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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 has reduced CFU-C and HSC.Gab2−/−STAT5ab/ and littermate control mice derived from Gab2+/−STAT5ab+/ inter-cross were generated and characterized. A. Fetal liver cells were plated into methylcellulose medium for Wild-type N = 3; STAT5ab/ N = 3; Gab2−/− N = 3; STAT5ab/Gab2−/− 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/ N = 4; Gab2−/− N = 6; STAT5ab/Gab2−/− N = 5. C. The average number of long-term HSC (KLS CD150+CD48−) cells per FL were determined for Wild-type N = 3; STAT5ab/ N = 6; Gab2−/− N = 4; Gab2−/−STAT5ab/ N = 5.
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pone-0009152-g004: STAT5 and Gab2 double mutant fetal liver has reduced CFU-C and HSC.Gab2−/−STAT5ab/ and littermate control mice derived from Gab2+/−STAT5ab+/ inter-cross were generated and characterized. A. Fetal liver cells were plated into methylcellulose medium for Wild-type N = 3; STAT5ab/ N = 3; Gab2−/− N = 3; STAT5ab/Gab2−/− 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/ N = 4; Gab2−/− N = 6; STAT5ab/Gab2−/− N = 5. C. The average number of long-term HSC (KLS CD150+CD48−) cells per FL were determined for Wild-type N = 3; STAT5ab/ N = 6; Gab2−/− N = 4; Gab2−/−STAT5ab/ N = 5.

Mentions: Since STAT5ab/ mice were not alive at the newborn or adult stages of development, we analyzed E14.5 fetal liver (FL) where STAT5ab/ embryos can survive[29]. At E14.5, the expected Mendelian ratio (1/16) of double mutant was obtained (15/231), indicating the STAT5/Gab2 double mutant embryos have no overall survival disadvantage at this stage of development. However, the CFU-C frequency of STAT5ab/Gab2−/− FL was reduced to 24% of normal (N = 4) compared with STAT5ab/ or Gab2−/− FL whose CFU-C frequency were reduced to 58% and 39% of wild-type respectively (Fig. 4A). The Gab2−/−STAT5ab/ CFU-C frequency was significantly lower than either Gab2−/− or STAT5ab/ groups (P<0.006, Mann-Whitney U Test; P = 0.014, 2-way ANOVA). The specific types of myeloid CFU-C were not enumerated for these studies since prior work showed that STAT5 and Gab2 mutant mice have reduced numbers of all types with no particular bias toward a single lineage.


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 has reduced CFU-C and HSC.Gab2−/−STAT5ab/ and littermate control mice derived from Gab2+/−STAT5ab+/ inter-cross were generated and characterized. A. Fetal liver cells were plated into methylcellulose medium for Wild-type N = 3; STAT5ab/ N = 3; Gab2−/− N = 3; STAT5ab/Gab2−/− 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/ N = 4; Gab2−/− N = 6; STAT5ab/Gab2−/− N = 5. C. The average number of long-term HSC (KLS CD150+CD48−) cells per FL were determined for Wild-type N = 3; STAT5ab/ N = 6; Gab2−/− N = 4; Gab2−/−STAT5ab/ N = 5.
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Related In: Results  -  Collection

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pone-0009152-g004: STAT5 and Gab2 double mutant fetal liver has reduced CFU-C and HSC.Gab2−/−STAT5ab/ and littermate control mice derived from Gab2+/−STAT5ab+/ inter-cross were generated and characterized. A. Fetal liver cells were plated into methylcellulose medium for Wild-type N = 3; STAT5ab/ N = 3; Gab2−/− N = 3; STAT5ab/Gab2−/− 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/ N = 4; Gab2−/− N = 6; STAT5ab/Gab2−/− N = 5. C. The average number of long-term HSC (KLS CD150+CD48−) cells per FL were determined for Wild-type N = 3; STAT5ab/ N = 6; Gab2−/− N = 4; Gab2−/−STAT5ab/ N = 5.
Mentions: Since STAT5ab/ mice were not alive at the newborn or adult stages of development, we analyzed E14.5 fetal liver (FL) where STAT5ab/ embryos can survive[29]. At E14.5, the expected Mendelian ratio (1/16) of double mutant was obtained (15/231), indicating the STAT5/Gab2 double mutant embryos have no overall survival disadvantage at this stage of development. However, the CFU-C frequency of STAT5ab/Gab2−/− FL was reduced to 24% of normal (N = 4) compared with STAT5ab/ or Gab2−/− FL whose CFU-C frequency were reduced to 58% and 39% of wild-type respectively (Fig. 4A). The Gab2−/−STAT5ab/ CFU-C frequency was significantly lower than either Gab2−/− or STAT5ab/ groups (P<0.006, Mann-Whitney U Test; P = 0.014, 2-way ANOVA). The specific types of myeloid CFU-C were not enumerated for these studies since prior work showed that STAT5 and Gab2 mutant mice have reduced numbers of all types with no particular bias toward a single lineage.

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