TGF-β signaling in the control of hematopoietic stem cells.

From www.bloodjournal.org by guest on May 11, 2015. For personal use only.

Blood First Edition Paper, prepublished online April 1, 2015; DOI 10.1182/blood-2014-12-618090

TGF-β signaling in the control of hematopoietic stem cells

Ulrika Blank and Stefan Karlsson

Division of Molecular Medicine and Gene Therapy, Lund Stem Cell Center, Lund

University Hospital, Lund, Sweden

Corresponding author:

Ulrika Blank

E-mail: [email protected]

Phone: +46-46-2220589

Fax: +46-46-2220568

BMC A12, 221 84 Lund, Sweden

β

Short title: Regulation of HSCs by TGF-

Copyright © 2015 American Society of Hematology


Abstract Blood is a tissue with high cellular turnover, and its production is a tightly

orchestrated process that requires constant replenishment. All mature blood

cells are generated from hematopoietic stem cells (HSCs), which are the self-

renewing units that sustain life-long hematopoiesis. HSC behavior, such as self-

renewal and quiescence, are regulated by a wide array of factors, including

external signaling cues present in the bone marrow. The Transforming Growth

β (TGF-β) family of cytokines constitutes a multifunctional signaling

Factor-

circuitry, which regulates pivotal functions related to cell fate and behavior in

β signaling

virtually all tissues of the body. In the hematopoietic system, TGF-

controls a wide spectrum of biological processes, from homeostasis of the

immune system to quiescence and self-renewal of HSCs. Here, we review key

β and downstream signaling

features and emerging concepts pertaining to TGF-

pathways in normal HSC biology, featuring aspects of aging, hematological

disease, and how this circuitry may be exploited for clinical purposes in the

future.

2


Introduction Blood cell production takes place in the bone marrow (BM) of adult individuals,

where it originates from a rare pool of tissue-specific stem cells known as

hematopoietic stem cells (HSCs). HSCs function to sustain and regenerate the

entire blood system in an unbroken fashion throughout life, as they have the dual

capacity to self-renew and differentiate to all blood cell lineages

1,2.

Self-renewal

pertains to the ability of a stem cell to duplicate itself without losing

developmental potential. This capacity is crucial for maintenance of the stem cell

pool and may occur in a symmetric or asymmetric fashion

3.

Furthermore,

quiescence, or withdrawal from the cell cycle, is a feature of HSCs that provides

the blood system with a dormant HSC reservoir that retains the ability to self-

renew and replenish all blood lineages. Hematopoiesis is hierarchically

organized, with the most immature pool of HSCs at the top, giving rise to various

progenitor cells that progressively lose self-renewal capacity as they form

differentiating progeny that proliferate extensively, finally generating functional

blood cells at the bottom of the hierarchy (Figure 1). To rapidly tailor blood

production to acute changes, such as bleeding and infection, HSC behavior is

tightly regulated yet highly flexible

4.

Signals that promote quiescence, and cues

that stimulate proliferation and differentiation, provide a fine-balanced system

that safeguards against depletion and overproduction of HSCs. Disruption of

these regulatory mechanisms can result in blood disease, such as BM failure or

β (TGF-β) is the founding member of a

leukemia. Transforming Growth Factor-

large family of secreted polypeptide growth factors, consisting of over 30

members in humans, including activins, Bone Morphogenetic Proteins (BMPs),

and others

5.

β family constitutes a multifunctional set of cytokines that

The TGF-

3


regulate a bewildering array of cellular processes during development and

β members regulate tissue homeostasis and

beyond. In the adult organism, TGF-

β plays an important role in

regeneration. With respect to hematopoiesis, TGF-

regulating HSC behavior, particularly quiescence and self-renewal. Although

many members of this family function to regulate hematopoiesis, we focus this

β.

review mainly on TGF-

The basic elements of TGF-β signaling TGF-β ligands signal through cell surface serine/threonine kinase receptors,

known as type I and type II receptors

6.

In vertebrates seven different type I

receptors (ALK1-7) and five distinct type II receptors have been identified,

serving the entire family of ligands

6.

β signals mainly via the type I receptor,

TGF-

βRII, both of which are required for signaling

ALK5, and the type II receptor, T

activation. Following ligand binding and receptor phosphorylation, the SMAD

signaling circuitry becomes activated (Figure 2)

6.

The SMAD proteins are a

family of transcription factors consisting of eight members, SMAD1-8, which are

further subdivided into three classes based on structural and functional

properties

7.

Receptor-regulated SMADs (R-SMADs), SMAD1, 2, 3, 5, and 8, are

the only SMADs directly phosphorylated and activated by the kinase domain of

type I receptors. Upon phosphorylation, R-SMADs form a complex with the

common-SMAD (Co-SMAD), SMAD4, resulting in nuclear accumulation of

activated complexes. In the nucleus, R-SMAD-SMAD4 complexes cooperate with

transcriptional co-regulators that further define target gene recognition and

transcriptional regulation

7.

The inhibitory SMADs (I-SMADs), SMAD6 and

SMAD7, constitute the third class, which function to inhibit TGF-β signaling. TGF-

β/activin/nodal and BMP/GDF employ different subsets of R-SMADs. R-

4


SMAD2/3 specifically relay signals from TGF-β and activin receptors, whereas R-

SMAD1/ 5/8 primarily operate downstream of BMP receptors

6.

Alternative pathways The SMAD pathway forms a fundamental signaling module and is the most well

β. However, activation of other pathways,

studied circuitry downstream of TGF-

β activated kinase 1 (TAK1), a component of the mitogen-

most notably TGF-

activated protein kinase (MAPK) pathway, has been observed in other cell types

8,9.

Depending on context, TAK1 activates a host of downstream transducers,

including p38 and JNK

10.

Conditional deletion of TAK1 in mice, using the

Mx-Cre

β has not been firmly

driver, results in hematopoietic failure, but the link to TGF-

established

11.

Other non-SMAD circuitries regulated by TGF-β include ERK,

PI3K-AKT-FOXO, and RHO-like small GTPases, though the contribution of most of

10.

these mechanisms are not well defined in HSCs (Figure 2)

In addition,

γ (TIF1γ) partners with SMAD2/3 (see

transcriptional intermediary factor-1

separate section below)

12.

The TGF-β pathway is intimately linked with other

signaling circuitries, for readers interested in crosstalk mechanisms we refer to

the following papers

13-17.

TGF-β: inducer of quiescence TGF-β is catalogued as one of the most potent inhibitors of HSC growth

and a variety of culture systems support this notion

18-21.

in vitro

During homeostatic

conditions, the majority of HSCs are in a quiescent cell-cycle state

22,23.

Quiescence is related to maintenance of the HSC pool and loss thereof results in

exhaustion and erosion of HSC function

24.

β has been

Naturally, TGF-

hypothesized to be a cardinal regulator of HSC dormancy, maintaining a pool of

quiescent HSCs

in vivo

. Indeed, neutralization of TGF-β

5

in vitro

releases early


hematopoietic progenitor cells from quiescence

25-27.

Several molecular

mechanisms have been proposed to account for TGF-β-mediated growth

inhibition, including alterations in cytokine receptor expression and

Ink4b,

upregulation of cyclin-dependent kinase inhibitors (CDKIs), such as p15

Cip1,

p21

Kip1 26,28-34.

and p27

β can exert growth inhibitory actions

However, TGF-

Cip1 and

Kip1 35.

independent of p21

p27

+ cells,

In human CD34

Kip2,

cell-cycle arrest occurs through upregulation of p57

CDKI family

36.

Kip2 is

β -mediated

TGF-

another member of the

-

Similarly, p57

+

-

+

highly enriched in mouse CD34 Kit Lin Sca1

+

(KLS) cells as opposed to the more mature and actively cycling CD34 KLS

fraction

37.

Kip2 correlates with

Interestingly, a high level of p57

the activation

-

status of SMAD2/3, which are uniquely phosphorylated in freshly isolated CD34

+

KLS cells but not in CD34 KLS progenitors

Kip2 in

p57

-

CD34 KLS cells

38.

β upregulates

Additionally, TGF-

in vitro 38

. These findings point to a mechanism where

β functions to induce p57Kip2 within the most primitive HSC compartment,

TGF-

thus promoting their quiescent state

in vivo

(Figure 3).

Heterogeneity of the HSC pool Recently, evidence has accumulated suggesting that the adult HSC compartment

consists of a number of functionally distinct subsets with diverse self-renewal

and differentiation potentials

23,39-41.

Interestingly, discrete HSC subtypes

β, according to a model proposed by Challen and

respond differently to TGF-

colleagues

42.

β stimulates proliferation of myeloid-biased HSCs

Specifically, TGF-

(My-HSCs) whereas lymphoid-biased HSCs (Ly-HSCs) are growth inhibited

(Figure 3)

42.

β on HSCs is more nuanced than previously

Thus, the effect of TGF-

thought, such that proliferation is induced in certain HSC subtypes at certain

6


β represents a potential signal for

concentrations. However, though TGF-

differential regulation between HSC subtypes, there is currently no tangible

explanation for the underlying molecular mechanism. Another study found that

the RNA-binding protein MUSASHI-2 is required for HSCs to mount a

β at low concentrations 43. Loss of MUSASHI-2

proliferative response to TGF-

leads to impaired HSC quiescence, reduced My-HSC numbers and myeloid output

in vivo

Kip2 and

. These findings are coupled to significant reductions in p57

phosphorylated SMAD2/3 in HSCs, suggesting that MUSASHI-2 modulates the

β signaling circuitry 43. Most work regarding TGF-β in hematopoiesis has

TGF-

β1. However, TGF-β exists in three isoforms that are encoded by

focused on TGF-

separate genes,

Tgfb1-3

β 1-3 share significant sequence

. Although TGF-

homology and signal through the same receptor complex

44,45,

differences in

receptor affinity exist and varying responses have been reported. Most notably

β2, being growth inhibited at high

KLS cells exhibit a biphasic response to TGF-

doses and stimulated at low concentrations

46.

β

The bidirectional effects of TGF-

on proliferation vs. quiescence further substantiate the complexity of this

signaling pathway, and it remains to be clarified whether or not the SMAD

pathway is differentially regulated at high and low doses and between diverse

HSC subtypes. Additionally, it is conceivable that the bidirectional effects are a

β receptor expression among HSC subtypes,

function of contrasting TGF-

translating to dosage effects that generate qualitatively different responses. This

theory is corroborated by a recent study, which shows that a high level of ALK5

correlates with increased myeloid output

7

47.


TGF-β and the aging hematopoietic system The hematopoietic system declines functionally with age, a phenomenon that can

be traced back to alterations in the HSC compartment. The clonal composition of

the HSC pool changes over time, such that My-HSCs become more abundant at

the expense of Ly-HSCs

48.

Physiologically, this translates to a myeloid-biased

hematopoietic system with reduced lymphoid potential. Furthermore, the risk of

hematological malignancies, anemia, autoimmunity, and inflammatory disorders,

increases with age

48.

As TGF-β differentially regulates My- and Ly-HSCs, the

connection to aging is evident, especially since TGF-β is implicated in the aging

process of non-hematopoietic tissues

49,50.

A recent study, which profiled the

transcriptome, DNA methylome, and histone modifications in highly purified

young and old HSCs, found significant changes in the TGF-β pathway at the

transcriptional level

51.

In fact, the TGF-β pathway represented 19 percent of

differentially expressed genes in young vs. old HSCs. The study shows changes in

multiple layers of the TGF-β pathway, from extracellular regulators, to SMAD

transcription factors, and cofactors, resulting in an overall reduction of TGF-β

signaling in aged HSCs

51.

proliferation of My-HSCs

Since low concentrations of TGF-β stimulates

in vitro

, downsizing the TGF-β circuitry may provide a

more proliferative environment for My-HSCs, which can expand over time.

Similarly, the stimulatory effect of TGF-β2 on the much cruder KLS population

increases with age

52.

This is logical, as the aged KLS compartment should

contain a larger proportion of My-HSCs. However, the relationship between TGF-

β and aging is complex, as evidenced by a study of TIF1-γ. In HSCs, the

expression of TIF1-γ decreases with age, consistent with an accelerated aging

phenotype of TIF1-γ knockout mice

47.

Additionally, TIF1-γ controls the turnover

8


+ HSCs

of ALK5, such that ALK5

become more abundant with age

aged HSCs are more sensitive to the cytostatic effect of TGF-β

a reflection of more generous ALK5 expression.

47.

Interestingly,

in vivo,

possibly as

As TIF1-γ is itself part of the

signal transduction machinery downstream of TGF-β, the balance between TIF1-

γ and SMAD4-mediated transcriptional responses may play an additional role,

which was not investigated. In sum, perturbations of TGF-β signaling represent a

potential mechanism that contributes to HSC aging.

TGF-β and the bone marrow niche β in the regulation of HSCs, requires an

The quest to decipher the role of TGF-

understanding of the BM niches that house HSCs. Due to the anatomical

complexity of the BM, much of our knowledge concerning the HSC

microenvironment has for long remained nebulous and it is only in the last

decade that we have gained a more detailed appreciation of the regulatory

elements of the BM environment, although Ray Schofield proposed the concept

of the BM niche already as far back as 1978

53.

An increasing number of niche

components have now been identified, revealing a complex network of cell-cell

interactions, extracellular elements, signaling cues, and structures

54,55.

Together,

these entities create specialized niches that function to maintain and control self-

β is part of this microenvironment,

renewing HSCs in a coordinated manner. TGF-

as it is produced by a variety of cell types in the BM, and large quantities of latent

β are deposited into bone matrix 56,57. However, according to one study,

TGF-

surprisingly few BM cells exhibit significant phosphorylation of SMAD2/3 at

β is tightly regulated

steady state, suggesting that activation of latent TGF-

spatially

58.

Nonmyelinating Schwann cells, which ensheath peripheral nerves

and lay in parallel with blood vessels in the BM, are proposed as one of the major

9


β activation in the BM 58. These glial cells regulate the activation

sources for TGF-

β by binding latent TGF-β via integrin β8 on the cell surface, thus

process of TGF-

β to proteolytic cleavage by

promoting activation by exposing TGF-

metalloproteinases

58.

A sizable portion of HSCs is in direct contact with

nonmyelinating Schwann cells in the BM, and denervation results in loss of HSCs

β are megakaryocytes, a

and increased cell cycling. Another major source of TGF-

cell type that physically associates with approximately 20 percent of HSCs in the

BM

59,60.

HSCs in close proximity to megakaryocytes exhibit activation of

SMAD2/3 and ablation of megakaryocytes results in reduced phosphorylation of

SMAD2/3 as well as loss of quiescence and increased HSC proliferation

Importantly, conditional deletion of

increased cell cycling of HSCs

60.

Tgfb1

60.

in megakaryocytes results in

The anatomical relationship between

megakaryocytes and Schwann cells have not been investigated, but the

β provides an important

conclusion from these studies is that, indeed TGF-

quiescence signal to HSCs in the BM niche. Additionally, the conclusion must be

β production and

that more than one cell type in the BM contributes to TGF-

signaling in HSCs (Figure 4).

Lessons from in vivo models TGF-β can affect most cell types throughout the hematopoietic hierarchy, but the

response is modulated by context and differentiation stage of the target cell.

Therefore, TGF-β generates highly variable biological outcomes and can affect

proliferation, differentiation, and apoptosis in both positive and negative

directions

21,61-63.

β

Much of our knowledge of the physiological relevance of TGF-

has been gained by studying knockout mouse models. Based on these reports, we

know that TGF-β is a principal regulator of immune cell homeostasis and

10


function

in vivo

. Importantly, both

Tgfb1

-ligand and receptor knockout mice

develop a lethal inflammatory disorder

64-66.

With respect to hematopoiesis,

Tgfb1-

null mice exhibit enhanced myelopoiesis, suggesting that TGF-β acts as a

negative regulator of myelopoiesis

in vivo 65

. Upon analysis before the onset of

inflammation, a host of HSC properties are altered in

Most significantly, BM cells from

Tgfb1

knockout mice

67.

Tgfb1

-deficient neonates exhibit impaired

reconstitution ability upon transplantation, a finding attributed to defective

homing

67.

However, mice deficient in ALK5 display normal HSC self-renewal and

regenerative capacity

in vivo

, even under extreme hematopoietic stress with no

defects in homing capacity

68,69.

show increased HSC cell cycling

transplantation

58.

β RII conditional knockout mice

In contrast, T

in vivo

, and reduced regenerative capacity upon

Because of the severe inflammatory disease that develops in

β signaling deficient mouse models, most studies have employed different

TGF-

strategies to overcome disease progression, including a variety of immune-

deficient genetic backgrounds. This may account for the differences observed

βRII

between knockout models. Additionally, signals may emanate from T

independently of ALK5, as has been shown in non-hematopoietic cell types

where the cell polarity regulator partitioning defective 6 (PAR6) is

βRII 70,71. Further, the fact that TβRII is more highly

phosphorylated directly by T

expressed within HSCs compared to ALK5 might be an additional contributing

factor

57.

Thus, there are both overlapping and non-overlapping phenotypes

between knockout models and it appears to be critically important at which level

β signaling is disrupted. This notion is supported by a

and for how long TGF-

β was

recent study, in which a neutralizing antibody against active TGF-

administered to mice following 5-FU treatment

11

72.

β blockade after

TGF-


chemotherapy results in enhanced hematopoietic regeneration by delaying the

β-SMAD2-

return of HSCs to quiescence. These findings imply that the TGF-

Kip2 signaling axis

p57

is important for reestablishing quiescence of HSCs

following stress, once sufficient regeneration of the hematopoietic system has

been attained. Additionally, overexpression of SMAD4 sensitizes human cord

β , resulting in reduced regenerative

blood-derived candidate HSCs to TGF-

capacity

in vivo 73

. Together, these findings suggest that transient inhibition of

β might be a feasible strategy to enhance hematopoietic recovery in patients

TGF-

β coupled with a

following chemotherapy. Due to the multifaceted nature of TGF-

potentially complex set of redundant mechanisms, its role as a critical regulator

of HSC quiescence

in vivo

has been difficult to unveil. However, current dogma

β is indeed an important quiescence signal in vivo.

now suggests that TGF-

A role for SMAD signaling in self-renewal of HSCs β , two approaches

To block the SMAD signaling network downstream of TGF-

have been used: overexpression of the inhibitory SMAD7 and deletion of

Smad4

.

In murine HSCs, forced expression of SMAD7 results in increased self-renewal of

HSCs, indicating that the SMAD pathway negatively regulates self-renewal

74.

in vivo

Importantly, differentiation was unperturbed in this model, suggesting that

self-renewal is regulated independently of differentiation by SMAD signaling. In

contrast, overexpression of SMAD7 in human SCID repopulating cells (SRCs)

results in altered differentiation from lymphoid dominant engraftment toward

increased myeloid contribution

75.

Thus, in the xenograft model system, forced

expression of SMAD7 modulates differentiation of multipotent human SRCs.

Using a conditional knockout mouse model, disruption of the SMAD pathway at

the level of SMAD4 was investigated. Intriguingly,

12

Smad4

-deficient HSCs display


a reduced repopulative capacity of primary and secondary recipients, indicating

that SMAD4 is critical for HSC self-renewal

SMAD7 vs. deletion of

Smad4

in vivo 76

. Since overexpression of

is anticipated to yield similar hematopoietic

phenotypes, it is conceivable that SMAD4 functions as a positive regulator of self-

renewal independently of its role in the TGF-β pathway

13-15.

Alternatively,

SMAD7 may have unanticipated functions in an overexpression setting that

impinge positively on self-renewal.

Variations on TGF-β signaling: a role for TIF1γ SMAD4 has traditionally been viewed as the nexus of SMAD signaling as it

β/activin and BMP signaling

functions as a core component of both TGF-

γ 12. In human

branches. However, SMAD2/3 can also partner with TIF1

β balances erythroid differentiation

hematopoietic stem/progenitor cells, TGF-

with growth inhibition, in a mechanism dependent on competitive binding

γ to SMAD2/3 12. In response to TGF-β , the TIF1γ-

between SMAD4 and TIF1

SMAD2/3 complex stimulates erythroid differentiation whereas SMAD2/3 in

association with SMAD4 leads to growth inhibition of human hematopoietic

γ

progenitors (Figure 2). Interestingly, the zebrafish homolog of TIF1 , encoded by

moonshine

, is essential for blood formation with mutants displaying severe red

γ is required for erythroid development 77.

cell aplasia, indicating that TIF1

γ is implicated in regulation of transcription elongation of

Furthermore, TIF1

γ functions to release

erythroid genes. The proposed model suggests that TIF1

paused Pol II at erythroid genes by recruiting positive elongation factors to the

blood-specific transcriptional complex, thus promoting transcription

78.

also functions at the erythroid/myeloid lineage bifurcation by modulating

13

γ

TIF1


GATA1 and PU.1 expression

79.

γ and TGF-β signaling has

The link between TIF1

γ functions as a

been delineated in detail in embryonic stem cells. There, TIF1

γ

chromatin reader where TIF1 -SMAD2/3 recognizes certain repressive histone

marks, promoting a transition to open chromatin that allows SMAD4-SMAD2/3

to gain access to DNA

80.

γ is needed for certain SMAD4-SMAD2/3

Thus, TIF1

transcriptional responses.

TGF-β in hematopoietic disease

Leukemia β’s pronounced cytostatic effect on HSCs in vitro and the fact that

Despite TGF-

β pathway are frequently

mutations in genes encoding components of the TGF-

found in other neoplasms, such as pancreatic and colon cancer

81,82,

inactivating

mutations are relatively uncommon for this pathway in hematological

malignancies

SMAD4

and

83.

Nevertheless, a number of cases have been reported involving

TGFBR2

in patients with acute myelogenous leukemia (AML)

84-87.

For example, in a study investigating human AML genomes, various copy number

alterations were found recurrently modified, one of which represented the

SMAD4 88 TGFBR1 TGFBR2 deletion of

. Furthermore, reports of sporadic mutations in both

and

in lymphoid neoplasms exist, and loss of SMAD3 is

associated with T-cell acute lymphocytic leukemia (ALL)

for the SMAD3-deficiency is not known since the

no mutations could be detected in the

MADH3

SMAD3

89-91.

The mechanism

mRNA was present and

gene, which encodes SMAD3

91.

However, T-cell leukemogenesis is promoted in mice with haplo-insufficiency of

Smad3

and a complete deficiency of

because the

p27Kip1

p27Kip1 91

. These findings are interesting

gene is frequently mutated in pediatric ALL, due to

14


translocations and deletions or germline mutations

92,93.

β

Impaired TGF-

signaling in hematological malignancies can also be caused by suppression of

SMAD-dependent transcriptional responses by oncoproteins like TAX, EVI-1, and

AML1-ETO

94-96.

Similarly, downregulation of the transcription factor ZEB1 and

β 1-mediated growth

overexpression of SMAD7 contribute to resistance to TGF-

suppression in adult T-cell leukemia/lymphoma without known mutations in

β pathway genes 97. In addition, there are several reports on oncoproteins,

TGF-

which generate leukemia and simultaneously neutralize the growth inhibitory

signal of the SMAD pathway by binding to or interacting with SMADs. Fusion

oncoproteins, like TEL-AML1 and AML1-EVI1, bind to SMAD3, impairing both

β signaling and apoptosis of transduced HSCs in vitro 95,98-100. Furthermore,

TGF-

SMAD4 physically associates with HOXA9, reducing its ability to regulate

transcriptional targets in hematopoietic cells

in vitro 101 In vivo .

studies show

that in wild-type mice overexpressing HOXA9 or NUP98-HOXA9, SMAD4 binds

the oncoproteins and sequestrates them to the cytoplasm, suggesting that

SMAD4 plays a protective role against further promotion and growth of leukemic

cells

102.

Therefore, SMAD signaling can be reduced or neutralized in

hematopoietic malignancies, but in a majority of cases this is not due to

mutations in genes of the circuitry itself, but rather through altered expression

β

or function of co-factors and oncoproteins, or alternatively via loss of TGF-

target genes.

The diseased bone marrow microenvironment β plays a major role as tumor suppressor, TGF-β can paradoxically

Although TGF-

facilitate tumor growth, particularly in the later stages of disease. This is due to

effects on the tumor microenvironment and the immunosuppressive function of

15


β. Interestingly, recent findings show that there is considerable interplay

TGF-

between leukemic cells and the BM microenvironment. In fact, the BM

microenvironment undergoes substantial remodeling by signals from leukemic

cells

103.

Similarly, alterations in niche cells can trigger hematopoietic disease

and promote leukemia

104,105.

Furthermore, activation of the parathyroid

hormone (PTH) receptor specifically in osteoblastic cells results in remodeling of

the BM microenvironment

106.

The remodeled niche attenuates

BCR-ABL1

oncogene-induced chronic myeloid leukemia (CML)-like disease, via up-

β 1, whereas MLL-AF9 oncogene-induced AML is enhanced.

regulation of TGF-

These data differ from the findings by Naka

et al

β, via regulation

., in which TGF-

of AKT and FOXO3a, instead maintains leukemia-initiating cells in CML

107.

The

β dosage and context,

discrepancies may be attributed to differences in TGF-

β sensitivity across different types of leukemia vary.

indicating that TGF-

β is part of the leukemic BM

Nevertheless, the findings point to the fact that TGF-

niche and strategies to target the BM microenvironment may be a promising

approach to reduce certain types of leukemic stem cells in the future. On this

β has been proposed to contribute to myelofibrosis, which is a chronic

note, TGF-

myeloproliferative disease characterized by clonal proliferation and bone

marrow fibrosis

108.

Several cytokines are thought to contribute towards

accumulation of reticulin fibers in the BM of patients with myelofibrosis. These

β , fibroblast growth factor 2 (FGF-2), and platelet-derived growth

include TGF-

factor

109-112.

Some of the most compelling evidence for a prominent role of TGF-

β in myelofibrosis comes from a study using Tgfb1-null mice. To induce myelofibrosis, irradiated mice were transplanted with BM cells transduced with

16


vectors containing the thrombopoietin (TPO) gene. Importantly, prominent

myelofibrosis developed in mice receiving transduced wild-type cells but not

Tgfb1

-null cells

111.

β 1, produced by hematopoietic

These data indicate that TGF-

cells, is a crucial component in the development of myelofibrosis, and underlines

further the interplay between hematopoietic cells and the BM microenvironment.

TGF-β in the pathogenesis of MDS Myelodysplastic syndromes (MDSs) encompass a spectrum of clonal stem cell

disorders characterized by inefficient hematopoiesis and reduced peripheral

blood counts. Excessive activation of inhibitory pathways, such as TGF-β, has

been proposed to amplify the inefficient blood production inherent to MDS

+ BM

For example, SMAD2 is upregulated and over-activated in CD34

113.

progenitors

from MDS patients. Importantly, pharmacologic inhibition of the TGF-β pathway

in vivo

, using a small molecule inhibitor of ALK5, alleviates anemia in a mouse

model of MDS

114.

Furthermore, SMAD7 is reduced in BM progenitors from MDS

β signaling 115. The reduction of

patients, leading to over-activation of TGF-

SMAD7 is attributed to increased levels of microRNA-21 (miR-21), which binds

to the 3´UTR of

SMAD7 116

.

Administration of a chemically modified inhibitor of

miR-21 results in increased red blood cell counts, using the same mouse model

as mentioned above

116.

Together, these studies implicate TGF-β in the

pathogenesis of MDS and suggest that this pathway may be a realistic

therapeutic target in a subset of MDSs.

Therapeutic avenues and future perspectives HSCs are the therapeutic units of BM transplantations, and as such by far the

most widely used application of stem cell therapy to date. Despite this, strategies

to improve efficiency and applicability of HSC transplantations are needed,

17


including expansion of HSCs

ex vivo

and techniques to improve engraftment and

regeneration post transplantation. Although BMPs have been reported to

contribute to HSC expansion

ex vivo,

manipulating SMAD signaling alone is

117.

unlikely to result in effective expansion

Another approach, with substantial

β

promise that should be investigated further, is to transiently block TGF-

signaling

in vivo

chemotherapy

to boost regeneration and recovery of hematopoiesis following

72.

Similarly, a number of recent studies highlight the feasibility of

β pathway in hematopoietic disorders like MDS and

manipulating the TGF-

anemia

115,118,119.

In addition, the complexity of the SMAD pathway continues to

be exposed as new layers of regulatory mechanisms are found. For example, the

linker region of SMADs is subject to negative regulation by GSK3 (glycogen

synthase kinase 3), FGF, and EGF

120-123.

The importance of this type of crosstalk

in hematopoietic cells is unknown, but it is worth mentioning as megakaryocytes

β and FGF output, thereby regulating

were recently shown to balance TGF-

hematopoietic regeneration following 5-FU challenge

in vivo 60

. Thus,

β signaling is relevant to several aspects of hematopoiesis. In

manipulating TGF-

the future, more detailed mechanistic studies are required to precisely define

β signaling may be manipulated in relation to other signaling

how TGF-

circuitries, ultimately improving HSC regeneration and hematopoietic disease

vivo

.

18

in


Acknowledgments The authors apologize to those whose work was not cited due to space

limitations.

This work was supported by the European Commission (Stemexpand); Hemato-

Linné and Stemtherapy program project grants from the Swedish Research

Council; a project grant to S.K from the Swedish Research Council; Swedish

Cancer Society; Swedish Children Cancer Foundation; Clinical Research Award

from Lund University Hospital; and a grant from The Tobias Foundation

awarded by the Royal Academy of Sciences. (S.K.).

Authorship Contribution: U.B. and S.K. wrote the review, and U.B. designed the figures.

Conflict-of-interest disclosure: The authors declare no competing financial

interests.

Correspondence: Stefan Karlsson, Division of Molecular Medicine and Gene

Therapy, BMC A12, 221 84 Lund, Sweden; email: [email protected]; and

Ulrika Blank, Division of Molecular Medicine and Gene Therapy, BMC A12, 221

84 Lund, Sweden; email: [email protected].

19


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Figure legends Figure 1. The hematopoietic hierarchy

. Hematopoiesis is organized in a

hierarchical manner, with rare HSCs at the top that give rise to various types of

progenitor cells, which proliferate extensively, finally generating mature blood

cells at the bottom of the hierarchy. Long-term HSC (LT-HSC); short-term HSC

(ST-HSC); multipotent progenitor (MPP); common myeloid progenitor (CMP);

common lymphoid progenitor (CLP); granulocyte-macrophage-lymphocyte

progenitor (GMLP); granulocyte-macrophage progenitor (GMP); megakaryocyte-

erythrocyte progenitor (MEP); natural killer (NK). Red arrows indicate

stimulation of proliferation by TGF-β, whereas inhibition signs point to TGF-β’s

growth inhibitory effect, in specific cell types or lineages.

Figure 2. TGF-β signaling pathways.

β ligands bind type I and type II

TGF-

receptors at the cell surface. Subsequently, the type I receptor (ALK5) becomes

phosphorylated by the type II receptor. This leads to phosphorylation of SMAD2

and SMAD3, which form a complex with SMAD4. Activated complexes

accumulate in the nucleus where they cooperate with DNA-binding cofactors to

regulate target gene transcription. SMAD2 and SMAD3 also bind to TIF1γ. In ES

cells, SMAD2/3-TIF1γ recognizes specific chromatin marks, promoting access of

SMAD2/3-SMAD4 to otherwise repressed targets. TIF1-γ-SMAD2/3 promotes

erythroid differentiation whereas SMAD4-SMAD2/SMAD3 complexes inhibit

proliferation. In certain cell types Jun N-terminal kinase (JNK) and p38 are

phosphorylated by TAK1 and constitute, together with the phosphatidyl inositol

3-kinase (PI3K)-AKT-FOXO axis, extracellular signal-regulated kinase (ERK), and

β. Dashed line

PAR6, so-called non-canonical signaling responses to TGF-

indicates unclear molecular mechanism.

28


Figure 3. Bidirectional effects of TGF-β.

Low concentrations of TGF-β

stimulate proliferation of My-HSCs, whereas Ly-HSCs are growth inhibited. A

higher dose of TGF-β inhibits proliferation, irrespective of HSC subtype, and

Kip2.

induces quiescence via SMAD2/3-SMAD4-dependent expression of p57

Figure 4. TGF-β in the bone marrow niche.

HSCs reside in the bone marrow, in

β is activated

specialized niches that support and regulate HSC fate options. TGF-

β are

by Schwann cells that ensheath sympathetic nerves. Large amounts of TGF-

also produced by megakaryocytes. HSCs in close proximity to Schwann cells and

β and exhibit activation of SMAD2/3.

megakaryocytes become exposed to TGF-

29

Self-renewal

Figure 1

LT-HSC Self-renewal

Symmetric

ST-HSC Asymmetric

TGFß

MPP

Differentiation: Lineage specification

CMP

MEP

GMLP

GMP

CLP

Granulocyte

Erythrocyte Platelets

Dendritic cell Monocyte

NK cell

T lymphocyte B lymphocyte

Figure 2

TGFß

Cell surface

TßRII ALK5

P

RHO

PAR6

P

P

P

PI3K

P

SMAD2/3

SMAD7

Cytoplasm

ERK

TAK1 P

AKT TIF1γ

P

SMAD4

JNK

P

p38

P FOXO

Erythroid differention

Co-factor

SMAD4

SMAD2/3

TIF1γ

Nucleus

Growth inhibition

Figure 3 My-HSC

TGFβ concentration Low

High

Ly-HSC

HSC

SMAD4

Co-factor

SMAD2/3

Nucleus

p57Kip2

Quiescence

Figure 4

Blood vessel

Sympathetic nerve

Schwann cell

TGFβ HSC

TGFβ

Bone

ce

s

Osteoblast

Q uie

Megakaryocyte

nce

Support cell


Prepublished online April 1, 2015; doi:10.1182/blood-2014-12-618090

TGF-β signaling in the control of hematopoietic stem cells Ulrika Blank and Stefan Karlsson

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