Oncogene (2005) 24, 5751–5763
& 2005 Nature Publishing Group All rights reserved 0950-9232/05 $30.00
www.nature.com/onc
Autocrine transforming growth factor-b regulation of hematopoiesis: many
outcomes that depend on the context
Francis W Ruscetti*,1, Salem Akel2 and Stephen H Bartelmez3
1
Laboratory of Experimental Immunology, Center for Cancer Research, National Cancer Institute-Frederick, Frederick, MD
21702-1201, USA; 2Department of Medical Laboratory Sciences, Hashemite University, Zarqa, Jordan; 3Hemogenix, Inc., Colorado
Springs, CO, USA
Transforming growth factor-b (TGF-b) is a pleiotropic
regulator of all stages of hematopoieis. The three
mammalian isoforms (TGF-b1, 2 and 3) have distinct
but overlapping effects on hematopoiesis. Depending on
the differentiation stage of the target cell, the local
environment and the concentration and isoform of TGF-b,
in vivo or in vitro, TGF-b can be pro- or antiproliferative,
pro- or antiapoptotic, pro- or antidifferentiative and can
inhibit or increase terminally differentiated cell function.
TGF-b is a major regulator of stem cell quiescence, at
least in vitro. TGF-b can act directly or indirectly through
effects on the bone marrow microenvironment. In addition, paracrine and autocrine actions of TGF-b have overlapping but distinct regulatory effects on hematopoietic
stem/progenitor cells. Since TGF-b can act in numerous
steps in the hematopoietic cascade, loss of function
mutations in hematopoeitic stem cells (HSC) have
different effects on hematopoiesis than transient blockade
of autocrine TGF-b1. Transient neutralization of autocrine TGF-b in HSC has therapeutic potential. In myeloid
and erythroid leukemic cells, autocrine TGF-b1 and/or its
Smad signals controls the ability of these cells to respond
to various differentiation inducers, suggesting that this
pathway plays a role in determining the cell fate of
leukemic cells.
Oncogene (2005) 24, 5751–5763. doi:10.1038/sj.onc.1208921
Keywords: transforming growth factor-b1; hematopoiesis; autocrine; stem cells
complex series of events. Hematopoietic stem cells
(HSC), which can remain quiescent for months, must
retain the abilities to differentiate into all lineages, to
self-renew and to maintain long-term repopulation
marrow activity (LTMRA). Recent work has shown
that a variety of intrinsic transcription factors such as
Runx, PU.1, GATA1, c-myb and SCL as well as
external cellular and humoral factors present in the
bone marrow microenvironment are involved in hematopoietic regulation. In the last 30 years, we and numerous
others have shown that the growth and differentiation of
hematopoietic cells is positively and negatively regulated
by a number of distinct cytokines (Ogawa, 1993).
Constitutive hematopoiesis is regulated, in part, by the
balance of these opposing signals (Jacobsen et al., 1994).
It has been our hypothesis that transforming growth
factor-b1 (TGF-b1) is an essential regulator of hematopoiesis. Previous in vitro and in vivo work indicates that
TGF-b is a regulator of all stages of hematopoiesis (for
reviews, see Fortunel et al., 2000; Hu and Zuckerman,
2001). The function of TGF-b1 in this process is
underscored by the observation that TGF-b1-null mice
have a significant increase in mature myeloid cells and
rapid death due to inflammation (Shull et al., 1992;
Kulkarni et al., 1993; Letterio et al., 1994). We proposed
that TGF-b1 regulates both HSC quiesence and survival
through autocrine and paracrine mechanisms. Here, we
will review new data that suggest that TGF-b is not
essential for regulation of HSC quiescence, the difficulties involved in defining the outcomes of TGF-b
signaling due to crosstalk between signaling pathways
and whether the actions of autocrine TGF-b on
regulating the heterogeneous HSC compartment will
present new therapeutic opportunities.
Introduction
In hematopoiesis, the need to continuously generate
large numbers of maturing cells of eight distinct lineages
from small numbers of stem cells requires a highly
Quiescent HSC are directly and reversibly growth
inhibited in vitro by TGF-b
*Correspondence: FW Ruscetti, Leukocyte Biology Section, Laboratory of Experimental Immunology, Bldg 567, RM 251, NCI-Frederick,
Frederick, MD 21702-1201, USA; E-mail: [email protected]
The content of this publication does not reflect the views or policies of
the Department of Health and Human Services, nor does mention of
trade names, commercial products or organizations imply endorsement by the US Government.
In order to determine if TGF-bs directly regulated stem
cell proliferation in vitro (Keller et al., 1990), sequential
cell sorting of murine lin bone marrow cells was used
for low Rhodamine 123 (Rho, a mitochrondial DNA
dye) followed by low Hoescht 33342 (Ho, genomic
DNA binder) and c-kit þ , to isolate a purified HSC
Autocrine TGF-b1 regulation of hematopoiesis
FW Ruscetti et al
5752
population. This method has been recently shown to
isolate the most quiescent part of the side population
(SP) cells which contains most of the HSC (Bertoncello
and Williams, 2004). Ho/Rholow HSC are CD34, SCA1 þ , give long-term donor repopulation with 5–10 cells
(Wolf et al., 1993) and 93–100% of these cells can be
stimulated by a combination SCF, IL-3 and IL-6 to
develop high proliferative potential (HPP) macrocolony
(>100 000 cells) formation in vitro (Wolf et al., 1993;
Sitnicka et al., 1996). Functionally, Ho/Rholow HSC are
in the G0 state of the cell cycle and have no radioprotection activity. In single cell assays, the continuous
presence of exogenous TGF-b1 at 10 mg/ml directly
inhibited the in vitro cell division of essentially all Ho/
Rholow HSC between their 0–5th division (mean ¼ eight
cells) (Sitnicka et al., 1996). The addition of TGF-b1
neutralizing antibodies as late as 3 days after the
addition of exogenous TGF-b1 preserved the ability of
80% of the cells to make macroclones, indicating that
the effect of exogenous TGF-b1 was reversible and not
markedly apoptotic to HSC in the context of other
cytokines. Delayed addition of TGF-b1 until the HSC
had undergone three divisions (eight cells/well) was still
able to cause growth arrest of the cells at that stage.
Thus, TGF-b1 inhibits the in vitro growth of HSC in the
Ho/Rholow cell population.
Direct inhibitory effects of TGF-b1 on purified
CD34 þ human stem/progenitor cells has also been
reported. Using purified CD34high þ Lin (Lu et al.,
1993) and CD34 þ CD38 (Batard et al., 2000) stem/
progenitor cells, it has been demonstrated that TGF-b
inhibited 80–90% of the colony formation induced by
various cytokine combinations including early acting
cytokines (SCF, Flt-3 or TPO), differentiating cytokines
(GM-CSF, IL-3 or EPO) plus cell cycle accelerators
(IL-6). Recently, a CD34High þ , CD38, c-kitlow, IL-6Rlow
and Mpllow cell population was isolated from human
bone marrow progenitors (designated HPP-Q). These cells
possess many of the characteristics of murine Ho/Rholow
HSC. They are quiescent, yet can be stimulated to form
HPP macrocolonies (Fortunel et al., 1998). In single cell
assays, these cells can be growth arrested by TGF-b. It is
not known whether these cells can promote long-term
engraftment particularly after in vitro growth arrest with
TGF-b1.
The observation that proliferation of both human and
murine quiescent primitive cells can be rapidly growth
arrested by TGF-b could result in the cells maintaining
their stem cell nature. Ho/Rholow HSC cultured for 5
days with SCF, IL-3 and IL-6 showed 15% of donor
repopulation at 10 months compared to 60% using the
same number of uncultured cells. The ability of the
cultured cells to compete in a competitive repopulation
assay was detectable but less than the uncultured cells.
However, the same cells treated for 5 days with
cytokines and TGF-b had no activity. Similarly, Ho/
Rholow HSC stimulated to undergo cell division and then
growth arrested with TGF-b showed markedly reduced
engrafting potential (Wiesmann et al., 2000). In human
and murine gene transfer experiments, when TGF-b was
used to growth arrest the cells after retroviral infection,
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severely decreased repopulation was seen (Nolta and
Kohn, 1990). TGF-b mediated arrested growth at the
G1/S transition and G0 quiescence are not the same.
Throughout the G1/S transition, new mRNAs and
proteins are being made in contrast to little or no
synthesis in G0. Perhaps, this marcomolecular synthesis
results in the loss of ‘stemness’ in these cells even in the
presence of TGF-b1.
Evidence supporting this hypothesis was obtained
when 50 Ho/Rhlow HSC were expanded in liquid culture
with IL-3, SLF and IL-6 in the presence or absence of
TGF-b1. After an 8 day incubation, the number of
HPP-colony-forming cells (CFC) in control cultures
expanded from 35 to 450, the number of GM-CFC from
none to roughly 70 000, and the total cellularity
increased to approximately 300 000 (Sitnicka et al.,
1996). The addition of optimal doses of TGF-b to these
cultures completely inhibited the proliferation and
expansion of the initial cell input. In contrast, TGF-b
at 0.4 mg/ml stimulated an increase in HPP-CFC
expansion and an increase in total cellularity to
450 000 cells, relative to untreated cultures. These results
suggest that even when HSC are growth arrested, TGFb treatment results in subtle changes in differentiation
state. As discussed previously (Ruscetti and Bartelmez,
2001), TGF-b blocks HSC cell cycle progression at the
G1/S boundary not G0 making it likely that TGF-binduced HSC inhibition is not equivalent to in vivo
quiescence.
Stem cell regulation in TGF-b-null mice
The study of the development of the hematopoietic
tissue in vivo in the absence of endogenous TGF-b1 has
been greatly aided by the generation of a variety of
knockout mice. Homozygous TGF-b1 knockout mice
have a 50% intrauterine death rate because of severe
developmental retardation. A strain-dependent variation in survival occurs due to trans-placental transport
of maternal TGF-b1 (Bottinger et al., 1997). Progressive
wasting occurs with death by 5 weeks due a systemic
T-cell-dependent massive inflammatory syndrome. MHC
class II-dependent autoimmune disease also occurred.
Defective hematopoiesis results in plasmacytosis, myeloid hyperplasia, reduced numbers of erythroid cells as
well as a lack of Langerhans dendritic cells. These
defects correlated with the absence of TGF-b1 (Dickson
et al., 1995; Yaswen et al., 1996; Borkowski et al., 1997).
These mice still make adequate amounts of TGF-b2 and
b3, which shows that the isoforms are not equal in
function in vivo. The double TGF-b1/MHC class II
knockout mice lose the proinflammatory and autoimmune defects but still retain the massive myeloproliferative syndrome (Letterio et al., 1998). The phenotype
of the type II TGF-b receptor (TbRII)-null mice was
indistinguishable from that of TGF-b1 knockout mice
(Oshima et al., 1996). Attempts at isolating the quiescent
Ho/Rholow c-kit þ HSC from the TGF-b knockout mice
have resulted in a >90% reduction of these cells
Autocrine TGF-b1 regulation of hematopoiesis
FW Ruscetti et al
5753
Table 1
Stem cella progenitora phenotypes in TGF-b and Class II KO marrow
% c-kit+ in sample
% Rh low, Ho lowb
% Rh low, Ho low, c-kit+c
Littermate
Class II KO
TGF-b, Class II KO
Normal–3 months
10.271.2
11.9710.6
10.2
9.1
1.170.9
1.270.9
0.5
1.5
0.870.5
1.270.8
0
0.7
Type of mouse
Littermate
Class II KO
TGF-b, Class II KO
Normal–3 months
% SCA-1+ in sample
4.372.0
6.271.4
3.1
8.3
% Rh low, Ho low
1.170.9
1.270.9
0.5
1.5
% Rh low, Ho low, SCA-1+
4.771.8
6.172.6
1.9
0.8
Type of mouse
a
(a) Rh low, Ho low are stem cell enriched, (b) Rh low, Ho low, c-kit+ or SCA-1 + are highly enriched for stem cells, (c) c-kit+ contain most
progenitors. bThis is percent of all viable cells going to the sorter. cThis is the percent of Rh low Ho low cells that were c-kit+ or SCA-1+
indicating that autocrine TGF-b1 could be a major
regulator of stem cell quiescence (Table 1), although
other molecules such as interferon g (Yang et al., 2005b),
and TNFa (Jacobsen et al., 1992) also contribute to this
inhibition. It is not clear whether this lack of quiescent
HSC is due solely to the absence of TGF-b1 or
subclinical pathologies also contribute to it.
It has been proposed that loss of function mutations
using conditional deletions is the most accurate to study
the physiologic role of a certain gene or a pathway.
Given TGF-b’s complex interactions at multiple stages
of hematopoiesis, a dynamic cellular process which by
itself has many indirect and direct complex interactions
with the environment, drawing any clearcut conclusions,
may be a ‘leap of faith’. Using such a conditional model,
TbRI-null mice have normal hematopoiesis containing
normal progenitor numbers and rates of differentiation
(Larsson et al., 2001, 2003). Compared to littermate
controls, HSC from these mice have normal cell cycle
profiles and normal LTMRA following bone marrow
transplantation. Furthermore, HSC from these TbRInull mice have normal susceptibility to repeated 5FU
treatments. Similarly, hematopoietic failure occurred at
a stage identical to normal controls following serial
transplants (Larsson et al., 2005). These results clearly
show that TGF-b is not an essential regulator of HSC
quiescence and self-renewal during constitutive and
stress-induced hematopoiesis.
Stem cell quiescence is regulated by a cellular niche
How can the starkly opposing effects of TGF-b on HSC
in vivo and in vitro be explained? Numerous historical
studies have shown that bone marrow has an organized
and structured architecture in which close relationships
exist between HSC and the microenvironment (Lord,
1990). Recently, following transplantation, stem cells
showed selective redistribution and were highly enriched
in the endosteal region while the more mature progenitors were enriched in the central marrow region (Nilsson
et al., 2001). Using genetic techniques, it was subsequently noted that osteoblasts were a critical component
of the marrow microenvironment (Calvi et al., 2003;
Zhang et al., 2003). Elegant work by Arai et al. (2004)
showed that the receptor tyrosine kinase Tie2-expressing
HSC are quiescent, and adhere to the osteoblasts in the
endosteal niche. The interaction of Tie2 with its ligand
angiopoietin-1 (Ang-1) maintained the in vivo LTMRA
of the HSC. Ang-1 enhanced the ability of the Tie2 HSC
to become quiescent resulting in the protection of the
HSC compartment from myelosuppressive stress. These
cells are resistant to a myelosuppressive dose of 5FU.
Furthermore, Arai and his co-workers show that Tie2
signaling in HSC maintains LTMRA in vitro. Tie2 does
this by keeping the cells quiescent, clearly demonstrating
that HSC self-renewal capacity can be maintained by
preventing cell division. Other regulators of the cell cycle
such as p21 (Cheng et al. (2000a); p27 Cheng et al.
(2000b); Bmi-1(Park et al. (2004)) have been shown to
regulate HSC self-renewal (Cheshier et al., 1999). It is
essential for an accurate interpretation of the data
obtained from disrupting parts of the TGF-b pathway in
mice that one determines the effects of any deletion on
the HSC-microevironment interactions.
It is then reasonable to ask why not a nonessential
role for TGF-b in the same HSC self-renewal process?
When HSC/progenitor cells from TbRI-null mice
(Larsson et al., 2003) and TbRII-null mice (Fan et al.,
2002) were stimulated in vitro by a single cytokine, there
were significantly increased numbers of actively proliferating cells comparable to the numbers by transient
blockage of TGF-b1 (see later section). This suggests
that the differences lie in the responses to the microenvironment. Several studies have examined the effects
of TGF-b on hematopoietic cell growth in long-term
bone marrow cultures (LTBMCs). This in vitro culture
system contains a complex mixture of hematopoietic
cells and stromal accessory cells that can sustain the
growth and proliferation of primitive progenitors over
many weeks in culture (Dexter, 1979). Both mRNA for
TGF-b and active and latent TGF-b protein have been
detected in the stroma of LTBMCs (Cashman et al.,
1990; Eaves et al., 1991). Several conclusions have been
drawn from these studies. First, the addition of exogenous TGF-b at the initiation of the culture completely
arrests hematopoiesis, such that no clonogenic
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Autocrine TGF-b1 regulation of hematopoiesis
FW Ruscetti et al
5754
progenitors are found after 2 weeks (Waegell et al.,
1994); second, the addition of TGF-b1 inhibits the
increased cycling of primitive progenitors initiated in the
culture by the addition of IL-1 or a horse serum media;
third, the addition of neutralizing TGF-b1 antibodies 3
days after the addition of IL-1, when progenitors were
maximally cycling, allowed the cells to remain longer in
S phase, whereas progenitor cells in control cultures
underwent cell cycle arrest; and fourth, the addition of
neutralizing TGF-b antibodies to LTBMCs resulted in
an increased output of mature myeloid cells, mature
progenitors and day-14 CFUs. Thus, the production of
TGF-b by stromal cells results in a negative-feedback
loop to maintain stem cell quiescence and to inhibit
progenitor cell cycling. Does TGF-b1 regulate or
interact with the signaling pathways of Wnt (Staal and
Clevers, 2005), Notch (Stier et al., 2002; Mancini et al.,
2005) or Tie2 (Arai et al., 2004), thought to be involved
in stem cell renewal? Cytokines such as IL-1 and TPO
whose stromal production is increased by TGF-b1
(Sakamaki et al., 1999) transiently override this inhibitory effect. These results could be indirect since the
TGF-b plays a role in the development and function of
osteoblasts in the marrow niche (Lieb et al., 2004).
Osteoblasts and adipocytes develop from a common
progenitor from the bone marrow stroma/stem cells.
TGF-b plays a role in directing the cell fate of these stem
cells to become osteoblasts and in bone formation rather
than fat tissue formation (Moerman et al., 2004).
Smad crosstalk, differential effects of TGF-b isoforms
and other unsolved mysteries
Smad 2 and Smad 3 are intracellular signaling molecules
for TGF-b1. Smad3-null mice are viable demonstrating
defects in immune function (Yang et al., 1999a, b),
whereas Smad2-null phenotype is embryonically lethal
indicating that Smad 2 and Smad 3 regulate a
nonoverlapping set of genes. Recent studies have shown
that Smad3 is the major transcriptional activator for
TGF-b, while Smad2 is a transmodulator of this
transcriptional activity (Yang et al., 2003).
Previous studies suggested that Smad5 is involved in
the signaling pathway by which TGF-b inhibits proliferation of primitive human hematopoietic progenitor
cells, since suppression of Smad5 expression by antisense oligonucleotides reversed the inhibitory effects of
TGF-b on hematopoietic colony formation (Bruno
et al., 1998). While this study was one of the first to
suggest that Smad 5, typically activated by bone
morphogenetic proteins (BMPs), might be activated by
TGF-b, more recent studies have shown this to occur in
other cells as well, including intestinal epithelial cells
(Yue and Mulder, 2001) and endothelial cells (Oh et al.,
2000; Goumans et al., 2002). It has also been shown that
BMPs can regulate the development of human hematopoietic stem cells, presumably through the BMPactivated Smad proteins, Smad1 and Smad5, which
they expressed in HSC (Bhatia et al., 1999). In addition,
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several Smad-independent pathways have been identified (Engel et al., 1999; Yue and Mulder, 2001; Wilkes
et al., 2003). The TGF-b-activated receptor complex can
signal through AKT, mitogen-activated protein kinases
(MAPKs), the phoshoinositol-3-kinase (PI3K) and the
PP2A/p70s65K pathways (Wakefield and Roberts,
2002). AKT (Remy et al., 2004) and the tuberous
sclerosis complex 2 gene product (Birchenall-Roberts
et al., 2005) can both alter TGF-b signaling by binding
to Smad3. The numerous pathways involved in crosstalk
adds new complexity to the TGF-b regulation of
hematopoiesis.
The opposing effects of TGF-b1 and 2 on HSC
regulation are equally puzzling. Recently, it has been
shown that TGF-b2 has a positive regulatory role on
HSC/progenitor cells (Langer et al., 2004). Studying the
proliferation of these cells, it was found that TGF-b2
had a biphasic dose response (low concentrations were
stimulatory while high concentrations were inhibitory).
Furthermore, the number and repopulating ability of
the HSC in herterozygous null TGF-b2 mice were
significantly lower than the littermate controls. There
have been several reports that isoforms, both qualitatively and quantitatively differs in their effects on
progenitor cells (Ohta et al., 1987; Jacobsen et al.,
1991a, b, 1992) The reasons for these opposing properties of TGF-b1 and 2 on HSC regulation are not clear.
Even though the nonsignaling binding molecules,
betaglycan III (Stenvers et al., 2003) and endoglin
(Chen et al., 2002, 2003), can increase the binding
affinity of TGF-b2 but not b1 for the receptor on HSC ,
both isoforms use the same receptor activation and
Smad signals, leaving the biochemical mechanism
obscure.
Another mysterious mechanism involves the autocrine actions of TGF-b by hematopoietic stem/progenitor cells that can negatively regulate the cycling status of
progenitors (Hatzfeld et al., 1994; Sitnicka et al., 1996).
Specifically, the addition of TGF-b antisense oligonucleotides or TGF-b neutralizing antibodies enhanced
the numbers and size of most immature bone marrow
colonies from purified human or human stem/progenitor cells in response to various combinations of
cytokines. In some experiments, more than half of the
detectable CD34 þ CFC were maintained in a growth
factor unresponsive state by autocrine TGF-b. The
observations that neutralizing antibodies that prevent
binding to the cell surface receptor can have differential
effects on autocrine vs paracrine TGF-b remain a
mystery. The clearest example of this is the HSC from
Smad3-null mice which are still sensitive to growth
inhibition by exogenous TGF-b1 but are not growth
arrested by the autocrine pathway (Ruscetti, Roberts
and Letterio, unpublished results).
Furthermore, disruption of the TbRII gene in mice
leads to a lethal inflammatory disorder that is transplantable (Leveen et al., 2002). In an adoptive transfer,
TGF-b1 bone marrow reconstitutes B and T lymphoid
tissues in Rag2-null mice. Despite normal levels of TGFb1 expressed in all other tissues, the mice die from a
lethal inflammatory, showing an obligate autocrine
Autocrine TGF-b1 regulation of hematopoiesis
FW Ruscetti et al
5755
function for TGF-b1 in lymphoid homeostasis (Mamura
et al., submitted).
Autocrine TGF-b1 is a regulator of the in vitro survival of
murine HSC-LTRA
Autocrine TGF-b1 has direct effects on the function of
murine HSC in vitro. SCF or IL-3 as single agents
promoted HSC to survive as single cells or undergo only
a few (1–3) cell divisions. Addition of a neutralizing
anti-TGF-b1 monoclonal antibody, in conjunction with
SCF or IL-3, increased the proportion of HSC that
divided as well as increase the average clone size. More
striking was the effect of adding anti-TGF-b1 to single
HSC cultures in the absence of added growth factors;
where a high proportion of cells survived up to 14 days
as single cells compared to medium alone in which cell
survival was limited to a few days (Ruscetti et al., 1999).
Neutralizing of autocrine TGF-b1 was required because
a non-neutralizing antibody that binds with the same
affinity does not prolong survival and performing the
assay in serum-free conditions due to concerns about
TGF-b in sera (Dybedal and Jacobsen, 1995) did not
affect the results (Table 2). All cells began cell cycle
progression (measured as the time to first division) more
rapidly when treated with anti-TGF-b1. Cultures of 100
LTR-HSC were grown without cytokine or sera but in
the presence of anti-TGF-b1 for 5 days, then assayed in
a competitive repopulation assay which indicated that a
substantial proportion of the surviving cells retained
their LTR ability (Soma et al., 1996; Ruscetti et al.,
1999). The competitive repopulating units indicated a
relative enrichment of HSC compared to 105 normal
bone marrow cells. Moreover, we isolated stem/progenitor cells from TGF-b1-deficient mice. These progenitor
cells did not show any increase in survival after
neutralizing antibody treatment. Thus, neutralization
of autocrine TGF-b1 directly promotes the survival of
the HSC and preserves some LTMRA in culture.
Further, adoptive transfer experiments using marrow
from TGF-b1, MHC class II knockout mice suggest the
existence of significant autocrine TGF-b1 functions.
Transplantation of TGF-b1-null bone marrow into
lethally irradiated wild-type recipients has shown that
bone marrow is the major source of plasma TGF-b1
(recipients of TGF-b1-null donor marrow have fivefold
lower levels than recipients of wild-type marrow).
Inhibition of autocrine TGF-b1 in HSC by morpholino
antisense oligomers prior to transplant accelerates
engraftment while reducing stem cell numbers required for
long-term repopulation
Even a short (60 min) exposure of highly purified HSC
to a neutralizing anti-TGF-b1 antibody dramatically
reduced the time required for engraftment of HSC with
LTMRA (Bartelmez et al., 2000). Subsequent experiments had shown that morpholino antisense nucleotides
(MAS) to TGF-b1 or TGF-bRII induce HSC survival,
reduce the number of HSC required for hematopoietic
repopulation, and decrease the time to engraftment.
Donor-derived peripheral blood T cells, B cells, monocytes and neutrophils total blood count were assayed.
As expected, transplantation of 100 untreated LTRHSC or control-treated LTR-HSC (scramble MAS or
isotype antibodies) required competitor or support
marrow to prevent hematopoietic death due to radiation
and engrafted slowly over a period of 1.5–6 months:
donor cell chimerism and reached 30–40% of circulating
cells at B3 months. In contrast, 60 HSC exposed to
TGF-b MAS for 16 h engrafted rapidly to reach donor
cell chimerism of 50–60% by 3 weeks and 70–85% by
1.5 months. The early engraftment was predominantly
donor neutrophils followed by B cells and then T cells
by 1.5 months. The induction of rapid engraftment of
LTR-HSC by TGF-b MAS or neutralizing antibody did
not require any support cells nor impair their LTMRA
ability as measured by sustained high % donor chimeras
beyond 1 year post-transplant. Also, a blockade of HSC
autocrine TGF-b dramatically reduced the number of
HSC required to rescue a lethally irradiated congenic
recipient, while more than 1000 control LTR-HSC per
mouse were required for hematopoietic rescue, as few as
30 TGF-b MAS-treated cells would rescue a recipient
(Bartelmez et al., 2000). Translation of this ex vivo
method to human stem cell transplants could reduce the
time to engraftment plus lower the number of stem cells
required for a durable graft.
Table 2 Effects of Anti TGF-b antribody and serum on cytokine-dependent survival of purified quiescent murine Ho/Rholow stem cells
Stem cell factor
No antibody
Neutralizing anti TGF-b
Binding anti TGF-b
Serum-free, no antibody
Serum free, neutralizing anti-TGF-b
% Viable cells
Day 1
Day 2
Day 4
Day 7
Day10
Day 14
9276I
9871
9871
9772
9870I
8678
9473I
7973S
8173
9372
72715
9373
6570.9
70714
8876
4679
8673
4278
3676
7778
1773
8377
2377
1476
5977
1272
7478
973
674
4378
Murine Lin cells were further purified by FACS separation and seeded as single cells in Terasaki plates. A minimum of 1000 wells was scored per
group. Cultures were supplemented with SCF (20 ng/ml) or IL-3 (20 ng/ml), cytokine reported to protect quiescent marrow cells against death
(Keller et al., 1995) plus or minus anti-TGF-b (20 mg/ml, ID11 neuralizing or 2G1.12 binding only). Media were IMDM 12.5 % horse serum and
12.5% fetal calf serum; serum free was QBSF alone
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Autocrine TGF-b1 regulation of hematopoiesis
FW Ruscetti et al
5756
The mechanism by which a blockade of autocrine
TGF-b1 in quiescent HSC occurs remains unclear. Does
antibody and antisense treatment block TGF-b1mediated apoptosis in the absence of stromal or
cytokine factors? TGF-b1 has been shown to regulate
the cell surface expression of many cytokine receptors
(Dubois et al., 1990, 1994; Jacobsen et al., 1991a, 1992)
as well as Fas (Dybedal et al., 1997). Since antisense
treatment of 60 cells rescues a mouse from lethal
irradiation, the HSC must undergo differentiation to
short-term repopulating cells (Yang et al., 2005a, b). In
what progeny and at what time do the cells resume
producing autocrine TGF-b? Does this blockade of
autocrine TGF-b lead to better homing to the marrow
space (Quesenberry and Becker, 1998)? Transient
modulation of CD26, a cell surface molecule which
negatively regulates engraftment, greatly increases the
efficiency of bone marrow transplantation (Christopherson et al., 2004). It is somewhat puzzling that such a
brief treatment with antibody or antisense leads to such
a profound increase in engraftment. A brief 6 h
treatment with anti-TGF-b antibody significantly increased number and size of macrocolonies in a single cell
assays, indicating that the transient blockade of autocrine TGF-b in HSC sets in motion a cascade of
hematopoietic proliferation and differentiation events
that the presence of autocrine TGF-b in the progeny
cannot alter.
In vitro neutralization of autocrine TGF-b in umbilical
cord blood stem cells: improved routine engraftment of
UCB stem cells in allogeneic transplants?
Umbilical cord blood cells (UCB) are being used as a
source of stem cells in allogeneic bone marrow
transplants (Laughlin 2001; Sorrentino, 2004). The good
news is that because of the immaturity of the donor
immune system, the incidence and severity of graft vs
host disease is favorable compared to adult transplants.
The bad news is that time to recovery of the graft is
delayed leading to more failures. Translation of this
murine ex vivo method to human umbilical cord blood
transplants could reduce the time to engraftment plus
lower the number of stem cells required for a durable
graft. The development of methods for in vitro
neutralization of TGF-b in hematopoietic stem/progenitor cells is an important goal for clinical research, given
the therapeutic potential of these cells. The challenge is
to develop defined culture systems in which the cell
cycling of primitive quiescent stem/progenitor cells is
stimulated in presence of either TGF-b antibodies or
TGF-b antisense, while their maturation and senescence
are prevented (Hatzfeld et al., 1991, 1994, 1996;
Ploemacher et al., 1993; Pierelli et al., 2000).
In an attempt to define culture conditions to exploit
TGFb1’s autocrine effects on hematopoiesis, the effect
of blocking autocrine TGFb1 in serum-free SCF
cultures was measured. These conditions allowed
increased early erythroid development from primitive
Oncogene
human HSC (Akel et al., 2003). UCB CD34 þ CD38
Lin cells were cultured in serum-free conditions using
various combinations of SCF and TGFb1 neutralizing
antibody. Anti-TGFb1 augmented proliferation of
CD34 þ CD38 Lin cells (day 21) in SCF-stimulated
cultures. While SCF alone stimulated production of
tryptase þ mast cells, while cellsstimulated by SCF/antiTGFb1 were predominantly erythroid (CD36 þ CD14
and glycophorin A þ ). A distinct expansion of erythroid
progenitors (CD34 þ CD36 þ CD14) with the potential
to form erythroid colonies was seen revealing early
EPO-independent erythroid development (Figure 1). In
contrast, TGFb1 accelerated the conversion of large
BFU-Es into CFU-Es. Finally, TGFb1 accelerated Epoinduced terminal erythroid differentiation and improved
enucleation (from 773 to 2276%) in serum-free
conditions (Figure 2). Serum addition stimulated
enucleation (54718%) that was reduced to 26714%
with anti-TGFb1, suggesting that optimal erythroid
enucleation is EPO dependent requiring serum factors
including TGFb1. Thus, the presence or absence of
TGF-b can alter the differentiation outcomes in the
same cell lineages.
Efficient retroviral-mediated gene transfer into stem/
progenitor cells released from quiescence by anti-TGF-b
Efficient retroviral-mediated gene transfer as therapy for
human genetic diseases implies that the host cells are in a
cycling state, which is not normally the case for
primitive HSC. Since both autocrine and paracrine
TGF-b1 produced by hematopoietic progenitors are
partly responsible for their maintenance in a quiescent
state, the use of anti-TGF-b1 antibodies to neutralize
the bioactivity of endogenous TGF-b1 during the
retroviral infection procedure was attempted to increase
gene transfer into primitive stem/progenitor cells. This
strategy has been tested with success on hematopoietic
cells of several origins, including human UCB cells
(Imbert et al., 1998; Yu et al., 1998). The efficiency of
Figure 1 Effect of TGF-b1 on the ability of EPO to induce red cell
enucleation of human red blood cells in serum-free cultures. UCB
CD34 þ Lin cells were incubated for 21 days in SCF (50 ng/ml)
and anti-TGF-b antibody without EP0. Then EPO were added
with out TGF-b (left panel) and with TGF-b (right panel) and
incubated for 72 h
Autocrine TGF-b1 regulation of hematopoiesis
FW Ruscetti et al
5757
HL-60
Bipotential cell
1,25 VitD3
Smad2
TGF-β1
Smad2P
Smad2
t-RA
t-RA
*t-RA
CD14+
monocyte
CD15+
granulocyte
Figure 2 A schematic model of the mechanism by which autocrine
TGF-b regulates the ability of Vit D3 and ATRA to stimulate
terminal differentiation of human promyelocytic HL-60 cell line to
either monocytes or granulocytes
this method was improved by the coupled use of antiTGF-b1 blocking antibodies with antisense oligos
against the cyclin-dependent kinase inhibitor (CDKI)
p27kip1 (Dao et al., 1998). This approach has been
extended to other effectors involved in the regulation of
hematopoietic cell cycling by TGF-b1, such as the
CDKI p21cip1. Stier et al., 2003). Moreover, CD34 þ cells
produce TGF-b1 during retroviral transfection protocols, promoting a return to quiescence of the most
primitive stem/progenitor cells after gene transfer. As
discussed above, this G1/S-induced inhibition is not
equivalent to quiescence and may be detrimental to
maintaining the ‘stemness’ of the transduced cells.
TGF-b signaling pathway in leukemia: rare mutational
inactivation
A number of leukemic cell lines blocked in differentiation representing early stages of myelomonocytic development exist (Dexter et al., 1980 and Greenberger et al.,
1983). Some absolutely require the presence of growth
factors for their growth and survival while others are
growth factor independent. Regardless of the factor
used to stimulate cell growth of murine leukemic cell
lines, their growth is inhibited by TGF-b (Keller et al.,
1988), while differentiation-blocked (TGF-bR positive)
cell lines were insensitive to TGF-b effects (Hampson
et al., 1989). It has been proposed that cells with a more
transformed phenotype (differentiation blocked) become refractory to the effects of TGF-b and possibly
escape negative regulation. However, growth factorindependent cell lines which can form tumors in nude
mice representing a more transformed phenotype can be
both TGF-b sensitive and insensitive cell lines (Keller
et al., 1988; Sing et al., 1988). Furthermore, infection of
IL-3-dependent cell lines with oncogene containing
retroviruses (v-abl, v-src and v-fms), to abrogate their
requirement for growth factors, produces cell lines
retaining their TGF-b sensitivity in all cases. Consistent
with the results above, TGF-b is a potent inhibitor of
the growth of freshly aspirated human CML (Sing et al.,
1988; Algietta et al., 1989) and AML (Tessier and
Hoang 1988; Nara et al., 1989) even in the presence of
saturating concentrations of growth simulators.
It has been proposed that genomic instability plays a
role in the disease progression of CML However, unlike
colon cancer (Grady et al., 1999), alterations in the
microsatelite regions of the TbRII gene are not seen in
CML. However, in all phases of CML and Bcell-CLL,
there is a reduction in the number of TbRII transcripts
(Rooke et al., 1999). In the case of B-cell CLL, this leads
to 30% of the cases being insensitive to TGF-b. In those
cases, mutations have been found in the signal sequence
of the TbRI gene (Schiemann et al., 1999). Mutations in
the Smad pathway are rare in leukemia. However,
disruption of Smad transcriptional responsiveness has
been associated with leukemic transformation. AML-1,
a transcriptional coactivator, has a binding domain with
homology to FAST, a protein involved in Smad2
signaling. The most common translocation in AML
(t8;21) results in an AML-ETO fusion protein. The
physical association of this fusion protein with Smad
proteins leads to a repression of constitutive and TGF-b
inducible gene transcription (Jakubowiak et al., 2000).
The physical association of several oncoproteins with
Smad3 blocks the ability of TGF-b to downmodulate
c-myc in G1, which is necessary to halt cell cycle
progression. The ability of Evi-1(Kurokawa et al.
(1998), EIA, c-ski (Shi and Massague, 2003)) and Tax
to associate with Smad3 prevents the formation of the
transcriptional complex of Smad3/E2F and the corepressor p107(for review, see Kim and Letterio, 2003).
p107-null mice get a myeloid hyperplasia. Loss of
Smad3 in association with loss of p27 has been
associated with murine and human lymphoid leuekmias
(Wolfraim et al., 2004, see J Letterio, this volume).
Cell fate stimulated by differentiation inducers in myeloid
leukemia cells is determined by endogenous Smad
signaling
Using the model system of HL-60 cells, a human
myeloblastic leukemia with promyelocytic features, the
interplay of signals from all trans retinoic acid (ATRA),
which specifies differentiation to granulocytes, or TGFb1/Vitamin D3 (Vit D3), which specify commitment to
monocytic differentiation, is mediated, in part, through
a balance between protein serine/threonine phosphatase
activity and levels of phosphorylated Smad2 and Smad3
(Cao et al., 2003). Thus, Vit D3/TGF-b1 induces
phosphorylation of Smad2/3 and that addition of
ATRA together with TGF-b1 reduces the level of
phospho-Smad2/3 and the extent of monocytic differentiation. Conversely, okadaic acid, which inhibits
protein serine/threonine phosphatases and which enhances the level of phospho-Smad2/3 in cells treated
simultaneously with ATRA and TGF-b, pushes the
Oncogene
Autocrine TGF-b1 regulation of hematopoiesis
FW Ruscetti et al
5758
balance toward monocytic differentiation. Together
these data suggest that monocytic differentiation is
favored by lower protein phosphatase activity and/or
high levels of nuclear Smad2/3 (if the inducing agents
are TGF-b or Vit D3), and that granulocytic differentiation is favored by higher protein phosphatase activity
and/or reduced nuclear Smad2/3. In the case where
ATRA and either TGF-b or Vit D3 are acting on the cell
simultaneously, the induction of protein serine/threonine phosphatase activity by ATRA can modulate the
levels of phospho-Smad2/3 induced by TGF-b and
thereby control the partitioning between the granulocytic and monocytic pathways.
Most germane to our findings is the report that
ATRA elicits a transient and reversible interconversion
of the protein phosphatase 2A (PP2A) holoenzyme at
the G1/S boundary during ATRA-induced granulocytic
differentiation of HL-60 cells (Zhu et al., 1997). PP2A
accounts for the majority of the serine/threonine
phosphatases activity in most cells and is specifically
inhibited by low concentrations of okadaic acid (Sontag
2001). Although several other studies show downregulation of the catalytic subunit of PP2A beginning about
48 h after treatment with ATRA and continuing for 3–5
days, it is probably the transient changes in the
regulatory subunit at 18–24 h, which are predicted to
result in a change in substrate specificity, which are most
likely to affect levels of phospho-Smads at the times we
observed. In HL-60R cells, which are resistant to effects
of ATRA on granulocytic differentiation, cytosolic
PP2A but not PP1 activity is reduced by almost 50%
compared to wild-type HL-60 cells (Giehl et al., 2000).
Consistent with our hypothesis that the ability of ATRA
to reduce levels of phospho-Smad2/3 induced by TGF-b1
treatment may depend, in part, on alterations in phosphatase activity, ATRA is unable to decrease levels of
TGF-b -induced phospho-Smad2/3 in either mutant
HL-60R cells or in wild-type HL-60 cells treated with
okadiac acid . The ability of okadaic acid to induce both
phenotypic and functional attributes of monocytes, even
in the absence of ATRA, further suggests that reduction
in the levels of phosphatases, independent of Smad
phosphorylation, is sufficient to specify differentiation
to monocytes.
Vit D3 has been shown to induce an autocrine TGF-b
pathway in several different cell types. In U937 cells,
treatment with Vit D3 induces differentiation to CD14positive cells with phagocytic capacity and this has been
shown to result from activation of an autocrine TGF-b
pathway (Defacque et al., 1999). In HL-60 cells, the Vit
D3 analog, EB1089, has been shown to induce expression of both TGF-b receptors and TGF-b ligand, and its
antiproliferative activity is blocked by a TGF-b
neutralizing antibody (Omay et al., 1995). Our data
now extend these studies and show that in HL-60 cells,
the ability of Vit D3 to phosphorylate Smad2/3 and to
stimulate monocytic differentiation can both be blocked
by neutralizing antibodies to TGF-b, suggesting that Vit
D3 acts indirectly by activating signaling from either
autocrine or paracrine (exogenous) TGF-b in these cells.
Moreover, these data also show that reduction in levels
Oncogene
of Smad2/3 phosphorylation is sufficient to reduce the
commitment of these cells to differentiate to monocytes,
even in the absence of changes in the levels of
phosphatases. Whereas treatment with TGF-b alone
results in arrest of differentiation at the CD14-negative
promonocyte stage, Vit D3 can induce HL-60 cells to
express CD14 and differentiate to mature monocytes, an
effect that has been shown to be dependent on induction
of phosphatidylinositol 3-kinase activity (Danielpour,
1996). These additional effects of Vit D3 are therefore
probably independent of TGF-b, or possibly dependent
on synergistic interaction of the vitamin D receptor with
Smad3 to regulate expression of certain target genes
containing both Vit D3 response elements and Smadbinding elements as previously reported (Hmama et al.,
1999).
Thus, cellular levels of phosphatase activity and of
phosphorylated Smad2/3 induced by TGF-b can independently affect the commitment to differentiation.
However, in the particular context of treatment of cells
simultaneously with ATRA and TGF-b, these two
mechanisms are inter-related in that elevation of
phosphatase activity by ATRA appears to underlie the
decrease in the level of Smad2/3 phosphorylation
(Figure 2). It remains to be demonstrated whether this
unique mode of integration of signals from ATRA and
TGF-b will also be relevant for other myeloid leukemia
cells.
Induction of differentiation of erythroleukemia cells
occurs by mediating crosstalk between the Smad and
MAPK pathways
A distinct role for TGF-b and activin in erythropoiesis
has been reported in studies of primary and transformed
cells. However, these studies were not fully informative
about the signaling networks that mediate TGF-b/
activin effects. The importance of crosstalk between
receptor-activated Smad signaling and the MAPK
pathways in the regulation of erythroid differentiation
induced by TGF-b/activin has been studied (Akel et al.,
submitted). Treatment of cells with TGF-b/activin
resulted in inhibition of cell growth and led to erythroid
differentiation as evidenced by a significantly increased
proportion of Hb-containing cells. Changes in cell fate
were preceded by cytokine/receptor-mediated intracellular signaling, which involved activation of the
receptor-activated Smads and various cascades of
MAPK, ERK, p38 and JNK. Signaling was rapid
suggesting that this process might be directly related to
receptor signaling and not to transcriptional activation.
Direct activation of Smad2/3 by TGF-b type I receptors
is well established and recent studies have described
links between MKK4/JNK and MKK3/p38 activation
and TGF-bR signaling involving XIAP, HPK1 and
TAK1 (Engel et al., 1999; Hanafusa et al., 1999).
Although no clear link of the Ras-MEK-ERK pathway
with TGF-bR signaling has been described yet, active
ERK still may negatively or positively modulate
Autocrine TGF-b1 regulation of hematopoiesis
FW Ruscetti et al
5759
receptor Smad activation and nuclear translocation (de
Caestecker et al., 1998; Kretzschmar et al., 1999). Since
inhibition of TGF-b type I receptors abrogated TGF-b/
activin-induced activation of ERK, p38 and JNK
MAPKs in K562 cells, all these MAPK pathways are
probably linked to TGF-b type I receptors. Our results
are in accordance with the recent findings of DaCosta
Byfield et al. (2004) showing that SB505124, an inhibitor
of TGF-bRI signaling interferes with TGF-b-induced
activation of MAPKs. In K562 cells that represent
human leukemic hematopoietic progenitor cells (HPC),
we have explored the role of the TGF-b/activin-induced
signaling in mediating cell growth arrest. TGF-b/
activin-induced growth arrest was reversed in cells
pretreated with the inhibitor of TGF-b/activin type I
receptors and to a lesser extent with a p38 MAPK
inhibitor, but not with ERK inhibitors. Recently, it was
found that activation of p38 mediates growth inhibition
of normal HPC by TGF-b (Verma et al., 2002), thus the
suppressive effect of TGF-b in normal and leukemic
human HPC seems to involve activation of Smad2/3 and
p38, but not ERK.
Cooperation between these two pathways was studied
in EPO-independent erythroid differentiation. Two
categories of Epo-independent erythroid differentiation
were evaluated: cytokine induced (TGF-b/activin) and
chemically induced (HU and butyrate). Cytokineinduced erythroid differentiation was dependent on the
activation of p38. Either selective inhibition of p38 by
SB203580 or coinhibition of phosphorylation of Smad2/
3 and p38 MAPK by SB505124 was sufficient to prevent
the formation of glycophorin A þ Hb þ cells. Since it was
not possible to achieve selective inhibition of phosphorylation of Smad2/3 by SB505124 without inhibiting p38
MAPK in cells treated with TGF-b/activin, it remains
uncertain whether activation of Smad2/3 is a prerequisite for TGF-b/activin-induced erythrodifferentiation.
Unfortunately, we found that ectopic stable expression
of Smad7, an inhibitor of Smad2/3 phosphorylation,
also prevented phosphorylation of p38 MAPK in
response to TGF-b/activin treatment.
Okadaic acid-induced Smad2/3 and p38 activation in
K562 cells was coincident with the induction of
erythroid differentiation and promotion of differentiation induced by various other agents. It was found that a
selective inhibition by SB505124 of phosphorylation of
Smad2/3 but not of p38 MAPK in OA-treated cells was
sufficient to prevent OA-induced differentiation. This
indicates that activation of p38 MAPK is subthreshold
for differentiation and activation of Smad2/3 is essential
for erythroid differentiation.
HU and butyrates induce cytodifferentiation and
growth inhibition of a variety of tumor cells and have
been used in cytoreductive and differentiation therapy of
malignant disease (Sowa and Sakai, 2000; Tsimberidou
et al., 2002). The ability of these chemicals to induce
changes in gene expression is mediated in part by
changes in signal transduction pathways. Butyrate
caused sustained activation of JAK/STAT signaling in
murine erythroleukemia cells, moreover, erythodifferentiation of K562 cells by HU and butyrate involved the
phosphorylation of p38 and dephosphorylation of ERK
and JNK MAPKs (Witt et al., 2000; Park et al., 2001).
Smad activation is also involved in the regulation of
HU- and butyrate-induced erythroid differentiation of
K562 cells. Although no direct binding was described
between HU and butyrate and TGF-b receptors, results
of SB505124 indicate that both chemicals activate the
serine/threonine TGF-bRI kinase upstream of Smad2/3.
It remains unclear whether these chemicals directly or
indirectly (through autocrine TGF-b) regulate receptor/
smad Signaling. Attempts to block ligand induction by
anti-TGF-b antibody did not prevent HU/butyrateinduced erythroid differentiation. Given that these
agents induce phosphorylation of Smads so quickly, it
is possible that they, like OA, block phosphatase activity
which increases the strength of basal endogenous
signaling by autocrine TGF-b/activin in K562 cells
leading to erythrodifferentiation.
In erythro-megakaryocytic differentiation, several
reports demonstrate that ERK negatively regulates
erythroid differentiation, and that sustained activation
of this MAPK is sufficient to induce a differentiation
program along the megakaryocytic lineage (Radtke
et al., 2004). Consistent with these reports, we have
shown that ERK1/2 is transiently activated by TGF-b/
activin but reduced below basal level during erythroid
differentiation. Moreover, inhibitors of ERK1/2 enhanced erythroid differentiation in the absence and the
presence of TGF-b/activin stimulation. Similar synergism was reported between ERK inhibitors, and HU
and butyrate, showing that ERK negatively regulates
this Epo-independent hemoglobin synthesis (Racke
et al., 1997). Surprising, ERK inhibitors induced
Smad2/3 phosphorylation, which was dependent on
TGF-bRI signaling. Similar results were found in HL-60
and TF-1 cells, suggesting that crosstalk between the
Smad and the ERK pathways operate in hematopoietic
cells and can modulate cell differentiation. Yang et al.
(2003) showed that inhibition of ERK in mouse embryo
fibroblasts potentiates Smad signaling and activation of
Smad-dependent gene targets. In a recent study, ERK
inhibition increased both the basal and TGF-b-induced
Smad7 promoter activity in rat fibroblasts (Uchida
et al., 1993). Based on the findings of our group and
others, this unexplained effect might be related to the
fact that inhibition of ERK augmented Smad2/3
signaling, which by itself led to increased Smad7
transcripts as part of the negative feedback loop of
TGF-b/Smad signaling.
Regardless of where in the TGF-b signaling pathway
they act, OA, ERK inhibitors and chemical inducers all
act to change the intracellular signaling balance in favor
of differentiation. The biological effect of HU and
butyrate on erythrodifferentiation, growth inhibition,
cell cycle arrest at G1 phase and stimulation of fetal
hemoglobin synthesis shares some similarity with those
of TGF-b supporting the idea that these agents may
mediate their effects through common signal transduction pathways. Collectively, our data suggest that
simultaneous activation of Smad2/3 and p38 is required
during EPO-independent erythroid differentiation.
Oncogene
Autocrine TGF-b1 regulation of hematopoiesis
FW Ruscetti et al
5760
Levels of Smad7 regulate the signaling of Smad2/3 and
MAPKs and control erythroid and megakaryocytic
differentiation of erythroleukemia cells
During the last decade, mechanisms underlying the
complexity of the multiple TGF-b responses and their
dependence on the type of target cells and environment
began to be more apparent. Regulation of TGF-b/Smad
signaling by inhibitory Smads (I-Smads) and the crosstalk with other signaling pathways undoubtedly explained in part the pleiotropic effects of the TGF-b
family (de Caestecker et al., 1998; Kretzschmar et al.,
1999). Recently, it was shown that smad 7 can direct cell
fate decisions in hematopoietic cells (Chadwick et al.,
2003). I-Smads (Smad7 and Smad6) directly regulate
receptor-activated Smads. Increased expression of
I-Smads inhibits TGF-b, activin and BMP signaling
(Nakao et al., 1997; Afrakhte et al., 1998). Modulation
of Smad and MAPK signaling pathways and cell
responses by Smad7 were investigated in erythroleukemia cell lines. In one previous study by Kitamura et al.
(2000), it was shown that Smad7 is specifically absent in
erythroleukemia cells that differentiate in response to
activin. Herein, RT–PCR results revealed that Smad7
transcripts are present in all tested cell lines, albeit at low
levels. Disruption of endogenous Smad7 expression in
these cells by siRNA significantly improved cell
differentiation to physiological doses of TGF-b and
activin. This indicates that although Smad7 is expressed
at low levels in erythroleukemia cells, it still can act as a
physiological inhibitor for TGF-b/activin-induced erythrodifferentiation. However, in the presence of high
ligand stimulation, activation of R-Smads escaped the
inhibition by endogenous Smad7. The cellular response,
Smad signaling and expression of physiologically
relevant endogenous Smad7 emphasize that TGF-b/
activin signaling is intact in erythroleukemia cells. To
examine whether the balance between TbRI/Smad
activation and levels of Smad7 regulates cell responses
to TGF-b/activin, K562 cells were stably transfected
with plasmid containing Smad7. Cells overexpressing
Smad7 became resistant to TGF-b/activin-induced
Smad2/3 phosphorylation, erythrodifferentiation and
growth inhibition. The inhibitory activity of Smad7
might result from a direct association with ligandactivated type I TGF-b receptors that interfere with
receptor phosphorylation of Smad2/3. However, it is
still possible that Smad7 can form a complex with
receptor-activated Smads and compete with Smad4
for oligomer formation and thus prevent movement of
R-Smads into the nucleus as has been observed with
Smad6 (Massague, 1998, 2000).
The regulation of erythroid and megakaryocytic
differentiation by possible crosstalk between the Smad
and MAPK pathways was studied (Rouyez et al., 1997;
Herrera et al., 1998). The overexpression of Smad7
blocked the activation of different MAPKs. It is likely
that Smad7 may modulate certain cell responses by its
ability to block a specific signaling pathway. One of
these pathways is the p38 MAPK, which is known to
mediate different actions of TGF-b and activin on cell
Oncogene
growth, differentiation and apoptosis. Hemoglobinzation of K562 cells by TGF-b/activin was prevented using
either an inhibitor of p38 (SB203580) or an inhibitor of
TbRI (SB505124) (see last section). The profound
inhibitory effect of Smad7 on the activation of Smad2/
3 and p38 provides further evidence about the involvement of these two signaling cascades in TGF-b/activininduced erythroid differentiation. The regulatory effect
of Smad7 on p38 and erythroid differentiation is not
limited to TGF-b/activin stimulation. Induction of p38
activation and erythrodifferentiation using HU and
butyrate occurred in control K562 cells but not in
Smad7-transfected K562 cells. Collectively, these data
suggest that Smad7 may modulate the kinase activity of
p38 irrespective of the stimulus that triggers this p38
activation. However, it is not fully understood how
Smad7 inhibited p38 activation and other MAPK
proteins. Limited recent reports showed a link between
Smad7 and MAPK proteins. Ectopically expressed
Smad7 enhanced the coimmunoprecipitation of HAMKK3 and flag-tagged p38, suggesting possible direct
interaction between Smad7 and p38. Moreover, Smad7
has been shown to modulate apoptosis of certain cells
independent of TGF-b signaling but mediated by the
JNK pathway (Mazars et al., 2001).
Next, the impact of Smad7 expression on megakaryocytic differentiation was studied. K562/7 cells did not
have any basal activity of Smad2 and exhibited an
increase in cell size and nuclear lobulation compared to
control cells. Moreover, megakaryocytes generated from
K562/7 cells by TPO and PMA underwent enhanced
endomitosis and exhibited more nuclear lobulation than
control cells. This may be explained by the ability of
Smad7 to counteract the inhibitory signals induced by
autocrine TGF-b and other cytokines. The interference
of Smad7 with R-Smad activation reversed TGF-binduced growth arrest and enhanced megakaryocyte
polyploidy, suggesting that Smad signaling mediates
TGF-b-induced inhibition of megakaryocyte proliferation and terminal differentiation. Thus, modulation of
Smad7 expression could provide a basis for abrogating
the inhibitory effects of TGF-b on megakaryopoiesis.
Since activation of ERK MAPK mediates megakaryocytic differentiation of transformed cell lines and
enhances endomitosis in cultures of primary cells
(Rojnuckarin et al., 1995), we looked at whether ERK
mediates effects of Smad7 on terminal megakaryopoiesis. Expression and activation of ERK was not
enhanced in relation with high Smad7 levels; moreover,
inhibition of ERK signaling did not reduce polyploidization of K562/7 cells. Our results favor the idea that
Smad7 regulates megakaryopoieis independent of ERK.
Using the K562/7 model, we are currently investigating
the molecular basis of the Smad7 effect on megakaryocyte endomitosis (Kuter et al., 1992; Kalina et al.,
2001).
Acknowledgements
This project has been funded in whole or in part with Federal
funds from the National Cancer Institute, National Institutes
of Health, under Contract No. NO1-CO-56000.
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FW Ruscetti et al
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