Smad2/3-pathway ligand trap luspatercept enhances erythroid differentiation in murine β-thalassaemia by increasing GATA-1 availability

J Cell Mol Med. 2020 Jun;24(11):6162-6177. doi: 10.1111/jcmm.15243. Epub 2020 Apr 29.

Abstract

In β-thalassaemia, anaemia results from ineffective erythropoiesis characterized by inhibition of late-stage erythroid differentiation. We earlier used luspatercept and RAP-536 protein traps for certain Smad2/3-pathway ligands to implicate Smad2/3-pathway overactivation in dysregulated erythroid differentiation associated with murine β-thalassaemia and myelodysplasia. Importantly, luspatercept alleviates anaemia and has been shown to reduce transfusion burden in patients with β-thalassaemia or myelodysplasia. Here, we investigated the molecular mechanisms underlying luspatercept action and pSmad2/3-mediated inhibition of erythroid differentiation. In murine erythroleukemic (MEL) cells in vitro, ligand-mediated overactivation of the Smad2/3 pathway reduced nuclear levels of GATA-1 (GATA-binding factor-1) and its transcriptional activator TIF1γ (transcription intermediary factor 1γ), increased levels of reactive oxygen species, reduced cell viability and haemoglobin levels, and inhibited erythroid differentiation. Co-treatment with luspatercept in MEL cells partially or completely restored each of these. In β-thalassaemic mice, RAP-536 up-regulated Gata1 and its target gene signature in erythroid precursors determined by transcriptional profiling and gene set enrichment analysis, restored nuclear levels of GATA-1 in erythroid precursors, and nuclear distribution of TIF1γ in erythroblasts. Bone marrow cells from β-thalassaemic mice treated with luspatercept also exhibited restored nuclear availability of GATA-1 ex vivo. Our results implicate GATA-1, and likely TIF1γ, as key mediators of luspatercept/RAP-536 action in alleviating ineffective erythropoiesis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Activin Receptors, Type II / pharmacology*
  • Anemia / complications
  • Anemia / drug therapy
  • Animals
  • Bone Marrow Cells / drug effects
  • Bone Marrow Cells / metabolism
  • Cell Differentiation* / drug effects
  • Cell Line, Tumor
  • Cell Nucleus / drug effects
  • Cell Nucleus / metabolism
  • Cell Survival / drug effects
  • Disease Models, Animal
  • Erythroblasts
  • Erythroid Cells / drug effects
  • Erythroid Cells / pathology*
  • GATA1 Transcription Factor / metabolism*
  • Hemoglobins / metabolism
  • Immunoglobulin Fc Fragments / pharmacology*
  • Leukemia, Erythroblastic, Acute / pathology
  • Ligands
  • Mice, Inbred C57BL
  • Phosphorylation / drug effects
  • Reactive Oxygen Species / metabolism
  • Recombinant Fusion Proteins / pharmacology*
  • Signal Transduction*
  • Smad2 Protein / metabolism*
  • Smad3 Protein / metabolism*
  • Transcription Factors / metabolism
  • Up-Regulation / drug effects
  • Up-Regulation / genetics
  • beta-Thalassemia / complications
  • beta-Thalassemia / genetics
  • beta-Thalassemia / pathology*

Substances

  • GATA1 Transcription Factor
  • Hemoglobins
  • Immunoglobulin Fc Fragments
  • Ligands
  • RAP-536
  • Reactive Oxygen Species
  • Recombinant Fusion Proteins
  • Smad2 Protein
  • Smad3 Protein
  • Transcription Factors
  • Trim33 protein, mouse
  • luspatercept
  • Activin Receptors, Type II