J Korean Orthop Assoc.  2008 Dec;43(6):775-782.

Effect of Adrenomedullin on Proliferation and Mitogen-Activated Protein Kinase Activity in Osteosarcoma Cell Lines (MG63, U2OS)

• Kim JR
• Choi BW
• Park JH
• Kim KH

Abstract

PURPOSE: The aims investigated how how human osteosarcoma cell proliferation and the MAP kinases cascade are regulated, in the MG63 and U2OS human osteosarcoma cell lines after stimulating them with adrenomedullin (AM) with particular focus on extracellular signal-regulated kinase 1/2 (ERK 1 and 2) activation.
MATERIALS AND METHODS
A cell proliferation assay was used to examined the effects of AM on the osteosarcoma cell lines (MG63 and U2OS). The effects of AM on ERK1/2 were examed by Western blot analysis. The roles of ERK 1/2 in the AM-induced proliferative signaling pathways in the two cell types were examed using PD98059, a selective inhibitor of the mitogen activated protein-ERK kinase (MEK) pathway.
RESULTS
The addition of AM to the medium containing the osteosarcoma MG63 and U2OS cells induced proliferation in a dose-dependent manner. AM strongly activated ERK 1/2 mediated cell proliferation signaling, which was prevented using PD98059.
CONCLUSION
These results suggest that AM plays an important role in the proliferation of human osteosarcoma MG63 and U2OS cells, and ERK kinase pathway plays a signal transduction role in AM treated human osteosarcoma MG63 and U2OS cell lines.

Keyword

Adrenomedullin; Mitogen-activated protein kinase; Osteosarcoma cell lines; MG63; U2OS

MeSH Terms

Adrenomedullin
Blotting, Western
Cell Line
Cell Proliferation
Flavonoids
Humans
Osteosarcoma
Phosphotransferases
Protein Kinases
Signal Transduction
Adrenomedullin
Flavonoids
Phosphotransferases
Protein Kinases

Figure

• 

  Fig. 1 Cell numbers/well of the two osteosarcoma cell lines examined (MG63, U2OS) treated with adrenomedullin (AM) at various doses for 24 h. The data is expressed as the as means±SE of the treatment versus control. The experiments were performed in triplicates. *Significantly different from control.

• 

  Fig. 2 Time course of the effect of AM (50 nM) on the cell number/well for the MG63 and U2OS cell lines. The data is reported as the means means±SE. Significantly different from control: *p<0.01 and †p<0.03.

• 

  Fig. 3 Effects of AM on proliferation. The MG63 and U2OS cells grown in 96-well plates, were treated for 48 h with different concentrations of AM. The mitochondrial activities of the living cells were determined using a WST-1 assay and compared with those of the untreated controls; results shown are the mean±SE (n=4). The absorbance at 405 nm of the control cultures of MG63 cells and U2OS cells were 0.709±0.058 and 0.853±0.082, respectively. *(p<0.05) and †(p<0.01) indicate significant differences from the untreated controls cultures.

• 

  Fig. 4 Time course of the effects of AM (50 nM) on the cell proliferations of the MG63 and U2OS cells, as determined by the WST-1 assays. The data is reported as the mean±SE. Significantly different from the untreated control: *p<0.01 and †p<0.03.

• 

  Fig. 5 Inhibitory effect of PD98059 on AM-induced proliferation in the MG63 and U2OS cell lines. The serum-deprived MG63 and U2OS osteosarcoma cells were incubated in serum-free MEM with AM at 100 nM, or with a combination of AM (100) nM) and PD98059 (10 uM) for 3 days. The cells were counted using a hemocytometer. The cell proliferation is expressed as the cell numbers: *p<0.05.

• 

  Fig. 6 mmunoblots of ERK in the MG63 and U2OS cells. After SDS-PAGE and immunoblotting, the MG63 and U2OS cells were extracted and probed with the ERK antibodies. The results showed a strongly immunoreactive protein with a molecular weight of about 42 kDa. Treatment with 100 nM AM caused a two-fold increase in the phosphor-MAP kinase levels without affecting the total MAPK protein level.

• 

  Fig. 7 The effect of the ERK inhibitor, PD98059 on the effect of AM on MG63 and U2OS cells. The ERK MAP kinase in the osteosarcoma cell lines was inhibited by PD98059.

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