Immunolocalization of metalloproteinases and their inhibitor in the rabbit growth plate

Abstract

The synthesis and distribution of three metalloproteinases, collagenase, stromelysin, and gelatinase, and of the tissue inhibitor of metalloproteinase were examined in the distal femoral growth plate, the secondary center of ossification, and the perichondral ossification groove of Ranvier in newborn to six-week-old rabbits. Specific antisera to each of the enzymes and to the tissue inhibitor of metalloproteinase were used to identify their distribution in the extracellular matrix of the growth plate and to determine the associated tissues and cells that are responsible for their synthesis. Immunolocalization using tissue that was cultured in the presence of monensin to augment accumulation of intracellular antigen revealed that the growth-plate chondrocyte is responsible for the synthesis of the metalloproteinases and the tissue inhibitor of metalloproteinase, and that there is a unique pattern of synthesis in each zone. Chondrocytes of the resting and proximal proliferative zones were shown to synthesize and secrete all of the metalloproteinases and the tissue inhibitor of metalloproteinase. Synthesis of collagenase also was demonstrated in the remainder of the proliferative zone and in the most distal cells of the hypertrophic zone. The presence of collagenase in the distal cells suggests their involvement in vascular invasion. By culturing tissues in the presence of antibodies, we were able to demonstrate collagenase and the tissue inhibitor of metalloproteinase throughout the growth-plate matrix. Staining of the extracellular matrix, implying active collagenase, was also found in the matrix of the proliferative and hypertrophic zones, suggesting that degradation of tissue may occur at a distance from the cells that synthesize the enzyme. Simultaneous localization with two different antibodies demonstrated that growth-plate chondrocytes are capable of synthesizing collagenase and the tissue inhibitor of metalloproteinase, both independently of one another and coordinately. Stromelysin was found to be synthesized in all zones, implying that it plays an important role in degradation. Monocyte-conditioned media stimulates synthesis of collagenase in growth-plate cells, principally through the action of interleukin-1. All chondrocytes throughout the growth plate, including hypertrophic cells, can be stimulated to produce collagenase. These changes in metalloproteinase and in the tissue inhibitory of metalloproteinase in the growth plate are crucial to remodelling of the matrix during development, and the appearance of metalloproteinases and the tissue inhibitory of metalloproteinase in the secondary ossification center and groove of Ranvier indicates that changes at these sites are similar to remodeling in the growth plate.

Clinical relevance: The life cycle of the chondrocyte in the growth plate is central to the process of endochondral ossification, bone growth, and development. Our new data on the zonal synthesis of metalloproteinase and of th