Evidence supporting proteolytic cleavage of insulin-like growth factor binding protein-1 (IGFBP-1) protein in amniotic fluid

Abstract

Objective: The purpose of this study was to determine if: 1) insulin-like growth factor binding protein-1 (IGFBP-1) in amniotic fluid (AF) exhibited proteolytic cleavage in cases of intra-amniotic inflammation; and 2) if the matrix metalloproteinases (MMP-3, MMP-8, MMP-9) in AF are associated with the degradation of IGFBP-1 in AF.

Methods: AF samples (n=20) were obtained from preterm gestations with and without intra-amniotic inflammation. The form of IGFBP-1 in AF was assessed by Western blot analysis and AF MMP-8 concentration was measured by ELISA. Densitometric analysis of Western blot was performed and the fragmented/intact IGFBP-1 ratio was calculated. Proteolysis of AF IGFBP-1 by MMPs was evaluated by incubating AF with exogenous human MMP-3, MMP-8 or MMP-9, and by incubating recombinant human IGFBP-1 in AF with and without inflammation.

Results: 1) IGFBP-1 was present in AF without inflammation as an intact form; however, the fragmented form was dominant in AF with inflammation; 2) the ratio of fragmented/intact IGFBP-1 was significantly higher in AF with inflammation than in AF without inflammation; 3) a higher ratio of fragmented/intact IGFBP-1 was associated with a higher concentration of MMP-8; 4) in-vitro proteolysis experiments showed that AF IGFBP-1 was degraded by exogenous human MMP-3, MMP-8 and MMP-9; 5) recombinant human IGFBP-1 was fragmented in AF with inflammation, but not in AF without inflammation.

Conclusion: The fragmented form of AF IGFBP-1 was significantly increased in AF with intra-amniotic inflammation, and MMPs produced in AF with intra-amniotic inflammation were associated with the proteolytic change of AF IGFBP-1.

Figure 1

Western blot analysis of amniotic fluid (AF) IGFBP-1 protein; IGFBP-1 was present in AF without inflammation as an intact form of 30 kDa (lane 1–4), however intact form of IGFBP-1 decreased and fragments were seen at 21, 17 and 12 kDa in AF with inflammation (lane 5–8). Representative cases are shown.

Figure 2

Densitometric analysis of amniotic fluid (AF) IGFBP-1 protein; The ratio of fragmented/intact IGFBP-1 was significantly higher in AF with inflammation than that in AF without inflammation (for total fragment: median 0.41 [range, 0.13–0.69] vs median 3.0 [range, 1.17–8.77]; for 21 kDa fragment: median 0.33 [range, 0.13–0.49] vs median 1.57, [range, 0.73–3.75]; for 17 kDa fragment: median 0.05 [range, 0.00–0.15] vs median 0.60, [range, 0.21–3.35]; for 12 kDa fragment: median 0.06 [range, 0.00–0.15] vs median 0.34, [range, 0.17–1.67]. P<0.001, respectively). F/I ratio, IGFBP-1 protein fragmented/intact form ratio by densitometric analysis

Figure 3

Degradation of IGFBP-1 in amniotic fluid (AF) according to the AF MMP-8 concentration; (A) Representative cases are shown: (1)–(2) AF without inflammation and (3)–(7) AF with inflammation. AF with a higher degree of inflammation measured by AF MMP-8 concentration had a higher ratio of fragmented/intact IGFBP-1. (B) Strong correlation is shown between AF MMP-8 concentrations and the ratio of fragmented/intact IGFBP-1 in AF (r=0.86; p<0.001; Spearman rank correlation test). F/I ratio, IGFBP-1 protein fragmented/intact form ratio by densitometric analysis.

Figure 3

Degradation of IGFBP-1 in amniotic fluid (AF) according to the AF MMP-8 concentration; (A) Representative cases are shown: (1)–(2) AF without inflammation and (3)–(7) AF with inflammation. AF with a higher degree of inflammation measured by AF MMP-8 concentration had a higher ratio of fragmented/intact IGFBP-1. (B) Strong correlation is shown between AF MMP-8 concentrations and the ratio of fragmented/intact IGFBP-1 in AF (r=0.86; p<0.001; Spearman rank correlation test). F/I ratio, IGFBP-1 protein fragmented/intact form ratio by densitometric analysis.

Figure 4

rhIGFBP-1 proteolysis in amniotic fluid (AF) without (A) or with (B) inflammation; 100 ng of rhIGFBP-1 was incubated in 10 uL of AFs at 37°C. There was no change in the form of rhIGFBP-1 in AF without inflammation (A). However, intact rhlGFBP-1 (30 kDa) was gradually degraded in AF with inflammation and 12 kDa fragment increased (B). Independent experiments were done on 4 cases of AFs and representative cases were shown.

Figure 5

Proteolysis of amniotic fluid (AF) IGFBP-1 in the presence MMP-3 (B), MMP-9 (C) and MMP-8 (D); 2 uL of AFs were incubated with exogenous human MMP-3, MMP-8 or MMP-9 (enzyme/substrate molar ratio =1:1) in a solution of 50 mM Tris (pH 7.5) containing 150 mM NaCl, 10 mM CaCl2, and 0.05% Triton (final volume 20 uL) at 37°C for 12–24 hours. AF IGFBP-1 was degraded by exogenous human MMP-3, MMP-8 and MMP-9 in a time-dependent manner. However, there was no change in the form of AF IGFBP-1 in the control AF (A).