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 1

Preparation of MTI-II–based anti–NF-κB drugs.

Options: View larger image (or click on image) Download as PowerPoint
Preparation of MTI-II–based anti–NF-κB drugs. (A) Schematic representati...
(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.