Recent understanding of the impact of cytokine signaling pathways on HSC homeostasis is beginning to show positive and negative effects depending on the pathway and degree of activation. In particular, activation of the PI3-kinase/Akt/mTOR or the Akt/Foxo pathways can have negative consequences on HSC. Foxo3a is pro-apoptotic and must be held in check. Mice lacking PTEN
, or Foxos
have increased HSC proliferation, reduced survival, and loss of repopulating function. Reports of mutant mice lacking Caspase-3
or Foxo3a-mediated loss of negative feedback by Spred2
demonstrate that Erk pathway activation can also be detrimental to HSC. In contrast, steady-state levels of STAT5 activation positively regulate HSC homeostasis and repopulating function
We have previously reported studies of STAT5 or Gab2 knockout mice and characterized their individual role in hematopoiesis. STAT5 deficiency resulted in severe HSC level defects and was only modestly required in CFU-C
. Gab2 was critical for committed CFU-C and multipotent progenitor level response to hematopoietic cytokines and for long-term multilineage competitive repopulating ability of HSC
. Gab2 may lie upstream or downstream of STAT5 to promote hematopoiesis or it may function solely through ERK or AKT. Therefore, the studies in this report were designed to identify new Gab2-dependent HSC functions and to determine whether regulation of HSC activity by Gab2 could be accounted for solely by STAT5 in a linear signaling pathway. To do this, the effect of combined deficiency of STAT5 and Gab2 on hematopoiesis was tested following generation of compound mutant mice.
The defects observed in compound mutant mice were informative regarding the levels of hematopoiesis where STAT5 and Gab2 play non-redundant roles and the defects uncovered new functions for Gab2 that were not previously recognized. Using 2-way ANOVA analysis, strong evidence was obtained for non-redundant functions of STAT5 and Gab2 during hematopoiesis. We found that combined deficiency had no additional impact upon peripheral blood hematology or BM cellularity in heterozygous STAT5 mutants. However, combined deficiency resulted in declines in CFU-C and long-term repopulating activity relative to STAT5 or Gab2 deficiency alone. These defects were consistent with the reduced in vitro cytokine response, indicative of reduced expansion and differentiation.
Newborn engraftment in the absence of myeloablation to clear the niche and promote hematopoietic engraftment tests the ability of host HSC to compete with donor HSC for engraftment under very stringent conditions that are biased against the donor graft. In this setting, loss of one allele of host STAT5 was sufficient to promote donor engraftment and full deletion of Gab2 alone in the host permitted significant donor reconstitution. Interestingly, the observed defects for Gab2−/−STAT5ab+/null mice were greater than predicted for additive. It should also be pointed out that secondary transplantation showed roughly equivalent levels of reconstitution from primary recipients with single or compound mutants. Therefore, the increased donor chimerism associated with non-ablated engraftment may be more associated with selection at the BM multipotent progenitor fraction than with the HSC.
Since in prior studies, Gab2 deficiency was not associated with defects in HSC self-renewal, we wanted to explore whether evidence for Gab2 participation in signaling at the HSC level might be evident in the complete absence of STAT5. STAT5 is important for HSC self-renewal and promotes competitive repopulation ability
. Using serial transplantation, we were able to uncover important roles for Gab2 in HSC signaling. In our prior work
, STAT5-deficient HSC caused death in quaternary transplants. However, Gab2-deficient HSC permitted tertiary transplantation (data not shown
). The results of serial transplant using FL cells from mice completely lacking STAT5 and Gab2 were very striking. The combined defects in secondary transplant reported here indicate that HSC depletion occurs following the first transplant, despite reasonable engraftment of the granulocytic lineage. This type of secondary transplantation failure is comparable to a recent report of E12.5 FL cells lacking Wnt3a
. Therefore, Gab2 can play an important role in normal HSC self-renewal in a novel manner by facilitating the role of STAT5 in HSC function.
To better understand why the HSC number was decreased in the FL of STAT5/Gab2 double mutant mice, flow cytometry assay for apoptosis was performed. The results of this assay indicate that reduced survival can account for the reduction in HSC number. STAT5 is known to be an important regulator of survival signaling and Gab2 facilitates Akt-mediated survival signals. However, the striking reduction in survival signaling is novel and suggests either direct effects between downstream targets mediated by STAT5 and PI3-K pathways or cross-talk between these pathways could be critical for optimal survival signaling. In the context of competitive repopulation and self-renewal activities, additional studies will be required to identify whether defective engraftment is due to additional loss of proliferative or homing potential. Limitations in the number of double knockout FL that could be obtained prohibited assays requiring large HSC numbers. Conditional deletion of STAT5 in adult BM may provide a tool for future studies.
The unique roles for STAT5/Gab2 interaction during non-ablated engraftment indicate important function in generation and maintenance of HSC. The important role of STAT5 in many aspects of HSC function is now clear. However the mechanisms of action of STAT5 remain largely undefined. Defining and targeting interactive signaling nodes has become important area of research for stem cell transplantation and eradicating leukemic stem cells. The finding that Gab2 can have STAT5-independent functions in HSC indicates that activation of the PI3-K or MAPK pathways could be beneficial for steady-state HSC function. In addition, these Gab2 functions appear to synergize with STAT5 in the most critical aspects of HSC function, namely self-renewal as assayed by serial transplantation. A better understanding of the importance of STAT5/Gab2 signals may lead to new approaches for non-ablative HSC transplantation or therapeutics for hematologic malignancies where HSC survival and self-renewal are dysregulated.