Guanine nucleotide exchange factor Vav1 regulates perivascular homing and bone marrow retention of hematopoietic stem and progenitor cells

Abstract

Engraftment and maintenance of hematopoietic stem and progenitor cells (HSPC) depend on their ability to respond to extracellular signals from the bone marrow microenvironment, but the critical intracellular pathways integrating these signals remain poorly understood. Furthermore, recent studies provide contradictory evidence of the roles of vascular versus osteoblastic niche components in HSPC function. To address these questions and to dissect the complex upstream regulation of Rac GTPase activity in HSPC, we investigated the role of the hematopoietic-specific guanine nucleotide exchange factor Vav1 in HSPC localization and engraftment. Using intravital microscopy assays, we demonstrated that transplanted Vav1(-/-) HSPC showed impaired early localization near nestin(+) perivascular mesenchymal stem cells; only 6.25% of Vav1(-/-) HSPC versus 45.8% of wild-type HSPC were located less than 30 μm from a nestin(+) cell. Abnormal perivascular localization correlated with decreased retention of Vav1(-/-) HSPC in the bone marrow (44-60% reduction at 48 h posttransplant, compared with wild-type) and a very significant defect in short- and long-term engraftment in competitive and noncompetitive repopulation assays (<1.5% chimerism of Vav1(-/-) cells vs. 53-63% for wild-type cells). The engraftment defect of Vav1(-/-) HSPC was not related to alterations in proliferation, survival, or integrin-mediated adhesion. However, Vav1(-/-) HSPC showed impaired responses to SDF1α, including reduced in vitro migration in time-lapse microscopy assays, decreased circadian and pharmacologically induced mobilization in vivo, and dysregulated Rac/Cdc42 activation. These data suggest that Vav1 activity is required specifically for SDF1α-dependent perivascular homing of HSPC and suggest a critical role for this localization in retention and subsequent engraftment.

Fig. 1.

Dysregulated Rho GTPase activation in Vav1^−/− hematopoietic progenitors. (A) Vav1 activation as demonstrated by immunoprecipitation (IP) with a Vav1-specific antibody, followed by detection with phosphotyrosine (P-Tyr) antibody. WT or Vav1^−/− lineage-depleted cells were starved in 1% FBS for 6 h and stimulated with SCF + SDF1α (100 ng/mL each) for the indicated time points. WB, Western blot. (B) Vav3 phosphorylation in WT and Vav1^−/− progenitors, detected with a phosphospecific antibody. (C) Levels of active (GTP-bound) and total Rac and Cdc42 in WT or Vav1^−/− lineage-depleted cells. Cells were starved and stimulated as in A. GTP-bound Rho GTPases were precipitated with agarose-conjugated PAK1-p21-binding domain (PBD) and detected by Western blot. (D) Activation status of different signaling pathways in WT and Vav1^−/− progenitors, analyzed by Western blot with phosphospecific antibodies. Lineage-depleted cells were either freshly purified (B, baseline activation) or starved in 1% FBS for 6 h and then stimulated with SCF + SDF1α (100 ng/mL each) for the indicated time points. Arrows indicate phospho-Vav3 (B) and the different isoforms of phospho-PAK and phospho-JNK (D).

Fig. 2.

Abnormal SDF1α responses in Vav1^−/− HSPC. (A) Day–night variation in the number of CFC in peripheral blood of WT and Vav1^−/− mice. Blood was collected 4 or 16 h after the onset of light. Data represent mean ± SD, n = 4–6; *P < 0.05 (t test). NS, nonsignificant. (B) Numbers of CFC in peripheral blood of WT and Vav1^−/− mice, either untreated or treated with 5 mg/kg of AMD3100 for 1 h. Data represent mean ± SD, n = 6–7; *P < 0.05 (t test). (C) Numbers of CFC in peripheral blood of WT and Vav1^−/− mice, either untreated or treated with G-CSF at a daily dose of 200 μg/kg for 6 d. Data represent mean ± SD, n = 3; *P < 0.05, **P < 0.005 (t test). The graph shows one of two experiments that yielded similar results. (D–F) In vitro chemotaxis of WT or Vav1^−/− LSK cells on a fibronectin-coated coverslip in the presence of an SDF1α gradient, determined by time-lapse microscopy. (D) Paths followed by individual cells in 1 h. Representative fields (325 × 325 μm) from one out of three similar experiments are shown. (E) Net path length migrated in 1 h (mean ± SEM, n = 36–38 cells per genotype) and (F) speed of migration (mean ± SEM, n = 14–41). For E and F, one representative experiment of a total of three is shown. **P < 0.005 (t test).

Fig. 3.

Engraftment defect of Vav1^−/− BM cells. (A) Peripheral blood chimerism of lethally irradiated B6.SJL (CD45.1) mice transplanted with 3 × 10⁶ WT or Vav1^−/− (CD45.2) BM cells and an equal number of WT CD45.1 BM cells as competitors. (B) CD45.2 chimerism in BM and spleen for the recipient mice in A, killed 4 mo posttransplant. Data in A and B are expressed as the percentage of CD45.2⁺ cells in the nucleated fractions measured by flow cytometry. Mean ± SD, n = 5, *P < 0.005 (t test). Note: Chimerism for Vav1^−/− cells in BM and spleen was less than 2%. (C) Survival of lethally irradiated B6.SJL mice transplanted with 3 × 10⁶ WT or Vav1^−/− (CD45.2) BM cells in the absence of competitor cells; n = 10 mice per genotype. (D) Leukocyte counts in peripheral blood of the recipient mice in C surviving at each time point. Mean ± SD, n = 6–10, *P < 0.005 (t test).

Fig. 4.

Bone marrow homing and localization of Vav1^−/− HSPC. (A) Homing efficiency of WT and Vav1^−/− progenitors (CFC) to the BM of C57BL/10J recipients 16 h after transplant. Data represent mean ± SD, n = 5. (B) Number of DiD⁺ LSK cells detected by intravital microscopy in a 4 × 6 mm region of the calvarium of Col2.3-GFP mice 1 h after transplant, for an input of 30,000 LSK cells (mean ± SD, n = 3). (C–E) Distances (in μm) of transplanted LSK cells to nestin⁺ cells (C), osteoblastic cells (D), and endosteal surface (E) in the calvarium of Nestin-GFP (C) or Col2.3-GFP (D and E) mice, determined 1 h after transplant. Data represent measurements for individual LSK cells; horizontal lines represent the mean. C represents pooled data from three mice per genotype (n = 24–32); D and E represent data from one representative mouse per genotype, out of three analyzed (n = 32–34; total number of events analyzed, n = 77–98). *P = 0.005 (t test).

Fig. 5.

Reduced retention of Vav1^−/− HSPC in the BM. (A) Number of DiD⁺ LSK cells detected by intravital microscopy in a 4 × 6 mm region of the calvarium of Nestin-GFP and Col2.3-GFP mice 48 h after transplant (mean ± SD, n = 4–5) for an input of 30,000 LSK cells. *P < 0.05 (t test). (B–D) Distances (in μm) of transplanted LSK cells to nestin⁺ cells (B), osteoblastic cells (C), and endosteal surface (D) in the calvarium of Nestin-GFP (B) or Col2.3-GFP (C and D) mice, determined 48 h after transplant. Data represent measurements for individual LSK cells; horizontal lines represent the mean. B represents pooled data from three mice per genotype (n = 29–71); C and D represent data from one representative mouse per genotype, out of three analyzed (n = 38–59; total number of events analyzed, n = 102–139).