Zinc inhibits osteoclast differentiation by suppression of Ca2+-Calcineurin-NFATc1 signaling pathway

Kwang Hwan Park,Jae-Hyuck Shim,Hyun Gyu Lee,Dong Min Shin,Jin Woo Lee,Jeon Han Park,Seung-Hyun Kwon,Jae Myun Lee,Boryung Park,Dong Suk Yoon

doi:10.1186/1478-811x-11-74

Kwang Hwan Park, Jae-Hyuck Shim + Show 8 more

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https://doi.org/10.1186/1478-811x-11-74

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Abstract

BackgroundZinc, an essential trace element, inhibits osteoclast differentiation in vitro and in vivo. The molecular mechanism for the inhibitory effect of zinc, however, is poorly understood. The purpose of this study was to investigate the effect of zinc and determine its molecular mechanism on receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis in mouse bone marrow-derived monocyte cells (BMMs) and RAW264.7 cells.ResultsIn BMMs, zinc treatment during osteoclast differentiation decreased RANKL-induced osteoclast formation in a dose-dependent manner. We show that zinc suppressed the mRNA levels of nuclear factor of activated T-cells, cytoplasmic 1 (Nfatc1). Zinc also accumulated phospho-Nfatc1 (p-Nfatc1) in the cytosol in a dose-dependent manner and inhibited the translocation of Nfatc1 to the nucleus in RAW264.7 cells. Zinc suppressed the activities of Nfatc1 in the nucleus without changing the activities of NF-κB in RAW264.7 cells. In contrast, calcineurin activity decreased in response to zinc but its protein level was unchanged. RANKL-induced Ca2+ oscillations were inhibited by zinc treatment, but phospho-phospholipase Cγ1 (p-PLCγ1), the upstream signaling molecule of Ca2+ oscillations, was unaffected. Moreover, a constitutively active form of Nfatc1 obviously rescued suppression of osteoclastogenesis by zinc.ConclusionsTaken together, these results demonstrate for the first time that the inhibitory effect of zinc during osteoclastogesis is caused by suppressing the Ca2+-Calcineurin-NFATc1 signaling pathway. Thus, zinc may be a useful therapeutic candidate for the prevention of bone loss caused by NFATc1 activation in osteoclasts.

Highlights

Zinc, an essential trace element, inhibits osteoclast differentiation in vitro and in vivo
To investigate the molecular mechanism of the inhibitory effect of zinc on Receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation, we focused on Nfatc1, a master transcription factor of osteoclastogenesis [9,13,22,23,24]
Zinc inhibits osteoclast formation and fusion from bone marrow-derived monocyte cells (BMMs) To determine whether zinc is cytotoxic to BMMs and RAW264.7 cells, we first examined the cells’ viability using an EZcytox cell viability assay kit, which estimates the number of surviving cells using WST-1 (4-[3-(4iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate)

Summary

Introduction

An essential trace element, inhibits osteoclast differentiation in vitro and in vivo. The purpose of this study was to investigate the effect of zinc and determine its molecular mechanism on receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis in mouse bone marrow-derived monocyte cells (BMMs) and RAW264.7 cells. To identify novel genes involved in osteoclastogenesis, we used two sets of microarray data from Gene Expression Omnibus (GEO) DataSets, which were comparative microarrays in mouse bone marrow-derived monocyte cells (BMMs) stimulated with or without receptor activator of NF-κB ligand (RANKL) [12,13]. We performed statistical data analyses using the R program From these analyses, we found intersections between the two sets of data. Mt3, which is known to regulate the intracellular level of zinc, and other zincrelated genes were up-regulated (log ratio > 4.0) during osteoclast differentiation (Additional file 1: Table S1). We suggest that zinc may play an important physiologic role in osteoclastogenesis signaling pathways

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