Collagen fibers in cirrhotic liver tissue retain gelatinases
Thioacetamide-intoxicated rats developed liver cirrhosis with extensive deposition of scar tissue in expanding fibrotic septa showing typical extensive bridged fibrosis, in which collagen types I and III (CI and CIII) predominate (Figures and ). In in situ zymography with dye quenched (DQ)-gelatine, strong gelatinolytic activity was associated with these structures, as shown by the bright fluorescence aligned with fibrillar structures (Figures and ). In the liver, MMP-2 is mainly expressed by hepatic stellate cells, whereas Kupffer cells are the major cellular source for MMP-9. Human fibrotic tissue was stained with monoclonal antibodies specific for MMP-2 or MMP-9 and subjected to a stringent washing procedure (Figures ). Light MMP-2 labeling was detected in fibrotic septa, and more pronounced MMP-9-specific labeling was observed in the pericellular region of macrophage-like cells (Figure ). The ubiquitous fibrillar staining observed for MMP-2 and the pericellular deposition of MMP-9 suggested fibrillar collagens or associated ECM molecules to have the capacity to store MMPs. No significant binding was observed when sections were preincubated with the aminophenyl mercuric acetate (APMA)-activated form of MMP (actMMP)-2 or actMMP-9 (not shown). If sequential liver sections were preincubated with prodomain-containing proMMP-2 or proMMP-9, preferential staining of fibrotic septa was observed for both gelatinase proforms (Figures ), confirming the localization of gelatinolytic activity observed by in situ zymography.
Figure 1 Localization of gelatinolytic activity in fibrotic liver tissue. (A and B) Collagenous septa in fibrotic rat liver tissue were stained with Sirius red with rectangular detail that highlights collagen fibers in (B). Original magnification, ×20. (more ...)
ProMMP-2 and ProMMP-9, but not actMMP-2 and actMMP-9, strongly bind to immobilized native collagens, CI fragments and to Hyp-containing collagen analogs
To further elucidate the interaction of human ECM components with human pro- or actMMP-2 or pro- or actMMP-9, we studied the retention of recombinant [125I]-labeled and enzymatically active gelatinases (Figure ) by highly purified and well-characterized native fibrillar collagens, CI fragments and Hyp-containing collagen analogs. Serial dilutions of potential non-substrate-binding partners dotted to a nitrocellulose membrane with high protein-binding capacity showed that natural and synthetic collagen structures sufficiently bound proMMP-2 and proMMP-9 (not shown) (Figure ). In this qualitative analysis, comparable signal intensities were observed for proMMP-2 binding to 0.25 to 0.5 μg/dot CI or CIII and from 2 to 4 μg/dot of the tightly packed helical Gly-Pro-Hyp (GPO)10 (Figure , left). The binding efficiencies of proMMP-2 to α1(I)-derived CB fragments declined in the following order: CB7>CB6>CB8> > CB3. Regardless of the collagenous structure immobilized, only weak binding was found for actMMP-9 (not shown) and for actMMP-2 (Figure , right).
Figure 2 Quality control of [125I]-(un)labeled pro/actMMP-2/-9 and dose-dependency of pro/actMMP-2-binding to nitrocellulose-immobilized collagenous molecules. (A) Unlabeled or [125I]-labeled pro/actMMP-2/-9 (~1 ng) were quality-checked in substrate gel zymography (more ...)
In the next step, [125I]-labeled proMMP-2 and proMMP-9 were applied to microwell-immobilized collagen structures, and retained radioactivity was determined after thorough washing to estimate gelatinase binding (Table ). Again, proMMP-2 and proMMP-9 strongly bound to CI as well as to single chains of CI and CIII (range, 22% to 45%). Confirming the results from the dot-blot analysis, a maximum binding of only 19% was found for actMMP-2 to the α1(I) chain, whereas no binding of actMMP-9 to collagenous structures was observed. The Hyp-containing (GPO)10 peptide, which structurally resembles triple-helical collagen helices with high melting temperatures, was used to further elucidate the relevance of the GPO triplet for proMMP-2 and proMMP-9 binding. Triple-helical Gly-Pro-Pro (GPP)10, devoid of Hyp, and the linear Gly-Ala-Pro (GAP) peptide served as controls. Compared to the respective actMMP, proMMP-2 and proMMP-9 bound two- to sevenfold more strongly to (GPO)10, but both forms of the MMPs showed only marginal interactions with the control peptides (GPP)10 and linear GAP (Table ).
Binding of pro- or actMMP-2 or pro- or actMMP-9 to native collagens, single chains of CI, chain fragments and synthetic peptidesa
Repeated GPO peptides interfere with binding of proMMP-2 and proMMP-9 to CI
The kinetics of gelatinase binding to CI were determined by surface plasmon resonance (SPR) measurements. The linear control peptide GAP had no effect on the interaction of the MMPs with CI (not shown). As for the assumed binding competitor (GPO)10, Kd values were determined using standard conditions for SPR measurements (Table ). These nonactivating conditions without divalent metal ions are known to interfere with MMP activity and conformation. Since the Off rates for proMMP-2 and actMMP-2 binding to CI were in the same range (0.48 s-1), the reduced binding strength of actMMP-2 (Kd, 170 ± 5 nM) compared to proMMP-2 (Kd, 70 ± 3 nM) was due to differences in the On rates. The effects seen with MMP-9 were more dramatic, since actMMP-9 bound to CI (Kd, 870 ± 39 nM) sevenfold less effectively than proMMP-9 (Kd, 120 ± 9 nM).
Effect of soluble (GPO)10 on the binding of pro- or actMMP-2 or pro- or actMMP-9 to CIa
A 10-fold molar excess of (GPO)10 only slightly impaired the binding of proMMP-2 and actMMP-2 to CI but had more pronounced effects on proMMP-9 (Table ). Using MMP activity buffer conditions, addition of (GPO)10 abolished binding of proMMP-9 to CI (Figure ), and (GPO)10 treatment of proMMP-2 led to a reduction in resonance units below baseline levels (Figure ). Since under these conditions actMMP-2 effectively degraded the CI matrix with a final loss of about 400 resonance units after 60 s (not shown), the sensorgram of proMMP-2 in the presence of (GPO)10 (Figure ) was most likely due to (GPO)10-induced collagenolytic MMP-2 activity, which is absent with MMP-9 (Figure ).
Figure 3 Binding of pro/actMMP-2/-9 to CI in the presence or absence of 10-fold molar excesses of Gly-Pro-Hyp triplets (GPO)10 and MMP inhibitor. (A) ProMMP-9 and (B) proMMP-2 (25-150 nM) with or without a 10-fold molar excess of (GPO)10 in MMP activity buffer (more ...)
Binding of MMP-2 to CI strictly depends on the gelatinase activation status
Since Kd values for binding of proMMP-2 to CI in the presence of (GPO)10 could not be assessed under activating buffer conditions, we studied proMMP-2 and actMMP-2 binding to immobilized CI in MMP activity buffer containing Ro 28-2653 that specifically inhibits proMMP (auto)activation and MMP enzymatic activity. The effects of strong binding of latent gelatinases to CI reduced by MMP activation were observed to be highly aggravated using this optimized buffer. ProMMP-2 bound to CI within low nanomolar Kd values representing a 10-fold binding enhancement, whereas the reduction of binding upon MMP activation was elevated from 2.5- to 35-fold (Figures and ; Tables and ). Here the addition of (GPO)10 to proMMP-2 resulted in a 22-fold reduced affinity for CI (Figure ), which was in the same range observed for MMP-2 activation (Table ).
Effect of thermostability and Hyp content of collagen analogs on the binding kinetics of pro- or actMMP-2 to CIa
ProMMP-2 activation and actMMP-2 activity are impaired upon CI binding and are strongly enhanced in the presence of (GPO)10
To further investigate the activation of MMP-2 by (GPO)10, the cleavage of a short, gelatinase-specific substrate by proMMP-2 and actMMP-2 was measured with and without a 10-fold excess of (GPO)10. In addition, some experiments used wells coated with fibrillar CI to explore its effects on activation and activity of MMP-2. Conversion of the quenched substrate peptide over time depended on the activation state of MMP-2. Activation of proMMP-2 (Figure ) showed sigmoid and activity of actMMP-2 (Figure ) immediate exponential kinetics of substrate turnover, probably due to an activation lag phase for proMMP-2. There was a strong reduction in activity of both proMMP-2 and actMMP-2 due to association with CI, showing that binding to fibrillar collagen results in impaired (auto)activation and activity of the enzyme (Figure hatched curves). Either on bovine serum albumin (BSA) or on CI, addition of (GPO)10 to proMMP-2 (Figure , bold lines) resulted in the same plateau activity as actMMP-2 without (GPO)10 (Figure , thin line). Since substrate freshly added after 120 min did not alter the outcome and excluded substrate depletion (data not shown), an elevated substrate turnover in the presence of low molar excesses of (GPO)10 was suggested.
Figure 4 Activation and activity of pro/actMMP-2 in the presence of (GPO)10 and immobilized CI. (A) Activation of proMMP-2 (50 ng) or (B) activity of fully activated actMMP-2 alone or mixed with 10-fold molar excesses of (GPO)10 was determined in CI- or bovine (more ...)
The Hyp content defines the efficiency of a competitor for proMMP-2 binding to CI
Further studies on the proMMP-2 exosite ligand-binding structure focused on the role of the triple helix and the Hyp content of the repeated triplet. Midpoints of melting curves of (POG)10 and (PPG)10 occurred at 64°C and 43°C, respectively. The control peptides GAP and (POG)5 were confirmed to be found nonhelical even at 5°C (Figure ). Triple-helical (GPP)10 without Hyp residues was much less efficient in competing with proMMP-2 binding to CI than the nonhelical (POG)5 (Table ). The competition potency of (GPO)10 that was triple-helical and contained Hyp residues was comparable to that of (POG)5. These results emphasize the crucial role of GPO triplets for gelatinase binding and established a 10-fold molar excess of (POG)5 in relation to proMMP-2 as a minimum prerequisite for blockade of proMMP-2 binding to fibrillar CI.
Figure 5 Polarimetric determination of melting temperatures of collagen analogs. Spontaneous triple helix assembly of collagen analogs was assessed by the midpoints of melting curves in a temperature regimen from 8°C to 60°C for Gly-Ala-Pro (GAP (more ...)
(GPO)10 and the MMP-2 prodomain peptide P33-42 compete for binding to the CBD
Collagen analogs similar to (GPO)10, as well as the proMMP-2 prodomain-derived peptide P33-42, are known to bind with high affinity to the Col-modules of the CBD of MMP-2. Addressing mechanisms of (GPO)10-induced inhibition of proMMP-2 binding to fibrillar collagen and (GPO)10-induced enzymatic activation, we used (GPO)10 and P33-42 to interfere with proMMP-2 binding to CI and to modulate CBD-dependent DQ-gelatine degradation of MMP-2.
In SPR measurements P33-42 was evaluated as a competitor of proMMP-2 binding to immobilized CI. High molar excesses of P33-42 increased the Kd values of proMMP-2 binding to CI to up to 50% (Figure ), which is moderate in comparison to the 22-fold inhibition in the presence of (GPO)10 (Table ). On the other hand, (GPO)10 and P33-42 had different effects on both proMMP-2 activation and activity (Figures ). Substrate zymography showed a faint band of prodomain-free actMMP-2 in the presence of (GPO)10 which did not occur if proMMP-2 was treated with P33-42 (Figure ). In MMP activity assays with DQ-gelatine as a fluorogenic substrate, actMMP-2 used as positive control established a plateau level of substrate conversion of about 1,000 relative fluorescence units while baseline levels for proMMP-2 were at about 200 relative fluorescence units. Whereas DQ-gelatine cleavage was significantly enhanced by 50- to 100-fold molar excesses of (GPO)10, P33-42 had no activating effect but a slight inhibitory effect (Figure ). The 150-fold excesses of P33-42 or (GPO)10 in relation to proMMP-2 resulted in a more enhanced inhibitory effect of P33-42 and a (GPO)10-driven shift from an active MMP-2 to an enzymatically blocked enzyme (Figure ). To gain insights into the mechanism of action, equimolar mixtures of (GPO)10 and P33-42 were added to proMMP-2 before assaying DQ-gelatine degradation (Figure ). At 50- to 100-fold molar excesses compared to proMMP-2, P33-42 drastically diminished the activating effect of (GPO)10 on proMMP-2. In addition, at a 150-fold molar excess of the mixture to proMMP-2, the inhibitory effect of (GPO)10 on MMP-2 activity was diminished (Figure ). These findings indicated that both (GPO)10 and P33-42 compete for the same proMMP-2 exosite.
Figure 6 Comparison of the effects of MMP-2 prodomain-derived collagen-binding domain (CBD)-binding peptide (P33-42) and (GPO)10 on proMMP-2 binding and activation. (A) Up to 100-fold molar excesses of P33-42 were mixed with 100-250 nM proMMP-2 in MMP activity (more ...)
(GPO)10 prevents proMMP-2 binding and releases proMMP-2 in situ bound to fibrillar septa
To answer the question about the capacity of (GPO)10 to modulate proMMP-2 binding to fibrillar structures in situ, cryostat sections of cirrhotic liver tissue were treated with exogenous Cy2-labeled proMMP-2 (Figure ). Without (GPO)10, the fluorescence pattern (Figure ) reflected the extent of fibrillar collagenous structures (Figure ), confirming that proMMP-2 can be extracellularly stored in fibrotic liver tissue. Coincubation of (GPO)10 with Cy2-proMMP-2 (Figure ) or subsequent treatment of ECM-bound Cy2-proMMP-2 with (GPO)10 (Figure ) either prevented gelatinase binding or promoted effective release of the sequestered enzyme.
Figure 7 (GPO)10-mediated release of proMMP-2 sequestered in cirrhotic liver tissue. (A) Cryostat sections of cirrhotic human liver tissue were fixed, stained with Sirius red for collagens and counterstained with Haemalaun. (B-D) Serial sections of the same liver (more ...)