A Sub-Clone of RAW264.7-Cells Form Osteoclast-Like Cells Capable of Bone Resorption Faster than Parental RAW264.7 through Increased De Novo Expression and Nuclear Translocation of NFATc1
by
Laia Mira-Pascual 1, Anh N. Tran 1,2, Göran Andersson 1, Tuomas Näreoja 1,*
and Pernilla Lång 1,*
Laia Mira-Pascual 1, Anh N. Tran 1,2, Göran Andersson 1, Tuomas Näreoja 1,* and Pernilla Lång 1,*
1
Department of Laboratory Medicine, Division of Pathology, Karolinska Institutet, Alfred Nobels Allé, 8, SE-141 52 Stockholm, Sweden
2
Musculoskeletal Group, Institute of Medical Sciences, University of Aberdeen, Aberdeen AB 252ZD, UK
*
Authors to whom correspondence should be addressed.
Int. J. Mol. Sci. 2020, 21(2), 538; https://doi.org/10.3390/ijms21020538
Received: 9 December 2019 / Revised: 7 January 2020 / Accepted: 10 January 2020 / Published: 14 January 2020
(This article belongs to the Special Issue Osteoclast Multinucleation Mechanisms)
The murine macrophage cell line RAW264.7 is extensively used as a progenitor to study osteoclast (OC) differentiation. RAW264.7 is a heterogeneous cell line, containing sub-clones with different abilities to form OCs. The aim of this study was to identify characteristics within the heterogeneous RAW264.7 cells that define sub-clones with an augmented ability to form bone-resorbing OCs (H9), as well as sub-clones representing non-OCs (J8). RAW264.7 sub-clones were isolated by single cell cloning. Selection was based on TRAP/cathepsin K expression in sub-clone cultures without added RANKL. Sub-clones before and after differentiation with RANKL were assayed for multiple OC-characteristics. Sub-clone H9 cells presented a higher expression of OC-markers in cultures without added RANKL compared to the parental RAW264.7. After 6 days of RANKL stimulation, sub-clone H9 cells had equal expression levels of OC-markers with RAW264.7 and formed OCs able to demineralize hydroxyapatite. However, sub-clone H9 cells displayed rapid differentiation of OC already at Day 2 compared to Day 4 from parental RAW264.7, and when cultured on plastic and on bone they were more efficient in resorption. This rapid differentiation was likely due to high initial expression/nuclear translocation of OC master transcription factor, NFATc1. In contrast to H9, J8 cells expressed initially very low levels of OC-markers, and they did not respond to RANKL-stimulation by developing OC-characteristics/OC-marker expression. Hence, H9 is an additional clone suitable for experimental setup requiring rapid differentiation of large numbers of OCs.
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Keywords:
Osteoclast; RAW264.7; tartrate-resistant acid phosphatase; osteoclastogenesis; bone resorption; NFATc1; differentiation
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MDPI and ACS Style
Mira-Pascual, L.; Tran, A.N.; Andersson, G.; Näreoja, T.; Lång, P. A Sub-Clone of RAW264.7-Cells Form Osteoclast-Like Cells Capable of Bone Resorption Faster than Parental RAW264.7 through Increased De Novo Expression and Nuclear Translocation of NFATc1. Int. J. Mol. Sci. 2020, 21, 538.
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