title: RANKL expression is related to the differentiation state of human osteoblasts.
abstract: Human osteoblast phenotypes that support osteoclast differentiation and bone formation are not well characterized. Osteoblast differentiation markers were examined in relation to RANKL expression. RANKL expression was induced preferentially in immature cells. These results support an important link between diverse osteoblast functions. Cells of the osteoblast lineage support two apparently distinct functions: bone formation and promotion of osteoclast formation. The aim of this study was to examine the relationship between these phenotypes in human osteoblasts (NHBC), in terms of the pre-osteoblast marker, STRO-1, and the mature osteoblast marker, alkaline phosphatase (AP), and the expression of genes involved in osteoclast formation, RANKL and OPG. The osteotropic stimuli, 1alpha,25(OH)2vitamin D3 (vitD3) and dexamethasone, were found to have profound proliferative and phenotypic effects on NHBCs. VitD3 inhibited NHBC proliferation and increased the percentage of cells expressing STRO-1 over an extended culture period, implying that vitD3 promotes and maintains an immature osteogenic phenotype. Concomitantly, RANKL mRNA expression was upregulated and maintained in NHBC in response to vitD3. Dexamethasone progressively promoted the proliferation of AP-expressing cells, resulting in the overall maturation of the cultures. Dexamethasone had little effect on RANKL mRNA expression and downregulated OPG mRNA expression in a donor-dependent manner. Regression analysis showed that RANKL mRNA expression was associated negatively with the percentage of cells expressing AP (p < 0.01) in vitD3- and dexamethasone-treated NHBCs. In contrast, RANKL mRNA expression was associated positively with the percentage of STRO-1+ cells (p < 0.01). In NHBCs sorted by FACS based on STRO-1 expression (STRO-1bright and STRO-1dim populations), it was found that vitD3 upregulated the expression of RANKL mRNA preferentially in STRO-1bright cells. The results suggest that immature osteoblasts respond to osteotropic factors in a potentially pro-osteoclastogenic manner. Additionally, the dual roles of osteoblasts, in supporting osteoclastogenesis or forming bone, may be performed by the same lineage of cells at different stages of their maturation.
authors.affiliation: Department of Orthopaedics and Trauma, University of Adelaide, Adelaide, South Australia, Australia. gerald.atkins@adelaide.edu.au
authors.forename: Gerald J
authors.initials: GJ
authors.lastname: Atkins
authors.forename: Panagiota
authors.initials: P
authors.lastname: Kostakis
authors.forename: Beiqing
authors.initials: B
authors.lastname: Pan
authors.forename: Amanda
authors.initials: A
authors.lastname: Farrugia
authors.forename: Stan
authors.initials: S
authors.lastname: Gronthos
authors.forename: Andreas
authors.initials: A
authors.lastname: Evdokiou
authors.forename: Kate
authors.initials: K
authors.lastname: Harrison
authors.forename: David M
authors.initials: DM
authors.lastname: Findlay
authors.forename: Andrew C W
authors.initials: AC
authors.lastname: Zannettino
mesh_terms: D002352:Carrier Proteins / Q000187:drug effects / Q000235:genetics*; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D003907:Dexamethasone / Q000494:pharmacology; D005452:Fluoresceins / Q000494:pharmacology; D005456:Fluorescent Dyes; D005786:Gene Expression Regulation / Q000502:physiology*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000187:drug effects / Q000235:genetics; D006801:Humans; D008562:Membrane Glycoproteins / Q000187:drug effects / Q000235:genetics*; D010006:Osteoblasts / Q000166:cytology* / Q000187:drug effects; D010641:Phenotype; D053245:RANK Ligand; D012333:RNA, Messenger / Q000235:genetics; D053246:Receptor Activator of Nuclear Factor-kappa B; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013388:Succinimides / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects; D014807:Vitamin D / Q000494:pharmacology
chemical_list: C087165:5-(6)-carboxyfluorescein diacetate succinimidyl ester; D002352:Carrier Proteins; D005452:Fluoresceins; D005456:Fluorescent Dyes; D008562:Membrane Glycoproteins; D053245:RANK Ligand; D012333:RNA, Messenger; D053246:Receptor Activator of Nuclear Factor-kappa B; D013388:Succinimides; C505686:TNFRSF11A protein, human; C505688:TNFSF11 protein, human; D014807:Vitamin D; D003907:Dexamethasone; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases