Biochemical and immunohistochemical analysis of tissue inhibitor of metalloproteinases-1 in human sound dentin

Abstract

Objectives Matrix metalloproteases (MMPs) are a family of enzymes that operate a proteolytic activity at the level of the extracellular matrix. MMPs are regulated by tissue inhibitors of metalloproteinases (TIMPs) that can ubiquitously bind different enzyme forms. The study aims to identify a morfo-functional association between TIMP-1 and MMP-2 and -9 in human dentin. Materials and methods Proteins were extracted from demineralized human sound dentin powder and centrifuged to separate two aliquots with different molecular weights of proteins, higher and lower than 30 kDa. In each aliquot, the evaluation of the presence of TIMP-1/MMP-2 and TIMP-1/MMP-9 was performed using co-immunoprecipitation/immunoblotting analysis. The distribution of TIMP-1, in association with MMP-2 and -9, was investigated using a double immunohistochemical technique. Furthermore, the activity of TIMP-1 was measured by reverse zymography, where acrylamide gel was copolymerized with gelatin and recombinant MMP-2. Results Co-immunoprecipitation/immunoblotting analysis showed the association TIMP-1/MMP-2 and TIMP-1/MMP-9 in human sound dentin. Electron microscopy evaluation revealed a diffuse presence of TIMP-1 tightly associated with MMP-2 and -9. Reverse zymography analysis confirmed that TIMP-1 present in human dentin is active and can bind different MMPs isoforms. Conclusions The strict association of TIMP-1 with MMP-2 and -9 in situ appeared a constant finding in the human sound dentin. Clinical relevance Considering the role of TIMP-1, MMP-2, and MMP-9 within the connective tissues, clinically applicable protocols could be developed in the future to increase or decrease the level of TIMPs in human dentin to regulate the activity of MMPs, contributing to reduce caries progression and collagen degradation.

Matrix Metalloproteinases in Dental and Periodontal Tissues and Their Current Inhibitors: Developmental, Degradational and Pathological Aspects

“…MMP-2 is also found in mature dentine and plays a role in caries pathogenesis, MMP-3 is seen in predentine and contributes to dentine mineralization [ 34 ]. Recently, it has been demonstrated using electron microscopy and reverse zymography analysis, that in sound human dentine TIMP1 is closely related to MMP-2 and MMP-9 and can bind to different isoforms of MMPs [ 36 ].…”

The inhibitory effects of various ions released from S-PRG fillers on dentin protease activity

“…Although several MMPs are present in demineralized dentin, MMP-2 and MMP-9 are the most abundant MMPs in dentine 36) . A recent report also showed strict association of TIMP-1 and MMP-2 in demineralized dentin matrices 37) . Therefore, a partial initial inhibition obtained with strontium might be due to its effect on MMP-2-TIMP balance 36) .…”