Pentabromopseudilin: a myosin V inhibitor suppresses TGF-<b>β</b> activity by recruiting the type II TGF-<b>β</b> receptor to lysosomal degradation

<p>Pentabromopseudilin (PBrP) is a marine antibiotic isolated from the marine bacteria <i>Pseudomonas bromoutilis</i> and <i>Alteromonas luteoviolaceus</i>. PBrP exhibits antimicrobial, anti-tumour, and phytotoxic activities. In mammalian cells, PBrP is known to act as a reversible and allosteric inhibitor of myosin Va (MyoVa). In this study, we report that PBrP is a potent inhibitor of transforming growth factor-β (TGF-β) activity. PBrP inhibits TGF-β-stimulated Smad2/3 phosphorylation, plasminogen activator inhibitor-1 (PAI-1) protein production and blocks TGF-β-induced epithelial–mesenchymal transition in epithelial cells. PBrP inhibits TGF-β signalling by reducing the cell-surface expression of type II TGF-β receptor (TβRII) and promotes receptor degradation. Gene silencing approaches suggest that MyoVa plays a crucial role in PBrP-induced TβRII turnover and the subsequent reduction of TGF-β signalling. Because, TGF-β signalling is crucial in the regulation of diverse pathophysiological processes such as tissue fibrosis and cancer development, PBrP should be further explored for its therapeutic role in treating fibrotic diseases and cancer.</p

Additional file 1: of Nr2f1b control venous specification and angiogenic patterning during zebrafish vascular development

Supplementary Methods. Table S1: Primer/morpholino sequences used in this study. Figure S1: Knockdown efficiency and specificity of nr2f1b morpholinos. Figure S2: An increase in non-specific cell death after morpholino injection is not the cause of the observed vascular phenotype. Figure S3. Loss of nr2f1b results in pericardial edema, absent parachordal vessels, subintestinal vessels (SIV) mispattern and circulation defects. (DOC 2967 kb

Additional file 2: Figure S2. of Lactobacillus acidophilus attenuates Salmonella-induced intestinal inflammation via TGF-β signaling

Effect of L. acidophilus on SMAD3/4 transcriptional activity with active or inactive S. typhimurium. Human intestinal Caco-2 cells were transfected with luciferase reporter plasmid containing the promoter of the SMAD-binding site or CMV promoter overnight. The cells were then pretreated with L. acidophilus (MOI: 20) 1 h prior to infection with S. typhimurium (MOI: 10) or UV-inactivated S. typhimurium (MOI: 10) in antibiotic-free DMEM for 1 h at 37 °C. Then, the cells were washed twice with PBS and added to medium containing D-luciferin to monitor SMAD activity. The cells were washed twice with PBS, added to DMEM medium containing D-luciferin and antibiotics for signal measurement with a Luminometer. Data were analyzed with Prism 5, and the results are shown as the means ± SEM from three independent experiments. (TIFF 100 kb

Euphol decreased the abundance of TβR-I and TβR-II in Mv1Lu (A) and MKN45 (B) cells after.

<p>Mv1Lu and MKN45 cells were treated with several concentrations of euphol at 37°C for 48 hours, after which the cell lysates were subjected to Western blot analysis using anti-TβR-I, anti-TβR-II, TfR, EEA-1, caveolin-1, and anti-β-actin antibodies (A and C), followed by quantification by densitometry (B and D). The ratio of the relative amounts of TβR-I, TβR-II, and β-actin in cells treated without euphol was taken as 100% TGF-β receptor expression. The data are representative of a total of four independent analyses; values are mean ± s.d. significantly lower than control cells: *<i>P</i><0.05 versus control.</p

Immunofluorescent localization of TβR-II and caveolin-1(or flotillin-2) in Mv1Lu cells treated with and without euphol and TGF-β.

<p>Mv1Lu cells that transiently expressed TβR-II-HA were treated with or without 10 μg/ml euphol at 37°C for 1 hour, after which they were incubated with or without 100 pM TGF-β at 37°C for 30 minutes. The cells were then fixed with cold methanol and incubated with mouse anti-HA antibodies (Fig 5A and 5B, a-d), rabbit anti-caveolin-1 antibodies (Fig 5A, e-h), or rabbit anti-flotillin-2 antibodies (Fig 5B, e-h) followed by incubation with rhodamine-conjugated donkey anti-mouse antibodies or FITC-conjugated goat anti-rabbit antibodies. The fluorescence in the cells was measured using confocal fluorescence microscopy. Bar, 20 μm. The white arrows indicate colocalization of TβR-II-HA and caveolin-1 (or flotillin-2) at the cell surface (j) and in endocytic vesicles (l).</p

Euphol inhibits TGF-β-induced fibronectin expression.

<p>(A) AGS cells were treated with TGF-β (100 pM) ± euphol (5–40μg/ml) for 24 hours. Total RNA were isolated and the expressions of fibronectin were determined by RT-PCR. GAPDH was used as a loading control. (B) AGS cells were treated with TGF-β (100 pM) ± euphol for 48 hours. Total protein extracts from treated cells were Western blotted with anti-fibronectin or anti-β-actin monoclonal antibody. β-actin was used as a loading control; results were quantified by densitometry showing in lower panel. Data represent the means ± s.d. of three independent experiments *<i>P</i><0.01 versus TGF-β-induced expression.</p

Inhibition of TGF-β-induced transcriptional activation by euphol.

<p>(A) Mv1Lu cells with stable expression of the PAI-1 luciferase promoter were treated with increasing concentration of euphol. The gray bars in (A) represent the cells without TGF-β treatment. The black bars represent the cells treated with 100 pM TGF-β. (B,C,D) Mv1Lu cells were transfected with CAGA₁₂-Luc, collagen, or a fibronectin luciferase plasmid, and AGS (E) and MKN45 (F) gastric cancer cells were transfected with CAGA₁₂-Luc, Mv1Lu, AGS, and MKN45 cells were treated with TGF-β (100 pM, +β), euphol (Eu), or MβCD (CD). Luciferase activity was measured as described in the methods section. Columns show mean of three independent experiments performed in triplicated; bars indicate s.d.; *<i>P</i><0.05 (compare with TGF-β treatment), **<i>P</i><0.01.</p

Chemical structures of (A) cholesterol and (B) euphol.

<p>Chemical structures of (A) cholesterol and (B) euphol.</p

Cholest-4-en-3-one attenuates TGF-β responsiveness by inducing TGF-β receptors degradation in Mv1Lu cells and colorectal adenocarcinoma cells

<p><b>Purpose:</b> The transforming growth factor-beta (TGF-β) pathway is an important in the initiation and progression of cancer. Due to a strong association between an elevated colorectal cancer risk and increase fecal excretion of cholest-4-en-3-one, we aim to determine the effects of cholest-4-en-3-one on TGF-β signaling in the mink lung epithelial cells (Mv1Lu) and colorectal cancer cells (HT29) <i>in vitro</i>.</p> <p><b>Methods:</b> The inhibitory effects of cholest-4-en-3-one on TGF-β-induced Smad signaling, cell growth inhibition, and the subcellular localization of TGF-β receptors were investigated in epithelial cells using a Western blot analysis, luciferase reporter assays, DNA synthesis assay, confocal microscopy, and subcellular fractionation.</p> <p><b>Results:</b> Cholest-4-en-3-one attenuated TGF-β signaling in Mv1Lu cells and HT29 cells, as judged by a TGF-β-specific reporter gene assay of plasminogen activator inhibitor-1 (PAI-1), Smad2/3 phosphorylation and nuclear translocation. We also discovered that cholest-4-en-3-one suppresses TGF-β responsiveness by increasing lipid raft and/or caveolae accumulation of TGF-β receptors and facilitating rapid degradation of TGF-β and thus suppressing TGF-β-induced signaling.</p> <p><b>Conclusions:</b> Our results suggest that cholest-4-en-3-one inhibits TGF-β signaling may be due, in part to the translocation of TGF-β receptor from non-lipid raft to lipid raft microdomain in plasma membranes. Our findings also implicate that cholest-4-en-3-one may be further explored for its potential role in colorectal cancer correlate to TGF-β deficiency.</p

Nuclear Receptor Subfamily 2 Group F Member 1a (nr2f1a) Is Required for Vascular Development in Zebrafish

<div><p>Genetic regulators and signaling pathways are important for the formation of blood vessels. Transcription factors controlling vein identity, intersegmental vessels (ISV) growth and caudal vein plexus (CVP) formation in zebrafish are little understood as yet. Here, we show the importance of the nuclear receptor subfamily member 1A (nr2f1a) in zebrafish vascular development. Amino acid sequence alignment and phylogenetic analysis of nr2f1a is highly conserved among the vertebrates. Our in situ hybridization results showed <i>nr2f1a</i> mRNA is expressed in the lateral plate mesoderm at 18 somite stage and in vessels at 24–30 hpf, suggesting its roles in vasculization. Consistent with this morpholino-based knockdown of <i>nr2fla</i> impaired ISV growth and failed to develop fenestrated vascular structure in CVP, suggesting that <i>nr2f1a</i> has important roles in controlling ISV and CVP growth. Consequently, <i>nr2f1a</i> morphants showed pericardial edema and circulation defects. We further demonstrated reduced ISV cells and decreased CVP endothelial cells sprouting in <i>nr2f1a</i> morphants, indicating the growth impairment of ISV and CVP is due to a decrease of cell proliferation and migration, but not results from cell death in endothelial cells after morpholino knockdown. To test molecular mechanisms and signals that are associated with <i>nr2f1a</i>, we examined the expression of vascular markers. We found that a loss of <i>nr2f1a</i> results in a decreased expression of vein/ISV specific markers, <i>flt4, mrc1</i>, vascular markers <i>stabilin</i> and <i>ephrinb2.</i> This indicates the regulatory role of nr2f1a in controlling vascular development. We further showed that <i>nr2f1a</i> likely interact with <i>Notch</i> signaling by examining <i>nr2f1a</i> expression in <i>rbpsuh</i> morphants and DAPT-treatment embryos. Together, we show nr2f1a plays a critical role for vascular development in zebrafish.</p></div