Arhgap21+/- Mice Show Impaired Adhesion, Migration, Homing and Short Term Reconstitution of Hematopoietic Progenitor Cells

Abstract The main signaling pathway involved in migration, adhesion and homing of hematopoietic progenitor cells (HPC) is the CXCL-12, chemokine produced by bone marrow (BM) stromal cells that bind to its main receptor CXCR-4, expressed by HPC. ARHGAP21 is a RhoGAP member, negative regulators of RhoGTPAses. It was described that ARHGAP21 is involved in cellular migration and adhesion but its role in hematopoiesis is unknown. Researching Arhgap21 role in hematopoiesis, Arhgap21+/- mice were generated using Embryonic Stem cell obtained from GeneTrap consortium. Hematologic parameters were investigated by blood cell count, colony formation assay in methylcellulose medium and immunophenotipic characterization of hematopoietic populations. Transwell migration and adhesion of Lin- cells assay in the presence of CXCL12 were also performed. In vivo analysis of Arhgap21+/- HPC was achieved with homing and CFU-S assays in sublethally irradiated mice and hematopoiesis stress was generated by 5-fluoracil treatment. Arhgap21+/- mice presented more than 50% reduction in Arhgap21 expression (0.47 ± 0.13) in BM when compared to WT (1 ± 0.03) and live in normal conditions. On the other hand, Arhgap21 mice showed a reduction in BFU-E colonies and erythroid (Terr119+) committed cells in BM together with decreased peripheral blood (PB) Terr119+ cells and red blood cell number and increased medium corpuscular volume. In myeloid compartment, Arhgap21+/- mice presented less GM colonies and myeloid (Gr1/Mac1+) cells in BM followed by an increase of these cells in PB suggesting myeloid mobilization. Corroborating to this, whenArhgap21+/- BM was challenged for hematopoiesis stress with 5FU, the animals showed increased neutrophil number in peripheral blood 14 days (WT: 1887/mm3 ±721.9; Arhgap21+/-:3325/mm3 ±1640, p=0.02) and 21days (WT: 1264/mm3 ±635; Arhgap21+/-: 2182/mm3 ±854, p=0.01) after treatment, together with increased number of LSK cells in the BM (WT: 1.3% ±0.4; Arhgap21+/-: 1.9% ±0.3, p=0.006) 28 days after 5FU infusion. Erythroid and myeloid compartments reductions were observed together with an increase of Arhgap21+/- BM short term LSK( Arhgap21+/- : 0.34% ±0.13; WT: 0.18% ±0.07, p=0.03) and long term LSK (Arhgap21+/-: 0.003% ±0.001; WT: 0.002 ±0.0007, p=0.02) suggesting an attempt to restore normal hematopoietic levels. Arhgap21+/- HPC showed reduced CXCL-12-induced migration compared to WT (WT: 100%, Arhgap21+/-: 54.73% ± 13.57, p=0.01) in addition to decreased adhesion to fibronectin (Arhgap21+/-: 15% ± 3.8; WT: 27% ± 3.5, p=0.003) and a4b1 integrin expression (WT: 83.76 ± 4.35; Arhgap21+/-: 71.06 ± 7.00, p=0.008). In homing assay, the percentage of donor Lin- HPC from Arhgap21+/- mice that homed to BM (6.32 ± 2.41) or spleen (3.48 ± 1.57), were lower than those from WT mice (BM=10.09 ± 1.81;p=0.004; spleen=6.9 ± 1.48; p=0.0007) together with higher frequency of these cells in peripheral blood (WT: 6.39 ± 3.38; Arhgap21+/-: 13.95 ± 5.33, p=0.003), suggesting a retention of Arhgap21+/- HPC in the bloodstream, which inefficiently home to BM and spleen. Arhgap21+/- CFU-S capacity decline was also observed (Arhgap21+/-: 20.86 ± 2; WT: 29.29 ± 5.4 p=0.002). This is the first study showing that ARHGAP21 is involved in hematopoiesis and also shows that ARHGAP21 is an important protein for chemotaxis, adhesion, homing and short term reconstitution of HPC. Table. Hematological Parameters WT Arhgap21+/- Hemoglobin (g/dL) * 16.19 ± 0.35 15.62±0.32 RBC (10^6/uL) * 10.91 ± 0.3 10.46±0.25 Hematocrit (%) 54.78 ± 1.6 53.75± 1.3 MCV (fL) * 50 ±0.4 50.08±0.74 WBC (10^3/Ul) * 10.08 ±2 12.9±1.8 Platelets(10^3/uL) 1793 ±114.3 1770±96.5 Myeloid, Gr1+Mac1+ in BM (%) * 47.27 ±4.7 40.88 ±6.23 Erythroid, Terr119+ in BM (%) * 9.98 ±4.1 6.4 ±1.5 Short Term HSC, LSK in BM (%) * 0.18 ±0.07 0.34 ±0.13 Long Term HSC, LSK CD150+CD48- in BM (%) * 0.002 ±0.0007 0.003 ±0.001 Myeloid, Gr1+Mac1+ in PB (%) * 7.7 ±1.5 13.1 ±2.6 Erythroid, Terr119+ in PB (%) * 38.7 ±4.4 28.4 ±8.7 Myeloid Colonies, CFU-GM (number) 66.7 ±14.7 48.07 ±12.5 Erythroid Colonies, BFU-E (number) 5.5 ±2.2 3.2 ±1.5 Blood samples were collected from 9 WT and 8 Arhgap21+/- mice. RBC: red blood cell count; MCV: mean cell volume; WBC: White blood cell count. Hematopoietic populations were analyzed in flow cytometry and colony formation in methylcellulose assay. Data are expressed as mean ± standard deviation, analyzed by Student t test and considered statistically significant (*) if p ≤ 0.05. Disclosures No relevant conflicts of interest to declare.