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

Abstract

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.

Figure 1

Effect of MMP-9 deficiency on the resorption of different types of bones. Calvariae of E18 embryos and tibiae and metatarsals of E17 embryos were obtained from two and eight litters, respectively, of heterozygote parents. The embryos were phenotyped as MMP-9–positive (circles) or MMP-9–negative (triangles). The bone explants were cultured for the indicated times. Each point shows the mean ± SD of ⁴⁵Ca release determined in independent cultures of 41 MMP-9–positive and 23 MMP-9–negative metatarsals or tibiae, or 9 MMP-9–positive and 4 MMP-9–negative calvariae. Significant differences in ⁴⁵Ca release from MMP-9–positive and MMP-9–negative explants were seen only for the metatarsals (P < 0.05).

Figure 2

Effect of proteinase inhibitors on the resorption of metatarsals and tibiae. Tibiae and metatarsals of E17 embryos were obtained from three (A) or five (B) litters of heterozygote parents. The embryos were phenotyped as MMP-9–positive (circles) or MMP-9–negative (triangles). The bone explants were cultured in the absence (filled symbols) or presence (corresponding open symbols) of 2.5 μM BB94 (A) or of 48 μM E64 (B). Each point shows the mean ± SD of ⁴⁵Ca release determined in 14 independent cultures of MMP-9–positive and 12 independent cultures of MMP-9–negative explants, each with and without BB94 (A), or in 29 independent cultures of MMP-9–positive and 12 independent cultures of MMP-9–negative explants, each with and without E-64 (B). All the inhibitors significantly inhibited ⁴⁵Ca release, with the exception of E64 on MMP-9–negative metatarsals after 2 d of culture (P < 0.05).

Figure 3

Gelatin zymography of extracts of metatarsals at different developmental stages. Metatarsal triads were isolated from four mouse embryos at E15, E16, E17, and E18. Extracts were obtained and analyzed by zymography as explained in Materials and Methods. Each lane is representative of one embryo. Pro–MMP-9 and Pro–MMP-2 are the zymogens of MMP-9 and MMP-2, respectively.

Figure 4

In situ hybridization and immunostaining of MMP-9 in sections of diaphysis of developing metatarsals. Two sets of adjacent sections of E17 metatarsals were prepared (A, B and C, D). In each set, one section was hybridized with antisense MMP-9 probe (A), or immunostained for MMP-9 (C); their respective adjacent sections were stained both for CD34 immunoreactivity and TRAP activity (B and D). Strong hybridization signals (A) and MMP-9 immunoreactivity (C) are seen outside the calcified cartilage (cc) (arrows). Signals for TRAP activity (red) in the adjacent sections (B and D) (arrows) correspond to the localizations of the MMP-9 hybridization (A) and immunoreactivity (C) signals, respectively. CD34 signals (brown) (B and D) are abundant in the periosteal cell layer (po) and do not have clearly matching MMP-9 hybridization (A) or immunoreactivity signals (C). Bars, 50 μm.

Figure 5

Effect of MMP-9 deficiency on number and distribution of TRAP⁺ cells in developing metatarsals. Litters of E17, E18, and E19 embryos were obtained from MMP-9^+/− and MMP-9^−/− parents. The embryos were genotyped as MMP-9^+/− or MMP-9^−/−. Metatarsal triads were analyzed with respect to number of nuclei of TRAP⁺ cells localized outside or inside the calcified cartilage. Means ± SD were calculated for each age group and genotype. Total numbers of embryos were as follows: at E17, 12 MMP-9^+/− and 11 MMP-9^−/− (pool of 3 litters); at E18, 31 MMP-9^+/− and 20 MMP-9^−/− (pool of 7 litters); and at E19, 9 MMP-9^+/− and 8 MMP-9^−/− (pool of 3 litters). Mean counts outside the calcified cartilage are shown by a block to the left of the zero axis, and mean counts inside the calcified cartilage by a block to the right. Each horizontal bar resulting from the alignment of these two blocks represents the mean number of total nuclei in one section of a metatarsal triad. At E18 and E19, MMP-9^−/− and MMP-9^+/− metatarsals differed significantly with respect to both total number of nuclei of TRAP⁺ cells and their proportion in the calcified cartilage (P < 0.05). In contrast, at E17 no significant differences were found.

Figure 7

Sections through the diaphysis of MMP-9–positive and MMP-9–negative metatarsals showing invasion and lack of invasion, respectively, by (pre)osteoclasts and endothelial cells at E18. Sections of MMP-9–positive (A) and MMP-9–negative (B) metatarsals of E18 embryos were obtained as explained in Fig. 5 and stained for TRAP activity and CD34 immunoreactivity to visualize (pre)osteoclasts (red) and endothelial cells (brown), respectively. MMP-9–positive metatarsals show large mature osteoclasts and endothelial cells that have invaded calcified cartilage (cc). MMP-9–negative metatarsals do not show osteoclasts or endothelial cells in calcified cartilage, but show well (pre)osteoclasts in the bone collar (bc). Endothelial cells are also abundant in the periosteal cell layer (po) in both phenotypes. Bar, 50 μm.

Figure 6

Effect of MMP-9 deficiency on number of nuclei per TRAP⁺ cell. The number of nuclei per TRAP⁺ cell were calculated from the counts of cells and nuclei shown in Fig. 5, and are shown as mean ± SD.

Figure 9

Effect of blocking VEGF activity on recruitment of TRAP⁺ cells into developing metatarsals. (A) Dark field photographs of sections of E17 wild-type (+/+) and MMP-9 null (MMP-9^−/−) metatarsals hybridized with a ³⁵S-labeled VEGF antisense probe. (B) Adjacent sections of metatarsals cultured for 3 d in control IgG or mFlt-IgG stained with hematoxylin and eosin (upper panels) or for TRAP activity (lower panels). Bars, (A) 200 μm; (B) 320 μm.

Figure 8

Comparative effect of MMP-9 deficiency and of a general MMP inhibitor on osteoclast migration through type I collagen. Osteoclasts of MMP-9–positive and MMP-9–negative mice were cultured on collagen-coated filters in the presence and absence of 10 μM GM6001 (average of 227 osteoclasts/filter). Their invasion through the collagen was evaluated as explained in Materials and Methods, and is shown as means ± SD of three cultures. *Significant effect compared with osteoclasts of MMP-9–positive mice cultured without inhibitor (P < 0.05).

Figure 10

Effect of VEGF on osteoclast migration. Osteoclasts were cultured overnight on collagen-coated membranes of culture inserts (average of 1,032 osteoclasts/insert). These inserts were placed in 12-well plates containing the indicated concentration of VEGF. 100 ng/ml Flt-1/Fc was added to the culture inserts where indicated. The migrations were scored as explained in Materials and Methods, and are shown as mean ± SD of four cultures. *Significant effect compared with osteoclasts cultured without additive (P < 0.05).