Mechanism involved in enhancement of osteoblast differentiation by hyaluronic acid.
OBJECTIVE
Bone morphogenetic protein-2 (BMP-2) is expected to be utilized to fill bone defects and promote healing of fractures. However, it is unable to generate an adequate clinical response for use in bone regeneration. Recently, it was reported that glycosaminoglycans, including heparin, heparan sulfate, keratan sulfate, dermatan sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, and hyaluronic acid (HA), regulate BMP-2 activity, though the mechanism by which HA regulates osteogenic activities has not been fully elucidated. The aim of this study was to investigate the effects of HA on osteoblast differentiation induced by BMP-2.


METHODS
Monolayer cultures of osteoblastic lineage MG63 cells were incubated with BMP-2 and HA for various time periods. To determine osteoblastic differentiation, alkaline phosphatase (ALP) activity in the cell lysates was quantified. Phosphorylation of Smad 1/5/8, p38, and ERK proteins was determined by Western blot analysis. To elucidate the nuclear translocation of phosphorylated Smad 1/5/8, stimulated cells were subjected to immunofluorescence microscopy. To further elucidate the role of HA in enhancement of BMP-2-induced Smad signaling, mRNA expressions of the BMP-2 receptor antagonists noggin and follistatin were detected using real-time RT-PCR.


RESULTS
BMP-2-induced ALP activation, Smad 1/5/8 phosphorylation, and nuclear translocation were up-regulated when MG63 cells were cultured with both BMP-2 and HA. Western blot analysis revealed that phosphorylation of ERK protein was diminished by HA. Furthermore, the mRNA expressions of noggin and follistatin induced by BMP-2 were preferentially blocked by HA.


CONCLUSIONS
These results indicate that HA enhanced BMP-2 induces osteoblastic differentiation in MG63 cells via down-regulation of BMP-2 antagonists and ERK phosphorylation.
Division of Maxillofacial Diagnostic and Surgical Science, Department of Oral and Maxillofacial Surgery, Kyushu Dental College, Kitakyushu 803-8580, Japan.
Michinao
M
Kawano
Wataru
W
Ariyoshi
Kenjiro
K
Iwanaga
Toshinori
T
Okinaga
Manabu
M
Habu
Izumi
I
Yoshioka
Kazuhiro
K
Tominaga
Tatsuji
T
Nishihara
D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D055396:Bone Morphogenetic Protein 2 / Q000494:pharmacology; D002352:Carrier Proteins / Q000096:biosynthesis; D002454:Cell Differentiation; D002460:Cell Line; D015536:Down-Regulation / Q000187:drug effects; D038681:Follistatin / Q000096:biosynthesis; D006801:Humans; D006820:Hyaluronic Acid / Q000378:metabolism / Q000494:pharmacology*; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D010012:Osteogenesis / Q000187:drug effects*; D010766:Phosphorylation; D012333:RNA, Messenger / Q000096:biosynthesis; D051898:Smad1 Protein / Q000378:metabolism; D051902:Smad5 Protein / Q000378:metabolism; D051904:Smad8 Protein / Q000378:metabolism; D000099176:Noggin Protein
D055396:Bone Morphogenetic Protein 2; D002352:Carrier Proteins; D038681:Follistatin; D006820:Hyaluronic Acid; D020929:Mitogen-Activated Protein Kinase Kinases; D012333:RNA, Messenger; D051898:Smad1 Protein; D051902:Smad5 Protein; D051904:Smad8 Protein; D000099176:Noggin Protein