Rosiglitazone inhibits IL-8 and MCP-1 production via a PPARγ-dependent mechanism in LPS-stimulated HK-2 cells.

<p>(A, B) HK-2 cells were pretreated with rosiglitazone (5, 10, 20 µM) in the absence or presence of GW9662 (10, 30, 100 µM) for 2 hours and then treated with 1 μg/ml LPS for 4 hours. IL-8 and MCP-1 mRNA was analyzed by real-time PCR. (C, D) HK-2 cell were pretreated with 10 µM rosiglitazone in the absence or presence of 100 µM GW9662 for 2 hours and then treated with 1 μg/ml LPS for 24 hours. IL-8 and MCP-1 protein in cell supernatants was measured by ELISA. Results are shown as mean ± SD and representative of three independent experiments. *<i>P</i><0.05 compared to the control group; ^<i>#</i><i>P</i><0.05 compared to the LPS-treated group; ^<i>&</i><i>P</i><0.05 compared to LPS + RGL (10 µM)-treated group. </p

Cytotoxic assessment of RGL in HK-2 cells.

<p>HK-2 cells were treated with the indicated concentrations of RGL (0, 5, 10 , 20 μM) for 24 hours with or without 1 μg/ml LPS. HK-2 cells viability was assessed using an MTT assay, and the surviving cell values are expressed as a percent of control treated cells (no addition of RGL). Each value indicates the mean ± SD from three independent experiments.</p

Rosiglitazone fails to reverse LPS-induced NF-κB nuclear translocation.

<p>HK-2 cells were treated without or with 10 µM RGL for 2 hours and then stimulation with 1 μg/ml LPS for 30 minutes. Nuclear protein was extracted for immunoblotting or cells was processed for immunofluorescent staining as described in 'Material and Methods'. (A) Relative levels of p50 and p65 in nucleus are determined by densitometric analysis and are presented as the relative ratio of LaminB, respectively. The 2 lanes for the same group in the blot represent duplicate experiments. The column bar graph shows the means ±SD of values obtained by densitometric analysis of three independent experiments. *<i>P</i><0.05 compared to the control group. (B) Immunofluorescent staining shows that NF-κB p65 translocated into nucleus after treatment of 1 μg/ml LPS for 30 minutes and pretreatment of 10 µM RGL fialed to antagonize p65 nuclear translocation (Original magnification × 400). </p

Rosiglitazone inhibits NF-κB-DNA binding activity.

<p>(A) HK-2 cells were pretreated with 10 µM rosiglitazone in the absence or presence of 100 µM GW9662 for 2 hours and then treated with 1 μg/ml LPS for 30 minutes. Equal amounts of nuclear extracts were assayed for binding a biotin-labeled double-stranded NF-κB oligonucleotide as described in 'Material and Methods'. The positions of NF-κB p65 was indicated. The column bar graph shows the means ± SD of values obtained by EMSAs densitometric analysis from three independent experiments. *<i>P</i><0.05 compared to the control group; ^<i>#</i><i>P</i><0.05 compared to the LPS-treated group; ^<i>&</i><i>P</i><0.05 compared to LPS + RGL (10 µM)-treated group.</p

RGL inhibits LPS-induced IL-8 and MCP-1 expression in a NCoR-dependent manner.

<p>HK-2 cells were transfected with siNCoR to knock down NCoR, or transfected with sicontrol as controls. Two days after transfection, IL-8 and MCP-1 mRNA were quantified after incubated without or with 10 µM RGL for 2 hours followed by stimulation with 1 μg/ml LPS for 4 hours in HK-2 cells and HK-2/NCoR-knockdown cells. (A) Knockdown efficience of NCoR mRNA by siRNA. (B) The expression of IL-8 mRNA in different groups. (C) The expression of MCP-1 mRNA in different groups. The results are representative of three independent experiments. *<i>P</i><0.05 compared to the control group; ^<i>#</i><i>P</i><0.05 compared to the LPS-treated group.</p

SUMOylation of PPARγ by RGL antagonizes LPS-induced chemokine expression and NCoR degradation.

<p>HK-2 cells were transfected with siPIAS1 to knock down PIAS1, or transfected with sicontrol as controls. Two days after transfection, IL-8 and MCP-1 mRNA were quantified after incubated with 10 µM RGL for 2 hours followed by stimulation with LPS 1 μg/ml for 4 hours in HK-2 cells and HK-2/PIAS1-knockdown cells. Quantification of mRNA level was performed by real-time PCR. (A) Knockdown efficience of PIAS1 mRNA by siRNA. (B) The expression of IL-8 mRNA in different groups. (C) The expression of MCP-1 mRNA in different groups. (D) Two days after transfection, Nuclear protein was extracted after incubated with 10 µM RGL for 2 hours followed by stimulation with 1 μg/ml LPS for 30 minutes in HK-2 cells. NCoR protein were quantitated by immunoblotting analysis in different groups. The results are representative of three independent experiments. *<i>P</i><0.05 compared to the control group; ^<i>#</i><i>P</i><0.05 compared to the LPS-treated group. </p

Rosiglitazone inhibits NF-κB binding in IL-8/MCP-1 promoters.

<p>HK-2 cells were treated without or with 10 µM RGL for 2 hours and then stimulation with 1 μg/ml LPS for 60 minutes. The representative agarose gel show PCR products of DNA obtained after chromatin immunoprecipitation with anti-p65 antibody (IP:anti-p65), non -immunoprecipitated DNA (input), or normal mouse IgG (negative control) amplified with primers specific for NF-κB site in IL-8/MCP-1 promoter. </p