Immunoreactive collagenase and bone resorption.

1. Active mouse bone collagenase is excluded from its inhibitory antibody by preincubation of that antibody with various forms of inactive enzyme, e.g. 'procollagenase', some collagenase-inhibitor complexes or partially denatured or degraded collagenase. This property allows the detection of several enzymatically inactive forms of collagenase. 2. The accumulation of immunoreactive collagenase in the culture fluid of mouse bones occurred only in the presence of heparin and was not correlated with bone resorption induced by parathyroid hormone. These experiments provide further (see Lenaers-Claeys, G. and Vaes, G., Biochim. Biophys. Acta (1979) 584, 375-388), more conclusive evidence that the critical role in the resorption of the organic matrix of these explants may be due to another enzyme system than collagenase.

The simultaneous release by bone explants in culture and the parallel activation of procollagenase and of a latent neutral proteinase that degrades cartilage proteoglycans and denatured collagen.

1. A latent neutral proteinase was found in culture media of mouse bone explants. Its accumulation during the cultures is closely parallel to that of procollagenase; both require the presence of heparin in the media. 2. Latent neutral proteinase was activated by several treatments of the media known to activate procollagenase, such as limited proteolysis by trypsin, chymotrypsin, plasmin or kallikrein, dialysis against 3 M-NaSCN at 4 degrees C and prolonged preincubation at 25 degrees C. Its activation often followed that of the procollagenase present in the same media. 3. Activation of neutral proteinase (as does that of procollagenase) by trypsin or plasmin involved two successive steps: the activation of a latent endogenous activator present in the media followed by the activation of neutral proteinase itself by that activator. 4. The proteinase degrades cartilage proteoglycans, denatured collagen (Azocoll) and casein at neutral pH; it is inhibited by EDTA, cysteine or serum. Collagenase is not inhibited by casein or Azocoll and is less resistant to heat or to trypsin than is the proteinase. Partial separation of the two enzymes was achieved by gel filtration of the media but not by fractional (NH4)2SO4 precipitation, by ion exchange or by affinity chromatography on Sepharose-collagen. These fractionations did not activate latent enzymes. 5. Trypsin activation decreases the molecular weight of both latent enzymes (60 000-70 000) by 20 000-30 000, as determined by gel filtration of media after removal of heparin. 6. The latency of both enzymes could be due either to a zymogen or to an enzyme-inhibitor complex. A thermostable inhibitor of both enzymes was found in some media. However, combinations of either enzyme with that inhibitor were not reactivated by trypsin, indicating that this inhibitor is unlikely to be the cause of the latency.

(Pro)collagenase (matrix metalloproteinase-1) is present in rodent osteoclasts and in the underlying bone-resorbing compartment.

Osteoclasts resorb the extracellular matrix of bone by secreting enzymes and acid into a sealed-off compartment that they form upon attachment to the bone surface. Although the lysosomal cysteine proteinases can degrade collagen after the demineralization of bone at low pH, several lines of evidence suggest that collagenase (matrix metalloproteinase-1, EC 3.4.24.7) may also be involved in this process. The question of whether collagenase is present in the osteoclast and/or in the bone-resorbing compartment has however not been resolved. We have prepared an anti-mouse collagenase antiserum and affinity-purified an IgG fraction that specifically immunoblots and immunoprecipitates (pro)collagenase. Using these antibodies, we demonstrate by immunolocalization the presence of (pro)collagenase both in the osteoclasts and in the extracellular subosteoclastic bone-resorbing compartment. These specific localizations were observed not only in mice but also in rat and rabbit osteoclasts and using not only the antibody we have prepared but also antibodies raised in other laboratories against rat (Jeffrey et al., J. Cell. Physiol. 143, 396-403, 1990) and rabbit (Brinckerhoff et al., J. Biol. Chem. 265, 22262-22269, 1990) collagenase. Intracellular collagenase was observed in the osteoclasts whether the cells were plated on bone or cultured on glass coverslips. It is proposed that osteoclastic collagenase is secreted in the resorbing compartment where it may cooperate with the lysosomal cysteine proteinases in the degradation of the collagen component of the matrix during the resorption of bone.