Journal of Zhejiang University SCIENCE
(ISSN 1009-3095, Monthly)

2005   Vol. 6B   No. 7   p.699-704

Effects of IGF-II on promoting proliferation and regulating nitric oxide synthase gene expression in mouse osteoblast-like cell

SUN Wei-lian¹, CHEN Li-li¹, YAN Jie^†‡2, YU Zhong-sheng³

(¹Department of Stomatology, Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310009, China)
(²Department of Medical Microbiology and Parasitology, School of Medicine, Zhejiang University, Hangzhou 310006, China)
(³Affiliated Children’s Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China)
^†E-mail: yanchen@mail.hz.zj.cn
Received Dec. 1, 2004; revision accepted Jan. 14, 2005

Abstract: Objective: To investigate the effects of insulin-like growth factor II (IGF-II) on promoting cell proliferation, regulating levels of cellular nitric oxide (NO) and mRNA transcriptions of inducible nitric oxide synthase (iNOS) and endothelial NOS (eNOS) in mouse osteoblast-like cells. Methods: Mouse osteoblastic cell line MC3T3-E1 was selected as the effective cell of IGF-II. After the cells were treated with IGF-II at different concentrations for different time duration, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay was used to examine cell proliferation, and nitrate reductase method was applied to detect NO concentrations in cell culture supernatants and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was employed to determine transcription levels of cellular iNOS and eNOS mRNAs. Results: After the MC3T3-E1 cells were treated with IGF-II at concentration of 1 ng/ml for 72 h, 10 and 100 ng/ml for 24, 48 and 72 h respectively, all the MTT values increased (P<0.05 or P<0.01) with obvious dosage-time dependent pattern. NO levels of the MC3T3-E1 cells treated with 100 ng/ml IGF-II for 48 h, and with 1, 10 and 100 ng/ml IGF-II for 72 h were remarkably lower than that of the normal control, respectively (P<0.05 or P<0.01). After the cells were treated with 100 ng/ml IGF-II for 48 h cellular iNOS mRNA levels were significantly decreased (P<0.01). But the levels of eNOS mRNA in the cells treated with each of the used IGF-II dosages for different time duration did not show any differences compared with the normal control (P>0.05). Conclusion: IGF-II at different concentrations could promote proliferation of mouse MC3T3-E1 cell. This cell proliferation promotion was associated with the low NO levels maintained by IGF-II. Higher concentration of IGF-II could down-regulate iNOS gene expression at the level of transcription but not affect transcription of eNOS mRNA, which might be one of the mechanisms for IGF-II maintenance of the low NO levels in MC3T3-E1 cells.

Key words: Insulin-like growth factor II, Osteoblast, Proliferation, Nitric oxide synthase, Nitric oxide, Regulation
doi:10.1631/jzus.2005.B0699             CLC number: R781.4

^‡ Corresponding Author