Basic Study
Copyright ©The Author(s) 2020. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Stem Cells. Mar 26, 2020; 12(3): 222-240
Published online Mar 26, 2020. doi: 10.4252/wjsc.v12.i3.222
CR6-interacting factor-1 contributes to osteoclastogenesis by inducing receptor activator of nuclear factor κB ligand after radiation
Li-Xin Xiang, Qian Ran, Li Chen, Yang Xiang, Feng-Jie Li, Xiao-Mei Zhang, Yan-Ni Xiao, Ling-Yun Zou, Jiang F Zhong, Shengwen Calvin Li, Zhong-Jun Li
Li-Xin Xiang, Qian Ran, Li Chen, Yang Xiang, Feng-Jie Li, Xiao-Mei Zhang, Yan-Ni Xiao, Zhong-Jun Li, Laboratory Medicine Center, Department of Blood Transfusion, Lab of Radiation Biology, The Second Affiliated Hospital, Third Military Medical University, Chongqing, 400037, China
Qian Ran, Yang Xiang, Jiang F Zhong, Department of Otolaryngology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, United States
Ling-Yun Zou, Bioinformatics Center, College of Basic Medical Sciences, Third Military Medical University, Chongqing 400038, China
Shengwen Calvin Li, CHOC Children’s Research Institute, Children’s Hospital of Orange County, University of California, Irvine, CA 92868, United States
Author contributions: Li ZJ and Xiang LX conceived the study; Xiang LX designed and performed most experiments and data analysis; Chen L, Xiang Y, Li FJ, Zhang XM, Xiao YN, Zou LY, Zhong JF, Li SC, and Ran Q assisted with experiments and data analysis; Xiang LX wrote and edited the manuscript; Li ZJ supervised the study; all authors read and approved the final manuscript.
Supported by National Natural Science Foundation of China, No. 81502754 and No. 31571352; and Interdisciplinary and International Cooperation Projects of The Second Affiliated Hospital, Third Military Medical University, No. 2016YXKJC0.
Institutional review board statement: Not applicable.
Institutional animal care and use committee statement: All animal studies performed were approved by the Laboratory Animal Welfare and Ethics Committee Of the Third Military Medical University.
Conflict-of-interest statement: The authors report no conflicts of interest in this work.
Data sharing statement: No additional data are available.
ARRIVE guidelines statement: The authors have read the ARRIVE guidelines, and the manuscript was prepared and revised according to the ARRIVE guidelines.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See:
Corresponding author: Zhong-Jun Li, MD, Professor, Chief, Laboratory Medicine Center, Department of Blood Transfusion, Lab of Radiation Biology, The Second Affiliated Hospital, Third Military Medical University, Xinqiao Road, Chongqing 400037, China.
Received: January 23, 2020
Peer-review started: January 23, 2020
First decision: February 19, 2020
Revised: March 9, 2020
Accepted: March 15, 2020
Article in press: March 15, 2020
Published online: March 26, 2020
Research background

Radiation induces rapid bone loss and enhances bone resorption and adipogenesis, leading to an increased risk of bone fracture. Receptor activator of nuclear factor κB ligand (RANKL) provides the crucial signal to induce osteoclast differentiation and plays an important role in bone resorption. However, the mechanisms of radiation-induced osteoporosis are not fully understood.

Research motivation

Current treatment of osteoporosis is based mainly on inhibiting bone resorption or stimulating bone generation to increase bone mass, however, the side-effects of some drugs affect long-term administration and adherence. There is still a lack of effective preventive or therapeutic method for radiation-induced bone injury. Therefore, it is necessary to look for alternative treatments with high efficiency but few side effects.

Research objectives

In this stduy, we aimed to investigate the role of CR6-interacting factor-1 (Crif1) in osteoclastogenesis after radiation and its possible mechanism.

Research methods

C57BL/6 mice were exposed to Co-60 gamma rays and received 5 Gy of whole-body sublethal irradiation at a rate of 0.69 Gy/min. For in vitro study, mouse bone marrow mesenchymal stem/stromal cells (BM-MSCs) were irradiated with Co-60 at a single dose of 9 Gy. For osteoclast induction, monocyte-macrophage RAW264.7 cells were cocultured with mouse BM-MSCs for 7 d. ClusPro and InterProSurf were used to investigate the interaction interface in Crif1 and protein kinase cyclic adenosine monophosphate (cAMP)-activited catalytic subunit alpha (PRKACA) complex. Virtual screening using 462608 compounds from the Life Chemicals database around His120 of Crif1 was carried out using the program Autodock_vina. A tetrazolium salt (WST-8) assay was carried out to study the toxicity of compounds to different cells, including human BM-MSCs, mouse BM-MSCs, and Vero cells.

Research results

Crif1 expression increased in bone marrow cells after radiation in mice. Overexpression of Crif1 in mouse BM-MSCs and radiation exposure could increase RANKL secretion and promote osteoclastogenesis in vitro. Deletion of Crif1 in BM-MSCs could reduce both adipogenesis and RANKL expression, resulting in the inhibition of osteoclastogenesis. The deletion of Crif1 in RAW264.7 cells did not affect the RANK expression or osteoclast differentiation. Following treatment with protein kinase A (PKA) agonist (forskolin) and inhibitor (H-89) in mouse BM-MSCs, Crif1 induced RANKL secretion via the cAMP/PKA pathway. Moreover, we identified the Crif1-PRKACA interaction interface by in silico studies and shortlisted interface inhibitors through virtual screening on Crif1. Five compounds dramatically suppressed RANKL secretion and adipogenesis by inhibiting the cAMP/PKA pathway.

Research conclusions

Crif1 promotes RANKL expression via the cAMP/PKA pathway, which induces osteo-clastogenesis by binding to RANK on monocytes-macrophages in the mouse model. These results suggest a role for Crif1 in modulating osteoclastogenesis and provide insights into potential therapeutic strategies targeting the balance between osteogenesis and adipogenesis for radiation-induced bone injury.

Research perspectives

Because of the contribution of adipocytes to osteoporosis, future drug screening should target not only the regulation of the balance between bone formation and bone resorption but also the balance between osteogenic and adipogenic differentiation. Here, through screening, we identified five Crif1 inhibitors targeting Crif1-PRKACA interaction interface that could dramatically reduce RANKL secretion and adipogenesis. Our study provides insights into potential therapeutic strategies for radiation-induced bone injury.