Dephosphorylation of ERK1/2 is involved in the WIN-induced down regulation of MMP-9 mRNA, but not in the effect of WIN on MMP-9 protein.

<p>MMP-9 quantitative Real-time PCR (bar chart) and Western blot analyses of intracellular (MMP-9 cellular) and secreted (MMP-9 secreted) MMP-9 and for phosphorylated (pERK1/2) and unphosphorylated ERK1/2. The figure shows one representative analysis out of three. U937-macrophages were treated with WIN (4 µM) or the pharmacological ERK1/2 phosphorylation inhibitor U-0126 (50 µM). Lane1: control; lane 2: WIN-treatment; Lane 3: U0126-treatment; lane 4: WIN + U0126-treatment. WIN treatment resulted in a dephosphorylation of ERK associated with inhibition of secretion, intracellular accumulation and decrease of MMP-9 mRNA (lanes 1 and 2). Inhibition of ERK1/2 phosphorylation with U-0126 decreased the level of MMP-9 mRNA significantly, but did not affect MMP-9 protein (lane 3). Treatment with WIN and U-0126 together did not decrease MMP-9 mRNA further than caused by U-0126 alone (lines 3 and 4). Bar chart: Data are shown as means +/− SD n = 3. *p<0.01 vs. control, ^#p>0.05 according to Newman-Keuls Multiple Comparison test following ANOVA. Western blot: ß-actin 1 is from the same blot as p-EKR1/2, ß-actin 2 is from the same blot as MMP-9 and ERK1/2. Originally, the blots contained more samples. In order to arrange the figure for comparison with mRNA quantification, blots were cut and rearranged. Original lanes can be seen in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048272#pone.0048272.s001" target="_blank">Figure S1</a>.