Published online Mar 26, 2020. doi: 10.4252/wjsc.v12.i3.222
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
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.
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.
In this stduy, we aimed to investigate the role of CR6-interacting factor-1 (Crif1) in osteoclastogenesis after radiation and its possible mechanism.
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.
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.
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.
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.