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
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ABSTRACT |
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inducible nitric oxide synthase; antisense; peroxynitrite; osteoblast
Tumor necrosis factor (TNF)- and interleukin (IL)-1
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).
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MATERIALS AND METHODS |
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The responses to cytokine stimulation are variable among cells and tissues
(10,
24). We found a combination of
recombinant TNF- (10 ng/ml; Dainippon Pharmaceutical, Tokyo, Japan) and
IL-1
(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 -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 -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
-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.
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RESULTS |
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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- and IL-1
, 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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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- and
IL-1
(Fig. 3). In
contrast, NADPH diaphorase was less detected in the antisense lines.
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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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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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ALPase activity in transformed MC3T3-E1 cells. Stimulation by
TNF- and IL-1
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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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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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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DISCUSSION |
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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- and IL-1
, 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-
and
IL-1
, 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-
and IL-1
, 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.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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REFERENCES |
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