Multi-domain structure of proMMP-9.

<p>3D molecular structure of the full-length human proMMP-9 monomer. The catalytic site is formed by the Zn²⁺ -binding domain (orange) and the active site (yellow) and is highly conserved within the MMP family. MMPs are secreted as pro-enzymes, containing a propeptide domain (green) which interacts with the catalytic Zn²⁺ ion, thereby keeping the enzyme inactive. The fibronectin repeats (blue) are only present in gelatinases (MMP-2 and MMP-9) and assist the catalysis of large substrates such as gelatins. The O-glycosylated domain (black) is a unique domain that, as its name suggests, is flexible and heavily glycosylated. This domain lends the MMP-9 molecule a high degree of flexibility and the ability to reach cleavage sites on long substrates. Finally, the hemopexin domain (red) is present in several MMPs and has a range of functions including substrate binding, inhibitor binding and binding to cell surface receptors [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174853#pone.0174853.ref013" target="_blank">13</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174853#pone.0174853.ref014" target="_blank">14</a>].</p

qPCR analysis of LPS-challenged THP-1 cells, treated with minocycline, azithromycin, bortezomib and SB-3CT.

<p>Fold changes of <i>MMP2</i>, <i>MMP9</i>, <i>TIMP1</i> & <i>NFKBIA</i> mRNA levels are shown, relative to the levels from cells treated with LPS only and corrected towards the housekeeping gene <i>GADPH</i>. Individual data points are shown and the bars represent the mean values. Data were statistically analyzed with the use of a Bonferroni's multiple comparison test. *, p ≤ 0.05; **, p ≤ 0.01; *** p ≤ 0.001; n = 3.</p

Inhibition of proMMP-9 activation by MMP-3.

<p>(A) The influence of minocycline, azithromycin, bortezomib and SB-3CT on the activation of proMMP-9 into MMP-9, measured by assessing the gelatinolytic activity of proMMP-9 after incubation with a proMMP-9 activator (catalytic domain of MMP-3) in the presence of the compounds. Data were compared to a condition without compound and expressed as percentage of activated proMMP-9. Individual data points, each representing a separate experiment, are shown. The bars represent the mean value. Inhibition of proMMP-9 activation by SB-3CT was significantly different as determined with a Bonferroni's multiple comparisons test. ***, P ≤ 0.001. (B) Zymography analysis of proMMP-9 activation by cdMMP-3 in the presence of minocycline, azithromycin, bortezomib and SB-3CT. Zymograms, representative for three experiments, show the stepwise activation of proMMP-9, from the full-length enzyme (proMMP-9, blue), to the partially activated enzyme (proMMP-9’, orange) and the fully activated enzyme (MMP-9, red) as indicated by the arrows. (C) Densitometric analysis of the zymograms. The proportions of pro, intermediate and activated MMP-9 are shown as the percentages of the total MMP-9. Individual data points, each representing a separate experiment, are shown.</p

Zymography of THP-1 cells challenged with LPS and treated with minocycline, azithromycin, bortezomib and SB-3CT.

<p>(A) Overview image of the zymography analysis showing a compilation of representative zymography gels. Prior to zymography, loading volumes were corrected for the total protein content of each sample. Equal amounts of an internal loading control (±48 kDa MMP-9 mutant lacking the O-glycosylated and hemopexin domain [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174853#pone.0174853.ref014" target="_blank">14</a>]) were included to allow correction for sample processing and loading errors. hrMMP-9, human recombinant proMMP-9. (B) Densitometry analysis of proMMP-9 bands. (C) Densitometry analysis proMMP-2 bands. Individual data points are shown and the bars represent the mean value. Data were statistically analyzed using a Bonferroni's multiple comparison test. *, p ≤ 0.05; **, p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001; n = 2–3.</p

Summarizing table of the results.

<p>Summarizing table of the results.</p

The effect of minocycline, azithromycin, bortezomib and SB-3CT on MMP-9-mediated gelatinolysis.

<p>(A) gelatin zymography of MMP-9 mixtures (MMP-9 trimers and MMP-9 monomers). Each gel slice was incubated with a different concentration of each compound, as indicated on top of the lanes. Representative image of two independent experiments. (B) Percentage of inhibition of MMP-9 mediated gelatinolysis, as measured using a gelatin degradation assay. Compound concentration ranged from 1000 μM to 0.24 μM. Data combined from seven independent experiments, including different concentration ranges and a dose-response curve was fitted using non-linear regression. Higher concentrations of bortezomib and azithromycin were not tested in the gelatin degradation assay, due to solvent interference. Individual data points are shown. Statistical analysis was performed for the data at 125 μM by using a Bonferroni's multiple comparison test. **, p ≤ 0.01; **** p ≤ 0.0001.</p

The effect of minocycline, azithromycin, bortezomib and SB-3CT on cell viability of LPS-stimulated THP-1 cells.

<p>(A) The effect of LPS on cell viability evaluated by measuring cell metabolic activity (MTT assay). Stimulation of THP-1 cells with LPS had no significant effect on the cell viability as determined with a Mann—Whitney U test. Data represented as background-subtracted absorbance (570 nm– 630nm). Individual data points are shown and the bars represent the mean value. Ns, not significant; n = 5. (B) The effect of LPS stimulation of THP-1 cells in combination with minocycline, azithromycin, bortezomib and SB-3CT on cell viability. The cell viability is expressed as the percentage of cells compared to the condition (LPS only, no compounds). Individual data points are shown and the bars represent the mean value. Mann—Whitney U tests were used to compare with the control condition (LPS condition) *, p ≤ 0.05; **, p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001; n = 3–5.</p

Cell signaling events leading to reduced MMP-9 expression.

<p>(A) Western blot analysis of intracellular, phosphorylated NF-κB (Ser536) and caspase-4 in THP-1 cells stimulated with LPS and treated with 6 μM minocycline, 6 μM azithromycin, 2 μM bortezomib or no treatment (cells + LPS without any of the compounds). Western blot images show two samples per condition. Graphs are based on densitometry analysis of Western blot images (n = 3). (B) IL-1β quantification by ELISA. THP-1 cells were challenged with LPS and 20, 6 and 2 μM of minocycline or azithromycin. Bortezomib was used at concentrations of 20 μM, 2 μM, 200 nM and 20 nM. (C) qPCR analysis of THP-1 cells challenged with LPS after treatment with minocycline, azithromycin, bortezomib and SB-3CT. Fold changes <i>COX2</i> mRNA levels are shown, relative to cells treated with LPS only and corrected towards the housekeeping gene <i>GADPH</i>. (D) PGE₂ ELISA on cell supernatants of THP-1 cells stimulated with LPS and treated with the compounds. Individual data points are shown and the bars represent the mean value. Horizontal dotted line always indicates cells only treated with LPS. Data were statistically analyzed using a Bonferroni's multiple comparison test. *, p ≤ 0.05; **, p ≤ 0.01; *** p ≤ 0.001; n = 3.</p