Osteopontin is proteolytically processed by matrix metalloproteinase 9

Abstract

Osteopontin is robustly upregulated following myocardial infarction (MI), which suggests that it has an important role in post-MI remodeling of the left ventricle (LV). Osteopontin deletion results in increased LV dilation and worsened cardiac function. Thus, osteopontin exerts protective effects post-MI, but the mechanisms have yet to be defined. Matrix metalloproteinases (MMPs) regulate LV remodeling post-MI, and osteopontin is a known substrate for MMP-2, -3, -7, and -9, although the cleavage sites have not been mapped. Osteopontin-derived peptides can exert distinct biological functions that may depend on their cleavage sites. We mapped the MMP-9 cleavage sites via LC-MS/MS analysis using label-free and N-terminal labeling methods, and compared them with those of MMP-2, -3, and -7. Each MMP yielded a unique cleavage profile with few overlapping cleavage sites. Using synthetic peptides, we validated 3 sites for MMP-9 cleavage at amino acid positions 151-152, 193-194, and 195-196. Four peptides were synthesized based on the upstream- and downstream-generated fragments and were tested for biological activity in isolated cardiac fibroblasts. Two peptides increased cardiac fibroblast migration rates post-wounding (p < 0.05 compared with the negative control). Our study highlights the importance of osteopontin processing, and confirms that different cleavage sites generate osteopontin peptides with distinct biological functions.

Keywords: MMP-9; MPM-9; cardiac; cardiaque; cleavage sites; fibroblastes; fibroblasts; infarctus du myocarde; mass spectrometry; matrix metalloproteinases; myocardial infarction; métalloprotéinases matricielles; osteopontin; ostéopontine; peptides; proteomics; protéomique; sites de clivage; spectométrie de masse.

Fig. 1

Matrix metalloproteinase (MMP)-2, -3, -7, and -9 induced distinct profiles of osteopontin fragments. Representative picture of a silver staining gel visualizing multiple fragments generated by each of MMP-2, -3, -7, and -9, by comparison with intact mouse recombinant osteopontin control (mrOPN). The generated fragments do not follow the same pattern, creating a unique cleavage profile for each enzyme. Some fragments do overlap between all or some MMPs (close arrows), whereas others were conserved for each enzyme (open arrows).

Fig. 2

Matrix metalloproteinase (MMP) cleavage sites on osteopontin (OPN) were identified using label-free and N-terminal labeling methods. The graph displays the number of cleavage sites identified by either label-free or N-labeling methods, by both methods, and using synthetic OPN-peptides (LL and GL fragments). Cleavage sites were identified using LC-MS/MS.

Fig. 3

Visualization of matrix metalloproteinase (MMP)-2, -3, -7, and -9 relative cleavage sites on mouse recombinant osteopontin. Cleavage sites are identified with arrows. The MMP(s) responsible for cleavage is(are) identified by number, for example, -2 refers to MMP-2. Arrows are color coded depending on the identification method. Blue represents cleavage sites determined by both methods (label-free and N-labeling), yellow denotes sites identified only by the label-free method, and pink indicates cleavage sites determined by the N-labeling method only. Arrows with no label correspond to a site cleaved by all tested MMPs.

Fig. 4

Matrix metalloproteinase (MMP)-9 cleavage sites were validated using synthetic osteopontin (OPN)-peptides. Figure shows MS/MS results for MMP-9 cleavage site validation using synthetic peptides. (A) Fragment LL: The upper panel shows the synthetic Fragment LL identified at 35.18 min retention time. The middle panel shows the MMP-9-generated fragments with their retention time. Results show cleavage at 2 sites (AQ and LL; position 193-194 and 195-196 respectively). The lower panel is the MMP-9 only background. Peptide cleavage efficiency was ∼93% (n = 5 technical replicates) and was calculated using peak area. (B) Fragment GL: Upper panel shows synthetic peptide identified at 20.38 min retention time. Middle panel shows the MMP-9 generated fragments, with cleavage between GL (position 151–152), with their retention times. Lower panel is the MMP-9 only background. The peptide cleavage efficiency was ∼99% (n = 5 technical replicates) and was calculated using peak area.

Fig. 5

Osteopontin (OPN)-derived synthetic peptides stimulated wound healing rates in cardiac fibroblasts. Four peptides 15-amino acids long (OPN-p151, -p152, -p195, and -p196) were synthesized based on the MMP-9 cleavage sites GL (151/152) and LL (195/196). Each peptide was designed upstream (p151 and p195) or downstream (p152 and p196) of the cleavage site, as shown on Table 1. Each OPN peptide was tested at 2 concentrations of 0.5 and 5.0 μmol/L, for biological effects on wounded isolated cardiac fibroblast (top and middle panels). Ten percent fetal bovine serum (FBS) was used as the positive control and 0.1% FBS was the negative control. Fibroblasts were cultured until confluent, then wounded and treated with or without peptide. The wound closing rates were recorded using electric cell-substrate impedance sensing. Wound healing was observed up to 48 h post-wounding. No significant effect was observed with any peptide during the first 24 h post-wounding OPN-p151 significantly increased wound healing 32 h post-wound, and this effect was maintained up to 48 h. OPN-p152 enhanced wound healing at 48 h post-wounding. The lower panel shows bar graphs for all OPN-peptides at concentrations of 5 μmol/L at 32 and 48 h post-wounding Both OPN-p195 and OPN-p196 had no effect on fibroblast healing rates. Data are the mean ± SEM, n = 4 biological replicates/group. Analysis was by 2-way ANOVA with Newman-Keuls multiple comparisons test; *, p < 0.05 compared with the 0.1% treatment group; #, p < 0.05 by comparison with all of the other groups.