Degradation of collagen in the bone‐resorbing compartment underlying the osteoclast involves both cysteine‐proteinases and matrix metalloproteinases

TLDR

The study aimed to determine where cysteine‑proteinases and matrix metalloproteinases act during osteoclast‑mediated bone collagen degradation by testing their inhibitors in a PTH‑stimulated mouse calvaria model. Using an in vitro PTH‑stimulated mouse calvaria system, the authors applied the CP inhibitor E‑64 and the MMP inhibitor Cl‑1 to assess their effects on collagen degradation. Inhibiting CPs or MMPs with E‑64 or Cl‑1 produced a tenfold increase in demineralized collagen adjacent to osteoclast ruffled borders, elevated osteoclast contact with these areas (60 % vs 5 %), and revealed that while demineralization proceeded, collagen degradation was strongly suppressed, demonstrating that both CPs and MMPs are required for collagen breakdown in the resorption lacuna.

Abstract

Abstract The site of action of cysteine‐proteinases (CPs) and matrix metalloproteinases (MMPs) in the degradation of bone collagen by osteoclasts was investigated by evaluating the effects of the CP‐inhibitor trans‐epoxy‐succinyl‐L‐leucylamido (4‐guanidino)‐butane (E‐64) and the MMP‐inhibitor N‐(3‐N‐benzyloxycarbonyl amino‐1‐R‐carboxypropyl)‐L‐leucyl‐O‐methyl‐L‐tyrosine N‐methylamide (Cl‐1) in an in vitro model system of PTH‐stimulated mouse calvaria. In the presence of each of the two inhibitors a large area of collagen free of mineral crystallites was seen adjacent to the ruffled border of the osteoclasts. Following a culture period of 24 h this area proved to be about 10 times larger in inhibitor‐treated explants than in controls. Moreover the percentage of osteoclasts in close contact with such demineralized bone areas appeared to be significantly higher in inhibitor‐treated explants than in control specimens (60% and 5%, respectively). These effects were not apparent when the osteoclastic activity was inhibited with calcitonin. No significant differences were found between the effects of the two inhibitors, E‐64 and Cl‐1. Our observations indicate that under the influence of inhibitors of MMPs and CPs demineralization of bone by osteoclasts proceeded up to a certain point whereas matrix degradation was strongly inhibited. It is concluded that within the osteoclastic resorption lacuna both CPs and MMPs participate in the degradation of the collagenous bone matrix.