RESUMO
Diamond-Blackfan anemia (DBA) is associated with developmental defects and profound anemia. Mutations in genes encoding a ribosomal protein of the small (e.g., RPS19) or large (e.g., RPL11) ribosomal subunit are found in more than half of these patients. The mutations cause ribosomal haploinsufficiency, which reduces overall translation efficiency of cellular mRNAs. We reduced the expression of Rps19 or Rpl11 in mouse erythroblasts and investigated mRNA polyribosome association, which revealed deregulated translation initiation of specific transcripts. Among these were Bag1, encoding a Hsp70 cochaperone, and Csde1, encoding an RNA-binding protein, and both were expressed at increased levels in erythroblasts. Their translation initiation is cap independent and starts from an internal ribosomal entry site, which appeared sensitive to knockdown of Rps19 or Rpl11. Mouse embryos lacking Bag1 die at embryonic day 13.5, with reduced erythroid colony forming cells in the fetal liver, and low Bag1 expression impairs erythroid differentiation in vitro. Reduced expression of Csde1 impairs the proliferation and differentiation of erythroid blasts. Protein but not mRNA expression of BAG1 and CSDE1 was reduced in erythroblasts cultured from DBA patients. Our data suggest that impaired internal ribosomal entry site-mediated translation of mRNAs expressed at increased levels in erythroblasts contributes to the erythroid phenotype of DBA.
Assuntos
Anemia de Diamond-Blackfan/genética , Anemia de Diamond-Blackfan/patologia , Biomarcadores/metabolismo , Diferenciação Celular , Eritroblastos/citologia , Polirribossomos/patologia , Biossíntese de Proteínas , Animais , Western Blotting , Proliferação de Células , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Eritroblastos/metabolismo , Feminino , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Camundongos , Camundongos Knockout , Mutação/genética , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Polirribossomos/genética , Polirribossomos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Ribossômicas/antagonistas & inibidores , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologiaRESUMO
The autonomic nervous system is a master regulator of homeostatic processes and stress responses. Sympathetic noradrenergic nerve fibers decrease bone mass, but the role of cholinergic signaling in bone has remained largely unknown. Here, we describe that early postnatally, a subset of sympathetic nerve fibers undergoes an interleukin-6 (IL-6)-induced cholinergic switch upon contacting the bone. A neurotrophic dependency mediated through GDNF-family receptor-α2 (GFRα2) and its ligand, neurturin (NRTN), is established between sympathetic cholinergic fibers and bone-embedded osteocytes, which require cholinergic innervation for their survival and connectivity. Bone-lining osteoprogenitors amplify and propagate cholinergic signals in the bone marrow (BM). Moderate exercise augments trabecular bone partly through an IL-6-dependent expansion of sympathetic cholinergic nerve fibers. Consequently, loss of cholinergic skeletal innervation reduces osteocyte survival and function, causing osteopenia and impaired skeletal adaptation to moderate exercise. These results uncover a cholinergic neuro-osteocyte interface that regulates skeletogenesis and skeletal turnover through bone-anabolic effects.