University of Groningen
Non canonical Wnt ligands and cytokine-driven myelopoiesis
Mastelaro de Rezende, Marina
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10.33612/diss.118670709
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158 159 Final Discussion
7. FINAL DISCUSSION
Upstream effectors of intracellular signaling are similar for IL-3 and GM-CSF
Stem cells were first described in the hematopoietic tissue1-3, in which stem cell biology
has been studied extensively. Maintenance of primitive cell potential, while continuously producing specialized and fully mature blood cells, is dependent on proliferation and differentiation. These processes are main events during hematopoiesis and rely on intricate regulatory mechanisms. Alterations in these mechanisms can cause cellular disruption and disease onset (Chapter 1). The extracellular environment as well as intracellular molecules are is critical in guiding signaling events. Cytokines constitute the main class of extracellular molecules involved in hematopoietic regulation4,5;
initially they were denominated “colony-stimulating factors (CSF)”5-7, due to the
induction of the formation of conglomerates consisting of similar cells (i.e. ‘colonies’) in semi-solid medium6,7.
Colonies develop due to the clonal expansion of one cell (hence colonies are also referred to as “colony formation units” – CFU), which has primitive characteristics and gives rise to others8. Only primitive cells can form colonies, as they have the ability
to self-renew and harbor relevant proliferative potential9, which are key features of
undifferentiated cells.
Introduction of the colony formation assay (CFU assay) established the hierarchical relations of hematopoietic cells and their progenitors10-12, and later on provided
evidence for the existence of primitive characteristics in cells obtained from patients with leukemia, the disease in which cancer stem cell research was initiated (Chapters 1 and 3). In addition, the CFU assay demonstrated the requirement of CSFs for colony formation, as no colonies were formed in the absence of these factors8,12,13.
Among the earliest described CSFs, GM-CSF and IL-3 have a broader activity, stimulating committed, multi-, and bipotent progenitors, as compared to M- and G-CSF, which only target the committed progenitors of monocytes/macrophages and granulocytes, respectively6. In our studies, we observed similar colony formation
potential for these cytokines (Chapter 2). There is evidence suggesting that IL-3 targets a slightly more primitive population than GM-CSF14,15. This was confirmed by
the results of our replating CFU assay (Chapter 5), showing that IL-3 induced colony formation for longer periods of time.
Divergent biological roles, and to an even lesser extent the molecular mechanisms involved, for IL-3 and GM-CSF have not been completely established but unveiling fine myeloid regulation as well as population-specific mechanisms may provide insight in the therapeutic relevance of HSCs and/or myeloid imbalances. In fact, this knowledge could even provide tools to fight aging-related changes, as ‘inflamm-aging’16
is an established event characterized by the predominance of specific inflammatory cytokines, including IL-3 and GM-CSF, in the aging individual16. In this context, gathering
information on pharmacological inhibition and protein activation is helpful, as inhibition analysis can assert if and to what extent a pathway is activated.
Similar to the structure of trees, activation of intracellular signaling is outlined in a non-linear fashion, with embranchments representing activation of multiple pathways17, converging to downstream elements17. As funnels, upstream signals are
more likely to affect multiple and even distinct pathways and subsequent cellular outcomes than downstream signals.
JAK kinase activity targets cytoplasmic tyrosines in the unspecific receptor β-chain and its inhibition completely abrogates GM-CSF18,19 and IL-3 activity20-22. At this point,
no difference was observed between IL-3 and GM-CSF, presumably due to the crucial requirement of JAK kinase for both IL-3 and GM-CSF receptor activity and subsequent signaling.
Tyrosine residues in the cytoplasmic portions of the β-chain receptor (Tyr577, Tyr612, Tyr695, Tyr750, Tyr806, and Tyr866) can provide specificity to JAK signaling activation23. Dimerization of the β-chain is ligand-independent. However, only in the
presence of ligand, the α-chain couples to this complex, allowing JAK2 activation and signaling18,24. Thus, the short receptor subunit (the α chain) appears to be involved in
cytokine specificity and triggering of appropriate signaling pathways19,25-27. Interestingly,
in endothelial cells, GM-CSF activity seems to be related to α-chain activation, leading to p85 coupling and induction of PI3K signaling24,28. This evidence of PI3K activation
upstream of JAK2 is in line with our data, which revealed significant effects of PI3K inhibition on colony forming activities by IL-3 and GM-CSF (Chapter 2). Again, there were no apparent differences between IL-3 and GM-CSF, indicating similar routes of initial signal transduction.
Of note, wortmannin and Ly294002 have been described as STAT inhibitors as well, which might have a direct result of PI3K inhibition upstream of JAK24.
IL-3 and GM-CSF induce a distinct pattern of Ca2+ signaling responses
U73122, 2APB and chelerythrine represent other pharmacological agents that had strong (more than 85%) inhibitory effects on colony formation induced by both cytokines (Chapter 2), suggesting important participation of PLC, the IP3R, and PKC, respectively. It was demonstrated that both IL-3 and GM-CSF activate PLCγ2 in Lin-
Sca-1+c-Kit+ cells29; however, activation was transient in response to IL-3, whereas GM-CSF
induced a more sustained activation (up to 30 minutes)29. It is unknown whether
transient and sustained PLC activation result in different downstream effects. There is evidence that, depending on the stimulation, DAG (downstream effector of PLC) can
Chapter 7 Final Discussion
follow Ca2+ oscillation signals; displaying an oscillatory pattern or exhibiting a sustained
signal, may reflect on PKC activation30. PKC inhibition (by GF109203X) significantly
reduced IL-3 and GM-CSF stimulated formation of subtype specific and total number of colonies, indicating roles for PKC in this event. In addition, we observed differential PKC activation after 15 minutes of stimulation with IL-3 and GM-CSF (Chapter 2). Both cytokines activate the threonine 514 region of PKC in both populations (progenitors and stem cells), indicating priming of PKC activation31, but only IL-3 induced PKCser660
phosphorylation, indicating a phosphosite specific activation by IL-3.
Considering Ca2+ signaling, the IP3R is importantly involved in the frequency and
modulation of the Ca2+ signals32, which can directly influence PKC and CaMKII signaling30,
as well as cellular outcomes33. Interestingly, inhibition due to interference with Ca2+
signaling (using 2APB, BAPTA, calmidazolium and KN-62) seems to be more effective when GM-CSF is used as a stimulus (Chapter 2). This is emphasized by the observed CaMKII phosphorylation, reinforcing the importance of Ca2+ in GM-CSF signaling and
suggesting another difference between GM-CSF and the IL-3 mediated response.
Differential Ca2+ signaling may be reflected by MAPK pathway activation
There is evidence from the literature to suggest that MAPK pathways are activated by PKC and CAMKII29. Since we observed differential PKC activation and CAMKII
phosphorylation for IL-3 and GM-CSF, we postulated this could influence MAPK activation. Transient MEK1/2 phosphorylation in response to IL-3 stimulation culminates in strong ERK1/2 activation, whereas sustained MEK1/2 phosphorylation by GM-CSF leads to decreased ERK1/2 activation29. Unfortunately, we could only
observe ERK1/2 activation after IL-3 stimulation (Chapter 2), which may indicate lower sensitivity of our analysis.
Interestingly, the sustained MEK1/2 activation (as described by Leon and coworkers in 2011), would take place in the same scenario we proposed to be CAMKII-dependent. Different from PKC, the CAMKII activation signal does not oscillate in response to [Ca2+]
i variations, exhibiting a progressive activation pattern30. Extrapolating this data,
we hypothesize that progressive CAMKII activation leads to consistent activation of MEK1/2, triggering self-inhibitory mechanisms, such as feedback loop activation, and resulting in decreased ERK1/2 activation. On the other hand, the oscillatory PKC activation pattern30 (putatively driven by IL-3 stimulation), would lead to a transient
MEK1/2 activation29, without activation of such self-inhibitory mechanisms, and
activation of ERK1/2.
Mild ERK1/2 activation in response to GM-CSF might be caused by modulatory mechanisms in response to sustained pathway activation, whereas strong ERK1/2 activation may reflect a sharper, more pinpointed mechanism of action, in which the
‘on and off’ status are changed faster without the activation of additional feedback loops. We observed robust p38 phosphorylation in response to both cytokines after 15 minutes of stimulation in all populations, except for progenitors in response to GM-CSF (Chapter 2). MAPK signaling has been described to vary between c-Kit+ and
c-Kit- cells, in addition to being PI3K dependent in response to stem cell factor34. Indeed,
after stimulation with either IL-3 or GM-CSF, we observed a stronger effect of PI3K inhibition than of MEK and ERK inhibition, which may indicate an upstream role for PI3K in the Raf/MEK/ERK cascade. In addition, PTEN phosphorylation suggested that Akt and PI3K were not being inhibited, further supporting PI3K participation. Akt activity may reflect GSK3β phosphorylation35, which tended to increase after IL-3 treatment in
the progenitor population; GSK3β is related to numerous intracellular pathways36-40,
including Wnt signaling41-43. Noteworthy, this “population-specific” response highlights
the importance of sensitive techniques, as has been discussed for the leukemic population (Chapter 3).
Figure 6 of Chapter 2 summarizes the main observed differences between IL-3 and GM-CSF treatment. STAT5-independent mechanisms have been implicated in JAK2 activity18,44; this could apply to our results in which little STAT5 phosphorylation
was observed (Chapter 2), despite published data indicating this signal transducer is activated by both IL-3 and GM-CSF26.
Conversely, STAT3 activation was observed under almost all conditions studied (with the exception of HSCs treated with GM-CSF); this was somewhat unexpected as cytokines other than IL-3 and GM-CSF have been more predominantly associated with STAT3 activation45. On the other hand, STAT3 activation by GM-CSF has been shown
to decrease apoptosis45, a well-known effect of this cytokine in the hematopoietic
system46-48.
Alterations in Wnt signaling can contribute to hematopoietic imbalances and malignancies (Chapters 3 and 4). Pathways involving PKC, Ca2+ and PI3K have been
linked to Wnt signaling49-52. Therefore, differences observed in these pathways were
hypothesized to regulate (or be regulated by) Wnt signaling.
The relative functional contribution of Wnt signaling may account for (some) differ-ences between IL-3 and GM-CSF driven signaling
Wnt signaling is increasingly associated with hematopoietic regulation, and its β-catenin independent branch appears to be particularly important for myelopoiesis and aging-related imbalances53-57. Chapters 4, 5 and 6 provide further evidence for
this functional association.
Our comprehensive literature review (Chapter 4) revealed some intriguing elements about noncanonical Wnt signaling in the regulation of the hematopoietic
162 163
Chapter 7 Final Discussion
system. For example, it highlighted the increased response of LT-HSCs to Wnt5a, leading to quiescence in these cells58,59, which seems to be related to Cdc42 regulation
and apolarity development59. In addition, for the population representing the following
lower level of the hierarchic tree of differentiation, ST-HSCs respond to this ligand with proliferation57,60, which appears to be associated with the activation of canonical
rather than noncanonical Wnt signaling, although a myeloid skewing in differentiation, as occurs during aging, might be observed59,61. These differential cellular responses
indicate both canonical and noncanonical effects of Wnt5a, depending on the target population. As for Wnt ligand stimulation, the cytokines we studied also induced population-specific responses (Chapter 5), further reinforcing highly population-specific intracellular signaling. Interestingly, most studies on Wnt signaling in the hematopoietic system focus on the canonical branch, hampering the identification and establishment of roles for the noncanonical branch. Our review (Chapter 4) exposed numerous situations in which noncanonical Wnt signaling is active and could be of profound importance, as this pathway has relevant roles in hematopoietic ontogenesis, adult hematopoiesis and during aging, which is increasingly being associated with changes in Wnt signaling in this system.
In a heterogeneous environment as bone marrow, where a myriad of compounds simultaneously modulates cellular responses, it is essential that we have a complete overview of the occurring events. In this context, Chapter 5 describes the importance of the interaction between different compounds in modulating hematopoietic activity.
There is evidence to suggest that Wnt5a-induced signaling in the myeloid lineage is associated with Ca2+, PKC and PI3K activation, similar as to what we demonstrated
for IL-3 and GM-CSF. Therefore, we studied the effects of combined stimulation with Wnt ligands and cytokines on myeloid regulation. To our surprise, the prototypical noncanonical Wnt ligand (Wnt5a) showed no (additional) effects in the presence of IL-3 or GM-CSF in the CFU assay. Our investigation also included Wnt5b, as it is structurally close to Wnt5a62, although little is known about its roles in the
hematopoietic regulation. There is evidence for a role of Wnt5b in the regulation of megakaryopoiesis63,64, involving Ca2+ signaling63, but to our knowledge nothing is
currently known with regards to Wnt5b in the myelopoiesis field. Different from what we observed with Wnt5a, our results suggest striking effects of Wnt5b in the presence of IL-3 and GM-CSF in the CFU assay, in addition to potentially underpinning functional differences between IL-3 and GM-CSF (Chapter 5).
Our results not only indicate that the presence of Wnt5b determines the functional outcome of stimulation with cytokines, but also that there are differences between the impact of Wnt ligands and CT99021 (GSK3β inhibitor and β-catenin signaling activator) on the response induced by IL-3 and GM-CSF.
With IL-3 as stimulus, Wnt5b caused a decreased colony formation ratio, the opposite of what was observed in the presence of CT99021, suggesting that Wnt5b activates intracellular signaling not directly involving β-catenin. In addition, colonies could be successfully replated more times in the replating assay, indicative of primitive cell maintenance, which was confirmed by increased Ifitm-1 and GATA2 gene expression as compared to colonies exposed to IL-3 alone. On the other hand, stimulation with GM-CSF in the presence of Wnt5b resulted in the induction of differentiation, as demonstrated by an increase in colony formation and rapid exhaustion of cell potential. In addition, the Wnt5b effect was opposite to that observed in the presence of CT99021, again suggesting the involvement of β-catenin independent Wnt signaling. To our knowledge, this is the first study showing opposite effects of IL-3 and GM-CSF in myeloid regulation. It is likely that Wnt5b acts as a pivotal player underpinning our observed results by reinforcing pathways that are differentially activated by these cytokines.
In the presence of CT99021, IL-3 induced CFU growth, in a similar fashion as observed for GM-CSF+Wnt5b. This strongly suggests colony formation growth as a consequence of β-catenin signaling activation, which is in accordance with the literature60. It has been described that regarding IL-3, Wnt signaling is off65 and Wnt5b
does not seem to activate β-catenin. Conversely, with GM-CSF as a stimulus, Wnt5b may be activating β-catenin-dependent Wnt signaling on its own or sustaining GM-CSF-induced activity. Increased cyclinD1 gene expression in response to GM-CSF stimulation66 reinforces the hypothesis that GM-CSF activates β-catenin signaling, as
cyclinD1 is a well-known target gene downstream of β-catenin67-69.
It is debatable whether there is a differential distribution of cytokines and Wnt ligands in the bone marrow. In fact, even the existence of bone marrow niches (endosteal and vascular) is questionable70,71, with hematopoietic cells in diverse
maturation stages spreading across the bones71,72. In addition, active movement
by hematopoietic cells in the bone marrow73,74 hampers the understanding of how
extracellular signals modulate hematopoiesis. There is evidence that IL-3 has a higher activity on LT-HSCs in comparison with GM-CSF75,76, whereas GM-CSF is more active on
progenitors77,78. Therefore, together with our results, we propose that more primitive
HSCs, which are responsive to IL-3, have an intracellular environment unfavorable to β-catenin and Ca2+ signaling activation. On the other hand, with activation of Ca2+
signaling (as we observe after GM-CSF treatment), the intracellular environment of less primitive hematopoietic cells might be more favorable to Wnt/β-catenin independent signaling. This would explain why in the presence of Wnt5b, IL-3 drives maintenance of cell potential and GM-CSF triggers differentiation.
Chapter 7 Final Discussion
Wnt5 inhibition restores the potential of aged progenitors
The environment in which adult hematopoiesis occurs changes with aging; most of the hematopoietic balance is lost, with LT-HSCs prolonging quiescence and ST-HSCs proliferating to compensate for functional losses79,80. In addition, this environment
is characterized by an increase in β-catenin independent Wnt ligands, mainly the prototypical Wnt5a (Chapters 5 and 6), which really put our investigation on IL-3 and GM-CSF to the test.
In Chapter 6, we used CFU assays to show how Wnt5b treatment in young cells only partially mimicked the functional results of aged cells, and Wnt5 inhibition in aged cells mimicked the results observed in young cells just to a certain extent. Yet, Wnt5 inhibition in aged cells provided definitive evidence of this protein’s participation in myeloid regulation in the aging environment. Thus, Wnt5 inhibition by Box5 strikingly increased progenitor potential in aged cells, which seemed to be caused by upregulation of self-renewal and downregulation of commitment and differentiation.
Our results (Chapter 6) showed that Box5 induced upregulation of myc, E2F and Cdk1, which are genes associated with self-renewal81-84. Interestingly, both myc
and Cdk1 are targets of β-catenin signaling85,86, but they can be activated by other
pathways as well; this was likely the case in our experimental setup, since β-catenin was inhibited. Gene Set Enrichment Analysis of the obtained mRNA sequencing data also demonstrated that Box5 induced upregulation of the PI3K/Akt pathway, which may underpin its effects on gene expression85. Indeed, Akt is known to phosphorylate
and inhibit GSK3β40, and GSK3β inhibition is a hallmark for stem cell maintenance
(together with MTORC1 inhibition87).
p53 was amongst the most downregulated genes after Box5 treatment in old cells, and together with Notch1 and Fos, which were downregulated as well, it is involved in hematopoietic specification and lineage commitment88. In addition, STAT5,
a well-known driver of differentiation88,89, and mTORC1 were downregulated as well87.
Although strong evidence exists for differentiation arrest by Box5 treatment, other mechanisms might be involved in the maintenance of cell potential. Downregulation of hypoxia, TNFα and KRAS may be linked to β-catenin signaling inhibition, as these pathways are connected to Wnt/β-catenin in numerous systems90,91. β-Catenin
inhibition in aged animals treated with Box5 suggests activation of this pathway in the absence of the inhibitor, as proposed above, which also agrees with evidence from the literature indicating that activation of β-catenin is related to proliferation in hematopoietic progenitors92,93.
Noncanonical Wnt5b modulates myelopoiesis driven by IL-3 and GM-CSF
In conclusion, IL-3- and GM-CSF-driven myelopoiesis might not be as similar as it was considered to be over the last several years. Although they can produce similar cellular outcomes depending on the method used, specific intracellular signaling events triggered may differ at some point further up or down the tree, which can be highlighted in the presence of other compounds, such as Wnt ligands. These ligands appear to be important components of hematopoietic ontogenesis and modulation in adult life, as well as responsible for imbalances of senescence. Wnt5b is shown here as a critical modulator of myelopoiesis driven by IL-3 and GM-CSF stimulation. Of note, during aging, Wnt5a is likely to be involved in the developing imbalances as well. In addition, we reveal that activation of β-catenin signaling is not sufficient to fix the imbalances that occur during aging, as complete abrogation of Wnt5 activity was needed. Furthermore, the results provide evidence for the modulation of myelopoiesis by noncanonical Wnts, and point out differences between the response to cytokines and the distinct involvement of these Wnts. In addition, experimental tools and approaches implemented and/or introduced in our studies could prove useful for the investigation of hematopoietic senescence and (future) therapeutics.
166 167
Chapter 7 Final Discussion
CONCLUDING STATEMENTS
1. Hematopoietic regulation is essential for nutrient and oxygen distribution, as well as immune and inflammatory responses. These functions are exerted by short-lived cells, which need to be continuously produced by proliferation and differentiation of primitive cells;
2. Differences exist between the effects of IL-3 and GM-CSF on myelopoiesis, by targeting different hematopoietic cell fractions and distinct cellular outcomes;
3. Activation of PLC, PKC, Ca2+ and MEK/ERK signaling by IL-3 or GM-CSF stimulation
are involved in colony formation by these cytokines;
4. Wnt5a, the prototypical noncanonical ligand, is associated with Ca2+, PKC and
PI3K activation in the hematopoietic system;
5. Wnt5b, rather than Wnt5a, affects IL-3- and GM-CSF-driven myelopoiesis and is pivotal in the differential cellular outcomes primed by these cytokines;
6. Noncanonical Wnt signaling plays a central role in (the development of) hematopoietic imbalances in the aging individual;
7. Activation of canonical Wnt signaling (with CT99021) is not sufficient to completely abrogate the effects of aging on the cell potential of progenitors;
8. Abrogation of Wnt5 signaling (thus, both Wnt5a and Wnt5b) with Box5 completely restores progenitor cell potential in aged cells by stimulating self-renewal and inhibiting commitment and differentiation.
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