Glutathione S-transferase P1 suppresses iNOS protein stability in RAW264.7 macrophage-like cells after LPS stimulation

<div><p>Glutathione S-transferase P1 (GSTP1) is a ubiquitous expressed protein which plays an important role in the detoxification and xenobiotics metabolism. Previous studies showed that GSTP1 was upregulated by the LPS stimulation in RAW264.7 macrophage-like cells and GSTP1 overexpression downregulated lipopolysaccharide (LPS) induced inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression. Here we show that GSTP1 physically associates with the oxygenase domain of iNOS by the G-site domain and decreases the protein level of iNOS dimer. Both overexpression and RNA interference (RNAi) experiments indicate that GSTP1 downregulates iNOS protein level and increases S-nitrosylation and ubiquitination of iNOS. The Y7F mutant type of GSTP1 physically associates with iNOS, but shows no effect on iNOS protein content, iNOS S-nitrosylation, and changes in iNOS from dimer to monomer, suggesting the importance of enzyme activity of GSTP1 in regulating iNOS S-nitrosylation and stability. GSTM1, another member of GSTs shows no significant effect on regulation of iNOS. In conclusion, our study reveals the novel role of GSTP1 in regulation of iNOS by affecting S-nitrosylation, dimerization, and stability, which provides a new insight for analyzing the regulation of iNOS and the anti-inflammatory effects of GSTP1.</p></div

Mass spectrometry data and RNA sequencing data of the effect of Gstp1 on SHR-VSMCs

RNA sequencing analysis was performed to recapitulate possible changes in transcriptome profile caused by downregulation of Gstp1 in SHR-VSMCs. Total RNA was extracted from the cells using Trizol reagent. The concentration, quality and integrity were determined using a NanoDrop spectrophotometer (ThermoFisher Scientific, MA, USA). Total RNA required for single library construction was more than 1 μg. Sequencing libraries were generated using the TruSeq RNA Sample Preparation Kit (Illumina, San Diego, CA, USA). Library sequencing was performed on an Illumina Novaseq 6000 platform (Illumina, San Diego, CA, USA).Mass spectrometry analysis was performed to identify Gstp1 binding proteins in SHR-VSMCs. Proteins were immunoprecipitated using anti-Gstp1 antibody and incubated with Protein A/G PLUS-agarose beads for 4 h. Elution buffer (0.1M glycine–HCl) was used to elute antigen from antibody and 1M Tris–HCl was used for neutralization. After trypsin digestion, peptides were desalted using C18 StageTip(Thermo Fisher Scientific, OH, USA), and vacuum dried. The samples were analyzed by LC-MS/MS using an Easy nLC 1200 coupled to a QExactive HF mass spectrometer (Thermo Scientific, OH, USA). Sequence database searching and protein quantification raw mass spectrometry data were analyzed using the MaxQuant software suite (version 1.6.1.0).</p

Inducible HSP70 antagonized IL-1β-induced cytotoxic effect on HeLa cells and improved the cell survival.

<p>(A) HeLa cells were stimulated with IL-1β (10 ng/ml) for indicated times, HSP70, HSP27, HSP90, and β-actin levels were determined by Western blotting. (B) HeLa cells were transfected with pcDNA3.0 (−) or pcDNA3.0-Flag-HSP70 (+). After 48 h, cells with either baseline level of or overexpression of HSP70 were exposed to IL-1β(10 ng/ml) or CHX (1 µg/ml) or both for 24 h. Cell viability was subsequently detected by CCK8 assay as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050059#s2" target="_blank">Material and Methods</a>. Cell viability is shown relative to the untreated control. The experiment was independently repeated for three times and data were shown as mean ± SD; bars: SD. Significant difference was determined by student’s <i>t</i>-test comparing HSP70 transfected cells with vector transfected control exposed to both IL-1β and CHX, * <i>P</i><0.05.</p

Inducible HSP70 modulated IL-1β-induced activation of TAK1-NF-κB cascades, but not of TAK1-MAPKs.

<p>(A) HeLa cells were transfected with pcDNA3.0 (−) or pcDNA3.0-Flag-HSP70 (+) for 48 h and then stimulated with IL-1β (10 ng/ml) for indicated times. Cell lysates were prepared and subjected to immunoblotting with anti-IκBα, anti-phospho-p38 (p-p38, Thr180/Tyr182), and anti-phospho-JNK (p-JNK, Thr183/Tyr185) antibodies, respectively, to reveal the activation of NF-κB and MAPKs. Total cellular TAK1, p38, and JNK protein levels in cell lysates were also determined by immunoblotting with specific antibody respectively. (B) HeLa cells were either non-transfected (control) or transfected with pRNA-U6.1/neo vector or pRNA-U6.1/neo-HSP70i construct. After 48 h transfection, cells were exposed to IL-1β (10 ng/ml) for 12 h and cell lysates were subjected to immunoblotting with anti-HSP70 antibody to determine the efficiency of RNAi, GAPDH was used as an internal control. (C) HeLa cells were transfected with either pRNA-U6.1/neo vector or pRNA-U6.1/neo-HSP70i construct. After 48 h transfection, cells were stimulated with IL-1β (10 ng/ml) for indicated times. immunoblotting with anti-IκBα, anti-phospho-p38 (p-p38, Thr180/Tyr182), and anti-phospho-JNK (p-JNK, Thr183/Tyr185) antibodies, respectively, to reveal the activation of NF-κB and MAPKs. Total cellular TAK1, p38, and JNK protein levels in cell lysates were also determined by immunoblotting with specific antibody respectively. Equal loading protein was confirmed by GAPDH.</p

HSP70 overexpression inhibited IL-1β-induced production of iNOS.

<p>HeLa cells were transfected with pcDNA3.0 (−) or pcDNA3.0-Flag-HSP70 (+) for 48 h and then stimulated with or without IL-1β (10 ng/ml) plus CHX (1 µg/ml) for 4 h. Cells lysates were prepared and subjected to immunoblotting to measure protein level of iNOS. Antibody against Flag-tag was used to show the overexpression of HSP70 and equal loading protein was confirmed by GAPDH.</p

Increased expression level of HSP70 upon heat shock reduced TAK1 protein level and the association between HSP90 and TAK1.

<p>HeLa cells were either untreated (control) or exposed to a mild heat shock (42°C for 30 min) followed by recovery at 37°C for indicated time periods. Cell lysates were prepared and subjected to immunoblotting with indicated antibodies or immunoprecipitation with TAK1 antibody. The immunopellets were analyzed by immunoblotting with HSP90 antibody. Equal loading protein was confirmed by GAPDH. The numbers below each Western blot represent relative expression level normalized to GAPDH, which were determined based on the intensity of band. These experiments were independently repeated for three times, and the representative blots were shown.</p

HSP70 destabilized TAK1 at protein level via proteasome pathway.

<p>(A) HeLa cells were exposed to a mild heat shock (42°C for 30 min) followed by recovery at 37°C for indicated time periods. Total RNA was isolated and TAK1 mRNA was detected by RT-PCR using primers indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050059#s2" target="_blank">Material and Methods</a>. β-actin was used as control. (B) HeLa cells were transfected with pcDNA3.0-Flag-HSP70 (+) or pcDNA3.0 (−). After 48 h, cells were treated with 1 µg/ml cycloheximide (CHX) and harvested at indicated time points. TAK1 protein levels were determined by Western blotting, GAPDH was used as loading control, and antibody against Flag-tag was used to show the overexpression of HSP70. (C) HeLa cells were transfected with pcDNA3.0-Flag-HSP70 (+) or pcDNA3.0 (−). After 48 h, cells were treated with a proteasome inhibitor MG-132 at indicated concentrations for 6 h. Cells were collected and subjected to immunoblot analysis with TAK1 antibody. Equal loading protein was confirmed by immunoblotting with anti-GAPDH antibody.</p

Luteolin associated with Hsp90.

<p>A. Upper left picture showed the chemical structure of luteolin. Upper right figure displayed the structure of Hsp90 with its two ATPase sites (in green). The lower left picture showed the molecular modeling of binding sites in Hsp90 for luteolin performed by using Chemistry at HARvard Macromolecular Mechanics (CHARMm)-Version 33.1. The lower right figure displayed hydrogen bonds between Hsp90 and luteolin (in green). B. SPR analysis showed the interaction between different concentration of luteolin and Hsp90. C. HeLa cell were incubated with luteolin, geldanamycin, celastrol, ethanol, DMSO or left untreated. Pre-equilibrated γ-phosphatelinked ATP-Sepharose was used to pull down endogenous Hsp90. ATP-Sepharose beads bound Hsp90 was detected by immunoblotting.</p

Luteolin induced carcinoma cells apoptosis.

<p>A, Carcinoma cells (HeLa, HepG2), and normal cells (WRL-68, HEK293, XJH B) were treated with 20, 30, 40, 50, 60 µM luteolin for 24 h and followed by CytoTox-Glo™ cytotoxicity assays. The data above are plotted as “dead cell” rate versus the concentration of Luteolin. B. HeLa cells were treated with indicated concentration of luteolin for 24 h and subjected to immunoblot analysis for pro-caspase3 and PARP. C. HeLa cells were treated with indicated concentration of luteolin for 24 h and were harvested, and then stained with propidium iodidle and AnnexinV-FITC for detecting the apoptosis by flow cytometry. D. HeLa cells were transfected with indicated concentration of HA-Hsp90 and 24 h after transfection cells were treated with 50 µM luteolin for 24 h and were harvested, and then stained with propidium iodidle and AnnexinV-FITC for detecting the apoptosis by flow cytometry. E. HeLa cells were treated with 50 µM luteolin and 10 µM GA, for 24 h, and then cells were incubated with primary antibodies against PARP. HeLa cells were immunostained with anti-mouse alexa fluor 488 secondary antibody and then stained with DAPI and Alexa Fluor 555 phalloidin derivatives for labeling F-actin. The specimens were visualized by confocal laser scanning microscopy. Blue depicts the nucleus, red depicts localization of F-actin and green depicts localization of cleaved-PARP. F. HeLa cells were pretreated with 25 µM z-VAD-fmk for 1 h and then treated with 50 µM luteolin for 24 h followed by immunoblotting with pro-caspase3 and cleaved PARP antibodies.</p

Luteolin inhibits activation of STAT3 in carcinoma cells.

<p>A, HeLa cells were treated with indicated concentrations of luteolin for 24 h and then were subjected to Western blot for measuring protein levels of phosphor-STAT3 (tyr705), phosphor-STAT3 (ser727), STAT3 and c-myc respectively. B, HeLa cells were treated with 50 µM luteolin for different time and then were subjected to Western blot by using phosphor-STAT3 (tyr705), phosphor-STAT3 (ser727), STAT3 or c-myc antibody respectively. C, MCF-7 cells were treated with indicated dose of luteolin, and then subjected to Western blot for detecting protein levels of phosphor-STAT3 (tyr705), phosphor-STAT3 (ser727) and STAT3. D, Hep3B cells treated with indicated dose of luteolin and then subjected to Western blot for detecting protein levels of phosphor-STAT3 (tyr705), phosphor-STAT3 (ser727) and STAT3. E, HeLa cells were treated with indicated dose of luteolin respectively for 24 h and then were subjected used for measuring the nuclear and cytoplasmic fraction of STAT3. F, HeLa cells were transfected with STAT3 luciferase reporter plasmids for 24 hours and then treated with luteolin for 24 hours. Luciferase activity was measured by Luminometer TD-20/20. Values were obtained from three independent experiments. _*<i>P</i><0.05, **<i>P</i><0.001, compared with the control.</p