Effects of inhibition of PI3K/Akt1 and MEK on 7αOHChol-induced IL-8 expression.

<p>(A) THP-1 cells were treated with 7αOHChol for 48 h in the absence or presence of LY294002 or U0126 (10 μM each). Transcript levels of IL-8 were assessed by realtime-PCR. Data are expressed as the means ± SD (n = 3 replicates for each group). *** P < 0.001 vs. control; ## P < 0.01 vs. 7αOHChol; ### P < 0.001 vs. 7αOHChol. (B) THP-1 cells were infected with lentiviruses expressing Akt1 shRNA or control lentiviruses. After selection in the presence of puromycin, expression of Akt1 transcripts was examined by RT-PCR. (C) THP-1 cells infected with lentiviruses expressing Akt1 shRNA or control lentiviruses were stimulated for 48 h with or without 7αOHChol. Transcript levels of IL-8 were assessed by realtime-PCR. Data are expressed as mean ± SD (n = 3 replicates/group). * P < 0.05; ** P < 0.01; *** P < 0.001.</p

Effects of 7αOHChol on expression of IL-8.

<p>(A) THP-1 cells were incubated with 7αOHChol (5 μg/ml) for the indicated time periods. The levels of IL-8 transcripts were assessed by real-time PCR. Data are expressed as the means ± SD (n = 3 replicates for each group). * P < 0.05 vs. 0 h; *** P < 0.001 vs. 0 h. (B) THP-1 cells were incubated in the presence of the indicated amounts of 7αOHChol for 48 h. The levels of IL-8 transcripts were assessed by real-time PCR. Data are expressed as the means ± SD (n = 3 replicates for each group). *** P < 0.001 vs. 0 μg/ml. (C) THP-1 cells were treated for 48 h with 7αOHChol (5 μg/ml) and 27OHChol (2.5 μg/ml). The levels of IL-8 transcripts were assessed by realtime-PCR. Data are expressed as the means ± SD (n = 3 replicates for each group). *** P < 0.001 vs. control. (D) THP-1 cells were incubated for 48 h with or without 7αOHChol (5 μg/ml) in the absence or presence of CHX (1 μg/ml). Transcripts of IL-8 were amplified by RT-PCR (upper panel), and the levels of IL-8 transcripts were determined by real-time PCR (lower panel). Data are expressed as the means ± SD (n = 3 replicates for each group). *** P < 0.001 vs. control; ### P < 0.001 vs. 7αOHChol.</p

Roles of C5a receptor in on 7αOHChol-induced IL-8 expression.

<p>(A) THP-1 cells were incubated with or without 7-oxygenated cholesterol derivatives (5 μg/ml each) for 48 h. Transcripts of C5a receptor were amplified by RT-PCR. (B) THP-1 cells were incubated with or without 7-oxygenated cholesterol derivatives for 48 h, and surface expression of C5a receptor was analyzed by flow cytometry. (C) THP-1 cells were pre-treated for 2 h with the indicated amount of W-54011 and stimulated with 7αOHChol (5 μg/ml) for 48 h. Transcript levels of IL-8 were assessed by real-time PCR. Data are expressed as mean±SD (n = 3 replicates for each group). ***P < 0.001 vs. control. ### P < 0.001 vs. 7αOHChol.</p

7α-Hydroxycholesterol induces monocyte/macrophage cell expression of interleukin-8 via C5a receptor

<div><p>We investigated effects of 7-oxygenated cholesterol derivatives present in atherosclerotic lesions, 7α-hydroxycholesterol (7αOHChol), 7β-hydroxycholesterol (7βOHChol), and 7-ketocholesterol (7K), on IL-8 expression. Transcript levels of IL-8 and secretion of its corresponding gene product by monocytes/macrophages were enhanced by treatment with 7αOHChol and, to a lesser extent, 7K, but not by 7βOHChol. The 7-oxygenated cholesterol derivatives, however, did not change transcription of the IL-8 gene in vascular smooth muscle cells. 7αOHChol-induced IL-8 gene transcription was inhibited by cycloheximide and Akt1 downregulation, but not by OxPAPC. Expression of C5a receptor was upregulated after stimulation with 7αOHChol, but not with 7K and 7βOHChol, and a specific antagonist of C5a receptor inhibited 7αOHChol-induced IL-8 gene expression in a dose dependent manner. Pharmacological inhibitors of PI3K and MEK almost completely inhibited expression of both IL-8 and cell-surface C5a receptor induced by 7αOHChol. These results indicate that 7-oxygenated cholesterol derivatives have differential effects on monocyte/macrophage expression of IL-8 and C5a receptor and that C5a receptor is involved in 7αOHChol-induced IL-8 expression via PI3K and MEK.</p></div

Effects of 7-oxygenated cholesterol derivatives on expression of the IL-8 gene.

<p>(A) THP-1 cells and human aortic smooth muscle cells (HAoSMCs) were incubated with or without the indicated 7-oxygenated cholesterol derivatives (5 μg/ml each) for 48 h. Transcripts of IL-8 were amplified by RT-PCR. (B) THP-1 cells serum-starved for 24 h in RPMI 1640 containing 0.1% BSA (endotoxin free) were incubated with or without 7-oxygenated cholesterol derivatives for 48 h, and the levels of IL-8 transcripts were assessed by real-time PCR. The y-axis values represent fold increases of IL-8 mRNA levels normalized to GAPDH levels relative to those of THP-1 cells incubated carrier of oxygenated cholesterol derivatives (control). Data are expressed as the means ± SD (n = 3 replicates for each group). **P < 0.01 vs. control; ***P < 0.001 vs. control. (C) Conditioned media were harvested after treatment of THP-1 cells (2 x10⁵ cells/ml) with or without 7-oxygenated cholesterol derivatives (5 μg/ml each) for 48 h. The amount of CXCL8 secreted into the media was measured by ELISA. Data are expressed as the means ± SD (n = 3 replicates for each group). *P < 0.05 vs. control; *** P < 0.001 vs. control.</p

Effects of inhibitors of PI3K and MEK on expression of C5a receptor.

<p>THP-1 cells were treated with 7αOHChol for 48 h in the absence or presence of LY294002 or U0126 (10 μM each). The surface expression of C5a receptor was determined by flow cytometry.</p

Effects of dexamethasone (Dex) on CCL2 expression and monocytic cell migration.

<p>(A) Levels of CCL2 transcript were assessed by real-time PCR. The y-axis values represent increases in CCL2 mRNA levels normalized to GAPDH levels, relative to that of the non-treated THP-1 cells (control). Data are expressed as the means ± SD (n = 3 replicates for each group). (B) Culture media were isolated, and the levels of CCL2 protein in the media were measured by ELISA. Data are expressed as the means ± SD (n = 3 replicates for each group). (C) Monocytic cells were exposed to the conditioned media isolated above, and migration of monocytic cells was measured by chemotaxis assay. Data are expressed as the means ± SD (n = 3 replicates for each group). The results shown are the representative of three independent experiments. *** P < 0.001 vs. control; ** P < 0.01 vs. control; ### P < 0.001 vs. 27OHChol; ## P < 0.01 vs. 27OHChol.</p

Effects of dexamethasone (Dex) on expression of MMP-9 induced by 27OHChol.

<p>(A) Levels of MMP-9 transcript were assessed by real-time PCR. Data are expressed as the means ± SD (n = 3 replicates for each group). (B) Culture media were isolated, and the levels of MMP-9 in the media were measured by ELISA. Data are expressed as the means ± SD (n = 3 replicates for each group). The results shown are the representative of three independent experiments. *** P < 0.001 vs. control; ### P < 0.001 vs. 27OHChol; # P < 0.05 vs. 27OHChol. (C) The activity of MMP-9 secreted by cells was assessed by gelatin zymography. Control THP-1 cells were cultured for 48 h in the medium alone. Data are representative of three independent experiments.</p