Characterization of HA-induced potentiation of collagen-degrading enzymes and their role in underpinning CD44-mediated invasion of breast cancer cells. (A) Bar graphs showing the induction of cathepsin K (left panel) and MT1-MMP (right panel) mRNA expression after treatment with 100 μg/ml HA over a 12-hour period. All values were normalized against 18S mRNA expression, and fold-changes were calculated by comparison of mRNA levels in the absence of HA stimulation. Data shown is the mean ± SEM fold-change relative to control determined from three independent experiments. (B) A series of immunoblots confirming a time-dependent increase in the expression of cathepsin K (top panel) and MT1-MMP (bottom panel) expression in response to HA-stimulation (100 μg/ml). Equal protein loading in immunoblots was confirmed by reprobing the membranes for β-tubulin. (C) ELISA experiments conducted on MDA-MB-231Hi cells on 100 μg/ml HA stimulation demonstrate a time-dependent increase in cathepsin K secretion from 6,437 ± 855.0 pmol/L/106 cells to 10,649 ± 860.5 pmol/L/106 cells within 6 hours of HA treatment. Data points are the mean concentration of cathepsin K ± SEM relative to concentration of cathepsin K in untreated MDA-MB-231Hi cells. (D) Stimulation of MDA-MB-231Hi cells with HA (100 μg/ml) for 3 hours potentiates the extracellular collagenolytic activity of MDA-MB-231Hi cells by using the in vitro EnzChek Collagenase assay (P < 0.001; n = 5).