The data presented here demonstrate an essential role for Gsk3
in the maintenance of LT-HSCs. Gsk3
loss of function phenocopies Pten
mutations in HSCs/HPCs, supporting the hypothesis that GSK-3 functions downstream of PTEN to suppress mTOR-dependent HSC activation and lineage commitment. However, inhibition of GSK-3 also stabilizes β-catenin within HSCs/HPCs to induce a β-catenin–dependent increase in phenotypic HSCs, and β-catenin
KO accelerates the loss of LT-HSCs in Gsk3
-depleted BM, consistent with prior reports with activators of Wnt signaling (20
). Thus, Gsk3
functions in at least 2 apparently opposing processes within HSCs/HPCs (Figure ). These observations indicate that Gsk3
plays an essential role in regulating the balance between self renewal and lineage commitment in HSCs. These observations also support the hypothesis that the highly prevalent effects of lithium on hematopoiesis in bipolar patients are mediated by inhibition of GSK-3 and suggest a therapeutic approach, using currently approved GSK-3 and mTOR inhibitors, to expand HSCs in vivo.
GSK-3 functions in 2 major pathways to regulate HSC self renewal and lineage commitment.
KO in HSCs/HPCs leads to activation and subsequent depletion of HSCs, increased lineage commitment resembling myeloproliferative disorder, and leukemia (33
), and this is prevented by the mTOR inhibitor rapamycin, which suggests that PTEN-mediated suppression of mTOR is required for maintenance of quiescent HSCs. Similarly, deletion of the mTOR inhibitor Tsc1
shifts HSCs from a quiescent to a proliferative state and reduces HSC self renewal (35
). We show that reduced expression of Gsk3
, either through RNAi or by homozygous Gsk3b
KO, yielded a similar hematopoietic phenotype. This Gsk3
phenotype was also reversed by rapamycin, and GSK-3β phosphorylation increased in Pten
-depleted BM, which suggests that GSK-3β functions downstream of PTEN to antagonize mTOR activation and maintain stem cell self renewal. In support of this, GSK-3 has previously been shown to antagonize mTOR activation in HEK293T cells by phosphorylating Tsc2 (37
). Interestingly, that work showed that Wnts could activate mTOR by inhibiting GSK-3, suggesting a bifurcation of the canonical Wnt pathway that could activate distinct and potentially opposing processes (Figure ). Furthermore, very recent work suggests that Wnt signaling through mTOR may also cause epidermal stem cell exhaustion, and this can also be prevented by rapamycin (49
). Although it is not yet known whether Wnts activate mTOR in HSCs/HPCs, activation of both mTOR- and β-catenin–dependent processes could explain some of the conflicting reports on Wnt effects in hematopoiesis (18
), if differing experimental conditions bias the effects toward either mTOR- or β-catenin–dependent responses.
Although the Gsk3-depletion phenotype we observed was similar to the Pten KO, important differences should also be noted. Conditional deletion of Pten (or Tsc1) leads to rapid HSC exhaustion, whereas the reduction in HSCs observed with Gsk3-rnai became evident more slowly, through serial transplants and competitive repopulation assays. Indeed, the initial expansion in phenotypic HSCs was observed 4 months after primary transplant; the number of phenotypic HSCs declined to control levels in secondary recipients, and only fell below control levels in tertiary transplants (Figure C). We propose that the delay is because the activation of HSCs and their subsequent exit from the HSC pool are balanced by enhanced Wnt signaling, which would slow the rate of HSC activation and depletion (Figure ); this idea is supported by the more rapid decline in phenotypic HSCs in Gsk3-rnai;β-catenin KO BM. Pten deletion also leads to leukemia in a substantial fraction of animals, which we have not observed so far with the Gsk3-rnai–transplanted mice. However, PTEN regulates multiple downstream effectors in addition to GSK-3, and modulation of these pathways could contribute to the Pten HSC phenotype independently of GSK-3 function. It would be interesting to test whether the acute leukemia observed in Pten KO mice is blocked in mice expressing nonphosphorylatable mutants of Gsk3.
We found that β-catenin was required for the initial increase in phenotypic HSCs/HPCs in response to Gsk3
inhibition and for the maintenance of Gsk3
-depleted HSCs in long-term transplant assays. These observations are consistent with previous studies showing that activation of canonical Wnt signaling can promote HSC self renewal and proliferation ex vivo (20
). Although basal hematopoiesis was unaffected when β-catenin
was deleted in adult BM, consistent with previous reports (29
), inhibition of Gsk3
can be considered a Wnt gain of function. The issue of whether canonical Wnt signaling is required for basal HSC homeostasis remains controversial (18
). A recent report showed that canonical Wnt signaling can function in HSCs in the absence of β-catenin (24
), which suggests that loss of β-catenin does not necessarily block all Wnt signaling. In addition, conditional deletion of β-catenin
with cre recombinase driven by the vav promoter impairs LT-HSC self renewal (26
). As vav expression begins in utero, whereas Mx-cre
was used to delete β-catenin
in adult BM (present study and refs. 29
), a reasonable explanation for these differences is that the role of Wnt/β-catenin signaling in basal hematopoiesis depends on the developmental context. In support of an early requirement for Wnt signaling in developing HSCs, long-term reconstituting capacity in serial transplants is impaired in HSCs recovered from fetal liver of Wnt3a
KO embryos (27
Lithium’s effects on hematopoiesis have been known for decades and affect more than 90% of patients taking it, yet to our knowledge, the mechanism of lithium action in this setting had not previously been defined. Plausible targets of lithium in addition to GSK-3 include inositol monophosphatase, which may indirectly regulate inositol trisphosphate signaling, and related phosphomonoesterases that play important roles in cell metabolism (50
). Therefore, a priori, it should not be considered obvious that GSK-3 is the biologically relevant target of lithium in HSCs, and it is essential to validate GSK-3 as the target in this setting. In support of this hypothesis, structurally diverse GSK-3 inhibitors mimicked lithium effects on the HSC pool and on progenitor cells (Supplemental Figure 1 and refs. 10
). Importantly, we show here, for the first time to our knowledge, that depletion of Gsk3a
mimics lithium action in HSCs and HPCs as well as more differentiated myeloid cells. Although these observations suggest that targeting GSK-3 may be a fruitful approach to treating hypoproliferative hematopoietic disorders, the reduction in LT-HSCs with Gsk3
loss of function suggests this should be approached with caution. Lithium has, in fact, been tested in clinical trials to enhance hematopoietic recovery after myelosuppressive chemotherapy, but this approach has not seen wide use, perhaps in part because of the risk of lithium side effects in already critically ill patients, but also because of limited success in restoring hematopoiesis in patients with reduced numbers of HSCs. It is also possible that the β-catenin–dependent increase in HSCs in response to lithium is offset by activation of mTOR and exit from the HSC pool, which would be consistent with the increase in more differentiated hematopoietic cells (especially those of myeloid lineage) commonly observed with lithium. In this case, we suggest that the combination of lithium and rapamycin, both now in wide clinical use, might achieve a more marked and durable increase in HSCs.
In summary, we have provided evidence for dual functions of GSK-3 within hematopoietic cells. GSK-3 antagonizes the canonical Wnt pathway, and we showed here that inhibition of GSK-3 activated the pathway to enhance HSC self renewal. This response to GSK-3 inhibition required β-catenin, as phenotypic HSCs were reduced in β-catenin CKO BM in both primary and secondary transplant recipients of Gsk3-depleted BM. GSK-3 also antagonizes mTOR signaling, and we showed that inhibition of Gsk3 either by RNAi or by conventional gene KO activated mTOR (similar to Pten or Tsc1 KOs) and led to activation of HSCs, with an initial expansion of LSK cells followed by dramatic depletion of HSCs (as assessed by long-term reconstitution assays). That this was successfully prevented by treatment with rapamycin raises the intriguing possibility that the combination of lithium and rapamycin could be used to expand HSCs either in vivo or ex vivo in HSC transplants and in the therapy of hypoproliferative hematologic diseases.