The Nuclear Matrix Protein, NRP/B, Acts as a Transcriptional Repressor of E2F-mediated Transcriptional Activity

Background: NRP/B, a family member of the BTB/Kelch repeat proteins, is implicated in neuronal and cancer development, as well as the regulation of oxidative stress responses in breast and brain cancer. Our previous studies indicate that the NRP/B-BTB/POZ domain is involved in the dimerization of NRP/B and in a complex formation with the tumor suppressor, retinoblastoma protein. Although much evidence supports the potential role of NRP/B as a tumor suppressor, the molecular mechanisms of NRP/B action on E2F transcription factors have not been elucidated. Methods: Three-dimensional modeling of NRP/B was used to generate point mutations in the BTB/Kelch domains. Tet-on inducible NRP/B expression was established. The NRP/B deficient breast cancer cell line, MDA-MB-231, was generated using lentiviral shNRP/B to evaluate the effect of NRP/B on cell proliferation, invasion and migration. Immunoprecipitation was performed to verify the interaction of NRP/B with E2F and histone deacetylase (HDAC-1), and the expression level of NRP/B protein was analyzed by Western blot analysis. Changes in cell cycle were determined by flow cytometry. Transcriptional activities of E2F transcription factors were measured by chloramphenicol acetyltransferase (CAT) activity. Results: Ectopic overexpression of NRP/B demonstrated that the NRP/B-BTB/POZ domain plays a critical role in E2F-mediated transcriptional activity. Point mutations within the BTB/POZ domain restored E2-promoter activity inhibited by NRP/B. Loss of NRP/B enhanced the proliferation and migration of breast cancer cells. Endogenous NRP/B interacted with E2F and HDAC1. Treatement with an HDAC inhibitor, trichostatin A (TSA), abolished the NRP/B-mediated suppression of E2-promoter activity. Gain or loss of NRP/B in HeLa cells confirmed the transcriptional repressive capability of NRP/B on the E2F target genes, Cyclin E and HsORC (Homo sapiens Origin Recognition Complex). Conclusions: The present study shows that NRP/B acts as a transcriptional repressor by interacting with the co-repressors, HDAC1, providing new insight into the molecular mechanisms of NRP/B on tumor suppression

TRAF6 Mediates IL-1β/LPS-Induced Suppression of TGF-β Signaling through Its Interaction with the Type III TGF-β Receptor

Transforming growth factor-β1 (TGF-β1) is an important anti-inflammatory cytokine that modulates and resolves inflammatory responses. Recent studies have demonstrated that inflammation enhances neoplastic risk and potentiates tumor progression. In the evolution of cancer, pro-inflammatory cytokines such as IL-1β must overcome the anti-inflammatory effects of TGF-β to boost pro-inflammatory responses in epithelial cells. Here we show that IL-1β or Lipopolysaccharide (LPS) suppresses TGF-β-induced anti-inflammatory signaling in a NF-κB-independent manner. TRAF6, a key molecule in IL-1β signaling, mediates this suppressive effect through interaction with the type III TGF-β receptor (TβRIII), which is TGF-β-dependent and requires type I TGF-β receptor (TβRI) kinase activity. TβRI phosphorylates TβRIII at residue S829, which promotes the TRAF6/TβRIII interaction and consequent sequestration of TβRIII from the TβRII/TβRI complex. Our data indicate that IL-1β enhances the pro-inflammatory response by suppressing TGF-βsignaling through TRAF6-mediated sequestration of TβRIII, which may be an important contributor to the early stages of tumor progression

Concise Synthesis of Catechin Metabolites 5-(3′,4′-Dihydroxyphenyl)-γ-valerolactones (DHPV) in Optically Pure Form and Their Stereochemical Effects on Skin Wrinkle-Reducing Activities

A concise and scalable synthetic route for optically pure (4S) and (4R)-5-(3′,4′-dihydroxyphenyl)-γ-valerolactones (DHPVs), catechin metabolites, has been developed via the efficient construction of a γ-valerolactone moiety from hexenol. Noticeably, the different skin wrinkle-reducing activities of each metabolite were revealed via our unique syntheses of DHPVs in an enantiomerically pure form

Human artificial chromosome (HAC) vector with a conditional centromere for correction of genetic deficiencies in human cells

Human artificial chromosome (HAC)-based vectors offer a promising system for delivery and expression of full-length human genes of any size. HACs avoid the limited cloning capacity, lack of copy number control, and insertional mutagenesis caused by integration into host chromosomes that plague viral vectors. We previously described a synthetic HAC that can be easily eliminated from cell populations by inactivation of its conditional kinetochore. Here, we demonstrate the utility of this HAC, which has a unique gene acceptor site, for delivery of full-length genes and correction of genetic deficiencies in human cells. A battery of functional tests was performed to demonstrate expression of NBS1 and VHL genes from the HAC at physiological levels. We also show that phenotypes arising from stable gene expression can be reversed when cells are “cured” of the HAC by inactivating its kinetochore in proliferating cell populations, a feature that provides a control for phenotypic changes attributed to expression of HAC-encoded genes. This generation of human artificial chromosomes should be suitable for studies of gene function and therapeutic applications

TRAF6 mediates IL-1β or LPS-induced suppression of TGF-β1/Smad pathway.

<p>(<b>A</b>) HEK293 cells were treated with TGF-β1 (0.4 ng/ml) and/or IL-1β (2 ng/ml) as indicated. TGF-β-mediated Smad3 phosphorylation was demonstrated by anti-pSmad3 and total Smad3 antibodies. As a loading control, α-tubulin was used. (<b>B</b>) SBE-Luc assay was performed in HepG2 cells. These luciferase assays were normalized by the activities of co-transfected β-galactosidase. (<b>C</b>) TRAF6 or GFP was over-expressed in HEK293 cells by use of a lentiviral system. Cells were harvested after TGF-β1 addition for up to 6 hours followed by westernblotting to compare phospho-Smad2/3 levels. (<b>D</b>) TGF-β1 target genes, <i>CDKN2B</i>, <i>CDKN1A</i>, and <i>SMAD6</i>, were detected by quantitative RT-PCR using total RNA from vector-(GFP) or TRAF6-expressing HaCaT cells treated as indicated. Human <i>GAPDH</i> was used as a loading control. (<b>E</b>) FaO cells were infected with either control vector or Myc-TRAF6 on previous day and then treated with TGF-β alone or together with LPS up to 8 hours. Both floating and adherent cells were harvested to compare the induction of cleaved caspase-3. TGF-β-induced signal transduction was displayed by showing pSmad2 level. The results are representative of three independent experiments.</p

<i>Traf6</i>-deficient primary cells show a higher sensitivity to TGF-β1.

<p><i>Traf6</i>-deficient primary cells show a higher sensitivity to TGF-β1.</p

TRAF6 forms a complex with TβRIII in response to TGF-β1.

<p>(<b>A</b>) CAGA12-Luciferase assays were performed in HepG2 cells. The plasmids encoding HA-TβRIII, TRAF6, CAGA12-Luc, and Renilla-luc reporter gene were transfected as indicated and, on the next day, TGF-β1 (0.4 ng/ml) and/or IL-1β (20 ng/ml) was added for 16 hours. The obtained relative luciferase units(RLU) were normalized by renilla luciferase activities. (<b>B</b>) Using the control and TβRIII knock-down HaCaT cells, TGF-β1 (0.4 ng/ml) and/or IL-1β (20 ng/ml) were treated as shown for up to 3 hours. The level of total Smad3 and phospho-Smad3 protein was detected by immunoblotting. For the control of equal loading, β-actin was used. (<b>C</b>) HEK293 cells stably expressing HA-TβRIII were transfected with Myc-Traf6 plasmids and then treated with TGF-β1. Cells were harvested at various times and were subjected to immunoprecipitation with anti-HA antibody. Co-immunoprecipitated TRAF6 was detected with anti Myc antibody. (<b>D</b>) Complex formation ability between TβRIII and TRAF6 wild-type or the TRAF6 (C85A/H87A) E3 ligase mutant was compared after TGF-β stimulation for an hour. (<b>E</b>) According to the manufacturer's protocol, interaction was visualized by <i>in situ</i> proximity ligation assay (O-link) with proximity probes directed against TRAF6 and TβRIII using Alexa 555 labeling (red). Endogenous TRAF6 was co-localized with HA-TβRIII along the plasma membrane in the presence of TGF-β (red blobs). Bar = 2.5 µm. (<b>F</b>) Quantification of blobs per cell was carried out by semi-automated image analysis using the freeware software BlobFinder V 3.0. (<b>G</b>) HA-TβRIII-stably expressing 4T07 mouse mammary cancer cells were treated with TGF-β and LPS for one hour. Co-immunoprecipitation was carried out using anti-HA antibody to query interaction with endogenous TRAF6. The results are representative of three independent experiments.</p

Association of TRAF6/TβRIII is regulated by functional TβRII and TβRI.

<p>(<b>A</b>) Myc-TRAF6, HA-TβRIII, and/or Flag-TβRII were expressed in SNU638 human gastric cancer cells and treated with TGF-β1 for 60 min. Immunoprecipitation and immunoblotting were carried out to elucidate the requirement of TβRII for TRAF6/TβRIII interaction. (<b>B</b>) TRAF6 and TβRIII interaction in TβRI−/− MEFs in the presence or absence of exogenous TβRI expression. (<b>C</b>) TRAF6/TβRIII interaction in the presence of wild-type, kinase inactive (KR: K232R), and constitutively active (TD: T204D) TβRI in HEK293 cells. (<b>D</b>) Interaction between TRAF6 and TβRIII in the presence of IN-1130 (0.1 µM), a specific inhibitor of TβRI kinase. (<b>E</b>) Olink assay for TRAF6 and TβRIII interaction in the presence of TGF-β and/or IN-1130. Transfected cells were counterstained with Alexa 488-labeled HA antibody to HA-TβRIII (green), and the nuclei were stained with Hoechst 33342 (blue). Bar = 2.5 µm. (<b>F</b>) The number of <i>in situ</i> PLA signals (red blobs) per cell was counted by semi-automated image analysis. (<b>G</b>) Co-immunoprecipitation analysis was conducted to elucidate the function of TβRI in TRAF6/TβRIII interaction. TβRI mutants KR, TD, and TD (E161A) mutated in TRAF6 consensus binding motif in TβRI (TD) were used. The results are representative of three independent experiments.</p