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Research ArticleBone biology
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10.1172/jci.insight.171962
1Department of Orthopaedic Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
2Department of Orthopaedic Surgery, Nippon Life Hospital, Nishi-ku, Osaka, Japan.
3Department of Obstetrics and Gynecology, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
4Department of Orthopaedic Surgery, National Hospital Organization Osaka Minami Medical Center, Kawachinagano, Osaka, Japan.
5Department of Orthopaedic Surgery, National Hospital Organization Osaka Toneyama Medical Center, Toyonaka, Osaka, Japan.
6Department of Orthopaedic Surgery, Osaka Rosai Hospital, Kita-ku, Sakai, Japan.
7Department of Health and Sport Sciences, and
8Department of Musculoskeletal Regenerative Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
Address correspondence to: Kosuke Ebina, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan. Phone: 81.6.6210.8439; Email: k-ebina@ort.med.osaka-u.ac.jp.
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1Department of Orthopaedic Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
2Department of Orthopaedic Surgery, Nippon Life Hospital, Nishi-ku, Osaka, Japan.
3Department of Obstetrics and Gynecology, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
4Department of Orthopaedic Surgery, National Hospital Organization Osaka Minami Medical Center, Kawachinagano, Osaka, Japan.
5Department of Orthopaedic Surgery, National Hospital Organization Osaka Toneyama Medical Center, Toyonaka, Osaka, Japan.
6Department of Orthopaedic Surgery, Osaka Rosai Hospital, Kita-ku, Sakai, Japan.
7Department of Health and Sport Sciences, and
8Department of Musculoskeletal Regenerative Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
Address correspondence to: Kosuke Ebina, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan. Phone: 81.6.6210.8439; Email: k-ebina@ort.med.osaka-u.ac.jp.
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1Department of Orthopaedic Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
2Department of Orthopaedic Surgery, Nippon Life Hospital, Nishi-ku, Osaka, Japan.
3Department of Obstetrics and Gynecology, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
4Department of Orthopaedic Surgery, National Hospital Organization Osaka Minami Medical Center, Kawachinagano, Osaka, Japan.
5Department of Orthopaedic Surgery, National Hospital Organization Osaka Toneyama Medical Center, Toyonaka, Osaka, Japan.
6Department of Orthopaedic Surgery, Osaka Rosai Hospital, Kita-ku, Sakai, Japan.
7Department of Health and Sport Sciences, and
8Department of Musculoskeletal Regenerative Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
Address correspondence to: Kosuke Ebina, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan. Phone: 81.6.6210.8439; Email: k-ebina@ort.med.osaka-u.ac.jp.
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1Department of Orthopaedic Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
2Department of Orthopaedic Surgery, Nippon Life Hospital, Nishi-ku, Osaka, Japan.
3Department of Obstetrics and Gynecology, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
4Department of Orthopaedic Surgery, National Hospital Organization Osaka Minami Medical Center, Kawachinagano, Osaka, Japan.
5Department of Orthopaedic Surgery, National Hospital Organization Osaka Toneyama Medical Center, Toyonaka, Osaka, Japan.
6Department of Orthopaedic Surgery, Osaka Rosai Hospital, Kita-ku, Sakai, Japan.
7Department of Health and Sport Sciences, and
8Department of Musculoskeletal Regenerative Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
Address correspondence to: Kosuke Ebina, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan. Phone: 81.6.6210.8439; Email: k-ebina@ort.med.osaka-u.ac.jp.
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1Department of Orthopaedic Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
2Department of Orthopaedic Surgery, Nippon Life Hospital, Nishi-ku, Osaka, Japan.
3Department of Obstetrics and Gynecology, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
4Department of Orthopaedic Surgery, National Hospital Organization Osaka Minami Medical Center, Kawachinagano, Osaka, Japan.
5Department of Orthopaedic Surgery, National Hospital Organization Osaka Toneyama Medical Center, Toyonaka, Osaka, Japan.
6Department of Orthopaedic Surgery, Osaka Rosai Hospital, Kita-ku, Sakai, Japan.
7Department of Health and Sport Sciences, and
8Department of Musculoskeletal Regenerative Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
Address correspondence to: Kosuke Ebina, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan. Phone: 81.6.6210.8439; Email: k-ebina@ort.med.osaka-u.ac.jp.
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1Department of Orthopaedic Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
2Department of Orthopaedic Surgery, Nippon Life Hospital, Nishi-ku, Osaka, Japan.
3Department of Obstetrics and Gynecology, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
4Department of Orthopaedic Surgery, National Hospital Organization Osaka Minami Medical Center, Kawachinagano, Osaka, Japan.
5Department of Orthopaedic Surgery, National Hospital Organization Osaka Toneyama Medical Center, Toyonaka, Osaka, Japan.
6Department of Orthopaedic Surgery, Osaka Rosai Hospital, Kita-ku, Sakai, Japan.
7Department of Health and Sport Sciences, and
8Department of Musculoskeletal Regenerative Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
Address correspondence to: Kosuke Ebina, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan. Phone: 81.6.6210.8439; Email: k-ebina@ort.med.osaka-u.ac.jp.
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1Department of Orthopaedic Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
2Department of Orthopaedic Surgery, Nippon Life Hospital, Nishi-ku, Osaka, Japan.
3Department of Obstetrics and Gynecology, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
4Department of Orthopaedic Surgery, National Hospital Organization Osaka Minami Medical Center, Kawachinagano, Osaka, Japan.
5Department of Orthopaedic Surgery, National Hospital Organization Osaka Toneyama Medical Center, Toyonaka, Osaka, Japan.
6Department of Orthopaedic Surgery, Osaka Rosai Hospital, Kita-ku, Sakai, Japan.
7Department of Health and Sport Sciences, and
8Department of Musculoskeletal Regenerative Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
Address correspondence to: Kosuke Ebina, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan. Phone: 81.6.6210.8439; Email: k-ebina@ort.med.osaka-u.ac.jp.
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1Department of Orthopaedic Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
2Department of Orthopaedic Surgery, Nippon Life Hospital, Nishi-ku, Osaka, Japan.
3Department of Obstetrics and Gynecology, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
4Department of Orthopaedic Surgery, National Hospital Organization Osaka Minami Medical Center, Kawachinagano, Osaka, Japan.
5Department of Orthopaedic Surgery, National Hospital Organization Osaka Toneyama Medical Center, Toyonaka, Osaka, Japan.
6Department of Orthopaedic Surgery, Osaka Rosai Hospital, Kita-ku, Sakai, Japan.
7Department of Health and Sport Sciences, and
8Department of Musculoskeletal Regenerative Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
Address correspondence to: Kosuke Ebina, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan. Phone: 81.6.6210.8439; Email: k-ebina@ort.med.osaka-u.ac.jp.
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1Department of Orthopaedic Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
2Department of Orthopaedic Surgery, Nippon Life Hospital, Nishi-ku, Osaka, Japan.
3Department of Obstetrics and Gynecology, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
4Department of Orthopaedic Surgery, National Hospital Organization Osaka Minami Medical Center, Kawachinagano, Osaka, Japan.
5Department of Orthopaedic Surgery, National Hospital Organization Osaka Toneyama Medical Center, Toyonaka, Osaka, Japan.
6Department of Orthopaedic Surgery, Osaka Rosai Hospital, Kita-ku, Sakai, Japan.
7Department of Health and Sport Sciences, and
8Department of Musculoskeletal Regenerative Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
Address correspondence to: Kosuke Ebina, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan. Phone: 81.6.6210.8439; Email: k-ebina@ort.med.osaka-u.ac.jp.
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1Department of Orthopaedic Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
2Department of Orthopaedic Surgery, Nippon Life Hospital, Nishi-ku, Osaka, Japan.
3Department of Obstetrics and Gynecology, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
4Department of Orthopaedic Surgery, National Hospital Organization Osaka Minami Medical Center, Kawachinagano, Osaka, Japan.
5Department of Orthopaedic Surgery, National Hospital Organization Osaka Toneyama Medical Center, Toyonaka, Osaka, Japan.
6Department of Orthopaedic Surgery, Osaka Rosai Hospital, Kita-ku, Sakai, Japan.
7Department of Health and Sport Sciences, and
8Department of Musculoskeletal Regenerative Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
Address correspondence to: Kosuke Ebina, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan. Phone: 81.6.6210.8439; Email: k-ebina@ort.med.osaka-u.ac.jp.
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1Department of Orthopaedic Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
2Department of Orthopaedic Surgery, Nippon Life Hospital, Nishi-ku, Osaka, Japan.
3Department of Obstetrics and Gynecology, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
4Department of Orthopaedic Surgery, National Hospital Organization Osaka Minami Medical Center, Kawachinagano, Osaka, Japan.
5Department of Orthopaedic Surgery, National Hospital Organization Osaka Toneyama Medical Center, Toyonaka, Osaka, Japan.
6Department of Orthopaedic Surgery, Osaka Rosai Hospital, Kita-ku, Sakai, Japan.
7Department of Health and Sport Sciences, and
8Department of Musculoskeletal Regenerative Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
Address correspondence to: Kosuke Ebina, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan. Phone: 81.6.6210.8439; Email: k-ebina@ort.med.osaka-u.ac.jp.
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1Department of Orthopaedic Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
2Department of Orthopaedic Surgery, Nippon Life Hospital, Nishi-ku, Osaka, Japan.
3Department of Obstetrics and Gynecology, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
4Department of Orthopaedic Surgery, National Hospital Organization Osaka Minami Medical Center, Kawachinagano, Osaka, Japan.
5Department of Orthopaedic Surgery, National Hospital Organization Osaka Toneyama Medical Center, Toyonaka, Osaka, Japan.
6Department of Orthopaedic Surgery, Osaka Rosai Hospital, Kita-ku, Sakai, Japan.
7Department of Health and Sport Sciences, and
8Department of Musculoskeletal Regenerative Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
Address correspondence to: Kosuke Ebina, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan. Phone: 81.6.6210.8439; Email: k-ebina@ort.med.osaka-u.ac.jp.
Find articles by Nakata, K. in: JCI | PubMed | Google Scholar
1Department of Orthopaedic Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
2Department of Orthopaedic Surgery, Nippon Life Hospital, Nishi-ku, Osaka, Japan.
3Department of Obstetrics and Gynecology, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
4Department of Orthopaedic Surgery, National Hospital Organization Osaka Minami Medical Center, Kawachinagano, Osaka, Japan.
5Department of Orthopaedic Surgery, National Hospital Organization Osaka Toneyama Medical Center, Toyonaka, Osaka, Japan.
6Department of Orthopaedic Surgery, Osaka Rosai Hospital, Kita-ku, Sakai, Japan.
7Department of Health and Sport Sciences, and
8Department of Musculoskeletal Regenerative Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
Address correspondence to: Kosuke Ebina, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan. Phone: 81.6.6210.8439; Email: k-ebina@ort.med.osaka-u.ac.jp.
Find articles by Okada, S. in: JCI | PubMed | Google Scholar
1Department of Orthopaedic Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
2Department of Orthopaedic Surgery, Nippon Life Hospital, Nishi-ku, Osaka, Japan.
3Department of Obstetrics and Gynecology, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
4Department of Orthopaedic Surgery, National Hospital Organization Osaka Minami Medical Center, Kawachinagano, Osaka, Japan.
5Department of Orthopaedic Surgery, National Hospital Organization Osaka Toneyama Medical Center, Toyonaka, Osaka, Japan.
6Department of Orthopaedic Surgery, Osaka Rosai Hospital, Kita-ku, Sakai, Japan.
7Department of Health and Sport Sciences, and
8Department of Musculoskeletal Regenerative Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
Address correspondence to: Kosuke Ebina, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan. Phone: 81.6.6210.8439; Email: k-ebina@ort.med.osaka-u.ac.jp.
Find articles by Ebina, K. in: JCI | PubMed | Google Scholar
Published November 22, 2023 - More info
Published in Volume 8, Issue 22 on November 22, 2023
AbstractNF-κB is a transcription factor that is activated with aging. It plays a key role in the development of osteoporosis by promoting osteoclast differentiation and inhibiting osteoblast differentiation. In this study, we developed a small anti–NF-κB peptide called 6A-8R from a nuclear acidic protein (also known as macromolecular translocation inhibitor II, Zn2+-binding protein, or parathymosin) that inhibits transcriptional activity of NF-κB without altering its nuclear translocation and binding to DNA. Intraperitoneal injection of 6A-8R attenuated ovariectomy-induced osteoporosis in mice by inhibiting osteoclast differentiation, promoting osteoblast differentiation, and inhibiting sclerostin production by osteocytes in vivo with no apparent side effects. Conversely, in vitro, 6A-8R inhibited osteoclast differentiation by inhibiting NF-κB transcriptional activity, promoted osteoblast differentiation by promoting Smad1 phosphorylation, and inhibited sclerostin expression in osteocytes by inhibiting myocyte enhancer factors 2C and 2D. These findings suggest that 6A-8R has the potential to be an antiosteoporotic therapeutic agent with uncoupling properties.
Graphical Abstract
Introduction
Bone homeostasis is primarily maintained by the cooperation of osteoblasts and osteoclasts — a process known as bone coupling (1, 2). Bisphosphonates and denosumab are commonly used antiosteoporotic drugs. They mainly inhibit bone resorption by osteoclasts, although they also inhibit bone formation by osteoblasts via by inhibiting coupling. Therefore, the prolonged use of these drugs may increase the risk of serious adverse events such as osteonecrosis of the jaw and atypical femoral fractures (3). Regarding bone anabolic agents, teriparatide increases bone formation as well as bone resorption, leading to cortical porosity and transient loss of bone mineral density of the hip (4). Romosozumab is the newest bone anabolic agent that shows uncoupling bone effects by promoting bone formation and inhibiting bone resorption through Wnt signaling activation. However, due to concerns regarding cardiovascular events, the United States Food and Drug Administration warned against using romosozumab to treat patients with a history of cardiovascular events within 1 year (5). Thus, there remains a strong need for safer bone-uncoupling agents with other mechanisms of action.
Receptor activator of NF-κB ligand (RANKL) is widely known to play a crucial role in osteoclastogenesis (6–11). The expression of its receptor, which is the receptor activator of NF-κB (RANK), is induced in osteoclast precursors by macrophage colony–stimulating factor (M-CSF) (12–14). RANKL binds to RANK and activates various signaling cascades, and it consequently activates the NF-κB cascade and nuclear factor of activated T cells 1 (NFATc1), which is the main transcription factor of osteoclast differentiation (15).
Furthermore, NF-κB plays an inhibitory role in the proliferation and differentiation of osteoblasts by inducing Smurf1 activation, which increases Smad degradation (16–19), and by promoting the degradation of runt-related transcription factor 2 (Runx2) and β-catenin (20–23). Thus, NF-κB inhibition is considered a potential uncoupling therapeutic target that promotes bone formation and inhibits bone resorption.
Although there are several previous studies on NF-κB cascade inhibitors, none of the NF-κB cascade inhibitors has yet been used in clinical settings (24, 25). This is because most of the previous studies reported inhibition of the NF-κB pathway upstream of the cascade, which may lead to side effects such as hepatocyte apoptosis, hepatocellular carcinoma, inflammation of the colon, and immune system abnormalities (24, 26–32).
In our previous study, we found that macromolecular translocation inhibitor II (MTI-II) (33) — also known as Zn2+-binding protein (34) or parathymosin (35) — inhibits the transcriptional activity of NF-κB by directly binding to NF-κB after stimulation by tumor necrosis factor-α (TNF-α) (36). Furthermore, we synthesized 40A-8R, a peptide composed of 40 amino acids of the effector site of MTI-II fused with 8 arginine residues in the C-terminus (36). Oligoarginines were added to amino acids because arginine-rich peptides can be efficiently internalized into cells, and they are widely used as carriers for the intracellular delivery of bioactive molecules (37–39).
Systemic TNF-α levels were found to be elevated in ovariectomized mice (40–48), and TNF-α activates osteoclasts via NF-κB and promotes sclerostin production in osteocytes (49–51). Moreover, it has been reported that the activation of NF-κB inhibits osteoblast differentiation. Therefore, we hypothesized that MTI-II and related molecules are good candidates for bone-uncoupling antiosteoporotic treatment for ovariectomy-induced (OVX-induced) osteoporosis. However, long peptides, such as 40A-8R, are expensive; thus, their clinical application was considered unfeasible. Hence, we decided to create a small peptide molecule, the molecular size of which may be advantageous in terms of facilitating synthesis and reducing immunogenicity of the peptide, although 40A-8R showed no immune epitope based on a search of the immune epitope database (https://www.iedb.org/).
In the present study, we synthesized a peptide (6A-8R) similar to 40A-8R in terms of NF-κB inhibition but with a smaller molecular weight than 40A-8R. This study aimed to investigate the safety, efficacy, and molecular mechanisms of 6A-8R in OVX-induced osteoporosis in mice.
ResultsConstruction of anti–NF-κB peptide. Our previous study reported that 40A-8R — developed from the effector site of MTI-II (Figure 1A) — shows antiinflammatory effects in various animal models of inflammatory diseases (36).
Figure 1Preparation of MTI-II–based anti–NF-κB drugs. (A) Schematic representations of MTI-II, 40A-8R, and 6A-8R. A, amino acids; R, oligoarginine residues; NLS, nuclear localization signal. (B) Amino acid sequence of 40A and the candidate sequences of 12A and 6A in the active site. The 2 effector sequences are enclosed in a box. Monotonous runs of 6 or 8 arginine residues (6R or 8R) were added to the C-terminal region of each peptide. (C) NF-κB–induced luciferase activity was measured in HeLa cells transfected with MTI-II, 12A-6R, and 6A-6R expression vectors along with 2 luciferase reporter genes (κB-Luc2P and TK-hRLuc). Luciferase activity was measured after stimulation with TNF-α (1 ng/mL). Data are expressed as a ratio of κB-Luc2P activity to TK-hRLuc activity (internal control) and are presented as mean ± SD (n = 4 without TNF-α, n = 12 with TNF-α). NC, negative (empty vector) control. (D) NF-κB–induced luciferase activity was measured in HeLa cells transfected with luciferase reporter genes (κB-Luc2P and TK-hRLuc). After 10 hours of transfection, the cells were cultured with each concentration of 12A-8R and 6A-8R for 24 hours; subsequently, TNF-α (1 ng/mL) was added. Luciferase activity was measured 4.5 hours after stimulation with TNF-α. Data are expressed as a ratio of κB-Luc2P activity to TK-hRLuc activity (internal control) and are presented as mean ± SD (n = 4 without TNF-α, n = 12 with TNF-α). (E) Quantitative real-time PCR of mouse bone marrow mononuclear cells (BMMCs) and MC3T3-E1 cells. The relative gene expression of Mti-II with or without differentiating stimulations and 6A-8R (3 mg/mL) is plotted on the y axis. Data were statistically analyzed using 1-way ANOVA and Tukey-Kramer test. **P < 0.01; ****P < 0.0001. NS, not significant.
We further selected 10 candidates of the NF-κB inhibitor (Figure 1B) and made the expression vectors of these candidates with a 6-arginine residue (6R) sequence in the C-terminus. Transfections of these candidates into HeLa cells revealed that 2 out of the 10 candidates have NF-κB inhibitory activity (the effector sequences are enclosed in a box in Figure 1B). Figure 1C shows the effects of using the 2 candidates and MTI-II in the inhibition of NF-κB transcriptional activity. Supplemental Figure 1 (supplemental material available online with this article; https://doi.org/10.1172/jci.insight.171962DS1) shows the effects of using the other 8 sequences that were less effective in suppressing NF-κB. In contrast, the addition of 12A-6R and 6A-6R peptides had no effect on NF-κB transcriptional activity (data not shown). Thus, we hypothesized that 6 arginine residues may be insufficient to internalize these peptides into cells, and we explored peptides with 8 arginine residues (8R) to improve its cell-penetrating effect. Figure 1D shows that both 12A-8R and 6A-8R peptides (1.5 and 3.0 mg/mL) inhibit NF-κB transcription. These findings suggest that 8R (not 6R) is required to internalize the peptide into cells to show its efficacy. Furthermore, the efficacy was confirmed by Western blotting to detect cyclooxygenase 2 (COX2) protein expression in HeLa cells stimulated with TNF-α (1.0 ng/mL) (Supplemental Figure 2).
Expression of MTI-II mRNA in osteoblasts and osteoclasts. The quantitative real-time polymerase chain reaction (PCR) was used to determine the extent to which MTI-II mRNA was expressed in osteoclasts (mouse bone marrow mononuclear cells [BMMCs]) and osteoblasts (MC3T3-E1 cells) before and after inducing differentiation with or without 6A-8R (3.0 mg/mL; Figure 1E).
In mouse BMMCs, MTI-II gene expression tended to increase with the induction of differentiation. Furthermore, the gene was well expressed in MC3T3-E1 cells, regardless of differentiation induction. Moreover, the presence of 6A-8R (3.0 mg/mL) did not affect the expression levels of MTI-II in each cell type during differentiation induction (Figure 1E).
Effects of 6A-8R on ovariectomized mice. To evaluate the effects of administering 6A-8R to ovariectomized mice (Figure 2A), we first investigated the effect of 6A-8R on mouse body weight and found no significant differences between before and after 6A-8R administration (Figure 2B). Furthermore, following 6A-8R administration, there was no obvious damage to the liver or kidneys (Supplemental Figure 3).
Figure 2Effects of 6A-8R on ovariectomized (OVX) mice. (A) Schematic protocol of the animal experiment. Four milligrams of 6A-8R was intraperitoneally administered 5 days per week for 4 weeks, and samples (femurs) were collected. (B) Percentage changes in the body weight of the mice from baseline in each group. (C) Micro-CT images of the distal part of the femur on day 28 after OVX with or without 6A-8R administration. (D) Cancellous bone volume (BV)/tissue volume (TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp). Data are expressed as mean ± SD (n = 7 or 9) and were statistically analyzed using 1-way ANOVA and the Tukey-Kramer test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
For 4 weeks, 4 mg of 6A-8R was administered intraperitoneally for 5 days per week, and samples (femurs) were collected. The administration dose of 6A-8R was determined based on a previously described study (see Methods) (36).
Figure 2C shows representative results of micro-computed tomography (micro-CT) of cancellous bone in the distal part of the femur on day 28. The OVX groups had significantly lower bone volume (BV)/tissue volume (TV), trabecular number (Tb.N), and trabecular thickness (Tb.Th) of cancellous bone, and had significantly higher trabecular separation (Tb.Sp). The administration of 6A-8R significantly improved these parameters and tended to improve Tb.Th (Figure 2D). The findings regarding BV exhibited similarity to BV/TV, while no significant differences were observed among the groups in terms of bone marrow density (BMD) and cortical bone parameters (total area [Tt.Ar], cortical area [Ct.Ar], and Ct.Ar/Tt.Ar) (Supplemental Figure 4).
Figures 3 and 4 show the results of the histological examination of the distal part of the femur. The number of tartrate-resistant acid phosphatase–positive (TRAP-positive) cells was significantly higher in the OVX groups than in the Sham groups, according to the results of TRAP staining; however, the administration of 6A-8R significantly reduced the numbers of these cells (Figure 3, B and C).
Figure 3Histological and histomorphometric analysis of the distal part of the femur for osteoclasts in Sham-operated and OVX mice with or without intraperitoneal injection of 6A-8R (4 mg) 5 days per week for 4 weeks. (A) Histological findings of the distal part of the femur stained with hematoxylin and eosin. Scale bar: 1 mm. (B) TRAP staining. Scale bars: 1 mm (top and middle rows) and 200 μm (bottom row). (C) Plot of the number of TRAP-positive cells per unit trabecular surface. Data are expressed as mean ± SD (n = 7 or 9). (D) Histomorphometric findings of the distal part of the femur. Plots of the number of osteoclasts (Oc.N) (N/mm), number of multinucleated osteoclasts (M.Oc.N) (N/mm), and eroded surface (ES)/bone surface (BS) (%). Data are expressed as mean ± SD (n = 4) and were statistically analyzed using 1-way ANOVA and the Tukey-Kramer test. **P < 0.01; ***P < 0.001; ****P < 0.0001.
Figure 4Histological and histomorphometric analysis of the distal part of the femur for osteoblasts and osteocytes in Sham-operated and OVX mice with or without intraperitoneal injection of 6A-8R (4 mg) 5 days per week for 4 weeks. (A) Osteocalcin staining. (B) Sclerostin staining. Scale bars (A and B): 1 mm (top rows) and 200 μm (bottom rows). (C) Plot of the number of osteocalcin-positive cells per unit trabecular surface. (D) Plot of the number of sclerostin-positive cells per total osteocytes. Data are expressed as mean ± SD (n = 7 or 9). (E) Plots of the number of osteoblasts (Ob.N) (N/mm), bone formation rate (BFR)/bone surface (BS) (mm³/mm²/year), and mineral apposition rate (MAR) (μm/day). (F) Images of MAR under fluorescent light (white arrow, double-labeled surface). Scale bars: 10 μm. Data are expressed as mean ± SD (n = 4) and were statistically analyzed using 1-way ANOVA and the Tukey-Kramer test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
Furthermore, in the Sham and OVX groups, 6A-8R administration significantly increased the number of osteocalcin-positive cells (Figure 4, A and C).
The number of sclerostin-positive cells in the cortical bone was significantly higher in the OVX groups than in the Sham groups; moreover, 6A-8R administration significantly reduced the number of sclerostin-positive cells in the OVX groups (Figure 4, B and D).
The results of the histomorphometric analysis are shown in Figure 3D and Figure 4E. The OVX groups had significantly higher numbers of osteoclasts (N.Oc)/bone surface (BS) and multinucleated osteoclasts (N.Mu.Oc)/BS as well as more eroded surface (ES)/BS than the Sham groups, and 6A-8R administration reduced these parameters in the OVX groups (Figure 3D).
Although there were no differences in the number of osteoblasts (N.Ob)/BS between groups, 6A-8R administration tended to increase both bone formation rate (BFR)/BS and mineral apposition rate (MAR) in the Sham groups (Figure 4, E and F).
Furthermore, cavities within paraffin-embedded tissue sections were designated as adipose tissue and quantified employing ImageJ software (version 1.52q, NIH). While a tendency toward augmentation was observed with OVX, and conversely, a decline was noted with 6A-8R treatment, these differences did not attain statistical significance (Supplemental Figure 5).
Effects of 6A-8R on osteoclasts. To confirm the inhibitory effect of 6A-8R on NF-κB transcriptional activity in osteoclasts, we performed luciferase assays. Notably, 6A-8R (1.5 and 3.0 mg/mL) administration significantly reduced NF-κB transcription of osteoclasts induced by mouse BMMCs in a dose-dependent manner (Figure 5A).
Figure 5Effects of 6A-8R administration on osteoclasts. (A) Mouse bone marrow mononuclear cells (BMMCs) were transfected with a luciferase reporter gene (κB-Luc2P). After 24 hours of transfection, the cells were cultured with or without RANKL (50 ng/mL) for 6 hours together with indicated concentrations of 6A-8R. Data are expressed as mean ± SD (n = 3 or 6). (B) TRAP staining was performed, and the number of TRAP-positive cells was determined by microscopy. Scale bars: 100 μm. Data are expressed as mean ± SD (n = 3). (C) The bone resorption activity of osteoclasts was evaluated using an osteo-assay plate. Data are expressed as mean ± SD (n = 4). (D and E) Western blotting analysis of mouse BMMCs cultured with RANKL (50 ng/mL) with or without 6A-8R (3 mg/mL). (F) Changes in the expression of genes involved in osteoclast differentiation was assessed. Data are expressed as mean ± SD (n = 4). (G) Immunofluorescence microscopy analysis of p65 was performed on mouse BMMCs before and after stimulation with RANKL (50 ng/mL) and with or without 6A-8R (3 mg/mL). Red, p65 immunofluorescent staining; blue, 4′,6-diamidino-2-phenylindole (DAPI) nuclear staining. Scale bars: 20 μm. (H) CUT&RUN analysis of mouse BMMCs was performed 60 minutes after stimulation with RANKL (50 ng/mL) with or without 6A-8R (3 mg/mL). Data are expressed as mean ± SD (n = 4). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by 1-way ANOVA and the Tukey-Kramer test (A–C and F) or Mann-Whitney U test (H). NS, not significant.
Moreover, TRAP staining revealed that the administration of 6A-8R (1.5 and 3.0 mg/mL) significantly reduced RANKL-induced N.Mu.Oc in a dose-dependent manner (Figure 5B). Furthermore, a resorption pit assay revealed that the administration of 6A-8R (3.0 and 10 mg/mL) significantly suppressed RANKL-induced osteoclast resorption activity in a dose-dependent manner (Figure 5C).
Western blotting revealed that 6A-8R (3.0 mg/mL) promoted p65 phosphorylation (p-p65) and p50 induction in mouse BMMCs. However, there were no apparent changes in the amount or phosphorylation of IκBα (Figure 5D). In addition, 6A-8R inhibited the induction of c-Fos and NFATc1 (Figure 5E) and slightly inhibited the induction of p52 and RelB (Supplemental Figure 6). Given that p-p65/p50 is thought to exist in the nucleus without transcription of the target gene despite binding to DNA, it is plausible that expression of the NF-κB subunits is suppressed. Owing to the suppression of the turnover by negative feedback, the levels of expression of p-p65 and p50 increased. Notably, 6A-8R had no direct effect on IκBα, and there was no change in IκBα expression. The suppression of NF-κB by 6A-8R administration reduced the expression of c-Fos and NFATc1.
Figure 5F shows the results of quantitative real-time PCR of osteoclast-related mRNAs performed after 6A-8R (3.0 and 10 mg/mL) administration. The administration of 6A-8R significantly inhibited osteoclast-related mRNA (cathepsin K [Ctsk], Trap, Mmp9, Nfatc1, calcitonin receptor [Calcr], dendritic cell-specific transmembrane protein [Dcstamp], Atp6v0d2, integrin αv [Itgav], and β3 integrin [Itgb3]) expression in a dose-dependent manner. Furthermore, 6A-8R increased Fas-related mRNA (Fas, Fasl) expression and inhibited NF-κB–related mRNA (RelB, Nfkb1, Nfkb2) expression (Supplemental Figure 7). The effects of 6A-8R (3.0 mg/mL) on p65 nuclear translocation and on the interaction between p65 and DNA binding are shown in Figure 5, G and H.
Fluorescent immunostaining revealed that the administration of 6A-8R had no effect on the nuclear translocation of p65 (Figure 5G). Furthermore, evaluation of the association between p65 and IκBα or the TNF-α promoter region using CUT&RUN data analysis revealed that 6A-8R did not affect DNA binding of p65 (Figure 5H).
Effects of 6A-8R on osteoblasts. As shown in Figure 6A, 6A-8R (1.5 and 3.0 mg/mL) increased alkaline phosphatase (ALP) staining in MC3T3-E1 cells in a dose-dependent manner. Mineralization was also evaluated in the same manner and showed significant improvement with 6A-8R administration (Figure 6B). When MC3T3-E1 cells were incubated with TNF-α (1.0 ng/mL) in the presence of recombinant human bone morphogenetic protein-2, 6A-8R (3.0 mg/mL) upregulated p-p65 similarly to that seen in osteoclasts (Figure 6C). In contrast, no changes in p50 expression were observed (Figure 6C). IκBα induction was marginally inhibited by 6A-8R (Figure 6D). Furthermore, Smurf1 induction was inhibited by 6A-8R (Figure 6E); therefore, p-Smad1/5/9 was upregulated (Figure 6E) (52). There were no obvious changes in the expression of Runx2, β-catenin, or JunB as a result of the administration of 6A-8R (Supplemental Figure 8).
Figure 6Effects of 6A-8R on osteoblasts. (A) The effect of 6A-8R on alkaline phosphatase (ALP) of MC3T3-E1 cells stimulated with TNF-α (1 ng/mL) was evaluated using ALP staining. (B) The effect of 6A-8R on the mineralization of MC3T3-E1 cells stimulated with TNF-α (1 ng/mL) was evaluated using Alizarin red staining. Data (bottom) are expressed as mean ± SD (n = 3). (C–E) The effects of 6A-8R on the phosphorylation of p65, expression of p50, phosphorylation of IκBα, phosphorylation of Smad1, and expression of Smurf1 after TNF-α stimulation were analyzed using Western blotting. (F) The effects of 6A-8R on the gene expression of runt-related transcription factor 2 (Runx2), Osterix, activating transcription factor 4 (Atf4), and Alp were analyzed using quantitative real-time PCR. Data are expressed as mean ± SD (n = 4). (G and H) The effects of 6A-8R on the proliferation of mouse bone marrow mononuclear cells (BMMCs) and MC3T3-E1 cells were evaluated using water-soluble tetrazolium assay. Data are expressed as mean ± SD (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by 1-way ANOVA and the Tukey-Kramer test (B and F) or 2-tailed Student’s t test (G and H). NS, not significant.
According to quantitative real-time PCR, 6A-8R (1.5 and 3.0 mg/mL) inhibits the downregulation of the expression of Runx2, Osterix, and acti
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