MMP-13 enzyme activity assay

The specificity of the MMP-13 inhibitor was determined by addition of 0 (control), 0.5, 1, 2, 5, and 10 μM MMP-13i to samples containing 100 ng/ml of recombinant MMP-9 and MMP-13, which was diluted in assay buffer to a final dilution ratio of 1:100 (AnaSpec, San Jose, CA). A 96-well plate format was used with the Sensolyte™ 490 MMP-13 fluorimetric assay kit as per the manufacturer’s instructions (Anaspec). Zymogens or pro-MMP-9 and 13 were activated immediately prior to experimentation by incubation with 1 mM 4-aminopheylmercuric acetate (APMA) for 40 min (MMP-13) and 1 h (MMP-9) at 37°C. Diluted samples were incubated for 45 min in a black 96-well plate (Perkin Elmer, Waltham, MA) prior to the fluorescent intensity being measured by a spectrophotometer (Tecan Genios Spectrophotometer) at 360 nM (excitation) and 465 nM (emission).

To measure MMP-13 activity in DPCs, cultures were seeded (6×10⁴ cells per well) in a 6-well culture plate. At 72 h (experimental day 0), the cells were cultured for 24 h in supplemented mineralizing medium either containing 1μM SAHA or 2μM MMP-13i or a combination of both. Active MMP-13 activity was analysed at 2 time-points (48 h and 14 days) to reflect an early and late time point to coincide with the gene expression data. In the 48 h group and 14 day group, the HDACi-supplemented mineralizing medium was removed after 24 h prior to culture with an SAHA-free mineralizing medium for further 24 h or 13 days. In the MMP-13i samples the inhibitor supplemented the culture for the duration of the experiment. Two control groups consisting of supplemented α-MEM and supplemented mineralizing medium. At the designated time-point, the medium was aspirated and the cell monolayer gently washed twice in ice cold PBS (pH 7.4), prior to the addition of 300μl lysis buffer containing M-PER (Thermoscientific Pierce), halt protease inhibitor (Thermoscientific Pierce) and 1 mM PMSF (Thermoscientific Pierce). Harvested cells were collected. The Bradbury dye-binding method (Bio-Rad, Hemel Hempstead, Hertfordshire, UK) was used to equalize total protein concentrations in samples.

A 96-well plate format was used as before with the Sensolyte™ 490 MMP-13 fluorimetric assay kit according to the manufacturer’s instructions (Anaspec). Briefly, the pro-MMP-13 zymogen was activated immediately prior to experimentation in all samples by incubation with 1 mM APMA for 40 mins at 37°C. Fluorescent intensity was measured by a spectrophotometer (Tecan Genios Spectrophotometer) at 360 nM (excitation) and 465 nM (emission). For calculation of MMP-13 activity, each measurement was background-corrected to the average of the substrate controls. As the FRET substrate in the Sensolyte™ 490 MMP-13 kit can also be cleaved by MMP-1, −2, −3, −8, and −12 the MMP-13 specific inhibitor group was used to ascertain the effect of MMP-13 alone. Three independent experiments (n=3) were performed in triplicate for each experimental concentration at both time intervals.