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Osteoblast recruitment and bone formation enhanced by cell matrix-associated heparin-binding growth-associated molecule (HB-GAM).

Imai S, Kaksonen M, Raulo E, Kinnunen T, Fages C, Meng X, Lakso M, Rauvala H - J. Cell Biol. (1998)

Bottom Line: We show here that heparin-binding growth-associated molecule (HB-GAM), an extracellular matrix-associated protein that enhances migratory responses in neurons, is prominently expressed in the cell matrices that act as target substrates for bone formation.The HB-GAM transgenic mice develop a phenotype characterized by an increased bone thickness.HB-GAM may thus play an important role in bone formation, probably by mediating recruitment and attachment of osteoblasts/osteoblast precursors to the appropriate substrates for deposition of new bone.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, Shiga University of Medical Science, Shiga-ken, 520-2192, Japan. simai@belle.shiga-med.ac.jp

ABSTRACT
Bone has an enormous capacity for growth, regeneration, and remodeling. This capacity is largely due to induction of osteoblasts that are recruited to the site of bone formation. The recruitment of osteoblasts has not been fully elucidated, though the immediate environment of the cells is likely to play a role via cell- matrix interactions. We show here that heparin-binding growth-associated molecule (HB-GAM), an extracellular matrix-associated protein that enhances migratory responses in neurons, is prominently expressed in the cell matrices that act as target substrates for bone formation. Intriguingly, N-syndecan, which acts as a receptor for HB-GAM, is expressed by osteoblasts/osteoblast precursors, whose ultrastructural phenotypes suggest active cell motility. The hypothesis that HB-GAM/N-syndecan interaction mediates osteoblast recruitment, as inferred from developmental studies, was tested using osteoblast-type cells that express N-syndecan abundantly. These cells migrate rapidly to HB-GAM in a haptotactic transfilter assay and in a migration assay where HB-GAM patterns were created on culture wells. The mechanism of migration is similar to that previously described for the HB-GAM-induced migratory response of neurons. Our hypothesis that HB-GAM/N-syndecan interaction participates in regulation of osteoblast recruitment was tested using two different in vivo models: an adjuvant-induced arthritic model and a transgenic model. In the adjuvant-induced injury model, the expression of HB-GAM and of N-syndecan is strongly upregulated in the periosteum accompanying the regenerative response of bone. In the transgenic model, the HB-GAM expression is maintained in mesenchymal tissues with the highest expression in the periosteum. The HB-GAM transgenic mice develop a phenotype characterized by an increased bone thickness. HB-GAM may thus play an important role in bone formation, probably by mediating recruitment and attachment of osteoblasts/osteoblast precursors to the appropriate substrates for deposition of new bone.

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(A) Schematic diagram of the gene construct  used to produce transgenic  mice. HB-GAM cDNA was  cloned in the pHBApr-1-neo  vector, which contains the  promoter and the first intron  from human β-actin gene and  SV-40 polyadenylation signal.  (B) Expression of HB-GAM  in transgenic mice. (a) Western blot analysis of different  neonatal tissues (postnatal  day 1) from a transgene-positive mouse (lanes noted by  +) and its transgene-negative  littermate (noted by −). Lane  b, brain; lane h, heart; lane m,  femoral muscle; lane l, liver;  lane k, kidney. Note the intense transgene expression  in heart and muscle. (b)  Transgene expression in the  periosteum in comparison to  the heart of 1-yr-old mice.  Western blot analysis of tissues from two different transgenic lines (noted by +) and  their nontransgenic littermates (noted by −). HB-GAM expression in the  transgenic periosteum (p) is  even higher than that in the  heart (h). Lane c, control recombinant HB-GAM protein  (100 ng). (C) Gross appearance of bones form a 1-yr-old  transgene-positive mouse  (left in all panels) and those from its transgene-negative littermate (right). (a) Femora of transgene-positive (Tg) and transgene-negative  (Non-tg) mice. Note the ivory-like solid appearance of the transgene-positive femur, whereas the transgene-negative femur displays a  brownish surface because of the bone marrow structures that are seen through the thin cortical bone (arrow). (b) Ulnae. Brownish marrow structure is also seen in the transgene-negative ulna (arrow). (c) Scapulae (1) and humeri (2). The ivory-like appearance is particularly prominent in the scapular spinus of the transgene-positive mouse (arrow). Note the transparency of scapular plane in the transgene-negative bone (arrowhead).
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Figure 7: (A) Schematic diagram of the gene construct used to produce transgenic mice. HB-GAM cDNA was cloned in the pHBApr-1-neo vector, which contains the promoter and the first intron from human β-actin gene and SV-40 polyadenylation signal. (B) Expression of HB-GAM in transgenic mice. (a) Western blot analysis of different neonatal tissues (postnatal day 1) from a transgene-positive mouse (lanes noted by +) and its transgene-negative littermate (noted by −). Lane b, brain; lane h, heart; lane m, femoral muscle; lane l, liver; lane k, kidney. Note the intense transgene expression in heart and muscle. (b) Transgene expression in the periosteum in comparison to the heart of 1-yr-old mice. Western blot analysis of tissues from two different transgenic lines (noted by +) and their nontransgenic littermates (noted by −). HB-GAM expression in the transgenic periosteum (p) is even higher than that in the heart (h). Lane c, control recombinant HB-GAM protein (100 ng). (C) Gross appearance of bones form a 1-yr-old transgene-positive mouse (left in all panels) and those from its transgene-negative littermate (right). (a) Femora of transgene-positive (Tg) and transgene-negative (Non-tg) mice. Note the ivory-like solid appearance of the transgene-positive femur, whereas the transgene-negative femur displays a brownish surface because of the bone marrow structures that are seen through the thin cortical bone (arrow). (b) Ulnae. Brownish marrow structure is also seen in the transgene-negative ulna (arrow). (c) Scapulae (1) and humeri (2). The ivory-like appearance is particularly prominent in the scapular spinus of the transgene-positive mouse (arrow). Note the transparency of scapular plane in the transgene-negative bone (arrowhead).

Mentions: 12 transgenic founders were produced using the HB-GAM cDNA under a β-actin promoter (Fig. 7 A). Southern blotting and PCR were used to verify integration of the construct to genomic DNA of the founders and the first generation offspring. PCR analysis indicated that the transgene was steadily transmitted to the next generations in a Mendelian fashion (data not shown).


Osteoblast recruitment and bone formation enhanced by cell matrix-associated heparin-binding growth-associated molecule (HB-GAM).

Imai S, Kaksonen M, Raulo E, Kinnunen T, Fages C, Meng X, Lakso M, Rauvala H - J. Cell Biol. (1998)

(A) Schematic diagram of the gene construct  used to produce transgenic  mice. HB-GAM cDNA was  cloned in the pHBApr-1-neo  vector, which contains the  promoter and the first intron  from human β-actin gene and  SV-40 polyadenylation signal.  (B) Expression of HB-GAM  in transgenic mice. (a) Western blot analysis of different  neonatal tissues (postnatal  day 1) from a transgene-positive mouse (lanes noted by  +) and its transgene-negative  littermate (noted by −). Lane  b, brain; lane h, heart; lane m,  femoral muscle; lane l, liver;  lane k, kidney. Note the intense transgene expression  in heart and muscle. (b)  Transgene expression in the  periosteum in comparison to  the heart of 1-yr-old mice.  Western blot analysis of tissues from two different transgenic lines (noted by +) and  their nontransgenic littermates (noted by −). HB-GAM expression in the  transgenic periosteum (p) is  even higher than that in the  heart (h). Lane c, control recombinant HB-GAM protein  (100 ng). (C) Gross appearance of bones form a 1-yr-old  transgene-positive mouse  (left in all panels) and those from its transgene-negative littermate (right). (a) Femora of transgene-positive (Tg) and transgene-negative  (Non-tg) mice. Note the ivory-like solid appearance of the transgene-positive femur, whereas the transgene-negative femur displays a  brownish surface because of the bone marrow structures that are seen through the thin cortical bone (arrow). (b) Ulnae. Brownish marrow structure is also seen in the transgene-negative ulna (arrow). (c) Scapulae (1) and humeri (2). The ivory-like appearance is particularly prominent in the scapular spinus of the transgene-positive mouse (arrow). Note the transparency of scapular plane in the transgene-negative bone (arrowhead).
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Related In: Results  -  Collection

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Figure 7: (A) Schematic diagram of the gene construct used to produce transgenic mice. HB-GAM cDNA was cloned in the pHBApr-1-neo vector, which contains the promoter and the first intron from human β-actin gene and SV-40 polyadenylation signal. (B) Expression of HB-GAM in transgenic mice. (a) Western blot analysis of different neonatal tissues (postnatal day 1) from a transgene-positive mouse (lanes noted by +) and its transgene-negative littermate (noted by −). Lane b, brain; lane h, heart; lane m, femoral muscle; lane l, liver; lane k, kidney. Note the intense transgene expression in heart and muscle. (b) Transgene expression in the periosteum in comparison to the heart of 1-yr-old mice. Western blot analysis of tissues from two different transgenic lines (noted by +) and their nontransgenic littermates (noted by −). HB-GAM expression in the transgenic periosteum (p) is even higher than that in the heart (h). Lane c, control recombinant HB-GAM protein (100 ng). (C) Gross appearance of bones form a 1-yr-old transgene-positive mouse (left in all panels) and those from its transgene-negative littermate (right). (a) Femora of transgene-positive (Tg) and transgene-negative (Non-tg) mice. Note the ivory-like solid appearance of the transgene-positive femur, whereas the transgene-negative femur displays a brownish surface because of the bone marrow structures that are seen through the thin cortical bone (arrow). (b) Ulnae. Brownish marrow structure is also seen in the transgene-negative ulna (arrow). (c) Scapulae (1) and humeri (2). The ivory-like appearance is particularly prominent in the scapular spinus of the transgene-positive mouse (arrow). Note the transparency of scapular plane in the transgene-negative bone (arrowhead).
Mentions: 12 transgenic founders were produced using the HB-GAM cDNA under a β-actin promoter (Fig. 7 A). Southern blotting and PCR were used to verify integration of the construct to genomic DNA of the founders and the first generation offspring. PCR analysis indicated that the transgene was steadily transmitted to the next generations in a Mendelian fashion (data not shown).

Bottom Line: We show here that heparin-binding growth-associated molecule (HB-GAM), an extracellular matrix-associated protein that enhances migratory responses in neurons, is prominently expressed in the cell matrices that act as target substrates for bone formation.The HB-GAM transgenic mice develop a phenotype characterized by an increased bone thickness.HB-GAM may thus play an important role in bone formation, probably by mediating recruitment and attachment of osteoblasts/osteoblast precursors to the appropriate substrates for deposition of new bone.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, Shiga University of Medical Science, Shiga-ken, 520-2192, Japan. simai@belle.shiga-med.ac.jp

ABSTRACT
Bone has an enormous capacity for growth, regeneration, and remodeling. This capacity is largely due to induction of osteoblasts that are recruited to the site of bone formation. The recruitment of osteoblasts has not been fully elucidated, though the immediate environment of the cells is likely to play a role via cell- matrix interactions. We show here that heparin-binding growth-associated molecule (HB-GAM), an extracellular matrix-associated protein that enhances migratory responses in neurons, is prominently expressed in the cell matrices that act as target substrates for bone formation. Intriguingly, N-syndecan, which acts as a receptor for HB-GAM, is expressed by osteoblasts/osteoblast precursors, whose ultrastructural phenotypes suggest active cell motility. The hypothesis that HB-GAM/N-syndecan interaction mediates osteoblast recruitment, as inferred from developmental studies, was tested using osteoblast-type cells that express N-syndecan abundantly. These cells migrate rapidly to HB-GAM in a haptotactic transfilter assay and in a migration assay where HB-GAM patterns were created on culture wells. The mechanism of migration is similar to that previously described for the HB-GAM-induced migratory response of neurons. Our hypothesis that HB-GAM/N-syndecan interaction participates in regulation of osteoblast recruitment was tested using two different in vivo models: an adjuvant-induced arthritic model and a transgenic model. In the adjuvant-induced injury model, the expression of HB-GAM and of N-syndecan is strongly upregulated in the periosteum accompanying the regenerative response of bone. In the transgenic model, the HB-GAM expression is maintained in mesenchymal tissues with the highest expression in the periosteum. The HB-GAM transgenic mice develop a phenotype characterized by an increased bone thickness. HB-GAM may thus play an important role in bone formation, probably by mediating recruitment and attachment of osteoblasts/osteoblast precursors to the appropriate substrates for deposition of new bone.

Show MeSH
Related in: MedlinePlus