53 research outputs found

    Purified GST-tagged HSF1 was phosphorylated <i>in vitro</i> by mTOR.

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    <p>(A) <i>In vitro</i> phosphorylation was performed by mixing GST-tagged HSF1 with various concentrations of recombinant mTOR. Samples were then fractionated by 10% SDS-PAGE and western analysis performed to examine the levels of HSF1-phosphoserine 326 and total HSF1. The identities of the phosphopeptides were determined by isolating the mTOR phosphorylated GST-HSF1 with 10% SDS-PAGE, trypsin digestion of the GST-HSF1 band and identification of peptides by mass spectrometry and database analysis. (B) Protein sequence of HSF1 from amino acids 300–330. The mTOR phosphorylation site, <b>serine 326</b> is marked in bold and nearby serine phosphorylation sites that have been characterized (serines 303, 307, 320 and 326) are underlined.</p

    The role of mTOR in cellular resistance to proteotoxic stress.

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    <p>Control and mTOR knockdown HeLa cells were either untreated or treated with heat shock at 43°C for 1 hr and 2 hr. For thermal tolerance experiment, cells were treated with heat shock at 43°C for 1 hr and recovered at 37°C overnight. Cells were then treated with a second heat shock at 43°C for 2 hr followed by 37°C recovery. Nine days post-treatment, colonies were visualized by crystal violet staining. Colony counts were the averages calculated from three individual plates. Percentage of colonies formed was determined by comparison to the number of cells initially seeded in each treatment. Experiments were carried out three times, with reproducible findings.</p

    mTOR activity and serine 326 phosphorylation are required for the heat induced activation of the <i>hsp70.1</i> promoter.

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    <p>(A) Effects of 43°C heat shock on <i>hsp70.1</i> activity in HeLa cells without and with rapamycin treatment. Cells transfected with the pGL3-Hsp70-LUC reporter construct were treated with 30 nM rapamycin for 2 hr prior to receiving heat shock and luciferase activity was assayed at 24 hr. Data are means of triplicate assays +/− SD. (B) Effects of mTOR knockdown on activation of the <i>hsp70.1</i> promoter by heat shock. HeLa cells either stably expressing shRNA targeted to mTOR or control hairpin were transfected with the pGL3-Hsp70-LUC reporter construct followed by heat shock (effectiveness of mTOR knockdown is indicated in Fig. 4B). Luciferase activity in cells after 48 hr recovery at 37°C is the mean of triplicate assay and is plotted +/− SD (C) HeLa cells depleted of HSF1 by stable expression of shRNA targeting the factor were co-transfected with the pGL3-Hsp70-LUC reporter construct and expression plasmids encoding either wild type HSF1 or HSF1-S326A. Cells lysates were collected from cells that were either heat shocked or used as control, 48 hr post-heating and luciferase assays were then performed in triplicate and activity plotted as mean +/− standard deviation. Experiments were carried out in duplicate, reproducibly.</p

    mTOR regulates transcriptional and translational expression of heat shock proteins.

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    <p>Control and mTOR knockdown HeLa cells were treated with or without heat. (A) Real time quantitative RT-PCR analysis was performed for analysis of the expression levels of Hsp70, Hsp90 and Hsp110 mRNA. Fold change was calculated by normalization to β-actin levels, followed by comparison with the control untreated sample. (B) Expression levels of Hsp70, Hsp90 and Hsp110 were then determined 24 hr after recovery from heat shock, at 37°C by western blotting using anti-Hsp70, anti-Hsp90 and anti-Hsp110 antibodies. Levels of GAPDH expression were also measured as loading controls. Experiments were carried out three times with consistent results.</p

    Regulation of HSF1 serine 326 phosphorylation during stress.

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    <p>(A) Levels of HSF1-phosphoserine 326, total HSF1 and β-actin in HeLa cells treated with heat at 43°C for 30 min and recovered at 37°C for up to 24hr. (B) Levels of HSF1-phosphoserine 326, HSF1-phosphoserine 303, HSF1-phosphoserine 320, total HSF1, mTOR and β-actin in control and mTOR knockdown HeLa cells with or without heat shock at 43°C for 30 min. (C) Intracellular concentration of HSF1-phosphoserine 326, total HSF1, S6 kinase-phosphothreonine-389, total S6 kinase and β-actin, without or with heat shock in HeLa cells pretreated with mTOR inhibitors rapamycin (30 nM) and KU0063794 (2 µM) and kinase inhibitor staurosporine (100 nM) for 2 hr. Relative levels of HSF1-phosphoserine 326 in cells after the various treatments were determined by densitometric analysis of X-ray films, normalized to untreated cells (lane 1), and are indicated below the representation of the immunoblots. (D) HeLa cells were treated for 2 hr with stress inducers MG132 (5 µM), 17-AAG (2 µM), CdCl<sub>2</sub> (200 µM) and sodium arsenite (1 µM) prior to assay for HSF1-phosphoserine 326, total HSF1 and β-actin. Relative levels of HSF1-phosphoserine 326 in cells were determined by densitometric analysis as above, normalized to levels in untreated cells (lane 1), and are indicated below the representation of the immunoblots. Experiments were performed on at least three occasions with reproducible findings.</p

    Schematic diagram of the proposed pathway of LPS-SREC-I-TLR4 mediated activation of proinflammatory cytokine expression.

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    <p>This cartoon depicts LPS binding to SREC-I (1) followed by LPS-SREC-I complex recruitment of TLR4 into discrete lipid microdomains (thick line) (2). Localization of LPS-SREC-I-TLR4 complexes to such lipid microdomains then led to activation of downstream proinflammatory cytokine release through adaptor proteins MyD88 and TRIF. LPS exposure could thus mediate activation of the NF-kB and MAPK pathways. In addition, LPS has been shown to be recognized by CD14, the primary responder to the endotoxin, which then bound to TLR4 and triggered proinflammatory signaling through NF-kB and MAPK (3). We have additionally shown that LPS-SREC-I-TLR4 signaling led to activation of IRF3. Engagement of these signaling pathways could then lead to activation of transcription factors NF-kB, AP-1 and IRF3 that have been shown to function combinatorially in cytokine gene transcription.</p

    Ligand-bound SREC-I colocalized with TLR4 after LPS treatment.

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    <p><b>A,</b> SREC-I and TLR4 did not interact in the absence of LPS. Raw 264.7 cells were transfected with FLAG-SREC-I for 22 hours. Cells were then fixed and stained with anti TLR4 ab (green) and anti-FLAG ab (red). <b>B,</b> TLR4 colocalized with SREC-I in the presence of LPS. Raw 264.7 cells overexpressing FLAG-SREC-I were exposed to LPS (1 μg/ml) for 20–30 min at 4°C. Cells were then fixed and stained for TLR4 (green) and FLAG (red). Percent colocalization with or without LPS is shown in the adjacent histogram. <b>C, D,</b> LPS, TLR4 and SREC-I were internalized at 37°C. Raw 264.7 cells overexpressing FLAG-SREC-I were incubated with Alexa LPS (1 μg/ml) at 4°C for 20 mins and then medium was replaced with warm medium. Cells were then incubated at 37°C for 10–15 mins. Cells were fixed and stained for TLR4 (green, C, D) and FLAG-SREC-I (red in C, purple in D). Alexa LPS was shown in red (D). <b>E, F</b>, Endogenous TLR4 and SREC-I did not colocalize in the absence of LPS. Raw 264.7 cells were treated for 30 mins with LPS (1 μg/ml) and then fixed and stained for TLR4 (green) and SREC-I (red). <b>E,</b> TLR4 and SREC-I colocalize in the presence of LPS. Cells were labeled with LPS (1μg/ml) for 20–30 minutes at 4°C then fixed and stained for TLR4 (green) and SREC-I (red) (F). <b>G,</b> SREC-I expression level in Raw 264.7 cells and in cells overexpressing FLAG-SREC-I. <b>H,</b> SREC-I interacted with TLR4 physically in the presence of LPS (1 μg/ml). HEK 293 cells expressing FLAG-SREC-I and TLR4s were treated with or without Hsp90 or LPS. FLAG-SREC-I was then immunoprecipitated (IP) with FLAG ab. The IP complex was separated with SDS-PAGE and blotted for TLR4. The amount of FLAG-SREC-I immunoprecipitated was determined and β-actin was used as a loading control. All images were representative of 3 different planes from each sample. Each experiment was performed 3 times reproducibly. Scale bar, 5 μm.</p

    SREC-I supported LPS-TLR4 mediated NF-kB (phospho-p65).

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    <p><b>A,</b> Phospho-p65, (S536/Rel) level was increased in cells expressing SREC-I with TLR4 in the presence of LPS. HEK 293 cells expressing TLR4-MD2-CD14 and/or SREC-I, SREC-I only were treated with or without LPS (1 μg/ml) or Hsp90 for 5–7 hours. Cell lysates were then collected and SDS-PAGE was performed. Phospho-p65 levels were measured. Total level of p65 was measured in the same lysate. Total p65 level was determined. <b>B,</b> HEK 293 cells expressing SREC-I and TLR4 or TLR4 only were transfected with NF-kB-SEAP and incubated with LPS (1 μg/ml) for 5 hours. NF-kB activity was measured as instructed by NF-kB-SEAporter assay kit. <b>C,</b> Raw 264.7 cells were transfected with siRNA for SREC-I/TLR4 for 72 hours and incubated with LPS (1 μg/ml) with or without CD14 neutralizing peptide (inhibitor). Phospho-p65 level is increased with LPS incubation in cells expressing both TLR4 and SREC-I. <b>D,</b> Raw 264.7 cells were transfected with ctl (scr) siRNA or TLR4 siRNA/SREC-I siRNA for 72 hours. Cell lysates were isolated and later SDS-PAGE was performed. <b>E,</b> HEK 293 cells expressing TLR4, MD-2, CD14 or TLR4, MD-2, CD14 and SREC-I were incubated with LPS (1 μg/ml) for indicated time. Cells lysates were collected and equal amount of protein was loaded for SDS-PAGE experiment. For blocking CD14 activity, cells were treated with 10 μg/ml of anti CD14 neutralizing antibody. Error bars in graph show S.D. between three replicate experiments. <i>P</i> <0.0001 values were generated by ANOVA using the Bonferroni post-test.</p

    Rho GTPase activity was required for LPS-SREC-I-TLR4 induced proinflammatory cytokine release.

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    <p>BMDM cells from WT and TLR4 KO mice were transfected with siRNA SREC-I or control RNA. Cells were treated with or without Toxin B (2 ng/ml) and incubated withor without LPS. Experiments were performed in the presence of SR-A blocking antibodies. Cell media were collected, clarified by centrifugation and the IL-6 ELISA assay was performed. Experiments were repeated reproducibly 3 times. The height of the error bars represents the average of three independent measurements. The error bars represent one standard deviation from the mean. ***<i>P</i><0.0001 when compared to the control, and #<i>P</i><0.0001 when compared to the WT. Values were generated by ANOVA using the Bonferroni post-test.</p

    Intact lipid microdomains were essential for LPS-SREC-I-TLR4-induced MAPK signaling.

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    <p><b>A,</b> HEK 293 cells expressing TLR4-MD2-CD14, SREC-I-TLR4-MD2-CD14 were treated with or without CD14 neutralizing peptide (inhibitor). Cells were then incubated with LPS (1 μg/ml) for 3 hrs. Cell lysates were run on SDS-PAGE and then immunoblotted with phospho-specific antibodies for JNK, p38, p65 and anti-JNK ab, p38 ab and anti-p65 ab. Drugs including PP2 (10 μM Srcinhibitor), MβCD (MBD, 10 mM, cholesterol sequestering agent), TxB (2 ng/ml, Clostridium Toxin B) were added to inhibit the functions of Src kinase, lipid microdomain formation and Rho GTPase activities as indicated. Raw 264.7 cells were transfected with siRNA for TLR4/SREC-I and then treated as described above. These experiments were repeated reproducibly 2 times. <b>B,</b> Raw 264.7 cells expressing TLR4, SREC-I or TLR4 only was incubated with LPS (1 μg/ml) for 3 hrs. Cell lysates were collected and then subjected to SDS-PAGE and Western Blotting. <b>C, D,</b> Raw 264.7 cells were transfected with indicated siRNA for 72 hours. Cell lysates were collected and equal amount of protein was subjected to SDS-PAGE and Western Blotting. Expression of SREC-I and TLR4 is shown in Raw 264.7 cells.</p
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