Anti–NF-κB peptide derived from nuclear acidic protein attenuates ovariectomy-induced osteoporosis in mice
Kenji Takami, … , Seiji Okada, Kosuke Ebina
Kenji Takami, … , Seiji Okada, Kosuke Ebina
Published November 22, 2023
Citation Information: JCI Insight. 2023;8(22):e171962. https://doi.org/10.1172/jci.insight.171962.
View: Text | PDF
Research Article Bone biology

Anti–NF-κB peptide derived from nuclear acidic protein attenuates ovariectomy-induced osteoporosis in mice

Abstract

NF-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.

Authors

Kenji Takami, Kazuki Okamoto, Yuki Etani, Makoto Hirao, Akira Miyama, Gensuke Okamura, Atsushi Goshima, Taihei Miura, Takuya Kurihara, Yuji Fukuda, Takashi Kanamoto, Ken Nakata, Seiji Okada, Kosuke Ebina

×

Figure 5

Effects of 6A-8R administration on osteoclasts.

Options: View larger image (or click on image) Download as PowerPoint
Effects of 6A-8R administration on osteoclasts. (A) Mouse bone marrow mo...
(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.