Membrane-bound receptor activator of NF{K}B ligand (RANKL) activity displayed by osteoblasts is differentially regulated by osteolytic factors

Preetinder Singh, Amanda Gabrielle van der Kraan, Jiake Xu, Matthew T Gillespie, Julian Michael Warner Quinn

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28 Citations (Scopus)


Osteoclast formation is central to bone metabolism, occurring when myelomonocytic progenitors are stimulated by membrane-bound receptor activator of NFkappaB ligand (RANKL) on osteoblasts. Osteolytic hormones induce osteoblast RANKL expression, and reduce production of RANKL decoy receptor osteoprotegerin (OPG). However, rather than RANKL and OPG mRNA or protein levels, to measure hormonally-induced osteoclastogenic stimuli the net RANKL activity at the osteoblast surface needs to be determined. To estimate this we developed a cell reporter approach employing pre-osteoclast RAW264.7 cells transfected with luciferase reporter constructs controlled by NFkappaB (NFkappaB-RAW) or NFATc1 (NFAT-RAW)-binding promoter elements. Strong signals were induced in these cells by recombinant RANKL over 24h. When NFkappaB-RAW cells were co-cultured on osteoblastic cells (primary osteoblasts or Kusa O cells) stimulated by osteolytic factors 1,25(OH)(2) vitamin D(3) (1,25(OH)(2)D(3)) and prostaglandin E(2) (PGE(2)), a strong dose dependent signal in NFkappaB-RAW cells was induced. These signals were completely blocked by soluble recombinant RANKL receptor, RANK.Fc. This osteoblastic RANKL activity was sustained for 3 days in Kusa O cells; with 1,25(OH)(2)D(3) withdrawal, RANKL-induced signal was still detectable 24 h later. However, conditioned medium from stimulated osteoblasts induced no signal. TGFbeta treatment inhibited osteoclast formation supported by 1,25(OH)(2)D(3)-treated Kusa O cells, and likewise blocked RANKL-dependent signals in NFAT-RAW co-cultured with these cells. These data indicate net RANKL stimulus at the osteoblast surface is increased by 1,25(OH)(2)D(3) and PGE(2), and suppressed by TGFbeta, in line with their effects on RANKL mRNA levels. These results demonstrate the utility of this simple co-culture-based reporter assay for osteoblast RANKL activity.
Original languageEnglish
Pages (from-to)48 - 53
Number of pages6
JournalBiochemical and Biophysical Research Communications
Issue number1
Publication statusPublished - 2012

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