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

Li G, Wang Z, Miskimen KL, Zhang Y, Tse W, Bunting KD - PLoS ONE (2010)

Bottom Line: Grb2-associated binding (Gab) adapter proteins play major roles in coordinating signaling downstream of hematopoietic cytokine receptors.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.Furthermore, important synergy between STAT5 and Gab2 was observed in HSC self-renewal, which might be exploited to optimize stem cell-based therapeutics.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology-Oncology, Department of Medicine, Case Western Reserve University, Cleveland, Ohio, United States of America.

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.

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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.
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pone-0009152-g002: 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.

Mentions: Since peripheral blood counts and steady-state BM cellularity do not provide indicators of stress hematopoiesis, we next tested KLS cytokine stimulation which measures a combination of growth and differentiation response, which differs significantly from maintenance of the steady-state HSC pool in vivo. KLS cells were sorted into 96 well plates and stimulated with cytokines as previously described[26], [28] (Fig. 1). Significant declines in cytokine response were observed for deficiency of STAT5 or Gab2 alone, as well as further decreases in Gab2−/−STAT5ab+/ KLS cells relative to single mutants in cocktails containing IL-3, IL-6, and SCF (Gab2−/−STAT5ab+/ vs. Gab2−/−, P = 0.03; Gab2−/−STAT5ab+/ vs. STAT5ab+/, P<0.01, t-test) or with IL-3, SCF, Flt-3 ligand, and TPO (Gab2−/−STAT5ab+/ vs. Gab2−/−, P = 0.02; Gab2−/−STAT5ab+/ vs. STAT5ab+/, P<0.01, t-test). Notably these cytokine combinations induce the strongest proliferation and differentiation from purified KLS cells. To determine whether defects were also observed in vivo, BM cells were harvested and transplantation experiments performed. Two independent competitive repopulation experiments were performed using adult BM cells with the same results. The average results of the two transplantation experiments are shown in Fig. 2A. STAT5ab+/ and Gab2−/− BM had competitive multilineage HSC repopulating defects averaging 36.7±6.4% and 17.5±3.5% of wild-type respectively. Notably, Gab2−/−STAT5ab+/ BM cells had an average 9.5±1.7% of wild-type engraftment. The percentage of donor engraftment between single mutant (Gab2−/− or STAT5ab+/) and Gab2−/−STAT5ab+/ were significantly different (P<0.001 for STAT5ab+/Gab2−/− vs. STAT5ab+/ or Gab2−/−STAT5ab+/ vs. Gab2−/−, Mann-Whitney U test). The reduction of donor engraftment in Gab2−/−STAT5ab+/ mice was significant (P<0.001, 2-way ANOVA). Further multilineage analysis showed that the long-term reductions in repopulation occurred in a primitive cell type (Fig. 2B). STAT5ab+/ and Gab2−/− mice were significantly different for all lineages (P = 0.001 for Gr-1 and P<0.001 for all others; 2-way ANOVA).


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

Li G, Wang Z, Miskimen KL, Zhang Y, Tse W, Bunting KD - PLoS ONE (2010)

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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2818849&req=5

pone-0009152-g002: 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.
Mentions: Since peripheral blood counts and steady-state BM cellularity do not provide indicators of stress hematopoiesis, we next tested KLS cytokine stimulation which measures a combination of growth and differentiation response, which differs significantly from maintenance of the steady-state HSC pool in vivo. KLS cells were sorted into 96 well plates and stimulated with cytokines as previously described[26], [28] (Fig. 1). Significant declines in cytokine response were observed for deficiency of STAT5 or Gab2 alone, as well as further decreases in Gab2−/−STAT5ab+/ KLS cells relative to single mutants in cocktails containing IL-3, IL-6, and SCF (Gab2−/−STAT5ab+/ vs. Gab2−/−, P = 0.03; Gab2−/−STAT5ab+/ vs. STAT5ab+/, P<0.01, t-test) or with IL-3, SCF, Flt-3 ligand, and TPO (Gab2−/−STAT5ab+/ vs. Gab2−/−, P = 0.02; Gab2−/−STAT5ab+/ vs. STAT5ab+/, P<0.01, t-test). Notably these cytokine combinations induce the strongest proliferation and differentiation from purified KLS cells. To determine whether defects were also observed in vivo, BM cells were harvested and transplantation experiments performed. Two independent competitive repopulation experiments were performed using adult BM cells with the same results. The average results of the two transplantation experiments are shown in Fig. 2A. STAT5ab+/ and Gab2−/− BM had competitive multilineage HSC repopulating defects averaging 36.7±6.4% and 17.5±3.5% of wild-type respectively. Notably, Gab2−/−STAT5ab+/ BM cells had an average 9.5±1.7% of wild-type engraftment. The percentage of donor engraftment between single mutant (Gab2−/− or STAT5ab+/) and Gab2−/−STAT5ab+/ were significantly different (P<0.001 for STAT5ab+/Gab2−/− vs. STAT5ab+/ or Gab2−/−STAT5ab+/ vs. Gab2−/−, Mann-Whitney U test). The reduction of donor engraftment in Gab2−/−STAT5ab+/ mice was significant (P<0.001, 2-way ANOVA). Further multilineage analysis showed that the long-term reductions in repopulation occurred in a primitive cell type (Fig. 2B). STAT5ab+/ and Gab2−/− mice were significantly different for all lineages (P = 0.001 for Gr-1 and P<0.001 for all others; 2-way ANOVA).

Bottom Line: Grb2-associated binding (Gab) adapter proteins play major roles in coordinating signaling downstream of hematopoietic cytokine receptors.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.Furthermore, important synergy between STAT5 and Gab2 was observed in HSC self-renewal, which might be exploited to optimize stem cell-based therapeutics.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology-Oncology, Department of Medicine, Case Western Reserve University, Cleveland, Ohio, United States of America.

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.

Show MeSH