Genetic deletion of Cdc42GAP reveals a role of Cdc42 in erythropoiesis and hematopoietic stem/progenitor cell survival, adhesion, and engraftment

Abstract

Rho family GTPases are key signal transducers in cell regulation. Although a body of literature has implicated the Rho family members Rac1 and Rac2 in multiple hematopoietic-cell functions, the role of Cdc42 in hematopoiesis remains unclear. Here we have examined the hematopoietic properties and the hematopoietic stem/progenitor cell (HSP) functions of gene-targeted mice carrying null alleles of cdc42gap, a negative regulator of Cdc42. The Cdc42GAP-/- fetal liver and bone marrow cells showed a 3-fold increase in Cdc42 activity but normal Rac and RhoA activities, indicating that Cdc42GAP knockout resulted in a gain of Cdc42 activity in the hematopoietic tissues. Cdc42GAP-/- mice were anemic. The cellularity of fetal liver and bone marrow, the number and composition percentage of HSPs, and the erythroid blast-forming unit and colony-forming unit (BFU-E/CFU-E) activities were significantly reduced in the homozygous mice. The decrease in HSP number was associated with increased apoptosis of the Cdc42GAP-/- HSPs and the activation of JNK-mediated apoptotic machinery. Moreover, homozygous HSPs showed impaired cortical F-actin assembly, deficiency in adhesion and migration, and defective engraftment. These results provide evidence that Cdc42 activity is important for erythropoiesis and for multiple HSP functions, including survival, adhesion, and engraftment.

Figure 1.

Genetic deletion of Cdc42GAP and effects on Rho GTPase activities in fetal liver-derived hematopoietic cells and on hematopoietic organ cellularity. (A) Low-density fetal liver cells from WT (+/+) or homozygous (-/-) mice were immunoblotted with anti-Cdc42GAP monoclonal antibody for the detection of Cdc42GAP expression. (B) Low-density cells from E14.5 fetal livers of WT, heterozygous, or homozygous mice were subjected to effector domain pull-down assays, and the activities of Cdc42, Rac1, Rac2 (detected by GST-PAK1), and RhoA (detected by GST-Rhotekin) were examined and compared in the anti-Cdc42, Rac1, Rac2, or RhoA immunoblots. Blotting of the respective total-cell lysates was carried out in parallel. Relative amounts of GTP-bound form of the GTPases were quantified by densitometry measurements and normalized to those of the WT cells. (C-E) E14.5 embryos of WT (n = 20) or homozygous (n = 40) mice were compared for their fetal-liver weights (C), total fetal liver-cell numbers (D), and low-density fetal liver-cell numbers (E). (F) Bone marrow cell numbers from 3-day-old pups were quantified for the WT (n = 5) and homozygous (n = 5) mice.

Figure 2.

Effects of Cdc42GAP deletion on phenotypic HSP number and erythropoietic progenitor activities. (A) WT or homozygous E14.5 fetal liver (n = 20 for each group) cells were stained with lineage-FITC, Sca1-PE, and c-Kit-APC and were examined by flow cytometry for HSP number and percentage composition. (B-D) E14.5 fetal liver (n = 20 for each group) cells (B-C) or 3-day-old pup (n = 5 for each group)-derived bone marrow cells (D) were cultured in methylcellulose medium supplemented with 100 ng/mL SCF, 100 ng/mL IL-3, 4 U/mL EPO, and 100 ng/mL G-CSF for 7 days for the development of CFU-GM, BFU-E, or CFU-GEMM colonies (B) or with 100 ng/mL SCF and 4 U/mL EPO for 2 days for the growth of CFU-E colonies (C-D).

Figure 3.

Cdc42GAP regulates HSP proliferation and survival. Lin^-c-Kit⁺ HSPs from WT (n = 8) and homozygous (n = 9) mice were isolated from the respective low-density fetal liver cells by flow cytometry. After 6-hour starvation, the cells were stimulated with 100 ng/mL SCF. (A) At the indicated time points, the cell numbers of each genotype were quantified in parallel. (B) Cell-cycle progression after 24-hour stimulation by SCF was analyzed by PI/RNase staining and FACS. (C) Apoptotic-cell populations were determined by annexin-V and 7-AAD staining and FACS analysis 24 hours after SCF stimulation.

Figure 4.

Increased spontaneous apoptosis in the Cdc42GAP-deficient fetal livers. Female heterozygous mice were timed for pregnancy and intraperitoneally injected with 100 μg BrdU/g body weight at E14.5. One hour after BrdU incorporation, the mice were killed and then underwent transcardial perfusion. Fetal livers of the embryos were dissected and embedded in paraffin while genotyping was carried out in parallel. Five-micrometer sections were deparaffinized and examined by anti-BrdU (A) or TUNEL (B) immunofluorescence microscopy. Right panels show quantification of at least 6 fields of the anti-BrdU or TUNEL fluorescence section shown in the left panels. Data are representative of 3 independent measurements.

Figure 5.

Cdc42GAP deletion leads to JNK activation and increased apoptosis of HSPs. HSPs were cultured in the presence or absence of 10 μM JNK inhibitor SP600125 and were starved for 6 hours. Cells were stimulated with 100 ng/mL SCF for 10 minutes before they were subjected to Western blot analysis (A) or were analyzed for apoptosis by annexin-V- and 7-AAD-based FACS at different time points (B). (C) JNK siRNA-expressing retrovirus or the control retrovirus-treated HSPs were selected with puromycin (2 μg/mL) for 2 days to enrich the transduced cell population before they were subjected to apoptosis analysis.

Figure 6.

Cdc42GAP regulates HSP F-actin assembly, adhesion, and migration. (A) WT or homozygous LSK E14.5 fetal liver cells were isolated by FACS and cultured overnight in the chamber slides. Cells were stimulated with 100 ng/mL SCF or 100 ng/mL SDF-1α for 10 minutes and stained for F-actin with TRITC-conjugated phalloidin. (B-C) WT or homozygous low-density fetal liver (n = 10 for each group) cells were subjected to adhesion and migration assays in vitro. The percentages of the cells that adhered to an H-296 fragment of fibronectin after 1-hour incubation (B) and that migrated toward a 100 ng/mL SDF-1α gradient in 4 hours in a Transwell migration chamber (C) were calculated based on the colony-forming activities of the total input cells and the adherent or migrated cells assayed in a methylcellulose medium containing 100 ng/mL SCF, 100 ng/mL IL-3, 4 U/mL EPO, and 100 ng/mL G-CSF.

Figure 7.

Cdc42GAP regulates HSP engraftment. One million donor-derived fetal liver cells mixed with 1 million recipient-derived fetal liver cells from B6.SJL/BoyJ mice were injected into lethally irradiated B6.SJL/BoyJ mice. Chimerism in the peripheral blood of the recipients was examined at indicated times after transplantation. Numbers of each group of animals that underwent transplantation are indicated. Results are representative of 2 independent experiments.