First published online 15 December 2004
doi: 10.1242/jcs.01581
Journal of Cell Science 118, 147-156 (2005)
Published by The Company of Biologists 2005
The metalloproteinase MT1-MMP is required for normal development and maintenance of osteocyte processes in bone
Kenn Holmbeck1,
Paolo Bianco2,
Isabelle Pidoux3,
S. Inoue3,
R. C. Billinghurst3,*,
W. Wu3,
,
Kali Chrysovergis1,
Susan Yamada1,
Henning Birkedal-Hansen1,
and
A. Robin Poole3
1 Matrix Metalloproteinase Unit, National Institute of Dental and Craniofacial Research, Bethesda, MD 20892, USA
2 Universita `La Sapienza'; Parco Scientifico Biomedico San Raffaele, 00161 Rome, Italy
3 Joint Diseases Laboratory, Shriners Hospitals for Children and Departments of Surgery, Medicine, and Cell Biology/Anatomy, Faculty of Medicine, McGill University, Montreal, Quebec, H3G 1A6, Canada
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Fig. 1. Osteocyte processes are associated with collagen cleavage. Cortical bone sections stained with Bodian silver stain (A,B), Giemsa (C,D), and collagen-cleavage-specific antibody (E-I). (A) Osteocytes are surrounded by many well-defined cell processes emanating from the lacunae and extending into the pericellular bone matrix to generate a mesh-like appearance in this 30-day-old wild-type mouse. (B) At 40 days of age, the network is even more highly developed. The progressive development of osteocyte processes can be appreciated more easily in sections from wild-type mice stained with Giemsa (C,D). (C) Bone section from a 20-day-old wild-type mouse demonstrating weakly outlined osteocyte processes (arrows). (D) In bone from a 40-day-old wild-type mouse, processes now appear more defined as canal-like structures radiating from the osteocyte lacuna (arrows). (E) Collagen cleavage is modest in the cortex of 30-day-old wild-type mouse and more obvious at the periosteal surface (p and arrow). The endosteal surface (e) in this section displays no staining. (F) Conspicuous staining of the osteocyte processes in a 40-day-old wild-type mouse. Clearly outlined processes are radiating from the osteocytes. (H) High-power magnification of osteocytes and their associated processes radiating from lacunae (H). (I) Punctate staining demonstrates areas cross-sectioned osteocyte processes. (G) Negative control stained with peptide-absorbed antiserum. Bars, 10 µm (A-D,H,I), 30 µm (E-G).
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Fig. 2. Localization of MT1-MMP in bone. (A) Bright-field image of a bone section from a 20-day-old wild-type mouse hybridized to MT1-MMP-antisense RNA probe. Staining is intense in the periosteum (p), with multiple cells expressing MT1-MMP-encoding mRNA. Isolated osteocytes in the bone cortex (c) likewise display signal indicating expression of MT1-MMP. e, endosteum. (B) Serial section from the same animal hybridized with MT1-MMP-sense RNA probe. (C) Immunolocalization of MT1-MMP in cortical bone from a 30-day-old wild-type mouse demonstrating osteocytes and their cell processes expressing MT1-MMP. (D) Bone section from 40-day-old wild-type mouse demonstrating even more intense stain than that seen at day 30. (E,F) Negative controls. Bone sections from 30-day-old MT1-MMP-deficient mouse stained for MT1-MMP (E) and 40-day-old MT1-MMP-deficient mouse stained for MT1-MMP (F). Bars, 40 µm (A,B), 10 µm (C-F).
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Fig. 3. Immunolocalization of MMP-13 in bone. (A-E) Bone sections from 30- and 40-day-old wild-type mice stained with antibody specific for MMP-13. (A) Section of cortical bone from 30-day-old wild-type mouse demonstrating staining of the periosteal surface (p) as well as of osteocyte lacunae and cell processes radiating into the bone matrix. e, endosteal surface. (B) Bone section from 40-day-old wild-type mouse demonstrating intense staining for MMP-13 associated with both osteocytes and their processes. e, endosteal surface. (D,E) High-power magnification of bone from 40-day-old wild-type mouse demonstrating osteocytes and associated processes staining intensely for MMP-13. Notice the punctate staining pattern in E, which indicates cross-sectioned cell processes. (C) Negative control. Bars, 30 µm (A-C), 10 µm (D,E).
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Fig. 4. Localization of collagen cleavage and MMP-13 in MT1-MMP-deficient mice. (A-E) Immunolocalization of collagen cleavage in cortical bone. (F-J) Immunolocalization of MMP-13 antigen in cortical bone. Bone sections from MT1-MMP-deficient mice at 30 days (A,F) and 40 days (B-E,G-J). (A) Collagen cleavage is absent from the pericellular area of osteocytes in a 30-day-old MT1-MMP-deficient mouse but is obvious at the endosteal surface (e). (B) 40-day-old MT1-MMP-deficient mouse displaying a complete absence of osteocyte process staining but with collagen cleavage at the periosteal surface (p). (C) Negative control for collagen cleavage. (D) High-power magnification from a 40-day-old MT1-MMP deficient mouse displaying a complete absence of cell process staining but with staining of the periosteal surface (p). (E) High-power magnification of an additional section from the same animal demonstrating in detail the lack of collagen cleavage in osteocyte processes. (F) 30-day-old MT1-MMP-deficient mouse demonstrating MMP-13 reactivity in osteocytes and periosteal (p) and endosteal surfaces (e). Notice the absence of staining associated with osteocyte processes, as seen in wild-type animals (Fig. 3). (G) 40-day-old MT1-MMP-deficient mouse displaying staining of osteocytes for MMP-13 but no staining of cell processes. e, endosteum; p, periosteum. (H) Negative control for MMP-13 antigen. (I,J) High-power magnification from 40-day-old MT1-MMP-deficient mouse demonstrating the absence of cell-process-associated MMP-13 staining. Only osteocyte cell bodies display reactivity for MMP-13. Bars, 30 µm (A-C,F-H), 10 µm (D,E,I,J).
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Fig. 5. Osteocyte process formation in cortical bone is dependent on MT1-MMP. Cortical bone sections stained with Bodian silver stain (A,B) or Giemsa stain (C,D). (A) Bone from 30-day-old MT1-MMP-deficient mouse displaying the absence of clearly defined osteocyte processes. The conspicuous mesh-like appearance of the bone matrix seen in wild-type mice is absent from this sample (Fig. 1). (B) Bone from a 40-day-old MT1-MMP-deficient mouse displaying a similar absence of osteocyte processes despite more advanced age. (C,D) Osteocyte processes are absent from these Giemsa-stained bones from a 20-day-old (C) and a 40-day-old (D) MT1-MMP-deficient mouse. Bars, 10 µm (A-C).
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Fig. 6. Imaging of osteocytes by transmission electron microscopy. (A-D) 48-day-old wild-type (A,B) and MT1-MMP-deficient (C,D) littermates. (A) Osteocyte (os) residing in lacuna (Lac). Notice the well-defined cell processes and canaliculi seen in cross section (arrows). (B) Section of osteocyte intersecting two well-defined processes (P). The matrix around the processes is electron dense and granular in appearance. (C,D) Osteocytes from MT1-MMP-deficient littermate displaying canaliculus (Can). Bar, 1 µm.
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Fig. 7. High-power transmission electron microscopy images of osteocytes. Electron micrographs show higher magnification of perilacunar matrix organization of osteocytes in (A) wild-type and (B) MT1-MMP-deficient 48-day-old mice. Canaliculi (can), fine osteocytic processes (p) and lacunae (Lac) are indicated. (A) Collagen fibrils have lost their banded pattern and granular staining in the lacuna wall. (B) The collagen fibrils display characteristic cross-striated banding pattern and no evidence of fine granular material. Lacunae and fine cellular processes are more obvious in the wild-type than in the deficient mouse. Bar, 1 µm.
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© The Company of Biologists Ltd 2005
