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

Engsig MT, Chen QJ, Vu TH, Pedersen AC, Therkidsen B, Lund LR, Henriksen K, Lenhard T, Foged NT, Werb Z, Delaissé JM - J. Cell Biol. (2000)

Bottom Line: Hanahan. 2000.Cell Biol. 2:737-744).These observations identify specific actions of MMP-9 and VEGF that are critical for early bone development.

View Article: PubMed Central - PubMed

Affiliation: OSTEOPRO A/S and Center for Clinical and Basic Research, DK-2750 Herlev/Ballerup, Denmark. me@osteopro.dk

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.

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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).
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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).

Mentions: We compared the sensitivity of resorption of MMP-9–positive and MMP-9–deficient bones to various inhibitors of proteinases reported to be rate-limiting for resorption (Fig. 2). E-64, a cysteine proteinase inhibitor, reduced resorption of tibiae to similar levels in MMP-9–positive and MMP-9–deficient bones. These results suggest that the absence of effect of MMP-9 deficiency on resorption is not due to compensation by cysteine proteinases. A general MMP inhibitor produced 50% inhibition in the tibiae, and close to 100% in the metatarsal cultures, in accordance with our previous report that MMP activity is involved in resorption but is more critical for osteoclast recruitment (Blavier and Delaissé 1995). These levels of inhibition were not influenced by MMP-9 deficiency. MMP-9 deficiency did not affect either the levels of other MMPs in extracts of freshly isolated or cultured calvariae, tibiae, and metatarsals or in their conditioned medium, as far as can be detected by gelatin/casein zymography (not shown). We interpret these data to indicate that MMPs other than MMP-9 are involved in resorption of calcified tissue itself and suggest that, instead, MMP-9 may play a role in osteoclast recruitment.


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

Engsig MT, Chen QJ, Vu TH, Pedersen AC, Therkidsen B, Lund LR, Henriksen K, Lenhard T, Foged NT, Werb Z, Delaissé JM - J. Cell Biol. (2000)

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).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169432&req=5

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).
Mentions: We compared the sensitivity of resorption of MMP-9–positive and MMP-9–deficient bones to various inhibitors of proteinases reported to be rate-limiting for resorption (Fig. 2). E-64, a cysteine proteinase inhibitor, reduced resorption of tibiae to similar levels in MMP-9–positive and MMP-9–deficient bones. These results suggest that the absence of effect of MMP-9 deficiency on resorption is not due to compensation by cysteine proteinases. A general MMP inhibitor produced 50% inhibition in the tibiae, and close to 100% in the metatarsal cultures, in accordance with our previous report that MMP activity is involved in resorption but is more critical for osteoclast recruitment (Blavier and Delaissé 1995). These levels of inhibition were not influenced by MMP-9 deficiency. MMP-9 deficiency did not affect either the levels of other MMPs in extracts of freshly isolated or cultured calvariae, tibiae, and metatarsals or in their conditioned medium, as far as can be detected by gelatin/casein zymography (not shown). We interpret these data to indicate that MMPs other than MMP-9 are involved in resorption of calcified tissue itself and suggest that, instead, MMP-9 may play a role in osteoclast recruitment.

Bottom Line: Hanahan. 2000.Cell Biol. 2:737-744).These observations identify specific actions of MMP-9 and VEGF that are critical for early bone development.

View Article: PubMed Central - PubMed

Affiliation: OSTEOPRO A/S and Center for Clinical and Basic Research, DK-2750 Herlev/Ballerup, Denmark. me@osteopro.dk

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.

Show MeSH
Related in: MedlinePlus