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>.</p

Treatment with WIN reduced secretion and activity of MMP-9 in macrophageal differentiated U937 cells.

<p>(a) Western blot analysis of conditioned medium using antibodies against MMP-9 and MMP-12. WIN-treatment resulted in a significant decrease of secreted MMP-9, whereas MMP-12 secretion was not affected. Control cells were treated with vehicle. The figure shows one representative analysis out of three. (b) MMP-9 activity-ELISA of conditioned medium. Upon treatment with 2 µM WIN a reduction of MMP-9 activity was observed, after treatment with 4 µM WIN the reduction was even stronger. Control cells were treated with vehicle. Data are shown as means +/− SD, n = 3. *p<0.05 vs. control, **p<0.01 according to Newman-Keuls Multiple Comparison test following ANOVA. (c) Zymography of conditioned medium. Gelatinolytic activity was strongly decreased by 2 and 4 µM WIN. The figure shows one representative analysis out of three.</p

WIN reduced bone resorption and MMP-9-activity in a capsaicin sensitive manner.

<p>(a) Measurement of resorption activity of osteoclasts using crosslaps-ELISA of conditioned medium. Treatment with WIN (4 µM) reduced the osteolytic activity compared to control cells (vehicle treated). Additional treatment with capsaicin (CIC) antagonized this decrease. Data are shown as means +/− SD, n = 5. (b) MMP-9-activity-ELISA of conditioned medium of osteoclasts. Treatment with WIN (4 µM) decreased MMP-9-activity significantly compared to control cells (vehicle treated) and this decrease was antagonized by parallel treatment with CIC. Data are shown as mean +/− SD, n = 5. *p<0.05, ^#p>0.05 according to Newman-Keuls Multiple Comparison Test following ANOVA.</p

TRPV1 was involved in inhibition of secretion and intracellular accumulation of MMP-9 upon WIN-treatment.

<p>(a,b) Western blot analyses of U937-macrophage cell lysates (MMP-9 cellular) using MMP-9 antibody. (a) Treatment with the TRPV1 antagonist capsazepine (CZP) enhanced the WIN-induced size shift of MMP-9 from 85 to 92 kDa when given parallel to WIN and mimicked this effect when administered separately. The figure shows one representative analysis out of three. (b) Treatment with the TRPV1 agonist capsaicin (CIC) antagonized the WIN-induced size shift while it exhibited no effect when given alone. The figure shows one representative analysis out of three. (c) MMP-9 activity–ELISA of conditioned medium. The WIN-induced decrease of MMP-9 activity was intensified by CZP (10 µM), and antagonized by CIC (10µM). Data are shown as means +/− SD, n = 3. **p<0.001 *p<0.1 according to Newman-Keuls Multiple Comparison test following ANOVA.</p

Inhibition of MMP-9 secretion and activity and intracellular accumulation of MMP-9 in WIN-treated activated primary peripheral monocytes.

<p>(a) Western blot analysis of cell lysates (MMP-9 cellular) and conditioned medium (MMP-9 secreted) using anti-MMP-9 antibodies. The figure shows one representative analysis out of three. WIN inhibited MMP-9 secretion and induced an intracellular accumulation of 92 kDa MMP-9. (b) MMP-9 activity-ELISA of conditioned medium. Upon treatment with 2 and 4 µM WIN, a concentration-dependent reduction of MMP-9-activity was observed. Data are shown as means +/− SD n = 3. **p<0.01 vs. control according to Newman-Keuls Multiple Comparison test following ANOVA. (C) Zymography of conditioned medium. Gelatinolytic activity was inhibited by WIN-treatment. The figure shows one representative analysis out of three.</p

WIN down-regulated MMP-9 mRNA in U937-macrophages.

<p>(a) Quantitative real-time PCR of MMP-9 mRNA. 2 µM and 4 µM WIN decreased MMP-9 mRNA to 66% and 55% respectively. Data are shown as means +/− SD n = 3. **p<0.01 vs. control according to Newman-Keuls Multiple Comparison test following ANOVA. (b) Comparison of MMP-9 mRNA and secreted protein, assessed by real-time PCR (n = 3) and densitometry of Western blot analyses (n = 2). Values of mRNA and secreted MMP-9 Protein are given as percentage of the amounts that were measured in untreated cells at the corresponding time point. Secretion of MMP-9 protein was already decreased to 40% after 1 h, and remained in this range for 24 h. In contrast, no mRNA decrease was observed after 1 h, mRNA level decreased steadily to 55% after 24 h. Data are shown as means +/− SD, *p<0.05, **p<0.01 vs. 1 h according to Newman-Keuls Multiple Comparison test following ANOVA.</p

Inhibition of MMP-9 secretion and activity and intracellular accumulation of MMP-9 in WIN-treated osteoclasts, but not in microglia.

<p>(a) Western blot analysis of cell lysates (MMP-9 cellular) and conditioned medium (MMP-9 secreted). using anti-MMP-9-antibody and MMP-9-activity ELISA of conditioned medium (bar chart) from osteoclasts. Upon WIN treatment (4 µM), the amount of intracellular 92 kDa-MMP-9 was enhanced, while the amount of secreted MMP-9 and the activity of MMP-9 in the conditioned medium was decreased. (b) Western blot analysis of cell lysates (MMP-9 cellular) using anti-MMP-9-antibody and MMP-9 ELISA of conditioned medium (bar chart) from primary microglia. Size and amount of intracellular MMP-9 were not changed after WIN-treatment (4 µM). The amount of MMP-9 in the conditioned medium increased insignificantly. PC = positive control (U937 macrophages). The figure shows one representative analysis out of three. Data are shown as means +/− SD n = 3. *p<0.05, ^#p>0.05 vs. control according to unpaired t test.</p

WIN-induced regulation of MMP-9 was mediated by a specific binding site, which is different from CB1, CB2, and PPARy, and independent from pertussis-toxin.

<p>Western blot analysis of cell lysates (MMP-9 cellular) and conditioned medium (MMP-9 secreted) of U937-macrophages treated with the receptor-inactive WIN-enantiomer S(–)-[2,3-Dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1 naphthalenyl) methanone mesylat (WIN3), specific inhibitors for cannabinoid-receptors or pertussis toxin (PTX). Control cells were treated with vehicle. In each case the figure shows one representative analysis out of three. (a) Treatment with WIN3 demonstrated the specificity of the effect of WIN. (b) Inhibitors for CB1 (AM251) and CB2 (AM630) did not abolish the WIN-induced inhibition of secretion and intracellular accumulation of MMP-9. (c) Treatment with PTX did not abolish the WIN-induced effect. (d) Inhibition of PPARy with GW9662 had no influence on the effect of WIN on MMP-9.</p

Inhibition of MMP-9 secretion was accompanied by an intracellular accumulation of a 92 kDa-MMP-9.

<p>Western blot analysis of cell lysates (MMP-9 cellular) and conditioned medium (MMP-9 secreted) of U937-macrophages treated with WIN using anti-MMP-9 antibody. Control cells were treated with vehicle. (a) Upon 24 h treatment with WIN, the amount of MMP-9 in the cell lysate increased, whereas the amount of secreted MMP-9 in the conditioned medium decreased. The size of MMP-9 in the cell lysate shifted from 85 kDa to 92 kDa. Control cells were treated with vehicle. The figure shows one representative analysis out of three. (b) Kinetic analysis of MMP-9 after WIN-treatment throughout 24 h. The inhibition of MMP-9 secretion was accompanied by an accumulation of intracellular 92 kDa-MMP-9. The intracellular 85 kDa-MMP-9 disappeared with time. The figure shows one representative analysis out of three.</p

Glycosylation of intracellular 92 kDa-MMP-9 after WIN-treatment was different from the 85 kDa-MMP-9 in untreated U937-macrophages.

<p>The pictures show one representative analysis out of three. (a) Western blot analysis using anti-MMP-9 antibody of endoglycosidase H-digested cell lysates treated with or without WIN. The WIN-induced 92 kDa-MMP-9 was resistant to digestion, whereas the 85 kDa-MMP-9 from control cells loses 5 kDa upon digestion. (b) Digestion with N-glycosidase F resulted in a loss of 5 kDa in both MMP-9 forms.</p