Statins modulate transcriptional activity of heme-oxygenase-1 promoter in NIH 3T3 Cells

Statins, inhibitors of HMG CoA reductase, have pleiotropic effects independent of their capacity to lower cholesterol. Heme-oxygenase-1(HO-1) plays an important role as an anti-oxidant and anti-inflammatory enzyme. In the present study, we used NIH 3T3 cells which express HO-1 to investigate the molecular mechanisms of HO-1 induction by statins. Simvastatin or fluvastatin induced a significant increase in HO-1 protein expression and mRNA levels. Both statins stimulated activity of a mouse HO-1 promoter (-1,287 to +73 bp)/luciferase reporter gene, 3.25 ± 0.23 (Mean ± S.E.M., n = 15, P < 0.001, t-test) and 3.13 ± 0.33 (Mean ± S.E.M., n = 6, P < 0.001, t-test), respectively. This effect was more pronounced in the short proximal promoter than the full promoter of HO-1. Gel retardation experiments for C/EBP and upstream stimulatory factor (USF) DNA-binding activities using simvastatin- or fluvastatin-treated cells showed significant nuclear protein-DNA complexes which were supershifted with antibodies specific for C/EBP β and Î or USF-1 and USF-2. Point mutations of the proximal HO-1 promoter (-149 to +73 bp) for the myc/max which binds USF or the C/EBP binding sequences showed a reduction in statin-induced reporter activity whereas no role of the distal C/EBP binding elements located at -4 kb was observed. Moreover, overexpression of mutated C/EBP β and USF factor or the siRNA for both factors supported a role of these transcription factors in statin-dependent induction of HO-1, with a clearer effect for C/EBP

New analogues of 13-hydroxyocatdecadienoic acid and 12-hydroxyeicosatetraenoic acid block human blood platelet aggregation and cyclooxygenase-1 activity.

International audienceUNLABELLED: BACKGROUND: Thromboxane A2 is derived from arachidonic acid through the action of cyclooxygenases and thromboxane synthase. It is mainly formed in blood platelets upon activation and plays an important role in aggregation. Aspirin is effective in reducing the incidence of complications following acute coronary syndrome and stroke. The anti-thrombotic effect of aspirin is obtained through the irreversible inhibition of cyclooxygenases. Analogues of 12-hydroxyeicosatetraenoic acid and 13-hydroxyocatdecadienoic acid were shown previously to modulate platelet activation and to block thromboxane receptors. RESULTS AND DISCUSSION: We synthesized 10 compounds based on the structures of analogues of 12-hydroxyeicosatetraenoic acid and 13-hydroxyocatdecadienoic acid and evaluated their effect on platelet aggregation triggered by arachidonic acid. The structure activity relationship was evaluated. Five compounds showed a significant inhibition of platelet aggregation and highlighted the importance of the lipidic hydrophobic hydrocarbon chain and the phenol group. Their IC50 ranged from 7.5 ± 0.8 to 14.2 ± 5.7 μM (Mean ± S.E.M.). All five compounds decreased platelet aggregation and thromboxane synthesis in response to collagen whereas no modification of platelet aggregation in response to thromboxane receptor agonist, U46619, was observed. Using COS-7 cells overexpressing human cyclooxygenase-1, we showed that these compounds are specific inhibitors of cyclooxygenase-1 with IC50 ranging from 1.3 to 12 μM. Docking observation of human recombinant cyclooxygenase-1 supported a role of the phenol group in the fitting of cyclooxygenase-1, most likely related to hydrogen bonding with the Tyr 355 of cyclooxygenase-1. CONCLUSIONS: In conclusion, the compounds we synthesized at first based on the structures of analogues of 12 lipoxygenase metabolites showed a role of the phenol group in the anti-platelet and anti-cyclooxygenase-1 activities. These compounds mediate their effects via blockade of cyclooxygenase-1

Implication of P38 MAP kinase pathway in statin-dependent induction of HO-1.

<p>(A) Phosphorylation of P38 by 25 µM simvastatin at different times in RAW 264.7 cells. Results are representative of 3 experiments. (B) Effect of p38 MAP kinase inhibitor, SB203580 on HO-1 induction in RAW 264.7 and eMPM. Cells were pretreated for 30 minutes with 10 µM of SB203580 prior to the addition of 25 µM of simvastatin for 24 hours. Western blotting of HO-1 was performed as indicated earlier. Results are representative of 3 experiments.</p

HO-1 induction by statins in RAW 264.7 cells.

<p>Cells were incubated for 24 hours with simvastatin or fluvastatin. 20 µg of total proteins were loaded on a 12% SDS polyacrylamide gel and blotted for HO-1 and then stripped and blotted for β-actin. HO-1 band intensities were measured by densitometry and normalized to the intensity of β-actin bands. Fold increase was calculated as a ratio of the result of stimulated cells to that of the control cells. (A) Dose-response of simvastatin or fluvastatin in RAW 264.7 cells. The western blot is representative of 4 experiments with similar results. B corresponds to basal untreated cells. (B &C) Effect of statins on LPS induced HO-1 expression. RAW 264.7 cells were incubated for 24 hours with increasing concentrations of LPS alone or in the presence of 25 µM simvastatin (B) or 10 µM fluvastatin (C). Results are representative of 3 experiments.</p

Role of prenylation in simvastatin-dependent induction of HO-1.

<p>(A) RAW 264.7 cells were co-treated with 25 μΜ simvastatin and increasing concentrations of GGPP and FPP for 24 hours in the presence of 125 µM SPNO. Western blotting of HO-1 was performed as described in the legend for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064092#pone-0064092-g001" target="_blank">figure 1</a>. Results are representative of 3 experiments for GGPP and 2 for FPP. (B) RAW 264.7 cells were incubated with 10 µM of GGTI-286 or FTI-277 for 24 hours. Results are representative of 3 experiments. (C) RAW 264.7 cells were treated with 5 µg/ml of C3-exoenzyme and 1 µg/ml of CNF-1 for 24 hours. Results are representative of 3 experiments with similar results.</p

Role of nitric oxide and CCAAT/enhancer-binding protein transcription factor in statin-dependent induction of heme oxygenase-1 in mouse macrophages

The effect of statins on heme oxygenase-1 (HO-1) was compared in 2 murine cell lines, RAW 264.7 and J774A.1 cell lines, and in primary peritoneal macrophages of BALB/c or C57BL/6 mice. The role of endogenous nitric oxide and the type of transcription factors involved were explored. Simvastatin and fluvastatin induced HO-1. Pretreatment of cells with l-NMMA or 1400 W, two different nitric oxide synthase inhibitors, partially blocked statin-dependent induction of HO-1 in RAW 264.7 and J774A.1 but not in primary peritoneal macrophages. Induction of HO-1 by statins was dependent on p-38 MAP kinase activation in all types of macrophages. In RAW 264.7 cells, both statins increased the activity of reporter genes linked to the proximal 1.3 kbp promoter of HO-1 (EC of 1.4±0.3 μM for simvastatin and 0.6±0.03 μM for fluvastatin). This effect was significantly blocked by 1400 W (80±5.2% inhibition,