Distinct Mechanisms for Induction and Tolerance Regulate the Immediate Early Genes Encoding Interleukin 1β and Tumor Necrosis Factor α

Interleukin-1β and Tumor Necrosis Factor α play related, but distinct, roles in immunity and disease. Our study revealed major mechanistic distinctions in the Toll-like receptor (TLR) signaling-dependent induction for the rapidly expressed genes (IL1B and TNF) coding for these two cytokines. Prior to induction, TNF exhibited pre-bound TATA Binding Protein (TBP) and paused RNA Polymerase II (Pol II), hallmarks of poised immediate-early (IE) genes. In contrast, unstimulated IL1B displayed very low levels of both TBP and paused Pol II, requiring the lineage-specific Spi-1/PU.1 (Spi1) transcription factor as an anchor for induction-dependent interaction with two TLR-activated transcription factors, C/EBPβ and NF-κB. Activation and DNA binding of these two pre-expressed factors resulted in de novo recruitment of TBP and Pol II to IL1B in concert with a permissive state for elongation mediated by the recruitment of elongation factor P-TEFb. This Spi1-dependent mechanism for IL1B transcription, which is unique for a rapidly-induced/poised IE gene, was more dependent upon P-TEFb than was the case for the TNF gene. Furthermore, the dependence on phosphoinositide 3-kinase for P-TEFb recruitment to IL1B paralleled a greater sensitivity to the metabolic state of the cell and a lower sensitivity to the phenomenon of endotoxin tolerance than was evident for TNF. Such differences in induction mechanisms argue against the prevailing paradigm that all IE genes possess paused Pol II and may further delineate the specific roles played by each of these rapidly expressed immune modulators. © 2013 Adamik et al

Injectable Hydrogel Guides Neurons Growth with Specific Directionality

Visual disabilities affect more than 250 million people, with 43 million suffering from irreversible blindness. The eyes are an extension of the central nervous system which cannot regenerate. Neural tissue engineering is a potential method to cure the disease. Injectability is a desirable property for tissue engineering scaffolds which can eliminate some surgical procedures and reduce possible complications and health risks. We report the development of the anisotropic structured hydrogel scaffold created by a co-injection of cellulose nanofiber (CNF) solution and co-polypeptide solution. The positively charged poly (L-lysine)-r-poly(L-glutamic acid) with 20 mol% of glutamic acid (PLLGA) is crosslinked with negatively charged CNF while promoting cellular activity from the acid nerve stimulate. We found that CNF easily aligns under shear forces from injection and is able to form hydrogel with an ordered structure. Hydrogel is mechanically strong and able to support, guide, and stimulate neurite growth. The anisotropy of our hydrogel was quantitatively determined in situ by 2D optical microscopy and 3D X-ray tomography. The effects of PLLGA:CNF blend ratios on cell viability, neurite growth, and neuronal signaling are systematically investigated in this study. We determined the optimal blend composition for stimulating directional neurite growth yielded a 16% increase in length compared with control, reaching anisotropy of 30.30% at 10°/57.58% at 30°. Using measurements of calcium signaling in vitro, we found a 2.45-fold increase vs. control. Based on our results, we conclude this novel material and unique injection method has a high potential for application in neural tissue engineering

Distinct metabolic sensitivity for transcription elongation on <i>Il1b</i> and <i>Tnf</i> in murine bone marrow-derived monocytes.

<p>(A) P-TEFb ChIP for mouse RAW264.7 <i>Il1b</i> and <i>Tnf</i> genes in the presence of LY-294002 inhibition. (B) Pol II, PTEFb, S2P CTD Pol II, S2P CTD Pol II and H3K36me3 ChIP, as indicated, for 2DG-treated mouse BMDM. The BMDM were stimulated for indicated times with LPS plus or minus 3 h pretreatment with 2–DG.</p

Spi1 mediates monocyte-specific <i>IL1B</i> expression.

<p>(A) Spi1 ChIP for <i>IL1B</i> and <i>TNF</i> in control and LPS-treated THP-1 cells. (B) Transcription factor mRNA expression profiles in HEK293 and THP-1 cells. A third panel (and data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070622#pone.0070622.s005" target="_blank">Figure S5H</a>) displays ectopic mRNA expression of Spi1 in transfected HEK293. (C) IL1BXT-Luc reporter activity for ectopic expression of indicated factors in HEK293. (D) Endogenous <i>IL1B</i> mRNA expression in transfected HEK293. (E) ChIP for endogenous TBP, Pol II and H3 with ectopic Spi1 in HEK293. Vertical gray bars designating important gene landmarks are as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070622#pone-0070622-g003" target="_blank">Figure 3</a>.</p

Comparison of <i>IL1B</i> and <i>TNF</i> expression in monocytes.

<p>(A) Steady-state mRNA kinetics for <i>IL1B</i> and <i>TNF</i> transcripts in LPS stimulated THP-1, RAW264.7 and human peripheral blood mononuclear cells (hPBMC). Solid lines denote mRNA levels for primary LPS challenge. Squares show transcript levels following re-stimulation, as indicated by arrows. (B) Western blot for 30 KDa proIL-1β precursor protein. (C) Pol II ChIP throughout the <i>IL1B</i> and <i>TNF</i> loci in resting (black), 1 h (red) and 5 h (green) LPS stimulated THP-1, RAW264.7 and hPBMC cells. Vertical gray bars locate the positions of important gene landmarks. These include TATA box and the canonical Pol II pause position (approximately 30 bp upstream and 50 bp downsteam of TSS, respectively). (D) Pol II ChIP at promoter and downstream sites for <i>IL1B</i> and <i>TNF</i>. E. Schematic of <i>IL1B</i> and <i>TNF</i> gene structures showing exons (solid boxes), positions of ChIP amplicons (midpoint relative to TSS), and important transcription factor binding sites (<b>C</b>: C/EBPβ, <b>κ</b>: NF-κB and <b>S</b>: Spi1) within regulatory regions (open boxes).</p

Proposed mechanism for LPS mediates induction of <i>IL1B</i> and <i>TNF</i> in monocytes.

<p>(A) Summary of ChIP kinetics for some key features of <i>IL1B</i> and <i>TNF</i> in THP-1 monocytes. Pol II, TBP and Spi1 are as indicated. Histone modifications at specific locations detailed in the text are labeled. Key nucleosomes are designated by position relative to the TSS (−2, −1, +1). (B) Models for <i>IL1B</i> and <i>TNF</i> gene regulation. Red text highlights important distinctions between the two genes along the induction kinetic. Nucleosomes are marked with stars (acetylation) and spheres (trimethylation) representative of significant increases in modification. Darkly colored nucleosomes are likely to be less dynamic and suggestive of impediments to gene expression. The indicated locations of Pol II are represented by various levels of intensity, reflecting the relative degree of proposed dwelling on DNA. Arrowheads on Pol II represent the relative efficiency of elongation, as indicated by the length of the associated dotted line. (C) Schematic representation of the relationships between metabolic pathways involved in <i>IL1B</i> gene activation, summarizing key elements from this study and that recently reported elsewhere <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070622#pone.0070622-Sarkar1" target="_blank">[56]</a>.</p

Nucleosome positioning dynamics and modifications during <i>IL1B</i> and <i>TNF</i> induction.

<p>(A) Kinetic ChIP of histone 3 (H3) for <i>IL1B</i> and <i>TNF</i> in THP-1 and control Hut102 and HEK293 cells, as indicated. Key nucleosomes are designated by position relative to the TSS (−2, −1, +1). (B) ChIP for histone modifications at <i>IL1B</i> and <i>TNF</i>, as indicated for each cell line. All panels are similarly scaled with respect to spatial distribution along each gene, permitting comparative localization. For all panels, along with the three gene landmarks in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070622#pone-0070622-g002" target="_blank">Figure 2</a>, an additional vertical gray bar designates the approximate location of the far-upstream enhancer (−3000 bp) for <i>IL1B</i>.</p

LPS-activated C/EBPβ interaction with Spi1 differentiates induction of <i>IL1B</i> and <i>TNF</i>.

<p>(A) NF-κB and C/EBPβ ChIP for THP-1 cells, as indicated. (B) Effect of ectopic expression of IκBα super repressor (IκBαSR) on IL1B XT-Luc reporter activity in HEK293 cotransfected with indicated factors. (C) Effect of dnC/EBPβ titration on IL1B XT-Luc reporter activity in HEK293. (D) Effect of C/EBPβ and NF-κB binding site mutations on IL1BXT-Luc reporter activity in RAW264.7. (E) XT-Luc reporter activity, as indicated in HEK293 cotransfected with C/EBPβ, NF-κB and Spi1 siRNA. (F) P-TEFb and (G) BRD4 ChIP in THP treated, as indicated.</p

LPS-dependent p300 binding and transcription factor-mediated promoter-enhancer looping at <i>IL1B</i>.

<p>(A) Inducible p300 binding at <i>IL1B</i> and <i>TNF</i>. <b>(</b>B) Schematic representation of PCR primer pairs used for evaluating 3C ligation products (Left) and PCR assessment of 3C ligation restriction fragment products in the absence and presence of U0126 and MG132 inhibitors. (C) Effects of U0126 and MG132 inhibitors on Pol II ChIP for <i>IL1B</i> and <i>TNF</i>, as indicated. (D) Model for chromatin looping during activation of <i>IL1B</i>.</p

EGFR PHOSPHORYLATION OF DCBLD2 RECRUITS TRAF6 AND STIMULATES AKT-PROMOTED TUMORIGENESIS

BACKGROUND: Oncogenic EGFR-Akt signaling is aberrantly activated in human glioblastomas. Discoid, CUC and LCCL domain containing protein 2 (DCBLD2, also known as CLCP1 and ESDN) is a neuropilin-like membrane protein that is up-regulated in vascular injury and metastatic lung cancers. However, the role of DCBLD2 in cancer is unclear. METHODS: We examined the expression of DCBLD2 in TCGA and other data bases of clinical glioma specimens. We assessed the effects of knockdown of endogenous DCBLD2 and re-express mutated DCBLD2 in glioma cells expressing EGFRvIII in vitro and in vivo. We performed IP-WB analyses to examine EGFR-induced tyrosine phosphorylation and DCBLD2 association with TRAF6. We also examined the impact of TRAF6 in EGFRvIII_driven tumorigenesis and association of p-Y EGFR and p-Y750 of DCBLD2 with tumor prognosis. We used patient-derived glioma stem cells, primary shortern culture GBM cells and established glioma cell lines in vitro, orthotopic brain glioma xenografts, various biochemical methods and IHC analysis of de-identified clinical tumor specimens of glioma samples and HNC smaples in our study. RESULTS: We found that DCBLD2 is up-regulated gene in glioblastomas and head and neck cancers (HNCs) and is required for EGFR-stimulated tumorigenesis. EGFR induces tyrosine phosphorylation (p-Y) of DCBLD2 including in a Y750 residue within a binding motif for TNF receptor-associated factor 6 (TRAF6). Consequently, the induced p-DCBLD2Y750 recruits TRAF6 to DCBLD2. This association causes increased TRAF6 E3 ligase activity and subsequent activation of Akt, thereby enhancing EGFR-driven tumorigenesis. CONCLUSIONS: Our findings uncover a novel pathway by which DCBLD2 functions as a signal relay for oncogenic EGFR signaling in promoting tumorigenesis, a conclusion that is supported by the association of EGFR activation and DCBLD2 phosphorylation with poor prognoses for gliomas and HNCs. These data also nominate DCBLD2 and TRAF6 as attractive therapeutic targets for human cancers that are associated with EGFR activation. SECONDARY CATEGORY: n/a