MMP-7 Cleavage of Fetuin Inhibits Control of Mineralization

Calcifying atheromas are mineralized vascular plaques enriched with cholesterol and hydroxyapatite (HA) and are risk factors for cardiovascular disease. Fetuin, a naturally-occurring serum glycoprotein may inhibit mineralization, thereby preventing calcified atheroma formation. Matrix metalloproteinases (MMP) are endopeptidases increased in periodontitis that may affect the regulatory actions of fetuin. We hypothesized that degradation of fetuin by MMPs may affect the ability of fetuin to regulate mineralization.

Objectives:  To measure the effects of intact and MMP-degraded bovine fetuin on mineralization in a cell-free assay.

Methods:  HA crystals were grown in vitro in a calcium/phosphate medium (pH=7.4). HA content was quantified by spectrophotometry at 540 nm using Alizarin Red S stain and measured in the presence of intact bovine fetuin or fetuin digested with MMP-7 or MMP-3 (MMP:fetuin=1:60). Degradation of fetuin was confirmed by SDS-PAGE. Electron microscopy was used to characterize HA crystals. Controls included assays without essential ions and replacement of fetuin with bovine serum albumin. Experiments were repeated a minimum of 3 times with at least 8 values each. Means, standard errors of means and p values were computed.

Results:  HA mineralization was slightly increased at low fetuin concentrations (0-1 µM) but was strongly inhibited by concentrations above 1 µM. There was a 2-fold reduction of mineralization (p<0.0001) with 1.4 µM fetuin and virtually no detectable mineralization at ≥2 µM fetuin. In time-courses under constant conditions, mineralization peaked at 3.5 hours incubation and remained constant thereafter. Both MMP-7 and MMP-3 degraded fetuin after 24 hours of incubation. MMP-7-cleaved fetuin did not block mineralization while MMP-3-cleaved fetuin inhibited mineralization.

Conclusions:  The regulation of mineralization by fetuin is disrupted by cleavage of fetuin by MMP-7 but not by MMP-3. The increased expression of MMP-7 in periodontitis could increase the risk of calcified atheroma formation, in part rationalizing the epidemiological links between these diseases.