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.
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.
Nakashima
T
T
Department of Hospital Pharmacy, First Department of Internal Medicine, Nagasaki University School of Medicine, Sakamoto 1-7-1, Nagasaki, 852-8501, Japan.
Kobayashi
Y
Y
Yamasaki
S
S
Kawakami
A
A
Eguchi
K
K
Sasaki
H
H
Sakai
H
H
D000818:Animals; D015153:Blotting, Western; D002117:Calcitriol / Q000494:pharmacology; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D016207:Cytokines / Q000494:pharmacology*; D003907:Dexamethasone / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors; D051379:Mice; D010006:Osteoblasts / Q000187:drug effects / Q000378:metabolism; D053244:Osteoprotegerin; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism*; D018124:Receptors, Tumor Necrosis Factor / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012995:Solubility; D017154:Stromal Cells / Q000187:drug effects / Q000378:metabolism
D002352:Carrier Proteins; D016207:Cytokines; D004791:Enzyme Inhibitors; D006023:Glycoproteins; D008024:Ligands; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D053244:Osteoprotegerin; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D018160:Receptors, Cytoplasmic and Nuclear; D018124:Receptors, Tumor Necrosis Factor; D011993:Recombinant Fusion Proteins; C505686:TNFRSF11A protein, human; C506030:TNFRSF11B protein, human; C505688:TNFSF11 protein, human; C505687:Tnfrsf11a protein, mouse; C506031:Tnfrsf11b protein, mouse; C505689:Tnfsf11 protein, mouse; D003907:Dexamethasone; D008666:Metalloendopeptidases; D002117:Calcitriol