J Korean Orthop Assoc.  2003 Apr;38(2):133-141.

Effect of Orthovanadate on Collagen and Fibronectin Synthesis, and Alkaline Phosphatase Activity in MC3E3-E1 Osteoblast cells

Affiliations
  • 1Department of Orthopedic Surgery, School of Medicine, Kyungpook National University, Daegu, Korea. syukim@knu.ac.kr
  • 2Department of Biochemistry, School of Medicine, Kyungpook National University, Daegu, Korea.
  • 3Department of Orthopedic Surgery, School of Medicine, Yeungnam National University, Daegu, Korea.

Abstract

PURPOSE
The purpose: of this study was to know the effect of inhibition of protein dephosphorylation on the synthesis of collagen and fibronectin (FN), alkaline phosphatase (ALP) activity, and the formation of bone nodule in MC3T3-E1 osteoblasts using orthovanadate (OVA) which is a potent protein tyrosine phosphatase (PTPases) inhibitor. MATERIALS AND METHODS: The synthesis of collagen, noncollagenous protein (NCP), and percent collagen in MC3T3-E1osteoblasts with or without OVA treatment according to concentration and time sequence was determined by incorporation of [3 H]-proline, synthesis of FN by [35 S] methionine incorproated immunoprecipitation after treatment with 100 M OVA for 24 hours, mRNA expression of collagen and FN by Northern blotting, activity of ALP by spectrophotometric method, and formation of bone nodule by staining method. RESULTS: OVA increased collagen and NCP synthesis concentration dependently, until 12 hours in short-time culture, and time dependently through the differentiation until 29 days, however, there was no significant effect on the percent collagen production. OVA increased percent collagen synthesis significantly at 6 hours, and decreased in a long time culture. Total FN synthesis and FN synthesis in cell layer were increased by OVA, however, FN synthesis in medium was not changed. OVA decreased collagen mRNA level dose-dependently and increased the steady-state level of FN mRNA. OVA inhibited activity of ALP in both short and long-time culture. OVA inhibited bone nodule formation in MC3T3-E1 osteoblasts. CONCLUSION: These results indicate that the inhibition of PTPase by OVA increased the synthesis of collagen, FN, and decreased ALP activity and it resulted in the inhibition of bone formation in MC3T3-E1 osteoblast cells.

Keyword

Protein phosphorylation; Orthovanadate; Bone formation

MeSH Terms

Alkaline Phosphatase*
Blotting, Northern
Collagen*
Fibronectins*
Immunoprecipitation
Methionine
Osteoblasts*
Osteogenesis
Ovum
Protein Tyrosine Phosphatases
RNA, Messenger
Vanadates*
Alkaline Phosphatase
Collagen
Fibronectins
Methionine
Protein Tyrosine Phosphatases
RNA, Messenger
Vanadates
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