Impact of matrix metalloproteinases on inhibition of mineralization by fetuin.
OBJECTIVE
Human subjects affected by inflammatory diseases, such as periodontitis, may be at increased risk for the development of cardiovascular diseases and calcification of atheromas; however, the potential mechanisms have not been defined. Alpha-2-Heremans Schmid glycoprotein (fetuin A) is an abundant serum glycoprotein of ~49 kDa that inhibits ectopic arterial calcification. We examined whether matrix metalloproteinases (MMPs), which are increased in inflammatory diseases, including periodontitis, bind and degrade fetuin and alter its ability to inhibit calcification in vitro.


METHODS
Binding and cleavage of fetuin by MMPs were assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in-silico analyses and mass spectrometry. The effects of intact and MMP-degraded human fetuin on mineralization were measured in a cell-free assay.


RESULTS
From in-silico analyses and literature review, we found that only MMP-3 (stromelysin) and MMP-7 (matrilysin) were predicted to cleave human fetuin at levels that were physiologically relevant. In-vitro assays showed that MMP-7, and, to a lesser extent, MMP-3, degraded human fetuin in a time- and dose-dependent manner. Fetuin peptides generated by MMP-7 cleavage were identified and sequenced by mass spectrometry; novel cleavage sites were found. Hydroxyapatite mineralization in vitro was strongly inhibited by fetuin (> 1 μm), as was MMP-3-cleaved fetuin, while MMP-7-cleaved fetuin was threefold less effective in blocking mineralization.


CONCLUSIONS
MMP-7 and, to a lesser extent, MMP-3, affect the ability of fetuin to inhibit the formation of hydroxyapatite in vitro. These data suggest that the MMPs increased in inflammatory diseases, such as periodontitis, could affect regulation of mineralization and potentially enhance the risk of calcified atheroma formation.
Discipline of Periodontology, Faculty of Dentistry, University of Toronto, Toronto, ON, Canada.
R
R
Schure
K D
KD
Costa
R
R
Rezaei
W
W
Lee
C
C
Laschinger
H C
HC
Tenenbaum
C A
CA
McCulloch
D002474:Cell-Free System; D017886:Durapatite / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D013058:Mass Spectrometry; D019278:Matrix Metalloproteinase 3 / Q000378:metabolism; D020783:Matrix Metalloproteinase 7 / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects*; D018709:Statistics, Nonparametric; D061205:Vascular Calcification / Q000139:chemically induced* / Q000378:metabolism; D060749:alpha-2-HS-Glycoprotein / Q000009:adverse effects* / Q000037:antagonists & inhibitors* / Q000378:metabolism
D060749:alpha-2-HS-Glycoprotein; D017886:Durapatite; D019278:Matrix Metalloproteinase 3; D020783:Matrix Metalloproteinase 7