title: Evidence that receptor activator of nuclear factor (NF)-kappaB ligand can suppress cell proliferation and induce apoptosis through activation of a NF-kappaB-independent and TRAF6-dependent mechanism.
abstract: The receptor activator of NF-kappaB ligand (RANKL), a recently identified member of the tumor necrosis factor (TNF) superfamily, has been shown to induce osteoclastogenesis and dendritic cell survival. Most members of the TNF superfamily suppress cell proliferation and induce apoptosis, but whether RANKL does so is not known. We demonstrate that treatment of monocyte RAW 264.7 cells with RANKL induces dose-dependent growth inhibition (IC50 = 10 ng/ml) as determined by dye uptake and [3H]thymidine incorporation methods. Suppression of RANKL-induced NF-kappaB activation by dominant-negative IkappaBalpha or by the NEMO-peptide had no effect on RANKL-induced cell growth inhibition. Inhibition of RANKL-induced JNK activation, however, abolished the RANKL-induced apoptosis. Suppression of interaction of RANK with TRAF6 by TRAF6-binding peptide abrogated the anti-proliferative effects of RANKL, suggesting the critical role of TRAF6. Flow cytometric analysis of cells treated with RANKL showed accumulation of cells in G0/G1 phase of the cell cycle, and this accumulation correlated with a decline in the levels of cyclin D1, cyclin D3, and cyclin E and an increase in cyclin-dependent kinase inhibitor p27 (Kip). Flow cytometric analysis showed the presence of annexin V-positive cells in cultures treated with RANKL. RANKL-induced apoptosis was further confirmed using calcein AM/ethidium homodimer-1 dye and cleavage of poly(ADP-ribose) polymerase (PARP), procaspase 3, and procaspase 9; benzyloxycarbonyl-VAD, the pancaspase inhibitor, suppressed the PARP cleavage. Thus, overall, our studies indicate that RANKL can inhibit cell proliferation and induce apoptosis through a TRAF-6-dependent but NF-kappaB-independent mechanism.
authors.affiliation: Cytokine Research Section, Department of Bioimmunotherapy, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA.
authors.forename: Alok C
authors.initials: AC
authors.lastname: Bharti
authors.forename: Yasunari
authors.initials: Y
authors.lastname: Takada
authors.forename: Shishir
authors.initials: S
authors.lastname: Shishodia
authors.forename: Bharat B
authors.initials: BB
authors.lastname: Aggarwal
mesh_terms: D000818:Animals; D017304:Annexin A5 / Q000494:pharmacology; D017209:Apoptosis; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry / Q000502:physiology*; D018797:Cell Cycle Proteins / Q000378:metabolism; D016923:Cell Death; D002454:Cell Differentiation; D002455:Cell Division; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002470:Cell Survival; D004396:Coloring Agents / Q000494:pharmacology; D019938:Cyclin D1 / Q000378:metabolism; D056743:Cyclin D3; D019927:Cyclin E / Q000378:metabolism; D050760:Cyclin-Dependent Kinase Inhibitor p27; D016213:Cyclins / Q000378:metabolism; D004247:DNA / Q000737:chemistry; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D004789:Enzyme Activation; D005434:Flow Cytometry; D016193:G1 Phase; D020128:Inhibitory Concentration 50; D047908:Intracellular Signaling Peptides and Proteins; D048031:JNK Mitogen-Activated Protein Kinases; D008024:Ligands; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000502:physiology*; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D016192:Resting Phase, Cell Cycle; D048029:TNF Receptor-Associated Factor 6; D013778:Tetrazolium Salts / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology; D013997:Time Factors; D025521:Tumor Suppressor Proteins / Q000378:metabolism
chemical_list: D017304:Annexin A5; C534696:CCND3 protein, human; D002352:Carrier Proteins; C534697:Ccnd3 protein, mouse; C493961:Cdkn1b protein, mouse; D018797:Cell Cycle Proteins; D004396:Coloring Agents; D056743:Cyclin D3; D019927:Cyclin E; D016213:Cyclins; D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; D008562:Membrane Glycoproteins; D016328:NF-kappa B; D009363:Neoplasm Proteins; D010455:Peptides; D011506:Proteins; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; C120635:TAX1BP1 protein, human; D048029:TNF Receptor-Associated Factor 6; C505686:TNFRSF11A protein, human; C505688:TNFSF11 protein, human; D013778:Tetrazolium Salts; D013844:Thiazoles; C505687:Tnfrsf11a protein, mouse; C505689:Tnfsf11 protein, mouse; D025521:Tumor Suppressor Proteins; D019938:Cyclin D1; D050760:Cyclin-Dependent Kinase Inhibitor p27; D004247:DNA; D011065:Poly(ADP-ribose) Polymerases; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; C022616:thiazolyl blue