Abstract
Diamond Blackfan Anemia (DBA) is an inherited bone marrow failure syndrome with clinical features of red cell aplasia and variable developmental abnormalities. Most affected patients have heterozygous loss of function mutations in ribosomal protein genes but the pathogenic mechanism is still unknown. We generated induced pluripotent stem cells from DBA patients carrying RPS19 or RPL5 mutations. Transcriptome analysis revealed the striking dysregulation of the transforming growth factor β (TGFβ) signaling pathway in DBA lines. Expression of TGFβ target genes, such as TGFBI, BAMBI, COL3A1 and SERPINE1 was significantly increased in the DBA iPSCs. We quantified intermediates in canonical and non-canonical TGFβ pathways and observed a significant increase in the levels of the non-canonical pathway mediator p-JNK in the DBA iPSCs. Moreover, when the mutant cells were corrected by ectopic expression of WT RPS19 or RPL5, levels of p-JNK returned to normal. Surprisingly, nuclear levels of SMAD4, a mediator of canonical TGFβ signaling, were decreased in DBA cells due to increased proteolytic turnover. We also observed the up-regulation of TGFβ1R, TGFβ2, CDKN1A and SERPINE1 mRNA, and the significant decrease of GATA1 mRNA in the primitive multilineage progenitors. In summary our observations identify for the first time a dysregulation of the TGFβ pathway in the pathobiology of DBA.
Highlights
Diamond Blackfan Anemia (DBA) is an inherited bone marrow failure syndrome that presents in early childhood with macrocytic normochromic anemia and sometimes with variablePLOS ONE | DOI:10.1371/journal.pone.0134878 August 10, 2015TGFß Signaling in Diamond Blackfan Anemia collection and analysis, decision to publish, or preparation of the manuscript
We observed a significant increase of SERPINE1 (18.30±1.7 fold for DBA line with RPS19 mutation; 2.40±0.08 fold for RPL5 line) and TGFBI (8.58±0.34 fold for RPS19 line; 4.78 ±0.14 fold for RPL5 line) in both DBA induced pluripotent stem cells (iPSCs) lines
Based on the results we have presented here, we observed that the transforming growth factor β (TGFβ) signaling is dysregulated in the DBA iPSCs, and we hypothesize that ribosomal proteins (RP) haploinsufficiency leads to an increasing expression of downstream TGFβ targets through non-canonical signaling (Fig 7)
Summary
Diamond Blackfan Anemia (DBA) is an inherited bone marrow failure syndrome that presents in early childhood with macrocytic normochromic anemia and sometimes with variable. Phosphorylated receptor activated SMADs (R-SMADs) bind SMAD4 and are transported to the nucleus where they act as transcription factors to regulate TGFβ target genes such as those encoding extra-cellular matrix proteins[13]. In addition TGFβ can signal through SMAD-independent non-canonical pathways These include signal transduction through PI3K/AKT, small GTPases and the MAP kinases ERK or JNK/p38 [14]. In this paper we show that TGFβ signaling is dysregulated in DBA induced pluripotent stem cells (iPSCs). This identifies a new player in DBA that is implicated in the regulation of the hematopoietic stem cell and in erythroid differentiation and is highly drugable with several compounds already in clinical trial
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