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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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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/ vs. STAT5ab/ p<0.05; **: Gab2−/−STAT5ab/ vs. Gab2−/− P<0.05; ***: Gab2−/−STAT5ab/ vs. Wild-type P<0.05; +: Wild-type vs. STAT5ab/ 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/ N = 7; Gab2−/− N = 3; STAT5ab/Gab2−/− N = 4. C. A representative flow cytometry plot showing Annexin V and DAPI gating on KLS cells among the 4 groups.
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pone-0009152-g005: 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/ vs. STAT5ab/ p<0.05; **: Gab2−/−STAT5ab/ vs. Gab2−/− P<0.05; ***: Gab2−/−STAT5ab/ vs. Wild-type P<0.05; +: Wild-type vs. STAT5ab/ 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/ N = 7; Gab2−/− N = 3; STAT5ab/Gab2−/− N = 4. C. A representative flow cytometry plot showing Annexin V and DAPI gating on KLS cells among the 4 groups.

Mentions: To better understand why KLS cell numbers were reduced, FL lineage-negative and KLS populations were analyzed by flow cytometry to determine the fraction of Annexin V stained cells. A marked increase in early apoptosis as measured by increased Annexin V+DAPI− cells was observed for double mutant FL cells relative to wild-type littermate control (Fig. 5). The difference was significantly higher than either STAT5 or Gab2 mutant alone. Significantly reduced cell survival was observed in the KLS fraction (P = 0.02; 2-way ANOVA), but not in the LIN- fraction (P = 0.056; 2-way ANOVA), indicating non-redundant function in regulating survival occurred in primitive HSC, a finding that is consistent with the reduced KLS number only in the STAT5/Gab2 double mutant mice.


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)

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/ vs. STAT5ab/ p<0.05; **: Gab2−/−STAT5ab/ vs. Gab2−/− P<0.05; ***: Gab2−/−STAT5ab/ vs. Wild-type P<0.05; +: Wild-type vs. STAT5ab/ 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/ N = 7; Gab2−/− N = 3; STAT5ab/Gab2−/− N = 4. C. A representative flow cytometry plot showing Annexin V and DAPI gating on KLS cells among the 4 groups.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2818849&req=5

pone-0009152-g005: 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/ vs. STAT5ab/ p<0.05; **: Gab2−/−STAT5ab/ vs. Gab2−/− P<0.05; ***: Gab2−/−STAT5ab/ vs. Wild-type P<0.05; +: Wild-type vs. STAT5ab/ 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/ N = 7; Gab2−/− N = 3; STAT5ab/Gab2−/− N = 4. C. A representative flow cytometry plot showing Annexin V and DAPI gating on KLS cells among the 4 groups.
Mentions: To better understand why KLS cell numbers were reduced, FL lineage-negative and KLS populations were analyzed by flow cytometry to determine the fraction of Annexin V stained cells. A marked increase in early apoptosis as measured by increased Annexin V+DAPI− cells was observed for double mutant FL cells relative to wild-type littermate control (Fig. 5). The difference was significantly higher than either STAT5 or Gab2 mutant alone. Significantly reduced cell survival was observed in the KLS fraction (P = 0.02; 2-way ANOVA), but not in the LIN- fraction (P = 0.056; 2-way ANOVA), indicating non-redundant function in regulating survival occurred in primitive HSC, a finding that is consistent with the reduced KLS number only in the STAT5/Gab2 double mutant mice.

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