Enamel matrix derivative protein stimulated wound healing via phosphoinositide 3-kinase.
BACKGROUND
Enamel matrix derivative (EMD) protein has been clinically used for periodontal regeneration, but the molecular mechanisms are not clear. Previous studies suggested that the activation of phosphoinositide 3-kinase (PI 3-kinase) plays a key role in facilitating cell migration. Given that the migration of osteoblasts is one of the key steps in the wound-healing processes, we hypothesized that EMD protein would stimulate osteoblast migration by activating PI 3-kinase. In this study, we tested this hypothesis using MG-63 cells as model systems to evaluate mechanisms of migration by stimulation with EMD protein.


METHODS
Confluent MG-63 cells were mechanically scratched using a sterilized 1-mm pipette tip that removed the cells within a circular area. The wells were incubated for 24 hours in various stimulation conditions (25, 50, or 100 microg/ml EMD protein) with or without the PI 3-kinase inhibitor wortmannin (1, 10, and 100 nM) or LY294002 (1, 10, and 100 microM). Migrated cells in the wound section were counted by randomly selecting three areas from one well. The activation of PI 3-kinase by EMD protein was evaluated by the phosphorylation of Akt using Western blot analysis.


RESULTS
Although EMD protein did not affect proliferation, it enhanced migration into wounds on MG-63 cells. We showed that EMD protein enhanced the phosphorylation of Akt in a dose-dependent manner. We demonstrated that the PI 3-kinase inhibitors wortmannin and LY294002 blocked migration into wounds and the phosphorylation of Akt enhanced by EMD protein in MG-63 cells.


CONCLUSIONS
These results demonstrated that the activation of PI 3-kinase plays a key role in the EMD protein-stimulated migration of osteoblasts.
Department of Biochemistry, Osaka Dental University, Osaka, Japan. goda@cc.osaka-dent.ac.jp
Seiji
S
Goda
Yuki
Y
Kaneshita
Hiroshi
H
Inoue
Eisuke
E
Domae
Takashi
T
Ikeo
Joji
J
Iida
Naochika
N
Domae
D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002452:Cell Count; D002460:Cell Line; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D003746:Dental Enamel Proteins / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D009025:Morpholines / Q000494:pharmacology; D010006:Osteoblasts / Q000187:drug effects* / Q000201:enzymology; D019869:Phosphatidylinositol 3-Kinases / Q000187:drug effects*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000187:drug effects; D000077191:Wortmannin; D014945:Wound Healing / Q000187:drug effects
D000730:Androstadienes; D002867:Chromones; D003746:Dental Enamel Proteins; D004791:Enzyme Inhibitors; D009025:Morpholines; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt; D000077191:Wortmannin; C109069:enamel matrix proteins; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one