THE SYNTHESIS OF COLLAGENASE, GELATINASE-A (72 KDA) AND GELATINASE-B (95 KDA), AND TIMP-1 AND TIMP-2 BY HUMAN OSTEOBLASTS FROM NORMAL AND ARTHRITIC BONE

Bone resorption is a complex multistep process that involves removal o f both the organic and mineral constituents of bone matrix by proteoly tic enzymes synthesized by osteoblasts and osteoclasts. To further und erstand the role of matrix metalloproteinases (MMPs) and their specifi c inhibitors TIMPs (tissue inhibitor of metalloproteinases) in this pr ocess, human osteoblasts were obtained by sequential enzymatic digesti on from samples of bone from normal donors and patients with various f orms of arthritis; first passage cells were used in all experiments an d cultured on a type I collagen substratum. Collagenase was detected b y an ELISA in supernatants from unstimulated osteoblasts (range 12-730 ng/mL), although the levels did not appear to bear any relationship t o the age or clinical status of the patient; treatment with parathyroi d hormone (PTH; 2 units/mL) and 1,25-dihydroxyvitamin D-3 [1,25(OH)(2) D-3, 10 ng/mL] had no added effect, but mononuclear cell conditioned m edium (MCM; 5% v/v) and interleukin-1 alpha (IL-1 alpha; 1 ng/mL) both stimulated collagenase synthesis, in the case of MCM by two orders of magnitude. TIMP-1 was detected in unstimulated cultures by an ELISA ( range 320-590 ng/mL), the mean level being three-fold greater than for collagenase and was stimulated by 1,25(OH)(2)D-3 and MCM treatment. D egradation studies showed that, over a 120 h culture period, one third of the collagen substratum was degraded by unstimulated cells. PTH an d 1,25(OH)(2)D-3 had no effect on this endogenous level of lysis, but addition of MCM and IL-1 alpha resulted in a significant increase in c ollagen degradation. Zymography using SDS/PAGE containing gelatin prov ided evidence for the presence of progelatinase-A (72 kDa) and progela tinase-B (95 kD) in both treated and untreated cultures, with the acti ve species of gelatinase-A generated in all cases. Inhibitor gel zymog raphy confirmed that TIMP-1 was constitutively synthesized, but we cou ld find no evidence for TIMP-2 using this method; immunocytochemistry was subsequently used to detect the presence of TIMP2 in osteoblast mo nolayers. These findings provide additional evidence that human osteob lasts can synthesize collagenase and other MMPs, and support the hypot hesis that osteoblast-derived MMPs play an important role in bone reso rption, particularly in removal of the surface osteoid that precedes o steoclast attachment.