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.

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

Gobbi, Pietro;Maravic, Tatjana;Comba, Allegra;Mazzitelli, Claudia;Mancuso, Edoardo;Falconi, Mirella;Breschi, Lorenzo;Mazzoni, Annalisa
2021

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.
Gobbi, Pietro; Maravic, Tatjana; Comba, Allegra; Mazzitelli, Claudia; Mancuso, Edoardo; Falconi, Mirella; Breschi, Lorenzo; Mazzoni, Annalisa
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11585/798906
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