNFAIP2 promotes TNBC cell migration and invasion through Cdc42 and Rac1. Citation Format: Lin Jia, Rong Liu, Ceshi Chen. Kruppel-like factor 5 promotes breast cancer proliferation, migration and invasion partially through upregulating the transcription of TNFα-induced protein 2. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4042. doi:10.1158/1538-7445.AM2015-4042

The Effect of Progestins on Tumor Necrosis Factor α-Induced Matrix Metalloproteinase-9 Activity and Gene Expression in Human Primary Amnion and Chorion Cells In Vitro.

Current treatment modalities for preventing preterm premature rupture of membranes are limited, but progestins may play a role. Tumor necrosis factor α (TNFα) enhances matrix metalloproteinase-9 (MMP-9) gene expression and activity in fetal membranes, contributing to membrane weakening and rupture. We previously demonstrated that progestins attenuate TNFα-induced MMP-9 activity in a cytotrophoblast cell line. However, whether they have a similar effect in primary amnion and chorion cells of fetal membranes is unknown. In this study, we evaluated the effect of progestins on basal and TNFα-induced MMP-9 activity and gene expression in primary chorion and amnion cells harvested from the fetal membranes of term nonlaboring patients. Primary amnion and chorion cells were isolated from fetal membranes obtained from term uncomplicated nonlaboring patients following elective cesarean delivery (n = 11). Confluent primary amnion and chorion cell cultures were both pretreated with vehicle (control), progesterone (P4), 17α-hydroxyprogesterone caproate (17P), or medroxyprogesterone acetate (MPA) at 10 M concentration for 6 hours followed by stimulation with TNFα at 10 ng/mL for an additional 24 hours. Cell cultures pretreated with the vehicle only served as the unstimulated control and the vehicle stimulated with TNFα served as the stimulated control. Both controls were assigned a value of 100 units. Cell culture medium was harvested for MMP-9 enzymatic activity quantification using gelatin zymography. Total RNA was extracted for quantifying MMP-9 gene expression using real-time quantitative PCR. Basal MMP-9 activity and gene expression data were normalized to the unstimulated control. TNFα-stimulated MMP-9 activity and gene expression were normalized to the stimulated control. The primary outcome was the effect of progestins on TNFα-induced MMP-9 enzymatic activity in term human primary amnion and chorion cells in vitro. Secondary outcomes included the effect of progestin therapy on TNFα-induced MMP-9 gene expression and on basal MMP-9 activity and gene expression in primary amnion and chorion cells in vitro. Primary cells were harvested from 11 patients. Compared with the unstimulated control, TNFα increased MMP-9 activity (P = 0.005 versus control in primary amnion cells and P < 0.001 versus control in primary chorion cells) and MMP-9 gene expression (P = 0.030 versus control in primary amnion cells, P < 0.001 versus control in primary chorion cells). Compared with the unstimulated controls, MPA, but not P4 or 17P, reduced basal MMP-9 activity [mean difference (95% CI) -49.6 (-81.9, -17.3) units, P = 0.001] and gene expression [mean difference (95% CI) -53.4 (-105.9, -0.9) units, P = 0.045] in primary amnion cells. Compared with the stimulated control, MPA also reduced TNFα-induced MMP-9 activity [mean difference (95% CI) -69.0 (-91.8, -46.3) units, P < 0.001] and gene expression [mean difference (95% CI) -86.0 (-120.7, -51.3) units, P < 0.001] in primary amnion cells. Progestin pretreatment had no significant effect on basal or TNFα-induced MMP-9 activity and gene expression in primary chorion cells. The inhibitory effect of MPA on both basal and TNFα-induced MMP-9 activity and gene expression in primary amnion cells demonstrate a possible mechanism by which progestins may prevent fetal membrane weakening leading to preterm premature rupture of membranes.

Abstract 597: Negative regulation of TNFα-induced NFκB activation by XAF1 via inhibition of the TRADD/TRAF2/RIP1 signal complex

Abstract Introduction: X-linked inhibitor of apoptosis protein (XIAP)-associated factor 1 (XAF1) was originally implicated as a tumor suppressor due to its inhibitory effect on anti-apoptotic XIAP. It contains an N-terminus tumor necrosis factor (TNF) receptor associated factor (TRAF) Zn finger domain that is highly conserved in TRAF family members. XAF1 expression can be induced by TNFα; however, its role in the signal cascade initiated by TNFα/TNF receptor (TNFR) 1 has not yet been clearly elucidated. Here we report that XAF1 can negatively regulate TNFα-induced nuclear factor kappa B (NFκB) activation via inhibition of signaling molecule TRAF2 and signal complex formation downstream of TNFα/TNFR1. Methods: To investigate the effects of XAF1 on TNFα signal transduction machinery, we assessed TNF-induced expression of the NFκB response gene IL-8 by real-time PCR, and phosphorylation of inhibitor of NFκB (IκB) by immunoblotting with an anti-phospho-IκB antibody, in HEK293 cells with an inducible XAF1 expression vector. Interaction between TRAF2 or RIP1 and XAF1 was investigated by immunoprecipitation. NFκB transcriptional activation potential, induced by ectopic TRAF2 or RIP1 with or without co-expressed XAF1, was quantified by luciferase assays. Inhibition of TRAF2 ubiquitination by XAF1 was also assessed by immunoblot. Transient transfection of plasmids expressing TRADD, TRAF2, RIP1 and/or XAF1 followed by immunoprecipition was performed to further investigate the role of XAF1 on the signal complex formation. Results: Luciferase assays and real-time PCR studies showed that XAF1 inhibited TNFα-induced NFκB activation and IL-8 expression. Both TRAF2 and RIP1 interacted with XAF1 according to our immunoprecipitation results but only TRAF2 activity was inhibited by XAF1. Poly-ubiquitination of TRAF2 was attenuated when XAF1 was present. In addition, immunoprecipitation and immunoblot data showed that XAF1 interfered with RIP1/TRADD association and regulated TRADD and RIP1 turnover. Discussion: Our data demonstrated an inhibitory effect of XAF1 on TNFα-induced NFκB activation and gene expression. A possible mechanism may be via inhibition of TRAF2, an E3 ubiquitin ligase responsible for signal transduction leading to NFκB activation. XAF1 inhibited both TRAF2-mediated NFκB activation and TRAF2 ubiquitination, which is essential for its signal delivery function. XAF1 also interacted with RIP1 yet did not block RIP1-induced NFκB promoter activity. However, the binding of RIP1 to TRADD, an adaptor protein of TNFR1-mediated signal pathway, was disrupted by XAF1. Furthermore, degradation of TRADD and RIP1 was accelerated when XAF1 was present. These data suggested that XAF1 may act as a TNF-induced negative regulator of NFκB activation by inhibiting TRAF2 ubiquitination and by regulating the integrity of the signal complex of TRADD/TRAF2/RIP1. Citation Format: Boren Lin, Da Xu, Douglas W. Leaman. Negative regulation of TNFα-induced NFκB activation by XAF1 via inhibition of the TRADD/TRAF2/RIP1 signal complex. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 597. doi:10.1158/1538-7445.AM2014-597

PPARγ ligand ciglitazone inhibits TNFαα-induced ICAM-1 in human airway smooth muscle cells

Modification of human airway smooth muscle (ASM) function by proinflammatory cytokines has been regarded as a potential mechanism underlying bronchial hyperresponsiveness in asthma. Human ASM cells express intercellular adhesion molecule (ICAM)-1 in response to cytokines. Synthetic ligands for peroxisome proliferator-activated receptor (PPAR)γ reportedly possess anti-inflammatory and immunomodulatory properties. In this study, we examined whether ciglitazone, a synthetic PPARγ ligand, can modulate the basal and tumor necrosis factor (TNF)α-induced ICAM1 gene expression in human ASM cells. Human ASM cells were treated with TNFα. ICAM-1 expression was assessed by flow cytometry and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. PPARγ activity was inhibited by target-specific small interfering (si) RNA targeting PPARγ and GW9662, a PPARγ antagonist. Activity of nuclear factor (NF)-κB was assessed by using immunoblot analysis, immune-confocal images, and electrophoretic mobility shift assay (EMSA). By flow cytometry, ciglitazone alone had no effect on ICAM-1 expression in ASM cells, but inhibited ICAM-1 expression in response to TNFα (10 ng/ml) in a dose-dependent manner (1-10 μM). It also inhibited TNFα-induced ICAM1 gene expression by RT-PCR analysis. Knockdown of PPARγ gene by target-specific siRNA targeting PPARγ enhanced ICAM-1 expression and the inhibitory effect of ciglitazone on TNFα-induced ICAM-1 expression was reversed by PPARγ siRNA and GW9662. SN-50 (10 μg/ml), an inhibitor for nuclear translocation of NF-κB, inhibited TNFα-induced ICAM-1 expression. Ciglitazone did not prevent TNFα-induced degradation of the cytosolic inhibitor of NF-κB (IκB), but inhibited the nuclear translocation of p65 induced by TNFα and suppressed the NF-κB/DNA binding activity. These findings suggest that ciglitazone inhibits TNFα-induced ICAM1 gene expression in human ASM cells through the ligand-dependent PPARγ activation and NF-κB-dependent pathway.

The NFκB inhibitor parthenolide reduces cell proliferation and PGE2 synthesis in human endometriotic stromal cells and inhibits development of endometriosis in murine model

Parthenolide is a sesquiterpene lactone derived from the plant feverfew, which has been used in folk medicine for its anti-inflammatory action. We sought to evaluate effects of parthenolide on ESCs, and on endometriotic lesions in a murine model. Aim of this study was to investigate usefulness of parthenolide as a possible therapeutic agent for endometriosis Experimental study. Ectopic endometrial tissue from ovarian chocolate cysts was collected from premenopausal women. ESCs were isolated from these tissues. ESCs were pretreated with parthenolide and exposed to TNFα. After treatment with TNFα, the levels of IL-8 and COX-2 gene expression were evaluated by real-time RT-PCR. After adding TNFα, IL-8 and PGE2 protein expression were determined by ELISA. Cell proliferation after adding TNFα was assessed by BrdU-ELISA. TNFα-induced phosphorylation of signaling pathways was evaluated by Western blot analysis. As an endometriosis model, estradiol-treated ovariectomized mice were injected intraperitoneally with endometrial fragments of donor mice. After 4 weeks of parthenolide injections, the extent of endometriotic lesions was evaluated. With parthenolide pretreatment, TNFα-induced IL-8 gene and protein expression were significantly repressed. TNFα-induced COX-2 expression and PGE2 synthesis were also inhibited. Adding parthenolide significantly suppressed BrdU incorporation of ESCs. Pretreatment with parthenolide inhibited TNFα-induced IκB phosphorylation, although it did not attenuate ERK1/2, p38MAPK, Akt, and JNK1/2 phosphorylation. Using the mice endometriosis model, administrating parthenolide significantly reduced surface area and weight of endometriotic lesions. These findings suggest that parthenolide represses development of endometriosis possibly by suppressing peritoneal inflammatory status through the NFκB pathway.