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Am J Physiol Endocrinol Metab 285: E614-E621, 2003. First published March 25, 2003; doi:10.1152/ajpendo.00267.2002
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Targeting of iNOS with antisense DNA plasmid reduces cytokine-induced inhibition of osteoblastic activity

Takahiro Abe,1 Hisako Hikiji,1,3 Wee Soo Shin,2,3 Noboru Koshikiya,1,3 Sei-ichi Shima,1,3 Jumi Nakata,1,3 Takafumi Susami,1 Tsuyoshi Takato,1 and Teruhiko Toyo-oka2,3

Departments of 1Oral and Maxillofacial Surgery and 2Organ Pathophysiology and Internal Medicine and 3Health Service Centre, Faculty of Medicine, University of Tokyo, Tokyo 113-8655, Japan

Submitted 17 June 2002 ; accepted in final form 14 March 2003


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Proinflammatry cytokines, tumor necrosis factor-{alpha} combined with interleukin-1{beta}, induce excessive production of nitric oxide (NO) and its cytotoxic metabolite peroxynitrite (ONOO-) via inducible nitric oxide synthase (iNOS) in murine osteoblasts. In this study, to properly estimate the effects of antisense DNA of iNOS on osteoblastic activity, we produced transformed cell lines with antisense plasmid that specifically targets the iNOS gene for potential long-lasting inhibition. Transformed antisense cell lines were identified by 1) the detection of antisense transcripts, 2) the attenuated expression of iNOS protein, 3) the reduction of NO synthase activity, and 4) the level of NO production. These cell lines targeting iNOS, which showed decreased production of both NO and ONOO-, prevented the inhibition of osteoblastic differentiation as was assayed by the mRNA expression of type I collagen, alkaline phosphatase, osteocalcin, and Core binding factor in the presence of proinflammatory cytokines. Present results indicate that the antisense DNA plasmid of iNOS is potent to reduce the cytokine-induced inhibition of osteoblastic activity.

inducible nitric oxide synthase; antisense; peroxynitrite; osteoblast

NITRIC OXIDE (NO) produced from L-arginine by NO synthase (NOS) has diverse functions in a variety of organs (34, 35). So far, three isoforms of the NOS have been isolated, 1) endothelial (eNOS), 2) neuronal (nNOS), producing small quantities of NO in response to intracellular calcium ions, and 3) the inducible isoform (iNOS) expressed after exposure to bacterial endotoxin or inflammatory cytokines.

Tumor necrosis factor (TNF)-{alpha} and interleukin (IL)-1{beta} enhance bone resorption (4, 27, 36, 39-41) and may lead to inflammatory diseases such as rheumatoid arthritis and osteoporosis under several pathological settings (13). These cytokines are reported to cause iNOS gene expression (21, 24) and actual NO production (10, 24, 36). In contrast, NO itself enhances osteoblastic differentiation in vitro (20). Therefore, these contradictory results suggest that the bone-resorbing effect of cytokines is not mediated via NO per se (20, 21, 41). NO reacts with superoxide (O2-) to form the highly reactive intermediate peroxynitrite (ONOO-), a potent cytotoxic intermediate (26, 29, 44). ONOO-, which is produced during an inflammatory response, causes a variety of toxic effects, including lipid peroxidation and tyrosine nitration on several biomolecules (22, 26). We showed previously that the cytokines actually generate both NO and O2- in osteoblasts and that NO and O2- produce an even more toxic product, ONOO-, modifying osteoblastic differentiation (20). We have postulated that the cytokine-induced iNOS, not eNOS or nNOS, plays an important role in the inhibition of osteoblastic differentiation (20, 21).

The purpose of the present study is to examine effects of the specific inhibition of iNOS expression on osteoblastic cells and to inspect whether iNOS antisense plasmid prevents cytokine-induced reduction of osteoblastic activity. The biosynthesis of NO is competitively inhibited by several guanidine-substituted arginine analogs (5, 16). Although these chemical inhibitors of NOS are often used when inhibiting NOS and new-type inhibitors are being developed, they are not specific enough for each isoform of NOS and may have additional actions as analogs of essential amino acids (5, 33). In contrast, the antisense technique is specific for inhibiting the biosynthesis of a single protein. Further antisense plasmids have advantages over synthetic antisense oligonucleotides because oligonucleotides must be repeatedly added at high concentrations in culture medium and are not suitable for the long-term experiment (17). Antisense DNA plasmid, but not oligonucleotides, has the potential for long-lasting expression and thus may be used as a therapeutic approach to chronic disease. Here, we established stable transformants derived from osteoblastic MC3T3-E1 cells in which transfected plasmids continuously produced iNOS antisense RNA. With these cells, we investigated the specific effects of iNOS inhibition on alkaline phosphatase (ALPase) activity and levels of mRNA expression in type I collagen (COL I), ALPase, osteocalcin (OSC), and Core binding factor (Cbfa1), all of which are established indexes of osteoblastic differentiation (43).


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Cell culture. Murine osteoblastic MC3T3-E1 cells (RIKEN RCB1126) were cultured in {alpha}-MEM (GIBCO, Grand Island, NY) containing 10% FBS (JRH Bioscience, Lenexa, KS). The cells were incubated at 37°C in humidified air including 5% CO2 and passaged every 7 days. The medium was changed every 2-3 days. Cellular confluence was maintained throughout all treatment procedures.

The responses to cytokine stimulation are variable among cells and tissues (10, 24). We found a combination of recombinant TNF-{alpha} (10 ng/ml; Dainippon Pharmaceutical, Tokyo, Japan) and IL-1{beta} (10 ng/ml; Genzyme, Cambridge, MA) to be the sufficient concentration of these cytokines to stimulate the MC3T3-E1 cells (20).

Preparation of plasmids containing antisense or sense sequence of iNOS. Murine iNOS mRNA was isolated from MC3T3-E1 cells stimulated by cytokines and used to synthesize the first-strand cDNA with RT. cDNA was used as a template in a PCR with a primer (primer 1) designed from the sequence of murine macrophage iNOS (upper: 52-71 bp; lower: 264-245 bp; GenBank M84373 [GenBank] ; see Ref. 31). The 213-bp product, which covered the ATG initiation codon of the murine iNOS gene, was purified and subcloned into the plasmid vector pTARGET (Promega, Madison, WI) by blunt-end ligation.

After making large-scale preparation of the plasmids of interest by CsCl-ethidium bromide gradients, we performed three experiments to determine the orientation of the insert (antisense or sense direction with respect to the CMV promoter/enhancer). First, digestion with the restriction enzyme Hinc II (New England BioLabs, Beverly, MA) was carried out. Second, PCR was performed for the resultant plasmid with primer 1 and primer 2, which were designed from the sequence of pTARGET. Finally, the insert was identified by direct sequencing of the PCR products.

Stable transfection of MC3T3-E1 cells. MC3T3-E1 cells were transfected using a lipofectamine reagent (GIBCO) according to the manufacturer's instruction. Briefly, the transfection was conducted for 4 h at 37°C in 5% CO2 by adding 5 µg plasmid DNA (antisense, sense, and empty vector) to 20 µl of lipofectamine reagent in each well of six-well plates. At the end of 4 h of incubation, the culture medium was replaced with fresh 10% FBS containing {alpha}-MEM.

After 24 h, the medium was replaced with culture medium containing 0.5 mg/ml neomycin (G418; Wako Pure Chemical Industries, Osaka, Japan) to isolate stable transfectants in 10-cm dishes. After three more days, the medium was exchanged with fresh selection medium and then changed every 3 days thereafter until G418-resistant colonies appeared. Transfectants were selected as "positive" if they were resistant to 0.5 mg/ml G418. The lowest concentration of G418 used was that in which nontransfected MC3T3-E1 cells died within 10-14 days. Single colonies were isolated and expanded in culture. Transcription of the iNOS inserts in either the antisense or sense orientation was confirmed by RT-PCR with primer 2 in each transfectant. NADPH diaphorase staining (see below), as a marker of NOS activity, immunocytochemistry of the iNOS protein, and the Griess reaction for NO production were added to confirm positive cell lines.

Immunocytochemistry of iNOS and nitrotyrosine. MC3T3-E1 cells on eight-well chamber slides (LAB-TEK II; Nalge Nunc International, Rochester, NY) were cultured for either 24 h for iNOS staining or 48 h for nitrotyrosine (NT) staining in the medium with or without cytokines. After being fixed in an ethanol-acetone mixture, the endogenous peroxidase was inactivated by 3% H2O2 in methanol.

Anti-iNOS polyclonal rabbit antibody (Santa Cruz Biotech, Santa Cruz, CA) or anti-NT polyclonal rabbit antibody (Upstate Biotech, Lake Placid, NY) was used as the first antibody, and rabbit IgG was used as the negative control. Cells were treated with the blocking reagent (Histofine; Nichirei, Tokyo, Japan) for 20 min and then with iNOS antibody for 3 h or NT antibody for 5 h at room temperature. These cells were then incubated with the secondary antibody (Simple stain MAX PO reagent; Nichirei), which consists of amino acid polymers conjugated to peroxidase and anti-mouse/rabbit IgG that is reduced to its F(ab)' fragment, at room temperature for 30 min. The immunoproduct was visualized by 3,3'-diaminobenzidine (Simple stain DAB reagent; Nichirei) according to the manufacturer's instructions and photographed by a digital camera (AX80; Olympus, Tokyo, Japan). The stained intensity was measured by densitometry with graphic software (version 6; Adobe Photoshop, Mountain View, CA). Precision of the intensity measurement was evaluated by making an arbitrary selection in the staining area and performing a double-blind test.

NADPH diaphorase staining. NOS has an activity of NADPH diaphorase that has been employed for histochemistry (12). Cells were grown to 100% confluence and incubated with or without cytokines for an additional 24 h. After cytokine stimulation, these cells were washed by PBS including 0.1% CaCl2 [PBS(+)] and fixed in 2% formaldehyde. These cells were then washed three times and reacted in PBS(+) containing 1 mM {beta}-NADPH and 0.2 mM nitroblue tetrazolium (Sigma, St. Louis, MO) for 30 min at 37°C.

Measurement of NO and ALPase activity. MC3T3-E1 cells were grown to 100% confluence and incubated with or without cytokines for a further 24 h. Nitrate and nitrite are stable after being formed from NO. Nitrate in the sample was converted to nitrite with nitrate reductase and then measured by spectrophotometry after the Griess reaction (19, 21).

The level of ALPase activity in bone tissues reflects osteoblastic differentiation (43). MC3T3-E1 cells were cultured on 24-well plates and stimulated by cytokines for 48 h. The ALPase activity (Wako) was assayed as described previously (21) and normalized by protein amount measured by the Bradford method (Bio-Rad Laboratories, Hercules, CA; see Ref. 21).

Cell proliferation assay. For measurement of cell proliferation, MC3T3-E1 cells and transfected cell lines were plated at a density of 4 x 10 cells/well on 96-well plates. After 24 h, medium was replaced with {alpha}-MEM containing 10% FBS in the absence or presence of cytokines and cultured for three more days. The effects of iNOS antisense on proliferation with or without cytokines were determined by a tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] assay (Promega). Briefly, 20 µl of MTS solution reagent were added to 100 µl of culture medium of each well. After incubation for 4 h at 37°C, the absorbance was measured at 490 nm using a 96-well plate reader (PowerWave x340; Bio-Tek, Winooski, VT).

RT-PCR of COL I, ALPase, OSC, and Cbfa1 gene. The OSC message was detected by semiquantitative RT-PCR. MC3T3-E1 cells were cultured on 6-cm-diameter dishes and stimulated by the cytokines for 48 h. Total RNA was extracted by ISOGEN (Nippon Gene, Toyama, Japan), and 2 µg of total RNA were reverse transcribed using Moloney murine leukemia virus RT (Superscript; GIBCO) for OSC, 1 µg total RNA was reverse transcribed using Avian myeloblastis virus RT (Roche, Indianapolis, IN) for COL I, ALPase, and Cbfa1, and the cDNA served for the following PCR template.

The PCR reaction was carried out as described previously (20, 21). cDNA was amplified by Taq DNA polymerase (Perkin-Elmer and Roche) using the following primers: OSC, 5'-GCCCTCTCCAAGACATATA-3' and 5'-CCATGATCACGTCGATATCC-3'; COL I, 5'-ATGAGGACCCTCTCTCTGCT-3' and 5'-CCGTAGATGCGTTTGTAGGC-3'; ALPase, 5'-GTGTGAATTGTTGGGGCTTT-3' and 5'-ACCTGGGATGATTGAACTGG-3'; Cbfa1, 5'-TCTCTACTATGGTACTTCGT-3' and 5'-AAGATCATGACTAGGGATTG-3'; and internal standard gene (GAPDH), 5'-TGAAGGTCGGTGTGAACGGATTTGGC-3' and 5'-CATGTAGGCCATGAGGTCCACCAC-3'. The denaturing, annealing, and elongating conditions for the PCR reaction were 94, 50 or 57 or 60, and 72°C, respectively, with an initial 9-min denaturation and an additional 7-min extension step at 72°C. The PCR conditions were determined so that the band intensity showed a linear relationship with increases in the cycle number (26 cycles for OSC and ALPase, 30 cycles for COL I and Cbfa1). Bands were quantified by densitometry (Epi-Light UV FA500; Aisin Cosmos R&D, Tokyo, Japan), and the intensities were normalized with reference to GAPDH.

Statistics. All values are expressed as means ± SD. Statistical difference between values was examined by one-way ANOVA followed by Scheffé's multiple comparison test. P values <0.05 were considered to be statistically significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 DISCLOSURES
 REFERENCES
 
Stable gene expression of inserts. To ascertain whether the transformants express the transcripts of the inserts in a stable manner, RT-PCR was carried out using primers designed from the multiple cloning site of pTARGET vector. Without treatment of the cytokines, transcripts of the insert cDNA were detected constitutively, as bands, for either the antisense or sense RNA in transformed cells (Fig. 1), and they were not detected in the cell lines where an empty vector (pTARGET) was transfected. Bands were recognized in cell lines transfected with antisense or sense iNOS. These bands were detected in both sense and antisense orientations after three passages in a stable manner. Bands of GAPDH as an internal control were always recognized in all cells.



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  		Fig. 1. Detection of antisense or sense inducible nitric oxide synthase (iNOS) mRNA using RT-PCR. Expression of the antisense or sense iNOS mRNA without the stimulation of tumor necrosis factor-{alpha} (TNF-{alpha}) and interleukin-1{beta} (IL-1{beta}) is shown. Cells were grown to 100% confluence without cytokine stimulation, and RT-PCR was carried out as described under MATERIALS AND METHODS. Expected product sizes for the inserts (antisense or sense) and GAPDH were 404 and 988 bp, respectively. DNA size marker (lane 1), wild-type cells (lane 2), vector control transfected with empty vector plasmid in MC3T3-E1 cells (vector control line, lane 3), cell lines in which sense vector was transfected (sense lines, lanes 4-6), and cell lines in which antisense vector was transfected (antisense lines, lane 7-9).

 

Immunodetection of iNOS. iNOS expression in MC3T3-E1 cells was investigated by immunocytochemistry (Fig. 2). iNOS was not detected in negative controls that employed unimmunized IgG or unstimulated cells (Fig. 2, A, B, D, F, and H). After stimulation by TNF-{alpha} and IL-1{beta}, iNOS protein was recognized for wild-type cell lines in which vectors were not transfected (wild-type lines; Fig. 2C). For the cell lines in which an empty vector was transfected (vector control lines; Fig. 2E), and those in which a sense vector was transfected (sense lines; Fig. 2G), iNOS protein was also observed after cytokine stimulation. In contrast, for the cell lines in which iNOS antisense was transfected (antisense lines; Fig. 2I), iNOS protein was less detectable after cytokine stimulation. Densitometry of the staining revealed 14 ± 6, 13 ± 4, 12 ± 7, and 18 ± 6 in a wild-type cell line, a vector control line, a sense line, and an antisense line, respectively (Fig. 2, B, D, F, and H). After cytokine stimulation, the corresponding levels of staining intensity were 65 ± 8, 83 ± 12, 79 ± 18, and 18 ± 5 (Fig. 2, C, E, G, and I). These results indicate that the iNOS expression was selectively inhibited by iNOS antisense DNA plasmid and was effi-ciently reduced to the same level as without cytokine stimulation.



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  		Fig. 2. Immunocytochemistry of iNOS after cytokine stimulation. Cells were grown to 100% confluence with TNF-{alpha} (10 ng/ml) and IL-1{beta} (10 ng/ml) stimulation for 24 h. A: negative control in wild-type cells stained by control IgG; B: unstimulated wild-type cells; C: positive control in wild-type cells after cytokine stimulation; D: unstimulated, empty vector control line; E: empty vector control line after cytokine stimulation; F: unstimulated sense line; G: sense line after cytokine stimulation; H: unstimulated antisense line; I: antisense line after cytokine stimulation. Note that iNOS protein was selectively expressed in the cytoplasm and that the antisense line exhibits the production of iNOS after cytokine stimulation. Bar length, 100 µm.

 

NADPH diaphorase staining. NADPH diaphorase staining is an index of NOS activity (6). NADPH diaphorase was distinctly recognized in a wild-type cell line, a vector control line, and a sense line after the stimulation of TNF-{alpha} and IL-1{beta} (Fig. 3). In contrast, NADPH diaphorase was less detected in the antisense lines.



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  		Fig. 3. NADPH diaphorase activity after cytokine stimulation. NADPH diaphorase activity after cytokine stimulation for 24 h found in wild-type cells was reduced in antisense. WT, wild-type cells; Vec, empty vector control line; S, sense lines; A, antisense lines; -, unstimulated; +, cytokine stimulated. Bar length, 100 µm.

 

Effects of cytokines on NO production. Unstimulated MC3T3-E1 cells released a basal amount of NO detected as nitrate/nitrite (2.47 ± 0.48 ~ 3.17 ± 0.32 µM) in all cell lines (Fig. 4). After cytokine stimulation for 24 h, the wild-type cell line and the vector control line showed a significantly high level of nitrate/nitrite accumulation (48.1 ± 1.5 and 41.4 ± 2.2 µM, respectively). The sense plasmid-induced cell line also produced a high level of nitrate/nitrite accumulation. The mean of three sense lines was 44.5 ± 6.7 µM. On the other hand, the antisense lines produced only 22-34% NO compared with that of the sense lines in response to the cytokines (mean of 3 antisense lines was 12.1 ± 0.92 µM). These results indicate that the production of NO after cytokine stimulation was significantly suppressed in the antisense lines (P < 0.01).



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  		Fig. 4. Effects of cytokines on nitric oxide (NO) production in MC3T3-E1 cells. After cytokine stimulation for 24 h, NO is detected as nitrate/nitrite as described in MATERIALS AND METHODS. Open bars, NO production in unstimulated cells (-); filled bars, NO production in stimulated cells (+). Results are means ± SD; n = 12 experiments. *P < 0.01 relative to antisense lines A1-A3.

 

Effects of iNOS antisense on proliferation in the presence of cytokines. An MTS assay was used to analyze the effects of iNOS antisense on the proliferation of cells treated with cytokines. As shown in Fig. 5, antisense cell lines significantly promoted the growth even if they were treated with cytokines. Therefore, it is indicated that iNOS antisense partially attenuated the reduction of proliferation in the presence of cytokines.



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  		Fig. 5. Cell proliferation assay. Each cell line was incubated at a density of 4 x 10 cells/well in 96-well plates for 24 h and then cultured in the absence or presence of cytokines and cultured for 3 more days. For determination of cell proliferation, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), inner salt assay was performed on days 0, 1, 2, 3, and 4. Broken and solid lines represent absorbance of unstimulated (-) and stimulated (+) cells, respectively. Results shown are means ± SD; n = 10. *P < 0.01 relative to stimulated control.

 

ALPase activity in transformed MC3T3-E1 cells. Stimulation by TNF-{alpha} and IL-1{beta} reduced the ALPase activity in the wild-type, vector control, and sense-transduced lines compared with that in unstimulated cells (Fig. 6). The mean reduction in ALPase activity by cytokines in the sense lines was 59.2 ± 17% that of the unstimulated control (P < 0.01). In contrast, the ALPase activity of the antisense lines did not change significantly (mean of 3 cell lines was 111.5 ± 17%), indicating that the antisense lines did not influence the ALPase activity secondary to the cytokine stimulation.



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  		Fig. 6. Effects of cytokines on alkaline phosphatase (ALPase) activity. After cytokine stimulation for 48 h, relative ALPase activity was compared with unstimulated cells (-). Open bars, ALPase activity in unstimulated cells; filled bars, ALPase activity in stimulated cells. Results are means ± SD; n = 12. *P < 0.01 relative to unstimulated control.

 

Expression of marker genes on osteoblastic differentiation. To investigate the effects of iNOS antisense on the expression of the differentiation markers in osteoblasts, we performed semiquantitative RT-PCR using specific primers for COL I, ALPase, OSC, and Cbfa1. OSC mRNA was constitutively expressed in unstimulated cells (Fig. 7A, top). However, after cytokine stimulation for 48 h, the gene expression was reduced in the wild-type, vector control, and sense lines. The antisense cell lines meanwhile showed higher levels of gene expression compared with the sense lines. The relative gene expression levels after cytokine stimulation were compared with the unstimulated control level (Fig. 7A, bottom). The sense lines decreased to 52 ± 9% (mean of 3 cell lines) compared with the unstimulated control (P < 0.01), whereas the antisense increased to 227 ± 92% (means of 3 cell lines, P < 0.01). After the normalization with GAPDH, the sense line decreased to 44 ± 8% (mean of 3 cell lines) compared with the unstimulated control (P < 0.01), and the antisense increased to 284 ± 149% (mean of 3 cell lines, P < 0.01). Similarly, we assessed the expression of COL I, ALPase, and Cbfa1 using representative cell lines (S2 and A2; Fig. 7B). These results indicate that the antisense cell line prevented the reduction of the relative mRNA levels of ALPase and Cbfa1 after cytokine stimulation compared with the other controls. Although cytokines showed a variable tendency to inhibit COL I mRNA in the three control cell lines, all of the antisense cell lines prevented the inhibition of COL I mRNA with cytokines.



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  		Fig. 7. Expression of marker genes on osteoblastic differentiation. PCR conditions were determined such that the band intensity had a linear relationship with an increase in the cycle number [30 cycles for type I collagen (COL I), 26 cycles for ALPase, osteocalcin (OSC), and core binding factor (Cbfa1)]. A: iNOS inserts (see Fig. 1). B: COL I, ALPase, Cbfa1, and GAPDH. Expected product sizes for COL I, ALPase, OSC, Cbfa1, and GAPDH are 656, 373, 276, 373, and 988 bp, respectively. Results of OSC quantified by densitometry are shown in A, bottom. mRNA levels of OSC are compared with unstimulated control after normalization with GAPDH and quantification by image analysis. Values are means ± SD of combined results from 3 separate experiments. *P < 0.01 relative to unstimulated control.

 

Immunodetection of ONOO- by anti-NT antibody. The NT residue on protein is a stable product of ONOO- reaction (9). The wild type (Fig. 8C), the vector control line (Fig. 8E), and the sense line (Fig. 8G) showed an intense NT expression after cytokine stimulation, whereas the antisense line (Fig. 8I) did not exhibit elevated levels of NT expression (Fig. 8). These results were quantified by densitometry. The levels of staining intensity were 15 ± 2, 17± 7, 20 ± 8, and 21 ± 5 in the wild-type, vector control, sense, and antisense lines, respectively (Fig. 8, B, D, F, and H). After cytokine stimulation, the corresponding levels of staining intensity were 88 ± 20, 75 ± 7, 84 ± 4, and 33 ± 10 (Fig. 8, C, E, G, I). These results suggest that MC3T3-E1 antisense cell lines, which inhibit the expression of iNOS after cytokine stimulation, decreased the production of NO and ONOO- (P < 0.01).



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  		Fig. 8. Immunocytochemistry of nitrotyrosine (NT) after cytokine stimulation. After cytokine stimulation for 48 h, immunocytochemistry of NT was performed. NT residues are stable markers of ONOO- synthesis. A: negative control in wild-type MC3T3-E1 cells; B: unstimulated wild-type cells; C: positive control in wild-type cells after cytokine stimulation; D: unstimulated empty vector control line; E: empty vector control line after cytokine stimulation; F: unstimulated sense line; G: sense line after cytokine stimulation; H: unstimulated antisense line; I: antisense line after cytokine stimulation. Note that the antisense line after cytokine stimulation decreased the immunoproduct of NT. Bar length, 100 µm.

 


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
DISCLOSURES
 REFERENCES
 
The purpose of our investigation is to examine whether the specific inhibition of iNOS by antisense DNA plasmid would prevent the proinflammatory cytokine-induced reduction in osteoblastic differentiation. One essential drawback to using chemical inhibitors is its apparent inability to discriminate between the different isoforms of NOS and its additional functions (5). Moreover, although antisense oligonucleotides are generally used as antisense techniques to hybridize to specific RNA sequences, the antisense oligonucleotides may not always demonstrate a significant effect on the target mRNA for long-lasting analysis (17, 23, 28, 38) and must be added repeatedly to the culture medium at high concentrations. Furthermore, there are no decisive systems to deliver them efficiently to the target site through the cell membrane (17, 28). In addition, it has been found that antisense oligonucleotides have many effects other than the gene action, such that they may adsorb intracellular proteins and activate the immune systems (25, 42). For these reasons, the use of antisense plasmid was chosen for the direct blockade of the iNOS pathway, without impinging on constitutive NOS-dependent events, and to make it possible to select transformed cell lines for long-term analysis.

Previously, several groups have reported that, in murine macrophages and endothelial cells, iNOS is inhibited by plasmid DNA that directed the production of iNOS antisense RNA (7, 8, 37). However, we report on anti-inflammatory effects in osteoblastic cells with this iNOS antisense technique for the first time. We designed the 213-bp fragment of the iNOS region so that it covered the ATG initiation codon of the murine iNOS gene. Although further experiments are required to create increasingly efficient constructs, this antisense construct, including the noncoding region, demonstrated effective inhibition of iNOS gene expression and suppression of the biological function of NO.

Proinflammatory cytokines, such as TNF-{alpha} and IL-1{beta}, are well known to be the most potent stimulators of bone resorption (4, 27) and to induce high levels of NO production in bone (10, 24, 34, 40). Interestingly, several groups have shown that these high concentrations of NO inhibit osteoclast formation and activity, which elevate with cytokine stimulation (6, 30, 32, 36). In contrast, two- to threefold inhibition of OSC synthesis (14, 20, 21, 39) and the reduction of ALPase activity with a combination of the two cytokines, TNF-{alpha} and IL-1{beta}, have been confirmed in previous studies in osteoblasts (20, 21). Based on our experience with NO donor, it was also revealed that NO directly facilitated the levels of ALPase activity in osteoblastic cells (20). Despite the differentiation-enhancing effect of an NO donor, NO, especially derived from iNOS, appears to potentiate the inhibitory effects with a treatment of the two cytokines, TNF-{alpha} and IL-1{beta}, on osteoblast activity in vitro.

Recent studies in iNOS knockout (KO) mice by van't Hof et al. (40) have shown that activation of the iNOS pathway is essential for IL-1-stimulated bone resorption, both in vitro and in vivo. Their coculture studies indicate that osteoblasts are the main source of NO and that osteoblast-derived NO acts in a paracrine and autocrine fashion on the bone component to promote IL-1-induced bone resorption. Furthermore, Armour et al. (3) have shown that apoptosis of osteoblasts and osteocytes contributes to inflammation-induced bone loss and suggested that the deleterious effects of iNOS activation and inflammation on bone may be relatively specific for mature osteoblasts. These findings strongly suggest that iNOS activation in osteoblasts may contribute to inflammatory diseases, inducing bone loss by suppressing bone formation (2, 15, 18). Another study in eNOS KO mice has shown that osteoblasts derived from eNOS KO mice reduce rates of growth when compared with the wild type and are less well differentiated, as reflected by lower levels of ALPase (1). These data suggest that eNOS is essential for normal osteoblast differentiation and function. These data support our hypothesis that high levels of NO production and its resultant metabolite, ONOO-, through cytokine-induced iNOS, have an inhibitory effect on osteoblastic growth and differentiation even though NO per se has an enhancing effect. Although our data do not consider a role for eNOS as mediators of osteoblast differentiation due to focus on the iNOS pathway, eNOS mRNA expression is confirmed, at least under the experimental conditions described in this study (no data shown).

We documented previously that the marker NT was formed from ONOO- generated via NO and O2- after cytokine stimulation in osteoblasts, which provided a useful marker for ONOO-. As expected, by immunocytochemical analysis, confirming the cellular distribution, we showed that staining levels of not only iNOS but also NT tend to decrease because of blockade of the iNOS pathway. The formation of NT is widely believed to be a result of the attack on tyrosine by ONOO- (9), but it actually may be safer to conclude that the formation of NT is a result of the generation of reactive nitrogen species rather than ONOO- specifically, because other pathways of NO/ONOO- interaction have been proposed in a previous report (26). The effects of iNOS antisense may have different aspects of osteoblast function on growth and differentiation. We showed that the indexes of osteoblastic differentiation, COL I, ALPase, OSC, and Cbfa1, were upregulated in the antisense cell lines with cytokines. However, iNOS antisense only partially attenuated the reduction of proliferation in the presence of cytokines. These data seemed to suggest that iNOS antisense had a more profound effect of osteoblast differentiation than proliferation.

In conclusion, it was likely that the iNOS antisensetransfected cell lines, derived from osteoblastic MC3T3-E1 cells, produced substantially less NO and ONOO- after cytokine stimulation and also that the indirect inhibition of ONOO- and its cytotoxic effects resulted in the prevention of osteoblastic dysfunction. Further studies must be done to quantify this association. A recent study (11) also shows that inflammatory cytokines can indirectly induce ONOO- production and that ONOO- is at least partially responsible for proliferation and differentiation in human osteoblasts by pharmacological manipulation, which is also suggested in our studies (20, 21).

A large amount of NO derived from iNOS and tyrosine nitration has been detected in chronic inflammatory lesions (22, 44). In these pathological situations, blockade of the iNOS pathway by antisense may terminate the process of bone resorption. Therefore, targeting of iNOS with antisense DNA plasmid, although it is necessary to use higher transfection technologies such as a virus vector, may be potentially applicable to inflammatory conditions and supply therapeutic strategies for arthritis, periodontitis, and other pathological processes in inflammatory conditions.


   DISCLOSURES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
REFERENCES
 
This work was financially supported by a grant-in-aid from the Ministry of Education, Culture, Science, Sports and Technology, the Ministry of Health, Welfare and Labor in Japan, Uehara Memorial Foundation, and the Motor Vehicle Foundation.


   ACKNOWLEDGMENTS
 
We thank Dr. Mitsuhiro Kuwahara and Akiko Kurachi (University of Michigan) for critical reading of the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: H. Hikiji, Dept. of Oral and Maxillofacial Surgery, Faculty of Medicine, Univ. of Tokyo, 7-3-1, Hongo, Bunkyo-ku 113-8655, Tokyo, Japan (E-mail: hikiji-ora{at}h.u-tokyo.ac.jp).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 

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