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.

[1]  E. Sakai,et al.  Cell adhesion is a prerequisite for osteoclast survival. , 2000, Biochemical and biophysical research communications.

[2]  S. Morham,et al.  Prostaglandin G/H synthase-2 is required for maximal formation of osteoclast-like cells in culture. , 2000, The Journal of clinical investigation.

[3]  R. Derynck,et al.  Ectodomain shedding of TGF‐α and other transmembrane proteins is induced by receptor tyrosine kinase activation and MAP kinase signaling cascades , 1999, The EMBO journal.

[4]  C. Blobel,et al.  Metalloprotease-disintegrins: modular proteins capable of promoting cell-cell interactions and triggering signals by protein-ectodomain shedding. , 1999, Journal of cell science.

[5]  E. Jimi,et al.  Osteoclast differentiation factor acts as a multifunctional regulator in murine osteoclast differentiation and function. , 1999, Journal of immunology.

[6]  T. Martin,et al.  Modulation of osteoclast differentiation and function by the new members of the tumor necrosis factor receptor and ligand families. , 1999, Endocrine reviews.

[7]  L. Lum,et al.  Evidence for a Role of a Tumor Necrosis Factor-α (TNF-α)-converting Enzyme-like Protease in Shedding of TRANCE, a TNF Family Member Involved in Osteoclastogenesis and Dendritic Cell Survival* , 1999, Journal of Biological Chemistry.

[8]  H. Yasuda,et al.  RANK is the essential signaling receptor for osteoclast differentiation factor in osteoclastogenesis. , 1998, Biochemical and biophysical research communications.

[9]  Z. Werb,et al.  A Cellular Striptease Act , 1998, Science.

[10]  David C. Lee,et al.  An essential role for ectodomain shedding in mammalian development. , 1998, Science.

[11]  J. Tschopp,et al.  Conversion of Membrane-bound Fas(CD95) Ligand to Its Soluble Form Is Associated with Downregulation of Its Proapoptotic Activity and Loss of Liver Toxicity , 1998, The Journal of experimental medicine.

[12]  D. Lacey,et al.  Osteoprotegerin Ligand Is a Cytokine that Regulates Osteoclast Differentiation and Activation , 1998, Cell.

[13]  K Yano,et al.  Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[14]  S. Mochizuki,et al.  Identity of osteoclastogenesis inhibitory factor (OCIF) and osteoprotegerin (OPG): a mechanism by which OPG/OCIF inhibits osteoclastogenesis in vitro. , 1998, Endocrinology.

[15]  R. Dubose,et al.  A homologue of the TNF receptor and its ligand enhance T-cell growth and dendritic-cell function , 1997, Nature.

[16]  S. Mochizuki,et al.  Isolation of a novel cytokine from human fibroblasts that specifically inhibits osteoclastogenesis. , 1997, Biochemical and biophysical research communications.

[17]  G Shimamoto,et al.  Osteoprotegerin: A Novel Secreted Protein Involved in the Regulation of Bone Density , 1997, Cell.

[18]  H. Okamura,et al.  Interleukin-18 (interferon-gamma-inducing factor) is produced by osteoblasts and acts via granulocyte/macrophage colony-stimulating factor and not via interferon-gamma to inhibit osteoclast formation. , 1997 .

[19]  Nicole Nelson,et al.  A metalloproteinase disintegrin that releases tumour-necrosis factor-α from cells , 1997, Nature.

[20]  T. Martin,et al.  Modulation of osteoclast differentiation. , 1992, Endocrine reviews.

[21]  T. Yoshimori,et al.  Protein disulfide-isomerase in rat exocrine pancreatic cells is exported from the endoplasmic reticulum despite possessing the retention signal. , 1990, The Journal of biological chemistry.

[22]  M. Noshiro,et al.  Molecular cloning of vitamin D3 hydroxylases. , 1997, Methods in enzymology.