Localisation of matrix metalloproteinases and TIMP-2 in resorbing mouse bone.
There is strong evidence that matrix metalloproteinases (MMPs) play a crucial role during osteogenesis and bone remodelling. Their synthesis by osteoblasts has been demonstrated during osteoid degradation prior to resorption of mineralised matrix by osteoclasts and their activities are regulated by tissue inhibitors of metalloproteinases (TIMPs). For this study we developed and utilised specific polyclonal antibodies to assess the presence of collagenase (MMP13), stromelysin 1 (MMP3), gelatinase A (MMP2), gelatinase B (MMP9) and TIMP-2 in both freshly isolated neonatal mouse calvariae and tissues cultured with and without bone-resorbing agents. Monensin was added towards the end of the culture period in order to promote intracellular accumulation of proteins and facilitate antigen detection. In addition, bone sections were stained for the osteoclast marker, tartrate-resistant acid phosphatase (TRAP). In uncultured tissues the bone surfaces had isolated foci of collagenase staining, and cartilage matrix stained for gelatinase B (MMP9) and TIMP-2. Calvariae cultured for as little as 3 h with monensin revealed intracellular staining for MMPs and TIMP-2 in mesenchymal tissues, as well as in cells lining the bone plates. The addition of cytokines to stimulate bone resorption resulted in pronounced TRAP activity along bone surfaces, indicating active resorption. There was a marked upregulation of enzyme synthesis, with matrix staining for collagenase and gelatinase B observed in regions of eroded bone. Increased staining for TIMP-2 was also observed in association with increased synthesis of MMPs. The new antibodies to murine MMPs should prove valuable in future studies of matrix degradation.
Dew
G
G
Strangeways Research Laboratory, Worts' Causeway, Cambridge, UK.
Murphy
G
G
Stanton
H
H
Vallon
R
R
Angel
P
P
Reynolds
J J
JJ
Hembry
R M
RM
D000135:Acid Phosphatase / Q000032:analysis / Q000276:immunology; D000818:Animals; D000918:Antibody Specificity; D015153:Blotting, Western; D001862:Bone Resorption / Q000201:enzymology*; D002478:Cells, Cultured; D017364:Collagenases / Q000032:analysis* / Q000276:immunology; D005455:Fluorescent Antibody Technique; D007527:Isoenzymes / Q000032:analysis / Q000276:immunology; D053509:Matrix Metalloproteinase 13; D020778:Matrix Metalloproteinase 2 / Q000032:analysis / Q000276:immunology; D019278:Matrix Metalloproteinase 3 / Q000032:analysis / Q000276:immunology; D020780:Matrix Metalloproteinase 9 / Q000032:analysis / Q000276:immunology; D051379:Mice; D010010:Osteoclasts / Q000201:enzymology; D012886:Skull / Q000166:cytology / Q000201:enzymology*; D000071681:Tartrate-Resistant Acid Phosphatase; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000032:analysis* / Q000276:immunology
D007527:Isoenzymes; D019716:Tissue Inhibitor of Metalloproteinase-2; D000135:Acid Phosphatase; C000605511:Acp5 protein, mouse; D000071681:Tartrate-Resistant Acid Phosphatase; D017364:Collagenases; D053509:Matrix Metalloproteinase 13; C507081:Mmp13 protein, mouse; D019278:Matrix Metalloproteinase 3; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9