HNE-induced 5-LO expression is regulated by NF-{kappa}B/ERK and Sp1/p38 MAPK pathways via EGF receptor in murine macrophages.

AIMS: 5-Lipoxygenase (5-LO) has been suggested to be a modulator of atherosclerotic plaque instability and co-exists with 4-hydroxynonenal (HNE) in macrophages in atherosclerotic lesions. To determine the potential role for HNE in 5-LO expression, the molecular mechanisms of 5-LO expression were evaluated in HNE-stimulated macrophages. METHODS AND RESULTS: A genomic sequence of the promoter 2.0 kb upstream of the transcription initiation site was amplified, and a series of sequentially deleted fragments were then fused to a luciferase reporter gene. The promoter region 213 bp upstream of the transcription start site was responsible for the HNE-enhanced transcriptional activity of 5-LO. Site-directed mutagenesis of this region showed that the transcription factors, including stimulating protein 1 (Sp1) and nuclear factor-κB (NF-κB), were associated with up-regulation of HNE-induced 5-LO transcription. Moreover, the role of Sp1 and NF-κB in HNE-induced 5-LO expression was confirmed by siRNA knockdown of Sp1 and NF-κB. The HNE-enhanced Sp1 and NF-κB activities were attenuated by SB203580, a p38 mitogen-activated protein kinase (MAPK) inhibitor, and PD98059, an extracellular signal-regulated kinase (ERK) inhibitor, respectively. In addition, the HNE-enhanced phosphorylation of p38 MAPK and ERK was inhibited by AG1478, an epidermal growth factor receptor (EGFR) antagonist, but not by AG1295, a platelet-derived growth factor receptor (PDGFR) antagonist. CONCLUSION: 5-LO expression by HNE was regulated at the transcriptional level by the EGFR-mediated activation of Sp1/p38 MAPK and NF-κB/ERK pathways in macrophages, which may lead to the development of therapeutic interventions for regulating 5-LO expression in atherosclerosis.

Acrolein increases 5-lipoxygenase expression in murine macrophages through activation of ERK pathway.

Episodic exposure to acrolein-rich pollutants has been linked to acute myocardial infarction, and 5-lipoxygenase (5-LO) is involved in the production of matrix metalloproteinase-9 (MMP-9), which destabilizes atherosclerotic plaques. Thus, the present study determined the effect of acrolein on 5-LO/leukotriene B(4) (LTB(4)) production in murine macrophages. Stimulation of J774A.1 cells with acrolein led to increased LTB(4) production in association with increased 5-LO expression. Acrolein-evoked 5-LO expression was blocked by pharmacological inhibition of the ERK pathway, but not by inhibitors for JNK and p38 MAPK pathways. In line with these results, acrolein exclusively increased the phosphorylation of ERK among these MAPK, suggesting a role for the ERK pathway in acrolein-induced 5-LO expression with subsequent production of LTB(4). Among the receptor tyrosine kinases including epidermal growth factor receptor (EGFR) and platelet derived growth factor receptor (PDGFR), acrolein-evoked ERK phosphorylation was attenuated by AG1478, an EGFR inhibitor, but not by AG1295, a PDGFR inhibitor. In addition, acrolein-evoked 5-LO expression was also inhibited by inhibition of EGFR pathway, but not by inhibition of PDGFR pathway. These observations suggest that acrolein has a profound effect on the 5-LO pathway via an EGFR-mediated activation of ERK pathway, leading to acute ischemic syndromes through the generation of LTB(4), subsequent MMP-9 production and plaque rupture.

Participation of 5-lipoxygenase-derived LTB(4) in 4-hydroxynonenal-enhanced MMP-2 production in vascular smooth muscle cells.

5-Lipoxygenase (5-LO) has been suggested as a modulator of atherosclerotic plaque instability, however, its role in MMP production in vascular smooth muscle cells (VSMC) is still unclear. Thus, this study investigated the role of 5-LO in HNE-enhanced MMP-2 production in VSMC, and the mechanisms by which this enzyme could be activated by HNE. VSMC stimulated with HNE (1 microM) produced MMP-2, which was markedly attenuated in 5-LO-deficient VSMC as well as in cells pretreated with a FLAP inhibitor, MK886, confirming a role for 5-LO metabolites in HNE-enhanced MMP-2 production. Related to these results, HNE increased nuclear translocation of 5-LO promoting 5-LO activity, which was attenuated not only by SB203580, a p38 MAPK inhibitor, but also by PD98059, an ERK inhibitor. In parallel, phosphorylation of p38 MAPK and ERK occurred as early as 15 min after exposure to HNE, suggesting a potential role for p38 MAPK and ERK pathways in HNE-induced activation of 5-LO. Among leukotriene (LT) receptor antagonists, U-75302, a BLT receptor antagonist, but not MK-571 and Rev-5901, cysLT receptor antagonists, showed an inhibitory effect on HNE-enhanced MMP-2 production. Moreover, MMP-2 production in VSMC was also significantly increased by LTB(4), but not by LTC(4) and LTD(4). Collectively, these data suggest that 5-LO mediates HNE-enhanced MMP-2 production via LTB(4)-BLT receptor pathways, consequently leading to atherosclerotic plaque instability.

4-Hydroxynonenal enhances MMP-9 production in murine macrophages via 5-lipoxygenase-mediated activation of ERK and p38 MAPK.

Exaggerated levels of 4-hydroxynonenal (HNE) and 5-lipoxygenase (5-LO) co-exist in macrophages in atherosclerotic lesions, and activated macrophages produce MMP-9 that degrades atherosclerotic plaque constituents. This study investigated the effects of HNE on MMP-9 production, and the potential role for 5-LO derivatives in MMP-9 production in murine macrophages. Stimulation of J774A.1 cells with HNE led to activation of 5-LO, as measured by leukotriene B(4) (LTB(4)) production. This was associated with an increased production of MMP-9, which was blunted by inhibition of 5-LO with MK886, a 5-LO inhibitor or with 5-LO siRNA. A cysteinyl-LT(1) (cysLT(1)) receptor antagonist, REV-5901 as well as a BLT(1) receptor antagonist, U-75302, also attenuated MMP-9 production induced by HNE. Furthermore, LTB(4) and cysLT (LTC(4) and LTD(4)) enhanced MMP-9 production in macrophages, suggesting a pivotal role for 5-LO in HNE-mediated production of MMP-9. Among the MAPK pathways, LTB(4) and cysLT enhanced phosphorylation of ERK and p38 MAPK, but not JNK. Linked to these results, a p38 MAPK inhibitor as well as an ERK inhibitor blunted MMP-9 production induced by LT. Collectively, these data suggest that 5-LO-derived LT mediates HNE-induced MMP-9 production via activation of ERK and p38 MAPK pathways, consequently leading to plaque instability in atherosclerosis.