Enzymes active in the areas undergoing cartilage resorption during the development of the secondary ossification center in the tibiae of rats ages 0-21 days: I. Two groups of proteinases cleave the core protein of aggrecan

The formation of a secondary ossification center in the cartilaginous epiph ysis of long bones requires the excavation of canals and marrow space and, therefore, the resorption of cartilage. On the assumption that its resorpti on requires the lysis of the major cartilage component aggrecan, it was not ed that the core protein may be cleaved in vitro by proteinases from two su bfamilies: matrix metalloproteinases (MMPs) and aggrecanases. Such cleavage results in aggrecan being replaced by a fragment of itself referred to as a "G1-fragment." To find out if this cleavage occurs in the developing epip hysis of the rat tibia, the approach has been to localize the G1 fragments. For this purpose two neoepitope antisera were applied, one capable of reco gnizing the MMP-generated G1-fragment that bears the C-terminus... FVDIPEN3 41 and the other capable of recognizing the aggrecanase-generated Gl-fragme nt that carries the C-terminus... NITEGE(373). With the aid of these antise ra, we report here that aggrecan cleavage is localized to newly developed s ites of erosion. Thus, at 6 days of age, canals allowing the entry of capil laries are dug out from the surface of the epiphysis in a radial direction (stage I), whereas immunostaining indicative of aggrecan cleavage by MMPs a ppears at the blind end of each canal. The next day, the canal blind ends f use to create a marrow space in the epiphysis (stage II), whereas immunosta ining produced by MMPs occurs along the walls of this space. By 9 days, clu sters of hypertrophic chondrocytes are scattered along the marrow space wal l to initiate the formation of the secondary ossification center (stage III ), where the resorption sites are unreactive to either antiserum. From the 9th to the 21st day, the center keeps on enlarging and, as the distal wall of the marrow space recedes, it is intensely immunostained with both antise ra indicating that both MMPs and aggrecanases are involved in this resorpti on. We conclude, that both enzyme subfamilies contribute to the lysis of ag grecan. However, the results suggest that the respective subfamilies target different sites and even stages of development in the tissue, suggesting s ome diversity in the mode of aggrecan lysis during the excavation of a seco ndary ossification center. (C) 2001 Wiley-Liss, Inc.