RANK/RANKL/OPG system in the intervertebral disc

Norihiko Takegami,Junichi Yamada,Koji Akeda,Akihiro Sudo,Koichiro Murata,Tomohiko Sano,Koichi Masuda,Jenny Huang

doi:10.1186/s13075-017-1332-y

Norihiko Takegami, Junichi Yamada + Show 6 more

Open Access

https://doi.org/10.1186/s13075-017-1332-y

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Abstract

BackgroundThe receptor activator of NF-κB ligand (RANKL), a member of the TNF ligand superfamily, is known to regulate bone metabolism. The expression of each component of the RANK/RANKL/osteoprotegerin (OPG) system in the intervertebral disc (IVD) has not been examined in detail. The purposes of this study were to examine the expression of the RANK/RANKL/OPG system and to evaluate the function of RANKL in the matrix metabolism of the rat IVD.MethodsSprague-Dawley, 12-week-old, male rats were used in this study. Anulus fibrosus (AF), nucleus pulposus (NP) and cartilaginous endplate (CEP) cells isolated from dissected thoracolumbar discs were monolayer-cultured. RANK/RANKL/OPG expression in rat IVDs was examined using real-time polymerase chain reaction (PCR) and immunohistochemical analysis (cultured cells and IVD tissues). To examine the effect of interleukin-1β (IL-1β) stimulation on the mRNA levels of RANK, RANKL and OPG, the cells were cultured with or without recombinant human IL-1β (rhIL-1β). To evaluate the effect of RANKL on the mRNA expression of catabolic factors (IL-1β, matrix metalloproteinase-3 (MMP-3) and MMP-13), the cells were cultured with RANKL in the presence or absence of rhIL-1β. The immunohistochemical expression of this system was also evaluated using human IVD tissues with different grades of degeneration.ResultsmRNA expression levels of RANK, RANKL, and OPG were clearly identified in AF, NP and CEP cells. Immunoreactivity to RANK, RANKL and OPG was distributed in the cell membranes and/or cytoplasm of the three types of cells. The mRNA level of RANKL was significantly upregulated by treatment with rhIL-1β of the three types of cells. Treatment with RANKL without rhIL-1β did not induce significant effects on the mRNA expression of catabolic factors by AF, NP and CEP cells. However, the expression was significantly upregulated by stimulation with RANKL in the presence of rhIL-1β. There was a general trend for more RANK/RANKL/OPG-positive cells in human IVD tissues in an advanced stage of degeneration compared to an early stage.ConclusionsOur study showed the possibility that the RANK/RANKL/OPG system may play a part in the process of intervertebral disc degeneration.

Highlights

The receptor activator of NF-κB ligand (RANKL), a member of the TNF ligand superfamily, is known to regulate bone metabolism
Immunohistochemical analysis of normal rat intervertebral disc (IVD) tissues Every component of the Receptor activator of nuclear factor kappa B (RANK)/RANKL/OPG system was identified in anulus fibrosus (AF), nucleus pulposus (NP) and cartilaginous endplate (CEP) tissues of normal IVDs from 12-week-old rats (Fig. 1)
Fluorescent immunohistochemical analysis of rat IVD cells Immunoreactivity to RANK, RANKL and OPG was clearly identified in monolayer cultures of rat AF, NP and CEP cells (Fig. 2)

Summary

Introduction

The receptor activator of NF-κB ligand (RANKL), a member of the TNF ligand superfamily, is known to regulate bone metabolism. The expression of each component of the RANK/RANKL/osteoprotegerin (OPG) system in the intervertebral disc (IVD) has not been examined in detail. Receptor activator of nuclear factor kappa B ligand (RANKL) is a member of the TNF ligand superfamily that is known to regulate bone metabolism [7, 8]. The binding of RANKL to RANK activates TNF receptor-associated factor (TRAF) 6, which stimulates the expression of proinflammatory cytokines through nuclear factor kappa B (NF-κB) pathways [9]. RANKL [15, 16] and OPG [17] have been shown to be expressed by the human IVD, and are considered to be associated with the progression of IVD degeneration

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