Matrix metalloproteinase 9 and vascular endothelial growth factor are essential for osteoclast recruitment into developing long bones.

Bone development requires the recruitment of osteoclast precursors from surrounding mesenchyme, thereby allowing the key events of bone growth such as marrow cavity formation, capillary invasion, and matrix remodeling. We demonstrate that mice deficient in gelatinase B/matrix metalloproteinase (MMP)-9 exhibit a delay in osteoclast recruitment. Histological analysis and specialized invasion and bone resorption models show that MMP-9 is specifically required for the invasion of osteoclasts and endothelial cells into the discontinuously mineralized hypertrophic cartilage that fills the core of the diaphysis. However, MMPs other than MMP-9 are required for the passage of the cells through unmineralized type I collagen of the nascent bone collar, and play a role in resorption of mineralized matrix. MMP-9 stimulates the solubilization of unmineralized cartilage by MMP-13, a collagenase highly expressed in hypertrophic cartilage before osteoclast invasion. Hypertrophic cartilage also expresses vascular endothelial growth factor (VEGF), which binds to extracellular matrix and is made bioavailable by MMP-9 (Bergers, G., R. Brekken, G. McMahon, T.H. Vu, T. Itoh, K. Tamaki, K. Tanzawa, P. Thorpe, S. Itohara, Z. Werb, and D. Hanahan. 2000. Nat. Cell Biol. 2:737-744). We show that VEGF is a chemoattractant for osteoclasts. Moreover, invasion of osteoclasts into the hypertrophic cartilage requires VEGF because it is inhibited by blocking VEGF function. These observations identify specific actions of MMP-9 and VEGF that are critical for early bone development.

Measurement of tumor load and distribution in a model of cancer-induced osteolysis: a necessary precaution when testing novel anti-resorptive therapies.

The Arguello model of cancer metastasis to bone has been used extensively to study breast cancer-induced osteolytic disease. The effects of therapy on skeletal disease and on tumour burden in soft organs are traditionally measured using radiography and/or time-consuming histomorphometry, respectively. The purpose of this study was to develop a sensitive and efficient method for evaluating tumour burden in vivo using MDA-231 cells transduced with the E. coli lacZ gene (MDA-231BAG). Osteolysis was measured by radiography and tumour burden was measured histomorphometrically or biochemically. In untreated mice, measurements of tumour burden in bone extracts using human cytokeratin-associated tissue polypeptide antigen (TPA) ELISA or E. coli beta-galactosidase (beta-gal) activity immunoassay reflected the extent of osteolytic disease as measured by radiography; however, tumour load could be detected before onset of osteolysis. When monitoring the effect of therapy (0.2 mg/kg ibandronate/day), radiography alone proved to be insufficient. Mice treated with the bisphosphonate ibandronate from time of inoculation with cancer cells had no radiologically visible signs of osteolysis but significant tumour load was measured in the bone extracts using these assays. Furthermore, beta-gal activity could be used as a measurement of tumour load in soft organs, and unlike other human breast cancer markers expressed by the MDA-231 cells in vitro, beta-gal activity was detected in the serum of mice with progressive disease. In conclusion, we describe an efficient model of breast cancer-induced osteolysis to quantify the effect of therapy on disease load and distribution, which could be beneficial in evaluating novel therapies for the treatment of the disease.

Proteinases in bone resorption: obvious and less obvious roles.

Bone resorption is critical for the development and the maintenance of the skeleton, and improper regulation of bone resorption leads to pathological situations. Proteinases are necessary for this process. In this review, we show that this need of proteinases is not only because they are required for the solubilization of bone matrix, but also because they are key components of the mechanism that determines where and when bone resorption will be initiated. Moreover, there are indications that proteinases may also determine whether resorption will be followed by bone formation. Some of the proteinases involved in these different steps of the resorption processes were recently identified, as for instance cathepsin K, MMP-9 (gelatinase B), and interstitial collagenase. However, there is also increasing evidence showing that the critical proteinase(s) may vary depending on the bone type or on other factors.