Limits...
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.

Show MeSH

Related in: MedlinePlus

(A) Transfilter haptotactic assay. (a) The lower  surface of the Transwell® filter (polycarbonate plastic)  was coated with HB-GAM,  and the cells that migrated  from the upper chamber to  the lower chamber were studied under microscopy. (b) After the migration, the number  of cells that migrated to the  lower surface was calculated.  (c) The osteoblast-type cells  (rat osteoblast-type cells  UMR-106, and human osteoblast-type cells, Saos-2, U-2  and KHOS/NP) migrated  through the pores. Less than  1% of the plated osteoblast-type cells migrated to the control matrices (nonspecific rat  IgG, 0.92%; bovine serum albumin, 0.89%). All the osteoblast-like cells migrated in a  dose-dependent manner, but  nonosteoblastic cells, 3T3 fibroblasts and N18 neuroblastoma cells, did not respond to  HB-GAM. (B) Northern blot  analysis of the rat osteoblastic  cell line, UMR-106. N-syndecan mRNA is seen at a similar  or even higher level as compared with that of the perinatal rat brain. No expression is  seen in the control cells (N18  and 3T3 cells). (C) HB-GAM– binding fractions isolated  from UMR-106 cells and rat  perinatal brain, and Western  blot analysis of the fractions.  Lane 1, HB-GAM–binding  fraction derived from the  brain; lane 2, the brain-derived  fraction treated with nitrous  acid; lane 3, the major HB-GAM–binding fraction from  UMR-106 eluted at 0.5 M  NaCl in the salt gradient; lane  4, the UMR-106–derived fraction treated with nitrous acid.  The HB-GAM–binding fractions isolated from UMR-106 cells have a molecular mass similar to those derived from the rat perinatal  brain. Lanes 5 and 6, Western blot using affinity-purified antibodies that bind to NH2-terminal peptide of N-syndecan. The samples in  lanes 3 and 4 are identified as N-syndecan. (D) Immunocytochemistry of N-syndecan in the UMR-106 cells. (a) N-syndecan of UMR-106 cells is immunostained (arrowheads). (b) Trypsin treatment clearly abolishes the immunostaining. (E) Inhibition of HB-GAM– induced recruitment of UMR-106 cells. (a) Recruitment of UMR-106 cells using 10 μg/ml HB-GAM is dose-dependently inhibited by  adding soluble HB-GAM or N-syndecan to the cell suspension. (b) Addition of herbimycin A, a general inhibitor for tyrosine kinases,  and PP1, a recently defined specific inhibitor for src-type tyrosine kinases, clearly interferes the cell recruitment induced by HB-GAM.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2132954&req=5

Figure 3: (A) Transfilter haptotactic assay. (a) The lower surface of the Transwell® filter (polycarbonate plastic) was coated with HB-GAM, and the cells that migrated from the upper chamber to the lower chamber were studied under microscopy. (b) After the migration, the number of cells that migrated to the lower surface was calculated. (c) The osteoblast-type cells (rat osteoblast-type cells UMR-106, and human osteoblast-type cells, Saos-2, U-2 and KHOS/NP) migrated through the pores. Less than 1% of the plated osteoblast-type cells migrated to the control matrices (nonspecific rat IgG, 0.92%; bovine serum albumin, 0.89%). All the osteoblast-like cells migrated in a dose-dependent manner, but nonosteoblastic cells, 3T3 fibroblasts and N18 neuroblastoma cells, did not respond to HB-GAM. (B) Northern blot analysis of the rat osteoblastic cell line, UMR-106. N-syndecan mRNA is seen at a similar or even higher level as compared with that of the perinatal rat brain. No expression is seen in the control cells (N18 and 3T3 cells). (C) HB-GAM– binding fractions isolated from UMR-106 cells and rat perinatal brain, and Western blot analysis of the fractions. Lane 1, HB-GAM–binding fraction derived from the brain; lane 2, the brain-derived fraction treated with nitrous acid; lane 3, the major HB-GAM–binding fraction from UMR-106 eluted at 0.5 M NaCl in the salt gradient; lane 4, the UMR-106–derived fraction treated with nitrous acid. The HB-GAM–binding fractions isolated from UMR-106 cells have a molecular mass similar to those derived from the rat perinatal brain. Lanes 5 and 6, Western blot using affinity-purified antibodies that bind to NH2-terminal peptide of N-syndecan. The samples in lanes 3 and 4 are identified as N-syndecan. (D) Immunocytochemistry of N-syndecan in the UMR-106 cells. (a) N-syndecan of UMR-106 cells is immunostained (arrowheads). (b) Trypsin treatment clearly abolishes the immunostaining. (E) Inhibition of HB-GAM– induced recruitment of UMR-106 cells. (a) Recruitment of UMR-106 cells using 10 μg/ml HB-GAM is dose-dependently inhibited by adding soluble HB-GAM or N-syndecan to the cell suspension. (b) Addition of herbimycin A, a general inhibitor for tyrosine kinases, and PP1, a recently defined specific inhibitor for src-type tyrosine kinases, clearly interferes the cell recruitment induced by HB-GAM.

Mentions: To test the hypothetical osteoblast recruitment induced by HB-GAM, we first performed a haptotactic assay using several osteoblast-type cells. All of the studied osteoblast-type cells rapidly migrated to HB-GAM in a dose-dependent manner, whereas the control cells, N18 and 3T3 cells, did not respond to HB-GAM (Fig. 3 A). Thus, the migration-enhancing effect of HB-GAM appears to be a general finding in the case of osteoblast-type cells (osteogenic sarcoma-derived cell lines).


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) Transfilter haptotactic assay. (a) The lower  surface of the Transwell® filter (polycarbonate plastic)  was coated with HB-GAM,  and the cells that migrated  from the upper chamber to  the lower chamber were studied under microscopy. (b) After the migration, the number  of cells that migrated to the  lower surface was calculated.  (c) The osteoblast-type cells  (rat osteoblast-type cells  UMR-106, and human osteoblast-type cells, Saos-2, U-2  and KHOS/NP) migrated  through the pores. Less than  1% of the plated osteoblast-type cells migrated to the control matrices (nonspecific rat  IgG, 0.92%; bovine serum albumin, 0.89%). All the osteoblast-like cells migrated in a  dose-dependent manner, but  nonosteoblastic cells, 3T3 fibroblasts and N18 neuroblastoma cells, did not respond to  HB-GAM. (B) Northern blot  analysis of the rat osteoblastic  cell line, UMR-106. N-syndecan mRNA is seen at a similar  or even higher level as compared with that of the perinatal rat brain. No expression is  seen in the control cells (N18  and 3T3 cells). (C) HB-GAM– binding fractions isolated  from UMR-106 cells and rat  perinatal brain, and Western  blot analysis of the fractions.  Lane 1, HB-GAM–binding  fraction derived from the  brain; lane 2, the brain-derived  fraction treated with nitrous  acid; lane 3, the major HB-GAM–binding fraction from  UMR-106 eluted at 0.5 M  NaCl in the salt gradient; lane  4, the UMR-106–derived fraction treated with nitrous acid.  The HB-GAM–binding fractions isolated from UMR-106 cells have a molecular mass similar to those derived from the rat perinatal  brain. Lanes 5 and 6, Western blot using affinity-purified antibodies that bind to NH2-terminal peptide of N-syndecan. The samples in  lanes 3 and 4 are identified as N-syndecan. (D) Immunocytochemistry of N-syndecan in the UMR-106 cells. (a) N-syndecan of UMR-106 cells is immunostained (arrowheads). (b) Trypsin treatment clearly abolishes the immunostaining. (E) Inhibition of HB-GAM– induced recruitment of UMR-106 cells. (a) Recruitment of UMR-106 cells using 10 μg/ml HB-GAM is dose-dependently inhibited by  adding soluble HB-GAM or N-syndecan to the cell suspension. (b) Addition of herbimycin A, a general inhibitor for tyrosine kinases,  and PP1, a recently defined specific inhibitor for src-type tyrosine kinases, clearly interferes the cell recruitment induced by HB-GAM.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132954&req=5

Figure 3: (A) Transfilter haptotactic assay. (a) The lower surface of the Transwell® filter (polycarbonate plastic) was coated with HB-GAM, and the cells that migrated from the upper chamber to the lower chamber were studied under microscopy. (b) After the migration, the number of cells that migrated to the lower surface was calculated. (c) The osteoblast-type cells (rat osteoblast-type cells UMR-106, and human osteoblast-type cells, Saos-2, U-2 and KHOS/NP) migrated through the pores. Less than 1% of the plated osteoblast-type cells migrated to the control matrices (nonspecific rat IgG, 0.92%; bovine serum albumin, 0.89%). All the osteoblast-like cells migrated in a dose-dependent manner, but nonosteoblastic cells, 3T3 fibroblasts and N18 neuroblastoma cells, did not respond to HB-GAM. (B) Northern blot analysis of the rat osteoblastic cell line, UMR-106. N-syndecan mRNA is seen at a similar or even higher level as compared with that of the perinatal rat brain. No expression is seen in the control cells (N18 and 3T3 cells). (C) HB-GAM– binding fractions isolated from UMR-106 cells and rat perinatal brain, and Western blot analysis of the fractions. Lane 1, HB-GAM–binding fraction derived from the brain; lane 2, the brain-derived fraction treated with nitrous acid; lane 3, the major HB-GAM–binding fraction from UMR-106 eluted at 0.5 M NaCl in the salt gradient; lane 4, the UMR-106–derived fraction treated with nitrous acid. The HB-GAM–binding fractions isolated from UMR-106 cells have a molecular mass similar to those derived from the rat perinatal brain. Lanes 5 and 6, Western blot using affinity-purified antibodies that bind to NH2-terminal peptide of N-syndecan. The samples in lanes 3 and 4 are identified as N-syndecan. (D) Immunocytochemistry of N-syndecan in the UMR-106 cells. (a) N-syndecan of UMR-106 cells is immunostained (arrowheads). (b) Trypsin treatment clearly abolishes the immunostaining. (E) Inhibition of HB-GAM– induced recruitment of UMR-106 cells. (a) Recruitment of UMR-106 cells using 10 μg/ml HB-GAM is dose-dependently inhibited by adding soluble HB-GAM or N-syndecan to the cell suspension. (b) Addition of herbimycin A, a general inhibitor for tyrosine kinases, and PP1, a recently defined specific inhibitor for src-type tyrosine kinases, clearly interferes the cell recruitment induced by HB-GAM.
Mentions: To test the hypothetical osteoblast recruitment induced by HB-GAM, we first performed a haptotactic assay using several osteoblast-type cells. All of the studied osteoblast-type cells rapidly migrated to HB-GAM in a dose-dependent manner, whereas the control cells, N18 and 3T3 cells, did not respond to HB-GAM (Fig. 3 A). Thus, the migration-enhancing effect of HB-GAM appears to be a general finding in the case of osteoblast-type cells (osteogenic sarcoma-derived cell lines).

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