Jennifer J Trowbridge ¹, Randall T Moon ², and Mickie Bhatia ¹

¹ McMaster Stem Cell and Cancer Research Institute, Hamilton, Ontario L9G 4L6, Canada, and Department of Biochemistry, McMaster University, Hamilton, Ontario, Canada.

² Howard Hughes Medical Institute, Department of Pharmacology and Institute for Stem Cell and Regenerative Medicine, University of Washington School of Medicine, Seattle, Washington 98195, USA. mbhatia@mcmaster.ca

By revealing the unexpected consequences of activating Wnt–beta-catenin signaling in the hematopoietic system, two reports by Scheller et al. [1] and Kirstetter et al. [2] in this issue of Nature Immunology further our understanding of factors that regulate hematopoietic stem cells (HSCs). Using conditional expression of a dominant active form of beta-catenin, both groups demonstrate that beta-catenin signaling blocks differentiation of HSCs, thereby leading to loss of stem cell activity. These data illustrate the importance of Wnt–beta-catenin signaling in regulating HSCs, and underscore the requirement of maintaining tight control of Wnt signaling in stem cells, thereby opening new areas of investigation into both HSC biology and its relationship to leukemic transformation.

A longstanding question in HSC biology has been the identification of the crucial pathways that control the balance of self-renewal divisions and differentiation of HSCs during the lifetime of an organism. Over the past several decades, detailed studies have carefully evaluated the roles of so-called 'hematopoietic' cytokines on hematopoietic cells. A subset of these factors has been defined by their critical role in establishing and maintaining the hematopoietic system through regulation of HSCs. More recently, morphogenic signaling pathways involved in specification of the blood lineage during mammalian embryogenesis [3, 4] have been implicated in HSC self-renewal. These morphogenic signaling pathways, including the Wnt–beta-catenin [5, 6] and hedgehog pathways [7], seem to affect function in committed HSCs. Wnt–beta-catenin signaling was initially proposed to increase HSC self-renewal [5]. Perhaps paradoxically, however, conditional inactivation of beta-catenin did not affect hematopoiesis or HSC capacity [8]. Thus, the precise role of Wnt–beta-catenin signaling in HSC function and hematopoiesis is not clear.

In vertebrates, Wnt ligands activate a 'canonical' beta-catenin pathway or one or more beta-catenin–independent 'noncanonical' pathways. The Wnt–beta-catenin pathway is in a state of default repression at several levels. A complex of intracellular proteins, including GSK-3, axin and APC, function in phosphorylation of beta-catenin, thereby targeting it for degradation. Upon activation of Wnt signaling, mediated by soluble or extracellular matrix–associated Wnt ligands interacting with specific receptors (Frizzled receptors) and co-receptors (LRPs) on the cell surface, the degradation of beta-catenin is blocked. Consequently, cytoplasmic beta-catenin is stabilized and translocates into the nucleus, where it interacts with a family of Tcf-LEF transcription factors to drive target gene expression (Fig. 1). Mutations that block the phosphorylation and degradation of beta-catenin thus result in dominant transcription of target genes. Scheller et al. [1] and Kirstetter et al. [2] have engineered inducible forms of stabilized beta-catenin for expression in HSCs, which can be activated selectively to mimic activation of Wnt signaling. The precise subcellular localization of overexpressed beta-catenin in HSCs was not evaluated, though it is possible that this influences HSC behavior in vivo.

Figure 1. Impact of beta-catenin constitutive expression in HSCs.

Figure 1. Impact of beta-catenin constitutive expression in HSCs.

Beta-catenin, normally expressed in HSCs, is targeted for degradation by the proteasome unless modified by a variety of mechanisms, including phosphorylation, that can be induced by the binding of some Wnt ligands to their putative receptors, Frizzled (FZD). In this state, the majority of beta-catenin is localized outside the nucleus and cannot bind factors such as Tcf-LEF that repress transcription of Wnt target genes. Under these basal conditions, normal production of progenitor and mature blood cell occurs, with adequate self-renewal of the HSC to sustain hematopoiesis (left). However, when expression of constitutively active beta-catenin is induced in HSCs, beta-catenin accumulates and most likely enters the nucleus to bind Tcf-LEF, thereby releasing transcriptional repression and allowing activation of putative Wnt targets. This Wnt-independent beta-catenin overexpression causes multiple deleterious effects on the hematopoietic system, including reduced HSC self-renewal capacity and reduced production of progenitor and mature blood cells.

Considering the well-characterized role of the Wnt–beta-catenin signaling pathway in T and B cell development [9], the recent demonstration that beta-catenin is dispensable for the function of the adult hematopoietic system [8] was somewhat surprising. So, is Wnt–beta-catenin signaling important in the adult hematopoietic system and hematopoietic reconstitution from transplanted HSCs? The studies by Scheller et al. [1] and Kirstetter et al. [2] address this by showing that activated Wnt–beta-catenin signaling results in widespread hematopoietic abnormalities and death of mice within 2–3 weeks, most likely as a result of anemia. Specifically, these studies observe a block in differentiation, suggesting that appropriate regulation of Wnt–beta-catenin signaling is necessary for hematopoietic maturation.

The role of Wnt–beta-catenin signaling in self-renewal of HSCs has also been open for debate. One study showed that Wnt–beta-catenin activation through exposing cells to Wnt3a ligand can expand HSCs [10]. However, HSC self-renewal has been shown to be normal in HSCs lacking beta-catenin [8]. Scheller et al. [1] and Kirstetter et al. [2] have now shown that activation of Wnt–beta-catenin signaling results in accumulation of phenotypically defined HSCs and an increase in their proliferation. However, this seemingly expanded pool of HSCs was shown to have little or no stem cell potency in vivo. Thus, these cells are no longer functional HSCs based on the strictest operational definition. In support of this notion, several genes encoding proteins that have been shown to be important in HSC function, including Bmi-1, HoxB4, p21 and PU.1, were found to be downregulated in HSCs expressing stabilized beta-catenin, suggesting that the transcription programs of the putative HSCs were not engaged.

Taken together, the existing reports may suggest that beta-catenin is not strictly required for HSC function, but Wnt–beta-catenin is able to effect HSC self-renewal in a manner that is dependent on the extent of Wnt–beta-catenin signaling. In the absence of specific experimental means to measure and quantitatively compare Wnt–beta-catenin signaling within a given cell type, it is difficult to determine whether the discrepancy is simply a product of morphogenic characteristics of Wnt signaling. Such characteristics predict that identifying and then striking the right balance of Wnt–beta-catenin in HSCs is a goal for modulating their clinical utility.

In addition, when considering morphogenic pathways recently associated with HSC self-renewal, including Wnt signaling, it is important not to overlook so-called 'hematopoietic' cytokines. Over the past several decades, these factors have been found to be critical in hematopoiesis and for successful ex vivo culture of HSCs. The interaction and cooperation between cytokine-activated pathways and Wnt signaling most likely contributes additional complexity as compared with manipulating Wnt signaling alone. For example, the effect of HSC expansion by Wnt3a ligand [10] was demonstrated in the presence of the hematopoietic cytokine stem cell factor (SCF) and was seen largely in HSCs isolated from transgenic mice overexpressing the antiapoptotic gene Bcl2. How these pathways are modulated, how they may interact with one another, and which cell types respond to signaling will all contribute to the ultimate phenotype and function observed. Additionally, the role of the HSC environment within the bone marrow requires consideration. Indeed, a specialized niche is critical for proper HSC function [11] and populations of cells lining the bone marrow express various Wnt ligands, as originally shown by Hoffman's group [12]. It is possible that the niche itself provides the right balance of Wnt signals as compared to signals from other pathways.

In addition to normal adult hematopoiesis, Wnt–beta-catenin signaling has been implicated in cancer, including cancers of the blood system (leukemias). In chronic myelogenous leukemia (CML), activation of beta-catenin enhances self-renewal and leukemic potential of a subpopulation of hematopoietic progenitor cells [13]. In the studies by Scheller et al. [1] and Kirstetter et al. [2], there seems to be a differentiation block reminiscent of acute leukemia phenotypes; however, these studies did not observe malignant transformation of the hematopoietic system up to 6 months post-transplantation, but rather exhaustion and HSC failure. Together these studies illustrate the underlying complexity of HSC transformation and leukemogenesis, and they suggest that although Wnt–beta-catenin signaling may play a role in this process, activation of the pathway alone may not be sufficient for transformation to occur. Furthermore, the combined data raise the provocative notion that Wnt signaling may represent a biological separation of normal HSC self-renewal as opposed to leukemic stem cell transformation, which, if true, may lead to the development of methods to destroy leukemic stem cells while sparing normal HSCs.

On first viewing, the studies by Scheller et al. [1] and Kirstetter et al. [2] suggest a need for caution in regard to proposals for the therapeutic expansion of HSCs using Wnts [5]. However, it is necessary to remember that the two recent studies used a gene-targeting approach. Thus these new data reasonably predict that gene therapy through activation of Wnt–beta-catenin signaling might have serious and most likely negative consequences on the hematopoietic system. However, this approach differs, in regard to the strength and duration of the beta-catenin signal, from what may be achieved upon transient stimulation of Wnt signaling, such as that shown with purified Wnt-3a [10] or through in vivo administration of Wnts6 or a small-molecule inhibitor of GSK-3beta [14].

Clearly, a complete understanding of HSC regulation and control will require further investigation before the information in the studies by Scheller et al. [1] and Kirstetter et al. [2] can be used to improve hematopoietic reconstitution in the setting of clinical transplantation. These and earlier studies nevertheless demonstrate that the Wnt–beta-catenin pathway is a potent regulator of HSC function that is central to HSC biology. Much like hematopoietic cytokines, we are now off to address the remaining questions that are relevant to clinical applications and to the basic biology: How much Wnt? Which Wnt? When to Wnt? And for how long to Wnt? And did we forget to mention that some Wnts actually antagonize beta-catenin signaling?  [15, 16]. Stay tuned.

REFERENCES

   1. Scheller M, Huelsken J, Rosenbauer F, Taketo MM, Birchmeier W, Daniel G Tenen DG,  and Leutz A,  "Hematopoietic stem cell and multilineage defects generated by constitutive bold beta-catenin activation",  Nat. Immunol. 7, 1037–1047 (2006).

   2. Kirstetter P, Anderson K, Porse BT, Jacobsen SEW, and Nerlov C, "Activation of the canonical Wnt pathway leads to loss of hematopoietic stem cell repopulation and multilineage differentiation block",  Nat. Immunol. 7, 1048–1056 (2006).

   3. Dyer, M. , Farrington, S. , Mohn, D. , Munday, J. & Baron, M. Development 128, 1717–1730 (2001).

   4. Gering  M, and Patient R, "Hedgehog Signaling Is Required for Adult Blood Stem Cell Formation in Zebrafish Embryos",     Dev. Cell 8, 389–400 (2005).

   5. Reya, T. et al. Nature 423, 409–414 (2003).

   6. Murdoch, B. et al. Proc. Natl. Acad. Sci. USA 100, 3422–3427 (2003).

   7. Bhardwaj, G. et al. Nat. Immunol. 2, 172–180 (2001).

   8. Cobas, M. et al. J. Exp. Med. 199, 221–229 (2004).

   9. Timm A,  and Grosschedl R, "Wnt signaling in lymphopoiesis",    Curr. Top. Microbiol. Immunol. 290, 225–252 (2005).

  10. Willert, K. et al. Nature 423, 448–452 (2003).

  11. Calvi, L. et al. Nature 425, 841–846 (2003).

  12. Van Den Berg, D.J. , Sharma, A. , Bruno, E. & Hoffman, R. Blood 92, 3189–3202 (1998).

  13. Jamieson CHM, Ailles LE, Dylla SJ, Muijtjens M, Jones C, Zehnder JL, Gotlib J, Li K, Manz MG, Keating A, Sawyers CL, and Weissman IL,  "Granulocyte–Macrophage Progenitors as Candidate Leukemic Stem Cells in Blast-Crisis CML",  N. Engl. J. Med. 351, 657–667 (2004).

  14. Trowbridge J, Xenocostas A, Moon R. and Bhatia M, "Glycogen synthase kinase-3 is an in vivo regulator of hematopoietic stem cell repopulation",   Nat. Med. 12, 89–98 (2006).

  15. Veeman, M. , Axelrod, J. & Moon, R. Dev. Cell 5, 367–377 (2003).

  16. Moon, R. Wnt/beta-catenin pathway. Sci. STKE 2005, cm1 (2005).

Additional References:

1. Bilodeau M, and Sauvageau G, "Uncovering stemness".

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