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

Different cysteine proteinases involved in bone resorption and osteoclast formation

Cysteine proteinases, especially cathepsin K, play an important role in osteoclastic degradation of bone matrix proteins and the process can, consequently, be significantly inhibited by cysteine proteinase inhibitors. We have recently reported that cystatin C and other cysteine proteinase inhibitors also reduce osteoclast formation. However, it is not known which cysteine proteinase(s) are involved in osteoclast differentiation. In the present study, we compared the relative potencies of cystatins C and D as inhibitors of bone resorption in cultured mouse calvariae, osteoclastogenesis in mouse bone marrow cultures, and cathepsin K activity. Inhibition of cathepsin K activity was assessed by determining equilibrium constants for inhibitor complexes in fluorogenic substrate assays. The data demonstrate that whereas human cystatins C and D are equipotent as inhibitors of bone resorption, cystatin D is 10-fold less potent as an inhibitor of osteoclastogenesis and 200-fold less potent as an inhibitor of cathepsin K activity. A recombinant human cystatin C variant with Gly substitutions for residues Arg(8), Leu(9), Val(10), and Trp(106) did not inhibit bone resorption, had 1,000-fold decreased inhibitory effect on catbepsin K activity compared to wildtype cystatin C, but was equipotent with wildtype cystatin C as an inhibitor of osteoclastogenesis. It is concluded that (i) different cysteine proteinases are likely to be involved in bone resorption and osteoclast formation, (ii) cathepsin K may not be an exclusive target enzyme in any of the two systems, and (iii) the enzyme(s) involved in osteoclastogenesis might not be a typical papain-like cysteine proteinase

Potassium citrate prevents increased osteoclastogenesis resulting from acidic conditions: Implication for the treatment of postmenopausal bone loss - Fig 3

<p><b>A)</b> RANKL-mediated OC differentiation in acidic and neutral microenvironment. The bars represent the mean ± SEM of the total number of OC/cm² obtained from buffy-coat of four individuals and cultured at pH 7.4 and pH 6.9 with three different RANKL concentrations. Symbols (*) indicate statistically significant differences vs RANKL 50 ng/mL at pH 7.4. <b>B)</b> The representative pictures show that the size of TRAcP positive multinucleated cells and the differentiating potency of low RANKL concentrations are higher at pH 6.9 than at pH 7.4. Magnification x 10, Scale bar = 50 μm. <b>(C)</b> and <b>(D)</b> K citrate inhibits the OC differentiation induced at pH 6.9 by high (C: 50 ng/mL) and low (D: 0.5 ng/mL) RANKL doses. Results are expressed as a ratio between the number of OC counted in cultures treated with different K citrate concentrations and number of OC counted in the negative control (K citrate 0 mM, ratio = 1). The positive control is Alendronate 10^-5M. Mean ± SEM of data obtained from three different donors and two replicates for each condition. Symbols (*) indicate statistically significant differences vs negative control (pH 6.9, K citrate 0 mM, ratio = 1). In all histograms P values ≤ 0.05, ≤ 0.01 and ≤ 0.001 were highlighted by one, two or three symbols, respectively.</p