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RINF maintenance of SMAD7 sustains human erythropoiesis
blood and adult bone marrow. The shRNA-RINF-induced acceleration of erythropoiesis was supported by the reduction in cell size (not shown) and the more rapid downregulation of c-KIT and PU.1 mRNA in RINF knock- down cells (Figure 3D). The accelerated maturation was also characterized by a pronounced reduction of ProE cells (∼2.8-fold less) enumerated at day 11 (Figure 3B). Despite a weaker efficiency of shRINF#3 to knockdown RINF expression compared to shRINF#4 (Online Supplementary Figure S3B, Figure 3E, left panel), we also confirmed that a similar phenotype, i.e., accelerated mat- uration (as shown by increased benzidine staining) (Figure 3E, right panel) and a reduction in the total num-
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ber of RBC produced at day 17 (Figure 3F), was obtained with another shRNA sequence targeting RINF.
RINF controls erythroid maturation and red blood cell expansion in a transforming growth factor b-dependent manner
We then wondered whether TGFb, a well-known induc- er of erythroid maturation and inhibitor of RBC expansion, could be involved in the RINF-dependent phenotype. In agreement with an important role for autocrine TGFb sig- naling in the early stages of erythropoiesis, both TGFB1 and TGFBRI were highly expressed in erythroid progenitors (Figure 4A). To functionally validate an involvement of the
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Figure 1. RINF is expressed in early erythropoiesis and downregulated during maturation of human hematopoietic stem and progenitor cells. (A) Schematic representation of cell culture experiments. CD34+ cells (from cord blood or adult bone marrow) were first amplified for 7 days (D- 7 to D0, in black) in the presence of stem cell factor (SCF), interleukin-3 (IL3) and interleukin-6 (IL6). Cells were sorted for CD36 expression and erythropoietin (EPO) was added to the medium in order to trigger the late stage of erythropoiesis, a phase lasting 11-17 days (in red). (B) For mor- phological characterization, cells were cytospun and stained with May- Grünwald-Giemsa (MGG) and the repartition of cells among differentia- tion stages indicated in percentages (right panel): early progenitor (Prog), proerythroblast (ProE), early and late basophilic erythroblast (Baso), poly- chromatophilic erythroblast (Poly), orthochromatic erythroblast (Ortho), and reticulocyte (Retic). The scale bar represents 20 mm. The experiment presented here is representative of four distinct experiments which were performed on cells isolated from human cord blood. RINF protein was detected by western-blot analysis (lower panel). HSC70 protein was used as a loading control. For each lane, 40 mg of protein were loaded. Band intensities were quantified with the Multigauge software and the relative expression of RINF protein (normalized to the loading control) is indicat- ed above the image in percentages on day 3. (C) CXXC5 mRNA expres- sion was measured by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). CXXC5 mRNA expression is downregulated from the basophilic erythroblast stage. Here, CD34+ cells were isolated from adult human bone marrow, but a similar expression profile was observed with cells isolated from cord blood.
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