title: Membrane-bound receptor activator of NFκB ligand (RANKL) activity displayed by osteoblasts is differentially regulated by osteolytic factors.
abstract: Osteoclast formation is central to bone metabolism, occurring when myelomonocytic progenitors are stimulated by membrane-bound receptor activator of NFκB 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 NFκB (NFκB-RAW) or NFATc1 (NFAT-RAW)-binding promoter elements. Strong signals were induced in these cells by recombinant RANKL over 24h. When NFκB-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 NFκB-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. TGFβ 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 TGFβ, 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.
authors.affiliation: Prince Henry's Institute, Clayton, Australia. preetinder.singh@princehenrys.org
authors.forename: Preetinder P
authors.initials: PP
authors.lastname: Singh
authors.forename: A Gabrielle J
authors.initials: AG
authors.lastname: van der Kraan
authors.forename: Jiake
authors.initials: J
authors.lastname: Xu
authors.forename: Matthew T
authors.initials: MT
authors.lastname: Gillespie
authors.forename: Julian M W
authors.initials: JM
authors.lastname: Quinn
mesh_terms: D000818:Animals; D001681:Biological Assay; D002117:Calcitriol / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D018920:Coculture Techniques; D015232:Dinoprostone / Q000494:pharmacology; D017930:Genes, Reporter; D008156:Luciferases / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D016328:NF-kappa B / Q000378:metabolism; D010006:Osteoblasts / Q000187:drug effects / Q000378:metabolism*; D010014:Osteolysis / Q000139:chemically induced / Q000378:metabolism*; D053244:Osteoprotegerin / Q000378:metabolism; D011401:Promoter Regions, Genetic; D053245:RANK Ligand / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D016211:Transforming Growth Factor alpha / Q000494:pharmacology
chemical_list: D016328:NF-kappa B; D053244:Osteoprotegerin; D053245:RANK Ligand; D016211:Transforming Growth Factor alpha; D008156:Luciferases; D002117:Calcitriol; D015232:Dinoprostone