TIMP-3 inhibits the procollagen N-proteinase ADAMTS-2

Abstract

ADAMTS-2 is an extracellular metalloproteinase responsible for cleaving the N-propeptides of procollagens I-III; an activity necessary for the formation of collagenous ECM (extracellular matrix). The four TIMPs (tissue inhibitors of metalloproteinases) regulate the activities of matrix metalloproteinases, which are involved in degrading ECM components. Here we delineate the abilities of the TIMPs to affect biosynthetic processing of procollagens. TIMP-1, -2 and -4 show no inhibitory activity towards ADAMTS-2, in addition none of the TIMPs showed inhibitory activity towards bone morphogenetic protein 1, which is responsible for cleaving procollagen C-propeptides. In contrast, TIMP-3 is demonstrated to inhibit ADAMTS-2 in vitro with apparent Ki values of 160 and 602 nM, in the presence of heparin or without respectively; and TIMP-3 is shown to inhibit procollagen processing by cells.

Figure 1. TIMP-3 inhibits ADAMTS-2, but not BMP-1

(A) An autofluorogram is shown of ³H-labelled type II procollagen incubated with ADAMTS-2 without (0×) or in the presence of TIMP-3/ADAMTS-2 (1:1, 2:1, 5:1, or 10:1, mol/mol; labelled 1×, 2×, 5× and 10× respectively). SM, procollagen II starting material. (B) Autofluorograms are shown of ³H-labelled type I (left-hand panel) or type III (right-hand panel) procollagen incubated without (−) or in the presence (+) of ADAMTS-2 and TIMP-3. TIMP-3/ADAMTS-2 (50:1, mol/mol) when incubated together. (C) Western blot of TIMP-3 and ADAMTS-2 incubated together at ratios of 5:0, 0:1, 1:1, 2:1 and 5:1 (mol/mol), followed by immunoprecipitation with antibody against TIMP-3 (α-TIMP-3), and then Western blot analyses with α-TIMP-3 antibody, or with antibody against the protein C epitope (α-protein C), for detection of ADAMTS-2. (D) An autofluorogram is shown of ³H-labelled type I procollagen incubated with BMP1 alone (0×) or in the presence of a 50-fold excess (50×) of TIMP-3. SM, procollagen I starting material.

Figure 2. N-TIMP-3 inhibits ADAMTS-2 with an apparent K_i of 602 nM

(A) A constant amount of ADAMTS-2 (12 nM) was incubated with increasing amounts of N-TIMP-3 (0–1000 nM), prior to incubation with ³H-labelled type II procollagen, followed by SDS/PAGE and autofluorography. A Table of the percentage procollagen II processed at each concentration of N-TIMP-3 is shown below the autofluorogram. Data were plotted and an apparent K_i of 618 nM was calculated using GraphPad Prism 4 software. The K_i range was 411–826 nM and the standard error was 80.7 nM (95.5 confidence intervals). (B) A constant amount of ADAMTS-2 (4 nM) was incubated with increasing amounts of N-TIMP-3 (0–500 nM), prior to incubation with ³H-labelled type II procollagen, followed by SDS/PAGE and autofluorography. A Table of the percentage of procollagen II processed at each concentration of N-TIMP-3 is shown below the autofluorogram. Data were plotted and an apparent K_i of 602 nM was calculated using GraphPad Prism 4 software. The K_i range was 396–809 nM and the standard error was 84.4 nM (95.5 confidence intervals).

Figure 3. N-TIMP-3 inhibits ADAMTS-2 with an apparent K_i of 160 nM in the presence of heparin

(A) A constant amount of ADAMTS-2 (12 nM) was incubated with a constant amount of N-TIMP-3 (200 nM) and with increasing amounts of heparin, prior to incubation with ³H-labelled type II procollagen, followed by SDS/PAGE and autofluorography. The amount of heparin used is plotted against the ratio of procollagen II compared with the pCα1(II)-form (which retains the C-propeptide, while the N-propeptide has been removed). (B) A constant amount of ADAMTS-2 (12 nM) was incubated with increasing amounts of N-TIMP-3 (0–1000 nM) and 100 μg/ml heparin, prior to incubation with ³H-labelled type II procollagen, followed by SDS/PAGE and autofluorography. A Table of the percentage of procollagen II processed at each concentration of N-TIMP-3 is shown below the autofluorogram. Data were plotted and an apparent K_i of 160 nM was calculated using GraphPad Prism 4 software. The K_i range was 11.4 to 309.3 nM and the standard error was 60.9 nM (95% confidence intervals).

Figure 4. N-TIMP-3 inhibits the pNP activity of cells

N-TIMP-3 was added to the culture media of MEF cells, to a final concentration of 250 or 800 nM, in the presence of 0.01% dextran sulfate. Following SDS/PAGE and electrotransfer, pro-α1(I)-derived chains in cell layer samples were probed using an antibody directed against sequences in the pro-α1(I) C-telopeptide domain.