We reported previously that low-density lipoprotein (LDL)-containing immune complexes (LDL-IC) stimulated matrix metalloproteinase-1 (MMP-1) appearance in U937 histiocytes through Fc gamma receptor (FcR)-mediated extracellular signal-regulated kinase pathway. that plays a part in the disruption of atherosclerotic plaques.8 Trp53inp1 We discovered that LDL-IC stimulated appearance of matrix metalloproteinase-1 (MMP-1, interstitial collagenase), a proteinase in charge of the original cleavage of fibrillar interstitial collagen, through FcR-mediated extracellular signal-regulated kinase (ERK) pathway in both individual monocyte-derived macrophages and U937 histiocytes. We also noticed that both FcRI and FcRII had been mixed up in arousal. Furthermore, our outcomes demonstrated that IC which contain individual rabbit and IgG anti-human IgG also activated MMP-1 appearance, recommending that IC, of their antigen items irrespective, can handle stimulating MMP-1 secretion from U937 histiocytes by crosslinking FcRs. In a follow-up study, we exhibited that pretreatment of macrophage-like U937 cells with interferon- (IFN-) augmented LDL-IC-induced MMP-1 expression.9 Because MMP-1 released by macrophages has been implicated in the destabilization of atherosclerotic plaques10C14 these studies suggest that LDL-IC may contribute to plaque rupture. Although above studies have shown that LDL-IC up-regulate MMP-1 expression through FcR-mediated ERK signalling pathway, the molecular mechanisms involved in the gene expression remain unknown. The current investigation was undertaken to determine the < 005 was considered significant. Results LDL-IC stimulate MMP-1 transcriptionThe effect of LDL-IC on MMP-1 expression was re-examined at the beginning of this study to ensure that the response of U937 cells to LDL-IC was comparable to that we reported previously.8 As shown in Fig. 2, LDL-IC stimulated MMP-1 secretion and mRNA expression by U937 cells. MAPK/ERK kinase (MEK) inhibitor PD98059 abolished the activation of MMP-1 expression (Fig. 3a), suggesting that LDL-IC-stimulated MMP-1 expression is usually ERK-dependent. All these results are consistent with our previous reports.8 To determine whether the activation of MMP-1 mRNA expression by LDL-IC is caused by a transcriptional R1626 or post-transcriptional regulation, cells were pretreated with actinomycin D, a transcription inhibitor, before the activation with LDL-IC. Results showed that 5 g/ml of actinomycin D abolished the activation (Fig. 3b), indicating that LDL-ICs up-regulate MMP-1 expression by activating the transcription of MMP-1 gene. The transcriptional activation by LDL-IC was also indicated by the observation that LDL-IC stimulated MMP-1 promoter activity in cells transfected with a MMP-1 promoterCluciferase reporter construct (Fig. 4). Physique 2 Activation of MMP-1 secretion and mRNA expression by LDL-IC. U937 cells were incubated with or without 150 g/ml of LDL-IC for 24 hr. After the incubation, the conditioned medium was collected for quantitative analysis of secreted MMP-1 by ELISA … Physique 3 Inhibition of LDL-IC-stimulated MMP-1 expression by PD98059 and actinomycin D. (a) U937 cells were incubated for 24 hr with 150 g/ml of LDL-IC in the presence of increasing concentrations of PD98059 as indicated. After incubation, the conditioned … Physique 4 LDL-IC activate MMP-1 promoter activity in cells transfected with Construct 1. U937 cells were transiently transfected with MMP-1 promoter-reporter constructs and -galactosidase vector for 24 hr, and then stimulated without (control) or with … The ?3471 AP-1 and ?3836 Ets motifs Are essential for LDL-IC-stimulated MMP-1 expressionTo explore the transcriptional mechanisms by which LDL-IC activate MMP-1 expression, deletion analysis was performed by transfecting U937 cells with eight luciferase reporter constructs, which contain successively 5-deleted fragments of MMP-1 promoter region (Fig. 1), followed by treatment with LDL-IC or phorbol 12-myristate 13-acetate (PMA) as positive control. Results from luciferase activity assay showed that PMA stimulated luciferase activity in cells transfected with Construct 1 (?4334/+52) and 2 (?2685/+52) (Fig. 4). Interestingly, LDL-IC only stimulated luciferase activity in cells transfected with the Construct 1 (?4,334/+52), suggesting that this cis-acting elements that are responsive to LDL-IC are located between ?4334 and ?2685. By analysing the sequence of R1626 the region between ?4334 and ?2685, cis-acting elements AP-1 (?3471), Ets (?3836), and CREB (?3187) motifs were found. To determine whether these motifs are involved in the LDL-IC-stimulated MMP-1 expression, mutation analysis using Construct 9 (?3471 AP-1 mutant), Construct 10 (?3836 Ets mutant), and Construct 11 (?3187 CREB mutant)19 (Fig. 1) was performed. Results showed that this AP-1 mutation in Construct 9 and the Ets mutation in Construct 10, but not the CREB mutation R1626 in Construct 11, abolished LDL-IC-stimulated luciferase activity (Fig. 5a). The AP-1 mutation in Construct 9 also markedly inhibited the baseline level of luciferase activity (Fig. 5a), which is usually consistent with the deletion study showing that control cells transfected with Constructs 2C8, which do not have.