Attachment of human periodontal ligament cells to enamel matrix-derived protein is mediated via interaction between BSP-like molecules and integrin alpha(v)beta3.
BACKGROUND
Although enamel matrix-derived protein (EMD) can stimulate attachment of human periodontal ligament (HPDL) cells to the root surface, the biological mechanism of this phenomenon is unclear. The purpose of this study was to determine which molecules in EMD are involved in the attachment of HPDL cells, and which types of integrins on the cell surface mediate the interaction between the cells and EMD.


METHODS
HPDL explants were obtained from tooth surfaces extracted from 4 individuals, and cells taken from the individual explants were separately harvested and subcultured through as many as 5 passages. Cells were incubated on EMD-coated culture plates with and without neutral antibodies for integrins or RGD-sequence blocking peptides and stained with toluidine blue. Proteins in EMD that were able to induce cell attachment were identified by incubating sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) replicas with HPDL cells; the cell-binding regions were detected by staining the cells with toluidine blue. Characteristics of the cell-binding proteins in the EMD were identified by Western blot analysis.


RESULTS
It was shown that anti-alpha(v)beta3 antibody and GRGDSP peptide markedly reduced attachment of HPDL cells to EMD. When the cells were incubated with SDS-PAGE replicas, distinct cell attachment was observed at a molecular mass of approximately 55 kDa. The cell-binding ability of this protein was completely blocked by treatment with anti-alpha(v)beta3 antibody or GRGDSP peptide. In the Western blot analysis, the 55 kDa protein was recognized by anti-bone sialoprotein (BSP) antibody as a single band.


CONCLUSIONS
Our study provides the first evidence that the attachment of HPDL cells to EMD can be mediated by interaction between a BSP-like molecule and integrin alpha(v)beta3 on the cell surface.
Suzuki
N
N
Department of Biochemistry, Nihon University School of Dentistry, Tokyo, Japan. suzuki-n@dent.nihon-u.ac.jp
Ohyama
M
M
Maeno
M
M
Ito
K
K
Otsuka
K
K
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D002448:Cell Adhesion; D018929:Cell Culture Techniques; D004396:Coloring Agents; D003746:Dental Enamel Proteins / Q000302:isolation & purification / Q000502:physiology*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D058574:Integrin-Binding Sialoprotein; D008970:Molecular Weight; D009842:Oligopeptides / Q000037:antagonists & inhibitors; D010513:Periodontal Ligament / Q000166:cytology*; D011971:Receptors, Immunologic / Q000037:antagonists & inhibitors; D019030:Receptors, Vitronectin / Q000302:isolation & purification / Q000502:physiology*; D012795:Sialoglycoproteins / Q000302:isolation & purification / Q000502:physiology*; D013223:Statistics as Topic; D014048:Tolonium Chloride
D000911:Antibodies, Monoclonal; D004396:Coloring Agents; D003746:Dental Enamel Proteins; C546871:IBSP protein, human; D058574:Integrin-Binding Sialoprotein; D009842:Oligopeptides; D011971:Receptors, Immunologic; D019030:Receptors, Vitronectin; D012795:Sialoglycoproteins; D014048:Tolonium Chloride; C047981:arginyl-glycyl-aspartic acid; C058161:glycyl-arginyl-glycyl-aspartyl-seryl-proline