FIGURE 2.
Regulation of MMP expression in IVD cells by recombinant HTRA1. A and B, recombinant HTRA1 proteolytic activity was determined using soluble bovine BODIPY FL-labeled DQ-elastin (100 μg/ml) as a substrate. Digestion of the DQ-elastin yielded fluorescent fragments detectable at 530 nm by a fluorescence microplate reader. The amount of DQ-elastin digestion was measured at selected time points (0, 1, 2, 4, and 8 h) using a defined amount of HTRA1 (5 μg) (A) or was determined after incubation for 8 h with varying amounts of HTRA1 (0, 0.5, 1, 3, and 5 μg) (B). C–E, the effects of recombinant HTRA1 (5 μg/ml) on MMP expression levels in IVD cells after a 24-h incubation period were determined by qRT-PCR, and the -fold change as compared with untreated controls was determined using the 2−ΔΔCT method (n = 4–6 patients). F, a specific MMP-3 ELISA was used to investigate the effects of recombinant HTRA1 on MMP protein secretion in supernatants obtained from IVD cells. Shown are results of triplicate determinations ± S.D. (error bars). *, p < 0.01, as determined by one-way ANOVA.