NFkB decoy oligodeoxynucleotides ameliorates osteoporosis ... - Nature

Gene Therapy (2006) 13, 933–941 & 2006 Nature Publishing Group All rights reserved 0969-7128/06 $30.00 www.nature.com/gt

ORIGINAL ARTICLE

NFkB decoy oligodeoxynucleotides ameliorates osteoporosis through inhibition of activation and differentiation of osteoclasts H Shimizu1,2, H Nakagami1,2,3, I Tsukamoto4, S Morita5, Y Kunugiza6, T Tomita6, H Yoshikawa6, Y Kaneda3, T Ogihara2 and R Morishita1 1

Department of Geriatric Medicine, Osaka University Graduate School of Medicine, Osaka, Japan; 2Division of Clinical Gene Therapy, Osaka University Graduate School of Medicine, Osaka, Japan; 3Division of Gene Therapy Science, Osaka University Graduate School of Medicine, Osaka, Japan; 4Department of Food Science and Nutrition, Nara Women’s University, Nara, Japan; 5Department of Oral and Maxillofacial Surgery, Osaka Dental University, Osaka, Japan and 6Department of Orthopaedic Surgery, Osaka University Graduate School of Medicine, Osaka, Japan

The transcription factor, nuclear factor-kappa B (NFkB), is believed to play a pivotal role in osteoclast formation. In this study, we focused on NFkB decoy oligodeoxynucleotides (ODN) as a new therapeutic strategy to attenuate osteoporosis. Tartrate-resistant acid phosphatase (TRAP)-positive multinuclear osteoclasts formed in mononuclear cells including osteoclast precursors from neonatal rabbit bone marrow were increased in the presence of 1,25-dihydroxyvitamin D3, whereas transfection of NFkB decoy ODN decreased the number of TRAP-positive cells and attenuated RANKL and M-CSF-induced osteoclast formation. NFkB decoy ODN also inhibited the activity of osteoclasts, as assessed by pit formation. In rat overiectomized model of estrogen defi-

ciency, continuous administration of NFkB decoy ODN attenuated the increase of TRAP activity, accompanied by a significant increase in calcium concentration in tibia and femur and decrease in urinary deoxypyridinoline. In additional osteoporosis model using vitamin C-deficient rat, inhibition of NFkB by decoy ODN dramatically improved the bone length, weight, density as assessed by dual-energy X-ray absorptiometry. Overall, inhibition of NFkB by decoy strategy prevented osteoporosis through the inhibition of bone resorption. Targeting of NFkB might be potential therapy in various bone metabolic diseases. Gene Therapy (2006) 13, 933–941. doi:10.1038/sj.gt.3302711; published online 2 March 2006

Keywords: transcriptional factors; NFkB decoy; osteoclast; osteoblast; osteoporosis

Introduction Osteoclasts are monocyte/macrophage lineage multinucleated cells that play a critical role in bone absorption. Osteoclast differentiation is regulated by a variety of hormones, local factors and inflammatory cytokines, such as IL-1 and TNF-a.1–3 Osteoblasts/stromal cells express receptor activator of nuclear factor-kappa B (NFkB) ligand (RANKL, also called OPGL, TRANCE, and ODF) in response to several bone-resorbing factors including 1,25-dihydroxyvitamin D3 (vitamin D3) to support osteoclast differentiation from their precursors.4,5 Osteoclast precursors, which express receptor activator of NFkB, recognize RANKL through cell-to-cell interactions with osteoblasts/stromal cells, and differentiate into mature osteoclasts in the presence of macrophage-colony stimulating factor (M-CSF). Targeted disruption of either RANKL or RANK in mice causes lack of osteoclasts and an osteopetrotic phenotype.3,6 Osteoprotegerin (OPG), which acts as a decoy receptor Correspondence: Professor R Morishita, Division of Clinical Gene Therapy, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. E-mail: [email protected] Received 24 June 2005; revised 1 December 2005; accepted 2 December 2005; published online 2 March 2006

for RANKL, is known to be effective for inhibition of osteoclast differentiation.4 Although osteoclasts are the only cells responsible for bone resorption, it is well recognized that osteoclasts are derived from hemopoietic cells of the monocyte-macrophage lineage.7 Since hemopoietic monocytes and macrophages are present in almost all tissues, whereas osteoclasts are present only in bone, we speculated that some local factors or local mechanisms are involved in this tissue-specific localization of osteoclasts in bone. The process of osteoclast development consists of several steps: proliferation, differentiation, fusion, and activation of osteoclasts. The target cells of bone-resorbing factors, such as vitamin D3, PTH, and IL-11 are all osteoblastic cells, but not hemopoietic osteoclast precursors. Vitamin D3 induces osteoclast formation via the vitamin D3 receptor present in the nucleus of osteoblasts. Osteoclast precursors having the RANKL receptor recognize RANKL by cell–cell contact and differentiate into mature osteoclasts. Macrophage-colony stimulating factor (MCSF) produced by osteoblastic cells also appears to play an important role in the proliferation and differentiation of osteoclast progenitors. Thus, osteoblasts are important for osteoclast recruitment in two different ways: the production of M-CSF, and the production of a membrane-associated factor like RANKL commonly induced

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by several bone-resorbing factors.8,9 The transcription factor, nuclear factor-kB (NFkB) plays a pivotal role in the coordinated transactivation of cytokine and adhesion molecule genes involved in such conditions, as inhibition of NFkB by decoy oligodeoxynucleotides (ODN) was effective to treat a rheumatoid arthritis model.10 Although one of the possible mechanisms is through the inhibition of several inflammatory cytokines such as TNF-a, it is noteworthy to examine the effects of inhibition of NFkB on the differentiation and activation of osteoclasts. Thus, we examined the role of NFkB using decoy ODN on osteoclast activation in vitro as well as in vivo.

Results Transfection of NFkB decoy ODN into osteoclasts To clarify the inhibitory effects of NFkB decoy ODN on bone destruction in arthritis model,10 we initially focused on the differentiation of osteoclasts. We observed vitamin D3-induced osteoclast differentiation by Hoechst 33258 nuclear staining and tartrate-resistant acid phosphatase (TRAP) staining. After vitamin D3 treatment, several TRAP-positive multinuclear cells were formed, as shown in Figure 1a. To confirm the activation of NFkB in the process of osteoclast differentiation, we transfected the luciferase gene driven by NFkB binding site into cultured bone marrow cells from neonatal rabbits. As shown in Figure 1b, treatment with vitamin D3 significantly induced luciferase activity at 48 h after treatment, indicating an increase in NFkB activity (Po0.01). To examine whether activation of NFkB induced by vitamin D3 would mediate both differentiation and activation of osteoclasts, we examined the inhibitory effect of transfection of NFkB decoy ODN into osteoclasts. As shown in Figure 1c, transfection of FITClabeled ODN (1 mM) resulted in the detection of intense nuclear fluorescence at 1 and 7 days after vitamin D3 treatment. Multinuclear cells were also confirmed by Hoechst 33258 nuclear staining. Using this method, we elucidated the role of NFkB in the differentiation of osteoclasts. To confirm the inhibitory effect of NFkB decoy ODN on the activation of osteoclasts by NFkB, co-transfection of decoy ODN with the luciferase gene driven by the NFkB binding site was performed. Transfection of NFkB decoy ODN, but not scrambled decoy ODN, significantly inhibited the increase in luciferase activity at 4 days after vitamin D3 treatment in undifferentiated osteoclasts in a dose-dependent manner (Figure 1d, Po0.01). Effects of NFkB decoy ODN on differentiation of osteoclasts induced by vitamin D3 treatment Stimulation of mononuclear cells from rabbit bone marrow cells with vitamin D3 led to the differentiation of osteoclast cells, which were defined as multinucleated cells positively stained for TRAP. As vitamin D3 possibly stimulate osteoblasts in rabbit bone marrow cells that secrete various cytokines such as RANKL. TRAPpositive multinuclear osteoclasts formed in the culture were markedly increased in the presence of vitamin D3 (Figure 2a). In contrast, transfection of NFkB decoy ODN significantly decreased the number of TRAP-positive multinuclear cells in a dose-dependent manner as compared to scrambled decoy ODN (Figure 2a, Gene Therapy

Po0.01). To further confirm the inhibitory effects of NFkB decoy ODN in the different culture system, we also employed TRAP staining in M-CSF and RANKLinduced differentiation of rat osteoclasts. Of importance, transfection of NFkB decoy ODN also attenuated M-CSF and RANKL-induced osteoclast differentiation, as shown in Figure 2b. These results were in concert with the hypothesis that NFkB might be the major pathway involved in the process of osteoclast differentiation as M-CSF and RANKL-induced bone marrow cells and vitamin D3-induced bone marrow cells as well. We also confirmed that TRAP positive multinuclear cells transfected with FITC-labeled NFkB decoy ODN showed caspase-3 activation. In addition, the transfection of NFkB decoy ODN increased Annexin V positive cells in mononuclear cells including osteoclasts from rabbit bone marrow (Figure 3). These results suggest that NFkB decoy ODN induced apoptosis of TRAP positive multinuclear cells, which correspond to osteoclasts. Overall, the differentiation of osteoclasts was significantly inhibited by NFkB decoy ODN, partially due to the induction of apoptosis, because NFkB is well known to be a survival factor preventing cell death in various cells.11 To examine the activation of osteoclasts, we performed a pit formation on dentine slices, which was 150 mM thick, 6 mm in diameter generated from Mommoth dentin, seeded mononuclear cells from rabbit bone marrow. We transfected NFkB decoy ODN at 7 days. Although we confirmed the similar expression of TRAP positive cells on dentine slides in the treatment of both NFkB or scramble decoy ODN, the amount of positive pit staining were decreased in cells trasnfected with NFkB decoy ODN at 10 days (Figure 4a). We also employed a pit formation assay on human adult teeth, as an ex vivo culture system. Vitamin D3 increased the amount of positive pit staining at 3 days on dentine slices after the transfection of NFkB decoy, as shown in Figure 4b. Importantly, transfection of NFkB decoy ODN resulted in significant inhibition of osteoclast activity in a dose-dependent manner, as assessed by number of pits (Figure 4c). These results suggest that NFkB decoy ODN inhibited osteoclast activation induced by vitamin D3, in addition to differentiation.

Effects of NFkB decoy ODN on osteoporosis in rat ovariectomy model To further clarify the role of NFkB on the activation of osteoclasts, we employed rat ovariectomy model of estrogen deficiency as a model of osteoporosis. At 14 days after bilateral ovariectomy, serum estradiol level was significantly decreased in ovariectomy group, while there was no significant difference in body weight (Figure 5a). To confirm the transfection into the bone, FITC-labeled ODN was administered by osmotic minipomp. As shown in Figure 5b, fluorescence could be detected in the tibia. Consistent with the targeted cells, TRAP staining positive cells was also detected after ovariectomy (Figure 5c). Indeed, TRAP activity was significantly increased in tibia and femur of ovariectomized rats (Figure 5d). In contrast, the continuous treatment of NFkB decoy ODN using osmotic minipomp attenuated the increase in the TRAP staining positive area and TRAP activity in tibia and femur, but not scramble decoy ODN (Figure 5c and d). Inhibition of


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Figure 1 (a) Multinuclear cells are formed during differentiation of bone marrow cells. Representative phase contrast microscopic views showing differentiation of bone marrow cells induced by vitamin D3 after 7 days, with Hoechst 33258 staining, TRAP staining ( 40), and dual view of TRAP staining and Hoechst 33258 staining ( 100). (b) Effects of vitamin D3 on luciferase activity driven by NFkB binding sites in bone marrow cells. At 4 h or 24 h after vitamin D3 (1 108 M) treatment. *Po0.01 compared to control group. N ¼ 6 per group. (c) Fluorescence due to transfection of FITC-labeled NFkB decoy ODN (1 mM) in cells derived from bone marrow with Hoechst 33258 staining at 1 days (left panel: 40) and 7 days (right panel: 100) after vitamin D3 treatment. (d) Effect of NFkB decoy ODN on luciferase activity driven by NFkB binding sites in bone marrow cells at 4 days after vitamin D3 treatment. NFkB decoy ODN (1 mM, 0.5 mM) or scrambled decoy ODN (1 mM) were transfected with vitamin D3 treatment (1 108 M). *Po0.01 compared to control. wPo0.01 compared to vitamin D3. N ¼ 6 per group.

the activation of osteoclasts was also confirmed by the measurement of the calcium content in tissue. As shown in Figure 5e, the concentration of calcium in tibia and femur were decreased after ovarectomy, whereas the treatment of NFkB decoy ODN significantly attenuated them. These results were accompanied with the change of urinary deoxypyridinoline that were released from bone by processing of tissue collagen. The treatment of NFkB, but not scramble decoy, ODN ameliorated the ovariectomy-induced increase in urinary deoxypyridinoline (Po0.01, Figure 5f). These results suggest that the treatment of NFkB decoy ODN ameliorated the estrogen deficieny-induced osteoporosis.

Effects of NFkB decoy ODN on osteoporosis in vitamin C deficient rat To further confirm the therapeutic potential of NFkB decoy in osteoporosis, we employed osteogenic disorder Shionogi rat, which lack the key enzyme for ascorbate synthesis leading to decrease several hydroxylases such as proline-hydroxylase.12 After withdrawal of L-ascorbate-supplement diet, the body weight was dramatically decreased and the length of bone was shortened. However, the treatment of NFkB decoy ODN inhibited the decrease in the body weight (Figure 6a) and the length (control: 36.870.2, NFkB decoy: 32.670.2*, Scramble: 30.170.5 mm, *Po0.05 vs Scramble) and weight (control; Gene Therapy


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Figure 2 (a and b) Representative example of TRAP and Hoechst nuclear staining ( 40) and quantitative analysis of NFkB decoy ODN on number of TRAP-positive stained cells at 7 days in (a) vitamin D3-treated mononuclear cells from rabbit bone marrow and (b) RANKL and M-CSF-induced rat preosteoclasts. Control ¼ treated with only vitamin D3 (1 108 M), NFkB decoy (0.25 mM) ¼ treated with vitamin D3 and NFkB decoy ODN (0.25 mM), NFkB decoy (0.5 mM) ¼ treated with vitamin D3 and NFkB decoy ODN (0.5 mM), NFkB decoy (1 mM) ¼ treated with vitamin D3 and NFkB decoy ODN (1 mM), scrambled decoy (1 mM) ¼ treated with vitamin D3 and scrambled decoy ODN (1 mM). *Po0.01 compared to control. N ¼ 6 per group.

0.86670.01, NFkB decoy: 0.63970.019*, Scramble: 0.55770.010 g, *Po0.05 vs Scramble) of femur. The bone fracture was frequently found in proximal femur in scramble decoy ODN transfected groups, but not in NFkB decoy ODN. As shown in Figure 6b, femoral head (arrow) was preserved in only NKkB decoy ODN treated groups. Of importance, bone density analysis by dualenergy X-ray absorptiometry showed the significant increase in NFkB decoy ODN-transfected group in femur. These results suggest that the treatment of NFkB Gene Therapy

decoy ODN ameliorates osteoporotic changes in osteogenic disorder Shionogi rat.

Discussion Osteoclasts are differentiated from hematopoietic precursors of the monocyte/macrophage lineage that also give rise to macrophages or dendritic cells, which mediate immune responses. An essential transcription factor for inflammation, NFkB, plays a pivotal role in

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Figure 3 Annexin V and caspase-3 activity by transfection of NFkB decoy ODN in vitamin D3-treated mononuclear cells from rabbit bone marrow. ‘control’ ¼ treated with vitamin D3 (1 108 M), ‘scramble’ ¼ treated with vitamin D3 (1 108 M) and transfected with scrambled decoy ODN (1 mM), ‘NFkB decoy’ ¼ treated with vitamin D3 (1 108 M) and transfected with NFkB decoy ODN (1 mM), ‘FITC-NFkB decoy’ ¼ treated with vitamin D3 (1 108 M) and transfected with FITC-labeled NFkB decoy ODN (1 mM).

the coordinated transactivation of cytokine and adhesion molecule genes whose activation has been postulated to be involved in numerous diseases including arthritis. Numerous cytokines including interleukin-1, -2, -6, -8 and TNF-a, to name a few, are regulated by NFkB, whereas adhesion molecules, such as VCAM and ICAM are also known to be upregulated by NFkB. A critical role of NFkB in osteoclastogenesis has also been demonstrated from the phenotype of NFkB1 and NFkB2deficient mice. In addition, M-CSF and RANKL, which are regulated by NFkB, are known to be essential and sufficient for differentiation of osteoclast precursors into mature bone-resorbing osteoclasts. Nevertheless, as the role of NFkB in bone remodeling is not yet clarified, in this study, we investigated the role of NFkB in osteoclast activation as well as differentiation using rabbit bone marrow cells and human dentine. Osteoclasts are monocyte/macrophage lineage multinucleated cells that play a critical role in bone resorption. The receptor activator of NFkB ligand (RANKL) is a TNF-related cytokine which is critical for osteoclast differentiation from hematopoietic precursors.4,5 To find a new therapeutic strategy involving bone remodeling, we used a ‘decoy’ approach in this study, as synthetic ds DNA with high affinity for transcription factors can be introduced as ‘decoy’ cis-elements to bind transcription factors and block the activation of genes mediating diseases. Expectedly, transfection of NFkB decoy ODN into macrophages attenuated their differentiation into osteoclasts. Although RANKL is the sole factor responsible for inducing osteoclast differentiation, TNF-a is also known to induce osteoclast differentiation from M-CSFdependent bone marrow-derived macrophages in the absence of RANKL and osteoblasts/stromal cells.13 Since NFkB decoy ODN significantly suppressed the production of TNF-a, the inhibitory effects of NFkB ODN

might be mediated by inhibition of both RANKL and TNF-a. Together with our present results, NFkB might contribute to the differentiation of macrophages into osteoclasts through both RANKL-dependent and M-CSF-dependent pathways. In addition, the inhibition of NFkB by decoy ODN also inhibited the activation of osteoclasts, in a pit formation assay. Thus, inhibition of NFkB has therapeutic aspects in both the inhibition of differentiation of osteoclasts in a quantitative manner and the blockade of activation of osteoclasts in a functional manner (Figure 7). This might be an advantage over OPG in bone resorption therapy, because of their broad application. NFkB is also involved in the apoptosis cascade, and recent evidence suggests that it has a protective effect against TNF-a or Fas ligand stimulation. Of importance, the uptake of NFkB decoy ODN was relatively restricted in macrophage/monocyte cells in this study, because of phagocytotic activity. Consist with previous report,14 NFkB decoy ODN may selectively induce cell death in monocyte/macrophage cells and thus in osteoclasts of all their differentiation stages. These phenomena come out to be great advantages considering the side effect to other cell types especially to osteoblasts, since all decoy ODN used in this experiments were trasfected in naked style. Nonviral and free cationic liposome manipulations may provide more simple and safe clinical application. We also put an emphasis on the advantage to use a vitamin D3 stimulated bone marrow culture system to investigate osteoclastgenesis in vitro. Since so many signal pathways and cytokine networks are involved in bone remodeling and metabolic diseases, M-CSF and RANKL stimulated assays are simple and clear in a sense, however, may not reflect all reactions occur in vivo. Thus, vitamin D3 stimulated bone marrow cultures might provides natural environment to investigate more close to nature. To further confirm the role of NFkB in the bone remodeling, we evaluated the effects of NFkB decoy ODN on two different osteoporosis model. Rat ovariectomy model of estrogen deficiency was used as a model of osteoporosis, as the loss of estrogen at menopause is a major contributor to disease pathogenesis. Estrogen is a principal negative regulator of osteoclast activity, and osteoclasts are the main effector cells responsible for bone remodeling in osteoporosis.15 We also used osteogenic disorder Shionogi rat, which lack the key enzyme for ascorbate synthesis leading to decrease several hydroxylases, such as proline-hydroxylase. Of importance, the present study revealed the therapeutic efficacy of the inhibition of NFkB activation using decoy. Inhibition of NFkB activity by decoy strategy dramatically improved the bone length or weight and density. As approximately 100 million people are estimated to suffer from this disease worldwide,16 our results indicate that NFkB could be a candidate target to treat osteoporosis. In addition, the previous studies examined the treatment of RA with NFkB decoy ODN, on the basis that RA is a chronic inflammatory disease characterized by synovial proliferation mediated by overexpression of inflammatory cytokines, especially IL-1 and TNF-a, which are regulated by NFkB. Especially, it is believed that these cytokines play an important role in the pathogenesis of joint destruction in arthritic conditions.17,18 Biological agents targeting these cytokines have been successful in both experimental models and human trials of RA,19,20

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Figure 4 (a) Effect of NFkB decoy ODN on pit formation using densitine slide. Left panel shows TRAP staining and right panel shows pit formation in each treated group (upper 5, lower 40). ‘Scramble’ ¼ treated with vitamin D3 (1 108 M) and transfected with scrambled decoy ODN (1 mM), ‘NFkB decoy’ ¼ treated with vitamin D3 (1 108 M) and transfected with NFkB decoy ODN (1 mM). (b) Effect of NFkB decoy ODN on pit formation at 3 days by Vitamin D3-treated mature osteoclasts. Representative example ( 5) and quantitative analysis of NFkB decoy ODN on pit formation at 3 days by Vitamin D3-treated mature osteoclasts. Scrambled decoy (1 mM) ¼ treated with vitamin D3 (1 108 M) and scrambled decoy ODN (1 mM), NFkB Decoy (0.25, 0.5, 1 mM) ¼ treated with vitamin D3 (1 108 M) and NFkB decoy ODN (0.25. 0.5, 1 mM). Po0.01 vs scrambled decoy ODN.

especially regarding bone destruction. It was recently reported that a cell-permiable inhibitor of the IkB-kinase complex prevented inflammatory bone destruction.21 Indeed, in vivo transfection of NFkB decoy ODN by intraarticular injection into a collagen-induced rat RA model decreased the severity of hind-paw swelling.10 Histologic and radiographic studies showed marked suppression of joint destruction by transfection of NFkB decoy ODN, accompanied by suppression of the production of IL-1 and TNF-a in the synovium of arthritic joints. Intraarticular administration of NFkB decoy ODN prevented the recurrence of streptococcal cell wall-induced arthritis in treated joints.10 Interestingly, suppression of synovial cell proliferation was achieved by NFkB decoy ODN in synovial cells derived from RA patients, through Gene Therapy

inhibition of the production of IL-1b, IL-6, TNFa, ICAM-1 and MMP-1.10 The inhibitory effects on the differentiation to osteoclasts and the activation of osteoclasts by NFkB decoy ODN might contribute to the therapeutic effects on joint destruction. Our preliminary study also showed inhibitory effects of NFkB decoy ODN on bone destruction in a cynomolgus monkey CIA model, which shares various features with human rheumatoid arthritis as a preclinical model of arthritis. Based upon these data, clinical trials of the treatment of RA patients with NFkB decoy ODN started in 2003 at Osaka University. Although the clinical trial is ongoing, gene therapy based on NFkB decoy ODN appears to be useful for the treatment of bone-related diseases, including arthritis and OA and osteoporosis.


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Figure 5 Effect of NFkB decoy ODN in the model of ovariectomyinduced osteoporosis. (a) body weight and serum estradiol levels, (b) upper panel shows hematoxylin staining and lower panel shows fluorescence in the cryosection of proximal tibia ( 40) after treatment of FITC-labeled NFkB decoy ODN (30 mg/kg/h). (c) TRAP staining in distal femur at 14 days after treatment ( 100), (d) TRAP activities and (e) calcium concentration in proximal tibia and distal femur in four groups at 14 days after treatment. (f) Urinary deoxypyridinoline in four groups at 14 days after treatment, that was adjusted with urinary creatine concentration. Sham ¼ sham operation, OVX ¼ bilateral ovariectomy, NFkB ¼ bilateral ovariectomy and treated with NFkB decoy ODN (30 mg/kg/h), Scb ¼ scrambled decoy ODN (30 mg/kg/h) ¼ bilateral ovariectomy and treated with scramble decoy ODN (30 mg/kg/h). *Po0.05 vs sham. wPo0.05 vs scb ODN, N ¼ 8 per group.

Materials and methods Bone marrow cell culture for osteoclast differentiation Bone marrow cells were obtained from 3-day-old neonatal white rabbits as previously described.21 Briefly, rabbit bone marrow cells were flushed out from the femur and tibiae, collected into tubes, and washed twice with PBS. Mononuclear rich cell fraction were separated

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Figure 6 (a and c) Effect of NFkB decoy ODN on (a) the decrease in body weight after L-ascorbate withdrawal, and (c) bone density as assessed by Dual-energy X-ray absorptiometry analysis at 28 days after treatment in the model of osteogenic disorder Shionogi rats. (b) Representative pictures of femur in rats transfected with scramble and NFkB decoy ODN. Femoral head (arrow) was preserved in only NKkB decoy ODN treated groups. ‘NFkB decoy’ ¼ osteogenic disorder Shionogi rats fed with vitamin C-deficient diet and transfected with NFkB decoy ODN (30 mg/ kg/h), ‘Scramble’ ¼ osteogenic disorder Shionogi rats fed with vitamin C-deficient diet and transfected with scramble decoy ODN (30 mg/kg/h). ‘Control’ ¼ age matched Wister rats fed with vitamin C-deficient diet *Po0.05 vs scramble, N ¼ 6 per group.

from marrow cells and cultured (1 105 cells/well of 24-well plate) in a-MEM medium containing 10% fetal bovine serum. Cultures were fed every 3 days with a-MEM medium with vitamin D3 (1 108 M). Bone marrow cells were also transfected with the luciferase gene driven by NFkB binding site (BD Bioscience Clontech, Palo Alto, CA, USA) using lipofectAMINE 2000 (Invitrogen, Grand Island, NY, USA).22 Transfected cells were incubated for 4 h or 48 h with vitamin D3 (1 108 M), washed with PBS, and lysed for 15 min with 200 ml cell lysis buffer at 41C. Then, 20 ml cell extract was mixed with 100 ml luciferase assay reagent, and the light produced was measured for 2 s using a luminometer. Sequences of the NFkB or scrambled ODNs were as follows: NFkB decoy ODN: (consensus sequences are underlined) as previous described.10 50 -CCTTGAAGGGATTTCCCTCC-30 30 -GGAACTTCCCTAAAGGGAGG-50 Gene Therapy


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osteoclastogenesis Figure 7 Summary of the inhibitory effect by NFkB in osteoclastogenesis. Vitamin D3 induces osteoclast formation via the vitamin D3 receptor present in the nucleus of osteoblasts. Osteoclast precursors having the RANKL receptor recognize RANKL by cell–cell contact and differentiate into mature osteoclasts. The transcription factor, nuclear factor-kB (NFkB) plays a pivotal role in the activation of nuclear factor of activated T cells c1 (NFATc1), which regulate the osteoclastogenesis. NFkB decoy ameliorates the osteoclastogenesis through the inhibition of NFkB activity.

Scrambled decoy ODN: 50 -TTGCCGTACCTGACTTAGCC-30 30 -AACGGCATGGACTGAATCGG-30 Synthetic ODNs were washed with 70% ethanol, dried and dissolved in sterile Tris-EDTA (10 mM Tris, 1 mM EDTA) and the supernatant was purified over a NAP 10 column (Pharmacia, Piscataway, NJ, USA).

Tartrate-resistant acid phosphatase staining After vitamin D3 (1 108 M) treatment with scrambled or NFkB decoy ODN, cells were fixed on day 7 of culture with 4% paraformaldehyde in PBS for 10 min at room temperature before being stained for TRAP. Enzyme histochemical staining for TRAP was performed as previously reported.23 Annexin V, Caspase 3, and Hoechst 33258 nuclear staining After vitamin D3 (1 108 M) treatment with scrambled or NFkB decoy ODN, cells were stained on day 7 of culture with Hoechst 33258 (10 mM) for 10 min and analyzed under a nonconfocal fluorescence microscope (Olympus BX60) with excitation at 360 nm as previously described.24 Cells were also stained with fluorescein isothiocyanate (FITC)-labeled annexin V (MBL, Nagoya) and CPP32/Caspase-3 colorimetric protease assay kit (MBL, Nagoya) according to the manufacturer’s instructions. Assessment by osteoclast formation kit Osteoclast differentiation was also examined by rat osteoclast culture system kit obtained from Hokudo Company (Hokudo, Sapporo). Rat osteoclasts precursor cells seeded in a 24-well plate were incubated with M-CSF and RANKL-containing medium. After 7 days, TRAP staining and Hoechst 33526 nuclear staining was performed as previously reported.22 Gene Therapy

Pit formation The inhibitory effects of NFkB decoy ODN on pitforming activity were determined by the method previously reported.25 We used the mammoth dentin slice obtained from Hokudo Company (Hokudo, Sapporo) and human teeth from patients at Osaka Dental University after their agreement, cut them into 3–4 mm thick dentin slices with dental diamond saw and placed them on 24-well culture plates. Bone marrow cells (1 104 /ml) 6 days after vitamin D3 treatment were incubated on dentin slices for 6 h, and after washing with PBS, further incubated with vitamin D3 (1 108 M) and cotreated with NFkB decoy ODN or scrambled decoy ODN for 3 days. After removing organic substances with sodium hypocloride, the slices were stained with toluidine blue (Sigma Chemical Co.) to visualize resorption pits, and the number of round dots (osteoclast pits) in slices, which has been previously reported to correlate with osteoclast activity,25 as counted by light microscopic observation. Rat osteoporosis model by ovariectomy and osteogenic disorder Shionogi Female adult Wister Rats (10 weeks old) were purchased from SLC Japan (Shizuoka, Japan). After the rats were anesthetized with intraperitoneal ketamine (80 mg/kg) and xylazine (10 mg/kg), bilateral ovariectomy or a sham operation was performed and osmotic minipumps (Alzet model 2004; Alza Corp) containing either NFkB or scramble decoy ODN (infusion rate 30 mg/kg/h) were implanted. All experimental protocols were approved by the Nara Women’s University Standing Committee on Animals. Body weights of these mice were recorded during the 2 weeks. At 2 weeks after operation, they were deeply anesthetized and femurs, tibias, blood and urine were collected for biochemical analysis. Proximal tibia and distal femur were cut out and homogenized in 10 mM Triethanolamin Buffer (pH 7.5), and supernatants were exposed for TRAP activities measured by the method of Walter,26 and precipitates were hybridized by HCl (6 M) in 1051C for 24 h to measure hydroxyproline on the method by Bergmana,27 and Ca content were measured based on the OCPC method by Gitelman.28 Estradiol level in serum was measured by EIA (Mitsubishi Kagaku latron, Tokyo, Japan), and urinary deoxypyridinoline levels were measured on day 14 of the experiments by EIA (Metra Biosystems, Mountain View, CA, USA). Osteogenic disorder shionogi (ODS), a mutant strain of Wistar rats, lack the key enzyme for ascorbate synthesis. Male ODS rats (8 weeks old) were purchased from CLEA laboratory and fed L-ascorbic acid-supplement diet (800 mg/kg). After 1 week, the diet was changed into L-ascorbic acid deficient and osmotic minipumps containing either NFkB or scramble decoy ODN (infusion rate 30 mg/kg/h) were implanted. The body weight was measured before and 4 weeks after L-ascorbate acid withdrawal. At 4 weeks after operation, the tibia and femur were analyzed. Dual energy X-ray absorptiometry (DEXA) Bone density measurements were performed using a dual-photon X-ray absorbsiometry (DEXA) bone densitometry (GE-Lunar DPX-IQ, Madison, WI, USA). Highand low-beam energies for all scans were 80 and 35 kV,


respectively, at 0.5 mA as previously described.29 Bone mineral density (BMD) was obtained in g/cm2.

Statistical analysis All values are expressed as mean7s.e.m. Analysis of variance with subsequent Bonferroni’s/Dunnet’s test was employed to determine the significance of differences in multiple comparisons. Values of Po0.05 were considered to be statistically significant.

Acknowledgements We wish to thank Miss C Kato, N Yasumasa and R Hanayama for their helpful job. This work was partially supported by a Grant-in-Aid from the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (NIBIO), the Organization for Pharmaceutical Safety and Research, a Grant-in-Aid from The Ministry of Public Health and Welfare, a Grant-in-Aid from Japan Promotion of Science, and through Special Coordination Funds of the Ministry of Education, Culture, Sports, Science and Technology, the Japanese Government.

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