ABSTRACT
Receptor activator of nuclear factor kappaB-ligand (RANKL) transduces a differentiation signal appropriate to osteoclasts likely through induction a receptor homotrimer; however, biological importance of RANK-trimerizarion is unknown. To address the signaling mechanism of the RANK receptor, we analyzed the effect of two different types of homodimer inducers RANK-TM-FKBP36v and hEpoR-RANK-TM on osteoclastogenesis. Dimerizing component FKBP36v or extracellular portion of human erythropoietin receptor (hEpoR) was fused to RANK lacking the extracellular domain, and the dimerization of this fusion protein was induced by addition of the chemical inducer of dimerization AP20187 or erythropoietin, respectively. Such treatment resulted in induction of TRAP-activity, a marker of osteoclast in a dose dependent manner, with an efficiency equivalent to that of induction by RANKL. However, dimerized-RANK-induced osteoclasts showed relatively low levels of multinucleation, pit forming activity, and expression of calcitonin receptor and cathepsin K, compared with osteoclasts which were induced in the presence of RANKL. As expression of nuclear factor of activated T cells 1 (NFATc1) was also reduced in dimerized-RANK-induced osteoclasts, RANK oligomerization by RANKL is a critical event to generate fully matured osteoclasts through upregulation of NFATc1.
Subject(s)
Carrier Proteins/chemistry , Carrier Proteins/metabolism , Cell Differentiation/physiology , Membrane Glycoproteins/chemistry , Membrane Glycoproteins/metabolism , Osteoclasts/physiology , Signal Transduction/physiology , Tacrolimus/analogs & derivatives , Animals , Biomarkers , Carrier Proteins/genetics , Cell Line , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Dimerization , Erythropoietin/pharmacology , Humans , Macrophages/cytology , Macrophages/drug effects , Macrophages/physiology , Membrane Glycoproteins/genetics , Mice , NFATC Transcription Factors , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Osteoclasts/cytology , RANK Ligand , Receptor Activator of Nuclear Factor-kappa B , Receptors, Erythropoietin/genetics , Receptors, Erythropoietin/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Tacrolimus/pharmacology , Tacrolimus Binding Proteins/genetics , Tacrolimus Binding Proteins/metabolism , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
The 'ataxia telangiectasia mutated' (Atm) gene maintains genomic stability by activating a key cell-cycle checkpoint in response to DNA damage, telomeric instability or oxidative stress. Mutational inactivation of the gene causes an autosomal recessive disorder, ataxia-telangiectasia, characterized by immunodeficiency, progressive cerebellar ataxia, oculocutaneous telangiectasia, defective spermatogenesis, premature ageing and a high incidence of lymphoma. Here we show that ATM has an essential function in the reconstitutive capacity of haematopoietic stem cells (HSCs) but is not as important for the proliferation or differentiation of progenitors, in a telomere-independent manner. Atm-/- mice older than 24 weeks showed progressive bone marrow failure resulting from a defect in HSC function that was associated with elevated reactive oxygen species. Treatment with anti-oxidative agents restored the reconstitutive capacity of Atm-/- HSCs, resulting in the prevention of bone marrow failure. Activation of the p16(INK4a)-retinoblastoma (Rb) gene product pathway in response to elevated reactive oxygen species led to the failure of Atm-/- HSCs. These results show that the self-renewal capacity of HSCs depends on ATM-mediated inhibition of oxidative stress.
