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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 partially repopulate the myeloid lineage of primary recipients.Gab2−/−STAT5ab/ and littermate control embryos were used to derive FL cells. FL cells from 3 independent Gab2−/−STAT5ab/ 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/ N = 5; Gab2−/−STAT5ab/ N = 12. Each dot represents an individual mouse that was analyzed. Horizontal bars indicate the average for all mice analyzed per group.
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pone-0009152-g006: STAT5 and Gab2 double mutant fetal liver HSCs partially repopulate the myeloid lineage of primary recipients.Gab2−/−STAT5ab/ and littermate control embryos were used to derive FL cells. FL cells from 3 independent Gab2−/−STAT5ab/ 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/ N = 5; Gab2−/−STAT5ab/ N = 12. Each dot represents an individual mouse that was analyzed. Horizontal bars indicate the average for all mice analyzed per group.

Mentions: To determine whether double mutant FL HSC were capable of long-term engraftment of irradiated hosts, total FL cells from 3 independent embryos or different litters were transplanted into CD45.1 recipient mice at a donor to recipient ratio of 1 FL per 5 recipient mice. The percentage of donor CD45.2 engraftment was determined at times up to 16 weeks later (Fig. 6A). The donor engraftment was not significantly changed by combined STAT5ab/ and Gab2−/− genotype (P = 0.415; 2-way ANOVA). Differences between STAT5ab/ and Gab2−/−STAT5ab/ were not significant (P = 0.104; Mann-Whitney U test). When analyzing the overall contribution of the donor to host hematopoiesis, there was a trend toward lower levels of donor chimerism and 3/15 primary recipients did not survive transplantation. Importantly, defects in reconstitution obtained with STAT5ab/ FL cells were consistent with prior work showing declines in T and B lymphocytes (Fig. 6B). As expected, Gab2 deficiency alone did not have any impact upon hematopoietic reconstitution in the absence of a competitor. Differences between STAT5ab/ and Gab2−/−STAT5ab/ were not significant (P = 0.383; Mann-Whitney U test). Even in the complete absence of STAT5 and Gab2, no additional declines were observed in steady-state hematopoiesis in recipients of double mutant FL transplant relative to STAT5ab/ alone.


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 partially repopulate the myeloid lineage of primary recipients.Gab2−/−STAT5ab/ and littermate control embryos were used to derive FL cells. FL cells from 3 independent Gab2−/−STAT5ab/ 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/ N = 5; Gab2−/−STAT5ab/ N = 12. Each dot represents an individual mouse that was analyzed. Horizontal bars indicate the average for all mice analyzed per group.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2818849&req=5

pone-0009152-g006: STAT5 and Gab2 double mutant fetal liver HSCs partially repopulate the myeloid lineage of primary recipients.Gab2−/−STAT5ab/ and littermate control embryos were used to derive FL cells. FL cells from 3 independent Gab2−/−STAT5ab/ 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/ N = 5; Gab2−/−STAT5ab/ N = 12. Each dot represents an individual mouse that was analyzed. Horizontal bars indicate the average for all mice analyzed per group.
Mentions: To determine whether double mutant FL HSC were capable of long-term engraftment of irradiated hosts, total FL cells from 3 independent embryos or different litters were transplanted into CD45.1 recipient mice at a donor to recipient ratio of 1 FL per 5 recipient mice. The percentage of donor CD45.2 engraftment was determined at times up to 16 weeks later (Fig. 6A). The donor engraftment was not significantly changed by combined STAT5ab/ and Gab2−/− genotype (P = 0.415; 2-way ANOVA). Differences between STAT5ab/ and Gab2−/−STAT5ab/ were not significant (P = 0.104; Mann-Whitney U test). When analyzing the overall contribution of the donor to host hematopoiesis, there was a trend toward lower levels of donor chimerism and 3/15 primary recipients did not survive transplantation. Importantly, defects in reconstitution obtained with STAT5ab/ FL cells were consistent with prior work showing declines in T and B lymphocytes (Fig. 6B). As expected, Gab2 deficiency alone did not have any impact upon hematopoietic reconstitution in the absence of a competitor. Differences between STAT5ab/ and Gab2−/−STAT5ab/ were not significant (P = 0.383; Mann-Whitney U test). Even in the complete absence of STAT5 and Gab2, no additional declines were observed in steady-state hematopoiesis in recipients of double mutant FL transplant relative to STAT5ab/ alone.

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