Деякі проблеми використання тимчасово зайнятих земель

<div><p>Glucocorticoid induced-leucine zipper (GILZ) has been shown to be induced in cells by different stimuli such as glucocorticoids, IL-10 or deprivation of IL-2. GILZ has anti-inflammatory properties and may be involved in signalling modulating apoptosis. Herein we demonstrate that wildtype <em>Yersinia enterocolitica</em> which carry the pYV plasmid upregulated GILZ mRNA levels and protein expression in epithelial cells. Infection of HeLa cells with different <em>Yersinia</em> mutant strains revealed that the protease activity of YopT, which cleaves the membrane-bound form of Rho GTPases was sufficient to induce GILZ expression. Similarly, <em>Clostridium difficile</em> toxin B, another bacterial inhibitor of Rho GTPases induced GILZ expression. YopT and toxin B both increased transcriptional activity of the GILZ promoter in HeLa cells. GILZ expression could not be linked to the inactivation of an individual Rho GTPase by these toxins. However, forced expression of RhoA and RhoB decreased basal <em>GILZ</em> promoter activity. Furthermore, MAPK activation proved necessary for profound GILZ induction by toxin B. Promoter studies and gel shift analyses defined binding of upstream stimulatory factor (USF) 1 and 2 to a canonical c-Myc binding site (E-box) in the <em>GILZ</em> promoter as a crucial step of its trans-activation. In addition we could show that USF-1 and USF-2 are essential for basal as well as toxin B induced GILZ expression. These findings define a novel way of <em>GILZ</em> promoter trans-activation mediated by bacterial toxins and differentiate it from those mediated by dexamethasone or deprivation of IL-2.</p> </div

CHC prevents constitutive degradation and phosphorylation of IκBα by an IKKα-dependent mechanism.

<p>(<b>A</b>) Reduced level of IκBα after CHC knockdown in HeLa cells. Cell lysates from control or CHC siRNA transfected cells were analyzed by western immunoblotting using indicated antibodies. Actin is shown as a loading control (representative of 3 independent experiments). (<b>B</b>) Quantification of the level of <i>IκBα</i> mRNA by quantitative RT-PCR in control or CHC-depleted HeLa cells. <i>GAPDH</i> mRNA was used as an internal control for normalization (results are expressed as the mean ± SD of 3 independent experiments). (<b>C</b>) IKKα-depletion abolishes the constitutive degradation of IκBα induced by CHC knockdown. Lysates from cells transfected with different combinations of IKKα and CHC siRNAs were analyzed by immunoblotting using indicated antibodies. Total siRNA concentration was kept constant by adding appropriate amounts of control siRNAs. Actin is shown as a loading control (data representative of 2 independent experiments). (<b>D</b>) CHC prevents enhanced basal phosphorylation of IκBα at position serine 32. Lysates from control or CHC-depleted HeLa cells were analyzed by immunoblotting using the indicated antibodies. Actin is shown as a loading control. (<b>E</b>) Densitometric quantification of the p-IκBα/IκBα ratio (results are expressed as the mean ± SD of 3 independent experiments).</p

CHC regulates NF-κB activation independently of endocytosis and CLCa.

<p>(<b>A</b>) Uptake of Alexa 594-transferrin (Alexa 594-Tf) in cells transfected with control (left panels), AP2M1 (middle panels) or CHC (right panels) siRNAs; Scale bars, 10 µm. (<b>B</b>) Quantification of transferrin uptake by automated image analysis (results are expressed as the mean ± SD of 12 images; graph representative of 2 independent experiments). (<b>C</b>) AP2M1 knockdown fails to enhance IκBα degradation. Cell lysates from control, AP2M1 or CHC siRNA-transfected cells were analyzed by immunoblotting using indicated antibodies. Actin is shown as a loading control. (<b>D</b>) Densitometric quantification of the levels of IκBα shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017158#pone-0017158-g004" target="_blank">Figure 4C</a> (graph representative of 2 independent experiments). (<b>E</b>) AP2M1 knockdown fails to induce constitutive IL-8 expression. HeLa cells were transfected with control, AP2M1 or CHC siRNAs for 72 hours. Supernatants were collected to measure the concentration of IL-8 by ELISA (results are expressed as the mean ± SD of 3 independent experiments). (<b>F</b>) Inhibition of transferrin uptake after dynasore and PAO treatment. HeLa cells were left untreated (Ctrl) or treated with dynasore (80 µM) (Dyn) or PAO (5 µM) 10 minutes before and during the transferrin uptake assay (results are expressed as the mean ± SD of 18 images; graph representative of 2 independent experiments). (<b>G</b>) Dynasore and PAO fail to enhance basal degradation of IκBα. HeLa cells were pretreated for 10 minutes with dynasore (80 µM) or PAO (5 µM) and analyzed by western immunoblotting using an IκBα antibody. Actin is shown as a loading control (results representative of 2 independent experiments). (<b>H</b>) Long-term inhibition of endocytosis in dynasore-treated HeLa cells. Transferrin uptake in HeLa cells left untreated or treated with dynasore (80 µM) for 48 hours (results are expressed as the mean ± SD of 18 images; graph representative of 2 independent experiments). (<b>I</b>) Long-term inhibition of endocytosis fails to enhance the basal degradation of IκBα. Basal degradation of IκBα in HeLa cells left untreated or treated with dynasore (80 µM) for 48 hours. As positive control of the degradation of IκBα, cells were stimulated for 20 minutes with TNFα (results representative of 2 independent experiments). (<b>J</b>) CLCa knockdown fails to enhance IκBα degradation. Cell lysates from control, CLCa or CHC siRNA transfected cells were analyzed by immunoblotting using indicated antibodies. Actin is shown as loading control. (<b>K</b>) Densitometric quantification of IκBα levels shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017158#pone-0017158-g004" target="_blank">Figure 4F</a> (Graph representative of 2 independent experiments). (<b>L</b>) CLCa knockdown fails to induce constitutive IL-8 expression. HeLa cells were transfected with control, CLCa or CHC siRNAs for 72 hours and supernatants were collected to measure the concentration of IL-8 by ELISA (results are expressed as the mean ± SD of 3 independent experiments).</p

CHC prevents constitutive NF-κB p65 nuclear localization in unstimulated epithelial cells.

<p>(<b>A</b>) Effective knockdown of CHC after siRNA transfection. Lysates from HeLa cells transfected with control (Ctrl) or CHC siRNAs for 72 hours were analyzed by western immunoblotting using indicated antibodies. Actin is shown as a loading control. (<b>B</b>) Constitutive nuclear localization of p65 after CHC knockdown. HeLa cells were transfected with either control or CHC siRNA and p65 localization was visualized by immunofluorescence microscopy. White arrows indicate cells showing a clear nuclear localization of p65. Scale bars, 10 µm. (<b>C</b>) Quantification of the nuclear/cytosolic p65 intensity ratio in control and CHC siRNA transfected HeLa cells (results are expressed as the mean ± SD of 12 images; *p = 3.14E-07, graph representative of 3 independent experiments).</p

CHC prevents constitutive IL-8 expression in unstimulated epithelial cells.

<p>(<b>A</b>) Constitutive IL-8 expression after knockdown of CHC in HeLa cells. Cells were transfected with control or CHC siRNAs. After 72 hours, supernatants were collected and analyzed for their content in IL-8 by ELISA (results are expressed as the mean ± SD of 3 independent experiments). (<b>B</b>) Constitutive IL-8 expression after knockdown of CHC in MCF-7 cells. MCF-7 cells were treated as described in (A) (results are expressed as the mean ± SD of 3 independent experiments). (<b>C</b>) IKKα-depletion abolishes the constitutive secretion of IL-8 induced by CHC knockdown. HeLa cells were transfected with different combinations of IKKα and CHC siRNAs for 72 hours. Total siRNA concentration was kept constant by adding appropriate amounts of control siRNAs. Supernatants were collected to measure the concentration of IL-8 by ELISA (results are expressed as the mean ± SD of 3 independent experiments).</p

GILZ is expressed upon stimulation with C3 exotoxin or toxin B.

<p>(A) HeLa cells were incubated with toxin B (50ng/ml) for indicated the time spans and immunoblots were performed from whole cell lysates using anti-Rac1 mAb102 recognizing only non-glucosylated Rac-1, and an anti-Rac1 mAb antibody recognizing total Rac1 as well as antibodies recognizing RhoB, actin or GILZ. (B) Immunoblot of GILZ and actin expression upon stimulation of HeLa cells with C2IN-C3lim (100 ng/mL) + C2IIa (200 ng/mL) for indicated time spans. (C) To explore the expression of additional GILZ isoforms, HeLa cells were transfected with 7.5 nM siRNA specific for GILZ or control siRNA for 48 h and subsequently either left untreated or stimulated with <i>C. difficile</i> toxin B (50 ng/ml) or 100 µM DEX for 4 h. Arrows mark three GILZ isoforms which were inhibited by the used GILZ siRNA. Note that only isoform 1 was induced by the used stimuli. (D) Cells were stimulated with toxin B for 2 or 4 h to determine <i>GILZ</i> mRNA expression by real-time RT-PCR. Mean + SD of 2 independent experiments normalized to untreated. (E) To assay transcriptional activity of the GILZ promoter cells were transfected with a luciferase reporter under control of a 2088 bp <i>GILZ</i> promoter and co-transfected with pCMV-ß-gal (for standardization) 24 h before infection with a <i>Y. enterocolitica</i> pYV⁺ and various mutant strains or treatment with DEX or toxin B. Means + SD of 4 independent experiments normalized to untreated. Significant differences compared to untreated are indicated by asterisks (p<0.05).</p

<i>Yersinia enterocolitica</i> induces GILZ expression.

<p>HeLa cells were infected with a strain harboring the <i>Yersinia</i> virulence plasmid (pYV⁺) or plasmid cured strain (pYV⁻) with MOI 20 or stimulated with 100 µM DEX for indicated time intervals. The amount of GILZ in cytosolic proteins lysates of HeLa cells was detected by immunoblot at different time points. Actin was used as an internal standard. A representative experiment and quantification means + SEM normalized to untreated of at least 3 experiments are shown.</p

Role of myc-1 E-box in TF binding.

<p>Electromobility shift analyses were performed using a double-stranded oligonucleotide probe representing the <i>GILZ</i> promoter sequence −63 to −37 and for some experiments probes with mutations of the E-box cis-elements and the flanking cAMP response (Cre) elements as depicted in (A). HeLa cells were stimulated/infected for 0.5 h or indicated time intervals with toxin B (B, D, E) or <i>Y. enterocolitica</i> (C) and nuclear extracts of these cells were incubated with P³²-labeled GILZ−63/−37 probe. Subsequently band shift analyses were performed. (D) Nuclear extracts were pretreated with a 100-fold excess of indicated cold probes. (E) Nuclear extracts were pretreated with indicated antibodies. Anti-p65 antibody was used as a negative control.</p

Role of Rho GTPases and MAP kinases for GILZ expression.

<p>(A) Overexpression of RhoA or RhoB lowers basal GILZ levels. HeLa cells were co-transfected with the p2088 <i>GILZ</i> promoter luciferase reporter and pHM6 based plasmid for overexpression of the indicated Rho GTPases. Means + SD of 3 independent experiments normalized to untreated. Significant differences compared to control vector transfection are indicated by asterisks (p<0.05). In a control experiment, HeLa cells were transfected in the same setting and cell lysates were used for immunoblots to detect RhoA, RhoB, Cdc42 and Rac1 expression. (B) HeLa cells were transfected for 48 h with indicated concentrations of siRNA. Immunoblots were performed from cell lysates for RhoA and RhoB and from cytosolic extracts for GILZ. (C) Toxin B treatment leads to fast and transient MAPK phosphorylation. After treatment of HeLa cells with toxin B for the indicated time spans, levels of phosphorylated as well as total ERK and p38 were assayed by immunoblot. (C) Toxin B induced GILZ expression is mediated by both ERK and p38 MAPK. Cells were pretreated with MAPK phosphorylation inhibitors SB 202190 (p38) or PD 98059 (ERK) 2 h prior to toxin B stimulation and GILZ protein was detected by immunoblot analysis at 6 h or 24 h after stimulation.</p

Role of USF-1 and USF-2 for toxin B induced GILZ expression.

<p>(A) HeLa cells were transfected with siRNA silencing USF-1 or USF-2 or with control siRNA (siC) 48 h prior to toxin B treatment and USF-1/2 as wells as GILZ and actin expression was determined by immunoblot. (B) HeLa cells were transfected with empty vector or a dominant negative (DN) USF-1 mutant and subsequently <i>GILZ</i> mRNA or GILZ protein expression was determined by real time RT-PCR (one representative experiment performed in quadruplicates, means + SEM) or immunoblot.</p