Page 88 - Haematologica April 2020

Y. Xie et al.
tion, possibly due to mRNA instability. On the other hand, based on o-Dianisidine staining and WISH analysis of αe1-globin, a co-injection of constitutively active mutant RhoA Q63L mRNA was able to restore the erythropoiesis defect caused by arhgef12 MO (Figure 4A). These results indicate that Arhgef12 activates RhoA to control erythroid differentiation.
To further understand the molecular events down- stream of RhoA in erythropoiesis, the K562 cell line in which ARHGEF12 is important for its erythroid differen- tiation was examined. An antibody microarray screen found that phosphorylation of molecules in the p38 MAPK pathway was significantly decreased in the ARHGEF12 knockdown K562 cells (Online Supplementary Figure S6A). Western blotting confirmed this effect on p38 phosphorylation (Online Supplementary Figure S6B), sug- gesting that the p38 MAPK signaling pathway may con- tribute to the ARHGEF12-regulated erythropoiesis. Further confirmation using the p38 inhibitor SB202190 in zebrafish found that p38 inhibition resulted in a similar block of erythropoiesis as in the arhgef12 morphants at 4 dpf (Figure 4B). Application of anisomycin, a p38 MAPK
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activator, was able to restore the erythrocyte maturation in the arhgef12 morphants (Figure 4C). Thus, an ARHGEF12-RhoA-p38 pathway is likely to be involved in erythroid differentiation.
STAT1 expression can rescue the erythroid phenotype caused by arhgef12 knockdown in zebrafish
Human STAT1 produces two splicing variants that dif- fer at their carboxy terminus. Zebrafish has two ortholo- gous genes related to human STAT1: stat1a and stat1b. It has been shown that p38 MAPK-STAT1 pathways can regulate neutrophil development. Meanwhile, our anti- body microarray screen showed that the phosphorylation of STAT1 at serine (S) 727 was decreased approximately 2- fold in ARHGEF12 knockdown K562 cells (Online Supplementary Figure S6A). We thus further examined whether STAT1 may be downstream of p38 MAPK in reg- ulating erythropoiesis. We co-injected the HA-stat1a con- struct together with arhgef12 MO in zebrafish, and observed that the phenotype of erythropenia was restored (Figure 5A a-c, a’-c’) and αe1-globin expression recovered (Figure 5A e-g, e’-g’), compared with control embryos. A
Figure 2. rs10892563 may disrupt a GATA1-binding cis element and is related to chemotherapy-induced anemia. (A and B) rs10892563 is located at a binding site of ery- throid-specific transcription factor GATA1. Dual luciferase assay with the vector inserted with a stretch of sequence with the motif revealed that C allele of rs10892563 down- regulated GATA1 cis-transcriptional function compared to the major allele T in 293T cells; P=0.0038. (C and D) CD71-positive nucleated erythroid cells were sorted from banked bone marrow samples of acute lymphoblastic leukemia patients in remission state by fluo- rescence-activated cell sorting. The relative gene expression of ARHGEF12 was reduced in CC genotype at rs10892563 versus TT genotypes by quantitative real-time polymerase chain reaction assay; P=0.0088. (E) A total of 452 chil- dren with ALL enrolled in the Shanghai Children’s Medical Center-Acute Lymphoblastic Leukemia-2005 (SCMC-ALL-2005) protocol were genotyped targeting rs10892563. The average normal- ized red blood cell (RBC) transfu- sion units was significantly higher in patients with CC genotype than the CT genotype (P=0.0011) and the TT genotype (P<0.0001); each point represents one patient's record. (F) Distribution of RBC transfusions across different rs10892563 genotypes. All patients with CC genotype (n=34) need RBC transfusions, whereas in patients with CT and TT genotype, proportions of RBC transfusions were 61.5% and 71%, respectively. Concerning multiple red blood cell transfusions (MRT), the proportion in CC, CT and TT were 14.705%, 8.021%, and 4.762%, respectively (P<0.001).
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