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

Effects of IGF-II on promoting proliferation and regulating nitric oxide synthase gene expression in mouse osteoblast-like cell 1 first-author 1 2 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:yanchen@mail.hz.zj.cn †E-mail:yanchen@mail.hz.zj.cn yanchen@mail.hz.zj.cn 3 Department of Stomatology, Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310009, China Department of Stomatology, Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310009, China Department of Medical Microbiology and Parasitology, School of Medicine, Zhejiang University, Hangzhou 310006, China Department of Medical Microbiology and Parasitology, School of Medicine, Zhejiang University, Hangzhou 310006, China Affiliated Children’s Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China Affiliated Children’s Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China Objective: 2 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: 2 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: 2 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: 2 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. Insulin-like growth factor II Osteoblast Proliferation Nitric oxide synthase Nitric oxide Regulation INTRODUCTION 1 It is well known that the osteoblast is the major functional cell in bone formation. As an important signal molecule participating in cell proliferation and differentiation, nitric oxide (NO) is closely involved in bone remodelling in vivo and in vitro (van’t Hof and Ralston, 2001 2001 ; Ralston, 1997 1997 ; Otsuka et al., 1998 1998 ). Insulin-like growth factors (IGFs), recently identified polypeptides secreted by multiple types of mammal cells, have various bioactivities such as promoting cell proliferation and differentiation (Fournier et al., 1993 1993 ; Langdahl et al., 1998 1998 ) and can be divided into two types: IGF-I and IGF-II. Results of previous studies revealed that IGF-II has bioactivities promoting proliferation and differentiation of human osteoblasts and human osteosarcoma cells (Fournier et al., 1993 1993 ; Langdahl et al., 1998 1998 ) and regulating cellular NO levels (Kaliman et al., 1999 1999 ; Schini et al., 1994 1994 ; Hill et al., 1999 1999 ). Especially, this NO level regulating function of IGF-II exhibits certain cell specificity or distinct NO regulating effects for different cells and even the contrary (Kaliman et al., 1999 1999 ; Schini et al., 1994 1994 ; Hill et al., 1999 1999 ). But the effects of IGF-II on proliferation and cellular NO level in osteoblasts and its possible mechanism has so far remained unknown, so it is reasonable for us to speculate that IGF-II may up-or down-regulate cellular NO level in osteoblasts. In this study, we used mouse osteoblast-like cell line MC3T3-E1 as the effective cell and investigated the effects of IGF-II on promoting proliferation and regulating cellular NO level, inducing expression of inducible NO synthase (iNOS) and endothelial NOS (eNOS) by measuring of their mRNA transcription. MATERIALS AND METHODS 1 Cell line and culture 2 A mouse osteoblast-like cell line MC3T3-E1 was kindly offered by the Department of Medical Microbiology and Parasitology, School of Medicine, Zhejiang University. Medium used for the cell culture was α-minimum essential medium (α-MEM) (HyClone) supplemented with 10% fetal bovine serum (FBS) (Hyclone), 100 U/ml penicillin and 100 μg/ml streptomycin. Two-hundred μl of 1.25×10 4 ml −1 MC3T3-E1 cell suspension was inoculated into each of the wells in a 96-well plates and then the plate was pre-incubated at 37 °C in atmosphere condition of 5% CO 2 for 24 h. Medium in the plates was replaced with 250 μl of 2% FBS α-MEM containing different concentrations of recombinant human IGF-II (rhIGF-II) (R & D) with 1, 10 and 100 ng/ml (IGF-II groups) or the same volume of 2% FBSα-MEM without IGF-II (control groups), and then the plates were continuously incubated for 8, 24, 48 and 72 h, respectively. After stopping incubation, the supernatants of cultures in 7 repeated wells were collected for NO detection and cells in the same wells were used for MTT examination, and cells in the rest of the 8 repeated wells were used for total RNA preparation. Detection of cell proliferation 2 Cell proliferation was detected by MTT method which determines cell proliferation through measuring the absorbent value at OD 490 based on the fine positive linear correlation between OD 490 value and cell numbers. The MC3T3-E1 cells in 96-well plates above were washed twice with 0.01 mol/L PBS (pH 7.4). Into each of the wells in the plates was added 200 μl of FBS-free α-MEM medium and 20 μl of 5 mg/ml MTT solution (Amresco) and then was continuously incubated at 37 °C for 4 h. The medium in the wells was discarded and 150 μl of dimethyl sulfoxide (DMSO) (Sigma) was then added into each of the wells. The OD 490 value of each of the wells was detected by spectrophotometry. Detection of NO concentration 2 The collected supernatants from MC3T3-E1 cell cultures mentioned above were centrifuged at 2000 rpm for 5 min and the supernatants were then recovered. NO concentrations in the supernatants were detected by using NO Detection kit (JINMEI Biotech Co. Ltd.) based on nitrate reductase method. Nitrate in samples was first reduced into nitrite with nitrate reductase and NO concentration was then obtained through measurement of NO 2 − /NO 3 − proportion. RNA extraction and reverse transcriptase polymerase chain reaction (RT-PCR) 2 RNA Isolation Kit (TaKaRa) was used to prepare total RNA from the MC3T3-E1 cells mentioned above. RT-PCR Kit (TaKaRa) was used to detect mRNAs of the cellular iNOS and eNOS. The sequences of iNOS primers were (Togari et al., 1998 1998 ): (F) 5′-CCCTTCCGAAGTTTCTGGCAGCAGC-3′, (R) 5′-GGCTGTCAGAGCCTCGTGGCTTTGG-3′ (497 bp). The sequences of eNOS primers (Laufs et al., 2000 2000 ): (F) 5′-TTCCGGCTGCCACCTGATCCTAA-3′, (R) 5′-AACATATGTCCTTGCTCAAGGCA-3′ (340 bp). The housekeeping gene GAPDH (452 bp) was used to standardize the samples with equal cDNAs and its sequences of primers: (F) 5′-ACCACAGTCCATGCCATCAC-3′, (R) 5′-TCC ACCACCCTGTTGCTGTA-3′ (452 bp). The parameters for RT: 42 °C 50 min, 99 °C 5 min and 5 °C 5 min. The total reaction volume per PCR was 50 μl containing 0.8 μmol/L dNTPs, 0.5 μmol/L for each the iNOS primers, 0.05 μmol/L for each the GAPDH primers, 2.5 U Taq polymerase, 6 μl RT product as template and 10×PCR buffer 5 μl. The parameters for PCR: 94 °C, 5 min, ×1; 94 °C, 50 s, 60 °C, 50 s, 72 °C, 1 min, ×28; 72 °C, 8 min, ×1. For detecting eNOS mRNA, the reagents, reaction volume and RT-PCR parameters were the same as those for iNOS mRNA detection, except of 0.02 μmol/L for each the GAPDH primers and 34 amplification cycles. RT-PCR products were detected by electrophoresis in 3% agarose gel (Sigma) pre-stained with 0.5 μg/ml ethidium bromide. The target fragments were quantitatively analyzed by density scanning technique using Bio Imaging System (Synoptics Ltd. USA). Data analysis 2 All values obtained were expressed as the mean± SD . T -test and one-way analysis of variance was used to statistically analyze the data. RESULTS 1 Effect of IGF-II on promoting MC3T3-E1 cell proliferation 2 After MC3T3-E1 cells were treated with 1 ng/ml IGF-II for 72 h, 10 or 100 ng/ml IGF-II for 24 h, 48 h and 72 h, respectively, the MTT values were remarkably increased ( P < 0.05 or P < 0.01) in a marked time-dosage-dependant manner (Fig. 1 1 ). Effect of IGF-II on NO levels of MC3T3-E1 cells 2 Compared with the control group, NO level of the MC3T3-E1 cells treated with 100 ng/ml IGF-II for 48 h was remarkably decreased ( P < 0.01), and obviously lower NO levels were observed in the MC3T3-E1 cells treated with 1, 10 or 100 ng/ml IGF-II for 72 h ( P < 0.01, P < 0.05 and P < 0.01, respectively) (Fig. 2 2 and Fig. 3 3 ). Effects of IGF-II on iNOS and eNOS mRNA levels of MC3T3-E1 cells 2 iNOS mRNA levels of the MC3T3-E1 cells treated with 100 ng/ml IGF-II for 48 h were obviously lower than those of the control group and 1 ng/ml IGF-II group ( P < 0.01). After being incubated for 72 h, iNOS mRNA levels of the MC3T3-E1 cells treated with 100 ng/ml IGF-II were remarkably lower than those of the control group, 1 ng/ml and 10 ng/ml IGF-II groups ( P < 0.01) (Fig. 4 4 and Fig. 5 5 ). eNOS mRNA levels of the MC3T3-E1 cells treated with different dosages of IGF-II and time durations were similar to those of the control group ( P > 0.05) (data not shown). DISCUSSION 1 IGF-II is one of the most abundant growth factors in bone matrix and the effects of IGF-II on osteoblasts are being closely studied (Fournier et al., 1993 1993 ; Langdahl et al., 1998 1998 ). Over the past decade, the mitogenic effect of IGF-II and its mechanisms have been extensively investigated. However, its regulating effect on cellular NO was noticed only recently. NO is a very small lipophilic molecule which diffuses and reaches the cytoplasmic components, and results in the activation of diverse biological function, and IGF-II had a distinct effect on NO and iNOS gene expression in different cell types (Kaliman et al., 1999 1999 ; Schini et al., 1994 1994 ; Hill et al., 1999 1999 ). Our present study, is the first study showing that IGF-II downregulates the production of NO in the mouse preosteoblastic cell line, MC3T3-E1, via inhibiting mRNA transcription of iNOS gene. And in agreement with the general view, IGF-II has been shown to be a mitogenic factor, at concentration range of 1–100 ng/ml, IGF-II can obviously promote MC3T3-E1 proliferation in a dosage- and time-dependent manner. NO exerts biphasic effects on osteoblasts: at moderately low concentrations NO can maintain normal proliferation or promote proliferation, while at high concentrations, can inhibit proliferation and induce apoptosis (Kanamaru et al., 2001 2001 ; Mancini et al., 2000 2000 ; Armour et al., 2001a 2001a ). In our studies, 1–100 ng/ml IGF-II, especially 100 ng/ml IGF-II, with 48 h and 72 h treatment enable the MC3T3-E1 cells to maintain lower NO levels compared to the controls, and significantly decrease NO levels compared to the control group. These lower NO levels occurred in parallel with the IGF-II-induced cell proliferation, indicating that maintenance of moderate low NO level benefits cell proliferation. Extracellular regulated kinase (ERK), a key molecule in the mitogen-activated protein kinase (MAPK) pathway, can mediate signals of cell proliferation and differentiation, and moderate level of NO can activate ERK (Robinson and Cobb, 1997 1997 ; Jessop et al., 2002 2002 ). IGF-II exhibits high-affinity binding to IGF-I receptor (IGF-IR), which can start the MAPK pathway (Vincent and Feldman, 2002 2002 ). Thus, the mechanism of IGF-II promoting MC3T3-E1 cell proliferation probably involves activations of the ERK and MAPK pathway. No expression or low expression of iNOS in osteoblast was found under basal conditions. But activated iNOS induces a great quantity of NO. It was reported that NO production, bone mineral density decrease and osteoblast apoptosis in iNOS gene knockout mice were much lower than those in experimental inflammation-mediated osteoporosis mice (Armour et al., 2001a 2001a ). High levels of inducible NO were cytotoxic to osteoblasts and even inhibited bone formation (Mancini et al., 2000 2000 ; Armour et al., 2001a 2001a ). Significantly high levels of NO in lesions of localised bone destruction (Mancini et al., 2000 2000 ), rheumatoid arthritis (Gonzalez-Gay et al., 2004 2004 ), and in periodontal lesions, a disease accompanied by alveolar bone loss (Batista et al., 2002 2002 ) were also found. Thus, inducible NO is harmful. On the contrary, eNOS seems to play an important role in increasing osteoblast activity and promoting bone formation. For example, eNOS gene knockout mice had defective bone formation (Aguirre et al., 2001 2001 ; Armour et al., 2001b 2001b ). In this study, we found that 100 ng/ml IGF-II continuously inhibited mRNA transcription of MC3T3-E1 cell iNOS gene, whereas eNOS mRNA levels of the MC3T3-E1 cells treated with the different concentrations of IGF-II in all the tested durations were similar to those of the normal control. 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