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

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

World J Stem Cells. 2020 Mar 26;12(3):222-240. doi: 10.4252/wjsc.v12.i3.222.

Authors

Affiliations

¹ Laboratory Medicine Center, Department of Blood Transfusion, Lab of Radiation Biology, The Second Affiliated Hospital, Third Military Medical University, Chongqing, 400037, China.
² Bioinformatics Center, College of Basic Medical Sciences, Third Military Medical University, Chongqing 400038, China.
³ Department of Otolaryngology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, United States.
⁴ CHOC Children's Research Institute, Children's Hospital of Orange County, University of California, Irvine, CA 92868, United States.
⁵ Laboratory Medicine Center, Department of Blood Transfusion, Lab of Radiation Biology, The Second Affiliated Hospital, Third Military Medical University, Chongqing, 400037, China. johnneyusc@gmail.com.

Abstract

Background: Radiation induces rapid bone loss and enhances bone resorption and adipogenesis, leading to an increased risk of bone fracture. There is still a lack of effective preventive or therapeutic method for irradiation-induced bone injury. 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.

Aim: To investigate the role of CR6-interacting factor-1 (Crif1) in osteoclastogenesis after radiation and its possible mechanism.

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 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.

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. Deletion of Crif1 in RAW264.7 cells did not affect the receptor activator of nuclear factor κB 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-protein kinase cyclic adenosine monophosphate-activited catalytic subunit alpha 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.

Conclusion: Crif1 promotes RANKL expression via the cAMP/PKA pathway, which induces osteoclastogenesis by binding to receptor activator of nuclear factor κB 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.

Keywords: Bone; Bone marrow; Irradiation; Mesenchymal stem cells; Monocyte macrophage; Osteoporosis.