The activity, distribution, and colocalization of cathepsin K and matrix metalloproteases in intact and eroded dentin.

Abstract

This study aimed to assess the activity, distribution, and colocalization of cathepsin K (catK) and matrix metalloproteases (MMPs) in both intact and eroded dentin in vitro. Eroded dentin was obtained by consecutive treatment with 5% citric acid (pH = 2.3) for 7days, while intact dentin remained untreated. Pulverized dentin powder (1.0g) was extracted from both intact and eroded dentin using 5mL of 50mM Tris-HCl buffer (0.2g/1mL, pH = 7.4) for 60h to measure the activity of catK and MMPs spectrofluorometrically. In addition, three 200-μm-thick dentin slices were prepared from intact and eroded dentin for double-labeling immunofluorescence to evaluate the distribution and colocalization of catK and MMPs (MMP-2 and MMP-9). The distribution and colocalization of enzymes were analyzed using inverted confocal laser scanning microscopy (CLSM), with colocalization rates quantified using Leica Application Suite Advanced Fluorescent (LAS AF) software. One-way analysis of variance (ANOVA) was used to analyze the fluorescence data related to enzyme activity (α = 0.05). The activity of catK and MMPs was significantly increased in eroded dentin compared with intact dentin. After erosive attacks, catK, MMP-2, and MMP-9 were prominently localized in the eroded regions. The colocalization rates of catK with MMP-2 and MMP-9 were 13- and 26-fold higher in eroded dentin, respectively, than in intact dentin. Erosive attacks amplified the activity of catK and MMPs in dentin while also altering their distribution patterns. Colocalization between catK and MMPs increased following erosive attacks. CatK, MMP-2, and MMP-9 likely play synergistic roles in the pathophysiology of dentin erosion.