Gab2 promotes hematopoietic stem cell maintenance and self-renewal synergistically with STAT5

Abstract

Background: Grb2-associated binding (Gab) adapter proteins play major roles in coordinating signaling downstream of hematopoietic cytokine receptors. In hematopoietic cells, Gab2 can modulate phosphatidylinositol-3 kinase and mitogen associated protein kinase activities and regulate the long-term multilineage competitive repopulating activity of hematopoietic stem cells (HSCs). Gab2 may also act in a linear pathway upstream or downstream of signal transducer and activator of transcription-5 (STAT5), a major positive regulator of HSC function. Therefore, we aimed to determine whether Gab2 and STAT5 function in hematopoiesis in a redundant or non-redundant manner.

Methodology/principal findings: To do this we generated Gab2 mutant mice with heterozygous and homozygous deletions of STAT5. In heterozygous STAT5 mutant mice, deficiencies in HSC/multipotent progenitors were reflected by decreased long-term repopulating activity. This reduction in repopulation function was mirrored in the reduced growth response to early-acting cytokines from sorted double mutant c-Kit(+)Lin(-)Sca-1(+) (KLS) cells. Importantly, in non-ablated newborn mice, the host steady-state engraftment ability was impaired by loss of Gab2 in heterozygous STAT5 mutant background. Fetal liver cells isolated from homozygous STAT5 mutant mice lacking Gab2 showed significant reduction in HSC number (KLS CD150(+)CD48(-)), reduced HSC survival, and dramatic loss of self-renewal potential as measured by serial transplantation.

Conclusions/significance: These data demonstrate new functions for Gab2 in hematopoiesis in a manner that is non-redundant with STAT5. Furthermore, important synergy between STAT5 and Gab2 was observed in HSC self-renewal, which might be exploited to optimize stem cell-based therapeutics.

Figure 1. Combined deficiency of Gab2 with heterozygous STAT5 reduces KLS cell responses to early acting cytokines.

KLS cells were sorted by flow cytometry and plated in 96-well plates in the presence of various cytokine cocktails. Total nucleated cells were counted 6 days later. IL-3 indicates murine IL-3 (20 ng/ml); 6, human IL-6 (50 ng/ml); S, murine stem cell factor (50 ng/ml); F, murine Flt3-ligand (50 ng/ml); and T, murine thrombopoietin (50 ng/ml). The cell expansion in each group of cytokines was plotted relative to wild-type control cells for each stimulation experiment. The results are the average ± SD for 3 experiments. Absolute cell expansions were comparable to previous descriptions, .

Figure 2. Combined deficiency of Gab2 with heterozygous STAT5 leads to declines in adult HSC activity.

A. BM cells from 3–5 donor mice were collected and mixed 1:1 with wild-type competitor (CD45.1) and transplanted into lethally-irradiated hosts for competitive repopulation analysis. Recipient mice were bled 12 weeks later for flow cytometry analysis. Shown is the average of two independent experiments with 5 mice per group in each experiment. B. At 16 weeks post transplant, mice were bled again for multilineage analysis for the same two independent experiments. Peripheral blood leukocytes were stained with antibodies to multiple lineage markers including Gr-1, B220, Ter119, and CD4.

Figure 3. Combined deficiency of Gab2 with heterozygous STAT5 allows replacement of newborn HSC with a donor graft.

A. Pups generated from inter-cross of Gab2^+/−STAT5ab^+/null mice were injected intraperitoneally with 1×10⁷ CD45.1 positive BM cells 2–3 days after birth. At 4 weeks of age, the newly weaned mice were genotyped for STAT5 and Gab2. From multiple sets of experiments, totals of WT N = 22; STAT5ab^+/null N = 27; Gab2^−/− N = 9; and Gab2^−/−STAT5ab^+/null N = 5 mice were obtained and analyzed for evidence of long-term donor engraftment. B. At 16 weeks following injection, the mice were analyzed by flow cytometry of the peripheral blood leukocytes for engraftment with CD45.1 positive donor cells. Horizontal bars indicate the average for all mice analyzed per group. All comparisons were done by Mann-Whitney U test. C. BM was pooled from primary recipients and injected into 5 lethally-irradiated secondary recipients. Individual primary donors utilized for secondary transplantation are marked in panel B by open circles. The percentage of CD45.1 positive cells co-staining for Gr-1, B220, Ter119, or CD4 is shown from secondary transplantation and analysis 12 weeks later. The wild-type group was not included in the secondary transplantation since the primary recipients had little to no evidence of donor engraftment.

Figure 4. STAT5 and Gab2 double mutant fetal liver has reduced CFU-C and HSC.

Gab2^−/−STAT5ab^null/null and littermate control mice derived from Gab2^+/−STAT5ab^+/null inter-cross were generated and characterized. A. Fetal liver cells were plated into methylcellulose medium for Wild-type N = 3; STAT5ab^null/null N = 3; Gab2^−/− N = 3; STAT5ab^null/nullGab2^−/− N = 4. All 4 groups were significantly different from each other (P<0.005, Student's two-tailed t-test). B. The HSC enriched c-Kit⁺Lin⁻Sca-1⁺ (KLS) population per fetal liver was determined for Wild-type N = 3; STAT5ab^null/null N = 4; Gab2^−/− N = 6; STAT5ab^null/nullGab2^−/− N = 5. C. The average number of long-term HSC (KLS CD150⁺CD48⁻) cells per FL were determined for Wild-type N = 3; STAT5ab^null/null N = 6; Gab2^−/− N = 4; Gab2^−/−STAT5ab^null/null N = 5.

Figure 5. STAT5 and Gab2 double mutant fetal liver HSCs have reduced survival.

Analysis of the frequency of early apoptotic cells by the Annexin V - DAPI flow cytometry assay on E14.5 FL cells. Early apoptotic cells were defined as Annexin V⁺/DAPI⁻. A. Percentages of apoptosis in the lineage-negative fraction. B. Percentages of apoptosis in the KLS fraction. (*: Gab2^−/−STAT5ab^null/null vs. STAT5ab^null/null p<0.05; **: Gab2^−/−STAT5ab^null/null vs. Gab2^−/− P<0.05; ***: Gab2^−/−STAT5ab^null/null vs. Wild-type P<0.05; +: Wild-type vs. STAT5ab^null/null p<0.05). For panels A and B, the numbers of mice in each of the 4 groups were as follows: Wild-type N = 4; STAT5ab^null/null N = 7; Gab2^−/− N = 3; STAT5ab^null/nullGab2^−/− N = 4. C. A representative flow cytometry plot showing Annexin V and DAPI gating on KLS cells among the 4 groups.

Figure 6. STAT5 and Gab2 double mutant fetal liver HSCs partially repopulate the myeloid lineage of primary recipients.

Gab2^−/−STAT5ab^null/null and littermate control embryos were used to derive FL cells. FL cells from 3 independent Gab2^−/−STAT5ab^null/null embryos were each injected into 5 lethally-irradiated CD45.1 positive hosts to determine the hematopoietic repopulating potential. For other groups, 1 FL was injected into 5 recipient mice. A. The percentage of donor engraftment was determined by flow cytometry for expression of CD45.2. B. Peripheral lympho-myeloid blood donor contribution was determined by flow cytometry for Gr-1, B220, or CD4, 16 weeks later for three independent transplant experiments. The total number of mice from all 3 experiments was WT N = 5; Gab2^−/− N = 5; STAT5ab^null/null N = 5; Gab2^−/−STAT5ab^null/null N = 12. Each dot represents an individual mouse that was analyzed. Horizontal bars indicate the average for all mice analyzed per group.

Figure 7. STAT5 and Gab2 double mutant fetal liver HSCs are defective in repopulation of secondary recipients.

At 14 weeks following transplant, 3 mice from each group were euthanized from the cohorts of Fig. 6 (Wild-type, Gab2^−/−, STAT5ab^null/null, and 3 sets of Gab2^−/−STAT5ab^null/null). BM cells were pooled from these primary recipient mice and transplanted into lethally-irradiated secondary CD45.1 hosts at a ratio of 1 donor to 5 recipient mice. A. Overall mouse survival was monitored on daily basis for WT N = 5; Gab2^−/− N = 4; STAT5ab^null/null N = 5; and Gab2^−/−STAT5ab^null/null N = 15. B. Donor engraftment in the peripheral blood of secondary transplanted mice for four combinations of STAT5 and Gab2 genotype that survived past approximately 12 weeks for WT N = 5; Gab2^−/− N = 4; STAT5ab^null/null N = 5; and Gab2^−/−STAT5ab^null/null N = 4. C. The absolute number of Gr-1 positive cells was determined by antibody staining and flow cytometry combined with the total white blood cell count per µl of whole blood. Horizontal bars indicate the average for all mice analyzed per group.