RRC ID 71712
Author Nakashima T, Kobayashi Y, Yamasaki S, Kawakami A, Eguchi K, Sasaki H, Sakai H.
Title Protein expression and functional difference of membrane-bound and soluble receptor activator of NF-kappaB ligand: modulation of the expression by osteotropic factors and cytokines.
Journal Biochem Biophys Res Commun
Abstract A variety of humoral factors modulate the osteoclastogenesis. Receptor activator of NF-kappaB ligand (RANKL) expressed on osteoblast/stromal lineage cells plays a pivotal role to transduce an essential differentiation signal to osteoclast lineage cells through binding to its receptor, RANK, expressed on the latter cell population; however, the difficulty to detect RANKL protein expression hampers us in investigating the regulation of RANKL expression by humoral factors. To determine protein expression of RANKL, we have established a new method, named as a ligand-receptor precipitation (LRP) Western blot analysis, which can specifically concentrate the target protein by the use of specific binding characteristic between RANKL and RANK/osteoprotegrin (OPG). RANKL protein expression in the postnuclear supernatant was not detected by common Western blotting, but LRP Western blot analysis clearly showed that RANKL is produced as a membrane-bound protein on murine osteoblasts/stromal cells, and cleaved into a soluble form by metalloprotease. Cytokines stimulating the osteoclastogenesis, such as IL-1beta, IL-6, IL-11, IL-17, and TNF-alpha, increased the expression of RANKL with decrease of OPG expression in osteoblasts/stromal cells. In contrast, cytokines inhibiting the osteoclastogenesis, such as IL-13, INF-gamma, and TGF-beta1 suppressed the expression of RANKL and/or augmented OPG expression. Functional difference between membrane-bound and soluble RANKL was demonstrated, which showed that membrane-bound RANKL works more efficiently than soluble RANKL in the osteoclastogenesis developed from murine bone marrow cell culture. The present study indicates the usefulness of LRP Western blot analysis, which shows that the modulation of osteoclastogenesis by humoral factors is achieved, in part, by regulation of the expression of RANKL and OPG in osteoblast/stromal lineage cells.
Volume 275(3)
Pages 768-75
Published 2000-9-7
DOI 10.1006/bbrc.2000.3379
PII S0006-291X(00)93379-5
PMID 10973797
MeSH Animals Blotting, Western Calcitriol / pharmacology Carrier Proteins / genetics Carrier Proteins / metabolism* Cells, Cultured Cytokines / pharmacology* Dexamethasone / pharmacology Enzyme Inhibitors / pharmacology Gene Expression Regulation / drug effects Glycoproteins / genetics Glycoproteins / metabolism* Humans Ligands Membrane Glycoproteins / genetics Membrane Glycoproteins / metabolism* Membrane Proteins / genetics Membrane Proteins / metabolism Metalloendopeptidases / antagonists & inhibitors Mice Osteoblasts / drug effects Osteoblasts / metabolism Osteoprotegerin RANK Ligand Receptor Activator of Nuclear Factor-kappa B Receptors, Cytoplasmic and Nuclear / genetics Receptors, Cytoplasmic and Nuclear / metabolism* Receptors, Tumor Necrosis Factor / genetics Receptors, Tumor Necrosis Factor / metabolism* Recombinant Fusion Proteins / metabolism Solubility Stromal Cells / drug effects Stromal Cells / metabolism
IF 2.985
Human and Animal Cells ST2(RCB0224)