Collapse of Ribosome Biogenesis Induces Rapid Erythroblast Differentiation

Genetic insults of ribosomal protein (RP) genes including heterozygous mutations in Diamond Blackfan Anemia (DBA) or RPS14 haploinsufficiency in acquired 5q- syndrome selectively impair erythropoiesis suggesting that the integrity of ribosome biogenesis (RB) is fully required for the production of 10^e11erythrocytes per day in humans. Defective RB consecutive to RPL5, RPL11 mutation or RPS14 deletion is thought to induce cell cycle arrest and a various degree of apoptosis in erythroblasts (EB). However, recent data showed that, loss of RPL5/RPL11neither induces apoptosis nor arrests cell cycle, but hampers the progression through the G1 phase, in primary fibroblasts. Furthermore, RB coordinates cell cycle to cell growth for the maintenance of constant cell size. In this work, we investigate the role of RB in erythroid cell differentiation and cell size regulation.

We first analyzed the renewal of ribosome during in vitro differentiation of human EB derived from cytapheresis CD34⁺ cells and mouse erythroblasts derived from mouse fetal liver by a pulsed SILAC (Stable Isotopic Labeling by Amino acids in Culture cell) riboproteomic assay. Ribosome biogenesis dramatically decreases with the disappearance of proEB and basophilic EB and the onset of poly- and ortho-chromatophilic EB. Importantly, inhibition of RNA polI by CX-5461 in proEB forced them to enter the final maturation steps with an increase of glycophorin A (GPA) expression. To study the effect of RPS14 heterozygous deletion on RB, UT7/EPO cell line was infected by a lentivirus containing an inducible GFP-shRNA RPS14. After a 48-h treatment with doxycyclin, Rps14 protein expression was reduced by half and sorted GFP-positive cells had an altered ribosome profile devoid of 40S small subunit or 80S entire particle. Consistently, RB inhibition induced a cell size reduction. Second, we compared RB level in cells responsive to SCF+EPO or EPO alone. RB was optimal when EB responded to SCF+EPO and this was correlated with cell size being higher in SCF+EPO-responsive cells compared to EPO-responsive cells. Both cytokines additively activate the cell growth regulator, p70S6K1. Third, inhibition of p70S6K1 by rapamycin, or a specific chemical S6K1 inhibitor significantly reduced RB as shown by a 50% decrease of ribosome renewal in pulsed-SILAC. Inhibition of RB by rapamycin led to a size reduction and to GPA acquisition, which are the features of erythroid cell differentiation.

Our data shows that the collapse of RB due to the loss of c-Kit and reduced activation of p70S6K1 is a key step for cell growth inhibition and induction of terminal differentiation in human or mouse erythroblasts.