|
|
 Previous Article | Table of Contents | Next Article 
Blood, 15 September 2000, Vol. 96, No. 6, pp. 2022-2036
REVIEW ARTICLE
Transforming growth factor- : pleiotropic role in the
regulation of hematopoiesis
Nicolas O. Fortunel,
Antoinette Hatzfeld, and
Jacques A. Hatzfeld
From the Laboratoire de Biologie des Cellules Souches
Somatiques Humaines, Centre National de la Recherche Scientifique,
Villejuif, France.
 |
Abstract |
Hematopoiesis is a remarkable cell-renewal process that leads to
the continuous generation of large numbers of multiple mature cell
types, starting from a relatively small stem cell compartment. A highly
complex but efficient regulatory network is necessary to tightly
control this production and to maintain the hematopoietic tissue in
homeostasis. During the last 3 decades, constantly growing numbers of
molecules involved in this regulation have been identified. They
include soluble cytokines and growth factors, cell-cell interaction molecules, and extracellular matrix components, which provide a
multifunctional scaffolding specific for each tissue. The cloning of
numerous growth factors and their mass production have led to their
possible use for both fundamental research and clinical application.
(Blood. 2000;96:2022-2036)
© 2000 by The American Society of Hematology.
| |
Introduction |
The regulation of hematopoiesis is a
complex process that has received much attention (for reviews see Moore
et al,1 1990; Metcalf,2 1993;
Ogawa,3 1993). Research continues to identify the various
components involved in this regulation (for review see Levesque et
al,4 1991). Many of the growth factors can now be cloned
for research and clinical purposes (for review see Simmons and
Haylock,5 1995).
Type  transforming growth factors (TGF-s) were discovered by De
Larco and Todaro6 in 1978. Originally called "sarcoma growth factors," they were first isolated from the supernatant fluids
of Moloney MuSV-transformed mouse 3T3 fibroblasts and described as a
family of growth-stimulating polypeptides. The further nomenclature "transforming growth factor" was adopted because of the ability of
these molecules to confer on untransformed indicator fibroblasts functional properties associated with neoplastic
transformation.6,7
At present, TGF-s are considered pleiotropic factors because
they have been shown to play a regulatory role in most processes linked
to the control of somatic tissue development and renewal. As pointed
out by Sporn and Roberts,8 TGF-s may be considered as
"prototypic multifunctional signaling molecules." Indeed, these factors can exert either a positive or a negative effect on
proliferation, differentiation, or cell death, depending on the
developmental stage of the target cell, its in vivo environment, or the
medium used for in vitro studies. As will be described in this review, this is particularly true in the hematopoietic system, where TGF-s play a pivotal role.
| |
Structure of TGF-s |
Latent and active forms
Three highly similar isoforms of TGF-, called TGF-1, -2,
and -3, were identified and cloned from mammals between 1985 and
1988.9-11 Although the regulatory role of these 3 isoforms may differ, it has been established that all 3 are involved in the
regulation of hematopoiesis. Two other isoforms, called TGF-4 and
-5, have been cloned, respectively, in the chicken12 and in xenopus.13 More recently, an mRNA encoding a new member
of the TGF- family, called endometrial bleeding associated factor (ebaf), has been identified in
mammals.14
TGF-s are synthesized as precursor proteins, which are
biologically inactive. They consist of pre-pro-peptides, which require a 2-step process to give rise to active TGF-s15 (for
reviews see Lawrence,16 1991; Gleizes et
al,17 1997). A first proteolytic cleavage leads to the
elimination of a hydrophobic signal peptide, in the N-terminal region
of the precursor protein, yielding pro-TGF-. A second cleavage
leads to the separation of the pro-region of the protein from the
TGF- mature peptide (Figure 1). In the
case of TGF-1, the entire precursor (pre-pro-peptide) is a
390-amino acid chain. The signal peptide corresponds to amino acids 1 to 29, the pro-region of the precursor to amino acids 30 to 278, and
the mature peptide to amino acids 279 to 390.15 The
bioactive forms of TGF-s (25 kd) are composed of 2 mature peptide
chains linked by disulfide bonds. TGF-s are usually produced as
homodimers (TGF-1.1, -2.2, -3.3), but natural heterodimeric
molecules have also been identified (TGF-1.2 and
-2.3).18
View larger version (32K):
[in this window]
[in a new window]
| Figure 1.
TGF-: structure, latency, activation, and
receptors.17,67
LAP indicates latency-associated peptide; LTBP, latent TGF- binding
protein; M6P/IGFII-R, mannose-6-phosphate/type II insulin-like growth
factor receptor; PLg, plasminogen; PL, plasmin; Smad, TGF- signal
transduction proteins; Anti-Smad, antagonistic Smad; Co-Smad,
common-partner Smad; R-Smad, receptor-regulated Smad; TGase,
transglutaminase; TR-I, -II, -III, TGF- receptor type I, II, III;
TSP, thrombospondin; uPA, urokinase plasminogen activator; and uPA-R,
uPA receptor.
|
|
Once synthesized and processed, TGF-s are released by cells as
latent complexes, which are biologically inactive. Two forms of latent
complexes have been described, the "small" and "large" latent
complexes, as shown in Figure 1. In the small latent complex, one
molecule of mature, active TGF- is noncovalently associated with one
disulfide-bonded pro-peptide dimer, called latency-associated protein
or LAP (74 kd in the case of TGF-1). In the large latent complex,
LAP is linked by disulfide bonds to one member of a family of
high-molecular-weight proteins (125-160 kd), called latent TGF--binding proteins or LTBPs.19,20 The cDNAs of
various related LTBPs have been cloned.21-23 In the
erythroleukemic cell line HEL, the synthesis of LTBPs has been found to
be coordinated with that of TGF- small latent complex to form the
large latent complex, which is then secreted by cells.24
The LTBPs confer to this complex the ability to associate with the
extracellular matrix, permitting the storage of TGF- (for reviews
see Munger et al,25 1997; Taipale and
Keski-Oja,26 1997). Because LTBPs exist in several
isoforms, the bioavailability of TGF- and its specific targeting to
different organs may be regulated in part by the formation of different
types of large latent complexes. It has also been suggested that LTBPs
participate in bone formation as structural matrix
proteins.27 The release of latent TGF- from the
extracellular matrix is triggered by proteolytic enzymes such as
chymase, elastase, and plasmin, which are able to cleave LTBPs.28-30
Activation of latent TGF-s
Extracellular activation of the TGF- latent complexes is
a critical process in the regulation of TGF- functions in vivo. The
interaction between TGF- and LAP is not covalent and can be
disrupted in vitro by heat treatment or acidification.31 Although physicochemical variables such as local
acidification32 or exposure to active oxygen
species33 may participate in the regulation of TGF-
activation, mechanisms involving proteolytic cleavage or conformational
modification of LAP are more likely to operate in vivo.
Different mechanisms of activation are presented in Figure 1. Plasmin
has been shown to promote the activation of latent TGF- by
proteolytic nicking within the N-terminal region of the
LAP.34,35 This disrupts noncovalent bonds and results in
the release of active TGF-.35 In monocytes,
macrophages, and endothelial cells, cellular activation of latent
TGF- has been reported to involve the mannose-6-phosphate/type II
insulinlike growth factor receptor (M6P/IGFII-R) and the urokinase
plasminogen activator receptor (uPA-R).36-38 One proposed
mechanism is that M6P/IGFII-R, which binds latent TGF-, complexes
with uPA-R. Plasmin would be generated locally from plasminogen through
the action of uPA and would allow the production of active TGF-.
Another enzyme, transglutaminase, has been identified as an effector
controlling both the deposition rate of LTBPs in the
matrix39 and the cell-surface activation of latent
TGF-.37,40 Transglutaminase-mediated activation of
latent TGF- depends on interactions with specific residues of
LTBP.41 Thrombospondin (TSP), a platelet  -granule and
extracellular matrix protein, has also been shown to promote activation
of latent forms of TGF-. In contrast to what has been described for
plasmin and transglutaminase, TSP-mediated activation of latent TGF- occurs through a cell- and protease-independent mechanism, as demonstrated by in vitro studies. This effector induces a
conformational change of LAP, which then results in the release of
active TGF-.42,43 The role of TSP in the activation of
latent TGF- in vivo has been demonstrated by the generation of
TSP-null mice.44 In this model, major
histologic abnormalities have been observed and correlated with a lack
of active TGF-. These defects could be reversed by a treatment that
activates TGF-. Regulation of the glycosylation of LAP has also been
proposed to participate in the control of TGF-
latency.45 Recently, activation of latent TGF- via an interaction with the integrin 5 6 has been reported, providing a
novel possible mechanism regulating the function of
TGF-.46
| |
TGF- signal transduction pathway |
Two families of serine/threonine kinase receptors form
heteromeric complexes
Among the several transmembrane or membrane-bound proteins
known to interact with TGF-s, the type I and type II TGF-
receptors (TR-I or ALK, and TR-II) are directly involved in
signal transduction. TR-I and TR-II represent 2 families of
transmembrane serine/threonine kinase receptors of 53 to 65 kd47-49 and 80 to 95 kd,50 respectively, that
interact and form heterotetrameric complexes. The mechanism by which
signaling by these 2 receptors occurs is now well
established.51 TGF- first binds to TR-II, which is a
constitutively active kinase. TR-I is then recognized and recruited
into the TGF-/TR-II complex and phosphorylated by TR-II.
Phosphorylation allows TR-I to propagate the signal to downstream
intracellular substrates.
Because TR-I and TR-II exist in multiple forms, it has been
proposed that homodimeric and heterodimeric forms of TGF- may induce
a specific response by interacting with different heterotetrameric receptor complexes of specific signaling capacities.52
Accessory receptors
In addition to TR-I and TR-II, accessory TGF- receptors,
not necessarily required for signal transduction, can be expressed at
the surface of cells responsive to TGF- (for review see Piek et
al,52 1999). The type III TGF- receptor (TR-III or
glycan), a 300- to 400-kd membrane-anchored
proteoglycan,53,54 and the 180-kd glycoprotein endoglin
could function as regulators of ligand access to the signaling
receptors. Although the precise roles of endoglin and glycan are not
fully understood, some of their properties suggest distinct functions
for these 2 TGF- receptors. First, glycan is able to interact
with TGF-1, -2, and -3,55 whereas endoglin
interacts with TGF-1 and -3 but not efficiently with
TGF-2.56 Second, the role of glycan could be to
present TGF-s to TR-II and facilitate their
binding,57,58 whereas endoglin appears to diminish rather
than enhance TGF- responses in certain cell types.59
Third, endoglin and glycan possess a specific cell-distribution
pattern, which may confer the ability of different cell types to
respond differentially to TGF-1, -2, and -3. For example,
endoglin is coexpressed with TR-I and TR-II on vascular
endothelial cells60,61 and on hematopoietic cells including
macrophages,62 erythroid cell subsets,63 and
B-cell precursors,64 whereas these cells express little or
no glycan. Marrow stromal cells may express both endoglin and
glycan,65 whereas none of these TGF- receptors
appear to be present on the cell surface of hematopoietic
progenitors including early colony-forming units
(CFU)-granulocyte/erythrocyte/monocyte/megakaryocyte (GEMM),
CFU-granulocyte/monocyte (GM), and burst-forming units-erythrocyte (BFU-E).63
Other cell-surface receptors have been identified for their ability to
bind TGF- and are classified as TR-IV to TR-VI (for review see
Massagué,66 1992). However, the function of these other receptor families in TGF- signaling remains to be clarified.
The Smad intracellular proteins
The intracellular TGF- signaling pathway involves the Smad
protein family as substrates for the signaling receptors (for review
see Massagué,67 1998; Piek et al,52
1999). This network involves the cooperation among 3 subclasses of Smad
proteins, which can be distinguished by distinct functions in TGF-
signal transduction. Briefly, a first group of Smads called
"receptor-activated"67 or
"receptor-regulated"52 Smads (R-Smads) are directly
phosphorylated by activated TR-I. Upon phosphorylation, R-Smads
interact with members of a second subclass of Smads called
"common-partner Smads" or Co-Smads, with which they form
heterodimeric complexes. R-Smad/Co-Smad complexes are translocated to
the nucleus, where they associate with DNA-binding partners and then
regulate the transcriptional response of the target genes. A third
subclass of Smads called "antagonistic"67 or
"inhibitory"52 Smads (Anti-Smads) prevents the
interaction between R-Smads and Co-Smads and participates in negative
feedback to repress TGF- responses. In the case of signal
transduction by TGF- in mammalian cells, R-Smads include Smad2 and
Smad3,68,69 Co-Smads include Smad4,69,70 and
Anti-Smads include Smad6 and Smad7.71-73 Smad5, otherwise
described as an R-Smad and involved in signal transduction by bone
morphogenetic proteins (BMPs),74 has been demonstrated to
mediate the inhibitory effect of TGF- on human hematopoietic
stem/progenitor cells.75 The specificity of the cellular
response depends on interactions between these different
possible partners.
| |
Studies of human malignancies involving TGF- reveal its
essential role in the control of hematopoiesis |
Studies of hematopoietic pathologies involving TGF- have
provided important evidence of its key role in the regulation of human
hematopoietic stem/progenitor cell quiescence, proliferation, and
differentiation. These human pathologies are often more informative than knockout mice as regards the role of this pleiotropic factor. Indeed, knockout mice often exhibit secondary disorders subsequent to
the accumulation of early defects during embryonic development. This
situation is also often observed in the case of genetic diseases, in
which more than one function or organ can be affected during development by a single mutation. In contrast, the pathologic situation
resulting from a single gene mutation occurring in the adult will
produce a clonal defect, allowing the precise evaluation of the role of
the mutated gene in a specific function. This is particularly true in
the case of various cancers.
Inactivation of the TGF- signaling cascade leads to malignant
transformation of early human hematopoietic cells
The inactivation of one of the various genes involved in the
TGF- signal transduction pathway may represent a possible mechanism by which some early hematopoietic progenitors, which are normally quiescent, escape from cell-cycling inhibition. Abnormalities in the
expression of TGF- receptors have been described in proliferative syndromes including both early myeloid76,77 and lymphocytic leukemia.78,79 In these pathologies, a selective advantage is given to the tumor cells by the loss of TR-I or TR-II
expression and by the fact that these cells continue to produce
TGF-1 to inhibit normal cell proliferation. Active TGF- present
in the bone marrow microenvironment and autocrine/paracrine TGF-1
secreted by normal and leukemic hematopoietic cells are able to exert a negative control on the growth of normal progenitors, but not on
leukemic cells, which have overcome TGF- regulatory signals. A loss
of sensitivity to the growth-inhibitory effect of TGF- due to an
inactivation of TR-II has also been described in the case of human
cutaneous T-cell lymphoma cells.80-82
A mutational analysis of the gene coding for the TGF-
signal transducer Smad2 has been performed on 50 primary lymphoid and myeloid leukemia cells, but no genetic defects were found in this gene.83 However, a larger panel of hematologic disorders
should be analyzed before excluding the possibility of various
mutations in Smad genes in some of these pathologies.
Blocking of TGF- signaling by repression of Smad3 activity has been
reported in chronic myeloid leukemia. In these cases, the dysregulation
was not due to mutations in the Smad3 gene, but was
correlated with an abnormal expression of Evi-1, a zinc-finger
oncoprotein that interacts with Smad3 and suppresses its
transcriptional activity.84 In hematopoietic cells, Evi-1
expression is normally restricted to a transient stage of myeloid
differentiation.85 Its constitutive expression can result
from chromosomal rearrangements and may contribute to leukemogenesis by
specifically blocking the growth-inhibitory signaling of
TGF-.86
Pathologic overproduction of TGF- induces bone marrow fibrosis
and decreases stem/progenitor frequency
In physiologic conditions, the amount of TGF- produced by bone
marrow stromal cells and hematopoietic cells should be adequate to
maintain homeostasis of the stem/progenitor cell compartment. In
pathologic situations, excessive production of TGF- by stromal cells
has been correlated with a failure of early hematopoietic progenitors
in the marrow. This situation has been described in the case of human
chronic idiopathic neutropenia, in which a drastic reduction in
CD34+ progenitor cell frequency is observed.87
The same phenomenon has been described for B-cell lymphocytic leukemia.
In this case, the pathogenesis is due to the proliferation of leukemic
cells, but also to an increased inhibition of normal progenitor cell growth, in response to excessive amounts of TGF-1 secreted by bone
marrow stromal cells.88
An abnormally elevated production of TGF- has also been shown to
contribute to the pathogenesis of leukemia by promoting the progression
of bone marrow fibrosis (for review see Le Bousse-Kerdiles and
Martyre,89 1999). In these pathologies, TGF- is often
secreted in excess by leukemic cells, monocytes, and megakaryocytes,
which results in stimulation of collagen synthesis in bone marrow
fibroblasts and deposition in the marrow.90-93 This
observation is in agreement with the fact that TGF- is able to
activate the promoter of the human type VII collagen gene through the
action of Smad3 and Smad4.94
Vascular pathologies involving TGF-
Defects in genes coding for a TGF- receptor have also been
correlated with the genetic diseases called hereditary hemorrhagic telangiectasia (HHT). These syndromes concern mainly endothelial cells
and are characterized by arteriovenous malformations and recurrent
hemorrhage. HHT syndromes were first found to be caused by mutations in
the endoglin gene95 but have also been correlated with
mutations in the TR-I
gene,96 suggesting that endoglin and TR-I act through a
common pathway to control blood vessel development and repair.
TGF- has also been reported to be involved in another vascular
disease, atherosclerosis, which has a multifactorial pathology implicating many other interacting phenomena. Atherosclerosis has been
associated with the presence of lipoprotein Lp(a), a glycoprotein that
has a structure similar to that of plasminogen. Lp(a) binds to the
membranes of endothelial cells and monocytes and thereby inhibits
plasminogen binding and the subsequent generation of plasmin by these
cells. Because plasmin is a potent activator of latent TGF- in vivo,
this results in an insufficient rate of activation of TGF- and, as a
consequence, in the migration and proliferation of smooth muscle cells
in the arterial intima (for review see Angles-Cano,97 1997).
TGF- and the control of hematopoietic stem/progenitor cell
proliferation: a model for other somatic cells?
We have reviewed above various studies showing that the
inactivation of the TGF- signaling cascade participates in malignant transformation of early hematopoietic cells, which then escape from
negative cell-cycle controls. It is interesting to note that the same
phenomenon has been described in cancers affecting various types of
nonhematopoietic somatic cells, which suggests that TGF- may act as
a cell-cycle inhibitor in several nonhematopoietic somatic tissues in
vivo. Indeed, although they probably do not represent the entire cause
of the pathology, mutations or genetic defects resulting in a lack of
TR-I or TR-II function are associated with the acquisition of a
transformed phenotype in several types of murine and human cancers,
including colon cancers,98,99 gastric
cancers,100,101 prostate cancers,102
pancreatic cancers,103 thyroid tumors,104
hepatic tumors,105,106 retinoblastoma,107,108 and lung adenocarcinoma.109 Moreover, the importance of
TGF- signaling for the control of normal somatic cell proliferation has been demonstrated in skin keratinocytes,110,111 cells
of the mammary gland, lung,112 and exocrine
pancreas113 with the use of transgenic mice expressing a
dominant-negative mutant TR-II (for review see Letterio and
Bottinger,114 1998).
In addition, elements of the TGF- signal transduction pathway
downstream to the TGF- receptors have been identified as potential targets for oncogenic transformation. Indeed, mutations or somatic alterations resulting in a disruption of the Smad signaling cascade have been observed in several tumor cells resistant to the
growth-inhibitory effect of TGF-1 (for review see Hata et
al,115 1998). Briefly, in these cells, insensitivity to
TGF-1 was reported to be due either to an inactivation of the
TGF- signal transducers Smad2 and Smad4116-118 or to an
enhanced expression of the TGF-1 signaling inhibitor
Smad6.119 These observations should promote a search for
Smad gene mutations in the hematopoietic system, especially in leukemia.
| |
TGF- and the regulation of murine hematopoiesis in vivo |
The role of TGF- in the regulation of hematopoiesis has also
been analyzed in vivo using different murine models (for review see
Bottinger et al,120 1997). However, the involvement of
this pleiotropic factor in the regulation of various nonhematopoietic tissue functions renders these studies sometimes difficult to interpret
because of possible indirect regulatory effects.
TGF- protects stem/progenitor cells from agents that selectively
kill cycling cells
Hematopoietic stem/progenitor cells are able to reconstitute the
pool of mature blood cells in the case of severe hematopoietic failure.
This implies that these cells can rapidly pass from a quiescent or slow
cycling state to active proliferation. This phenomenon can be observed
in mice treated with chemotherapeutic drugs, such as 5-fluorouracil
(5-FU), that selectively kill the cycling cells. Following the
administration of 5-FU, a dramatic decrease in hematopoietic progenitor
cell frequency in the bone marrow is observed, as only the most
primitive and quiescent cells remain unaffected by this treatment. A
few days later, the quiescent stem/progenitor cells enter a
hyperproliferative state, promoting hematopoietic reconstitution. This
experimental system has been used to evaluate the ability of TGF- to
affect hematopoietic stem/progenitor cell cycling. TGF-1 has been
shown to delay hematologic recovery after a sublethal injection of
5-FU.121 Moreover, both the TGF-1 and -2 isoforms
were able to protect hematopoietic stem/progenitor cells from a
treatment by a high dose of 5-FU,122 which demonstrates
the ability of these molecules to exert a negative control on the cell
cycle of primitive murine hematopoietic cells in vivo. It is important
to note that this effect was reversible,121,122 which
suggests that TGF- is not an inducer of cell death for primitive
stem/progenitor cells in vivo. This point will be discussed more
extensively later.
In vivo administration of TGF- in mice modulates hematopoietic
development in a lineage-specific manner
In vivo administration of TGF- in mice has also been performed
to study its specific effects on early and late progenitors and on the
different hematopoietic lineages. One approach, which was to test in
vitro the clonogenic capacity of hematopoietic progenitors after the
local administration of TGF-1 into the femur of mice, revealed a
preferential growth-inhibitory effect of this factor on the earlier
progenitors.123 A second approach was to perform
histologic analyses of hematopoietic tissues from mice treated with
TGF-1. Such studies revealed an inhibition of erythropoiesis and
thrombopoiesis in TGF-1-treated mice,124,125 whereas
granulopoiesis was stimulated.124-126 Although some of
these effects may be directly mediated, the possibility that
administration of exogenous TGF- in vivo may deregulate the
production of other factors involved in the control of hematopoiesis
should be taken into account. For example, an increased production of
tumor necrosis factor (TNF)- has been observed after the
administration of TGF- in mice.127
Hematopoiesis in knockout mice
An opposing strategy has been to study in vivo the development of
the hematopoietic tissue in the absence of endogenous TGF-, or in a
context in which the cell responsiveness to TGF- is abrogated. For
this purpose, a variety of knockout mice have been generated, in which
a targeted disruption of genes encoding a TGF- isoform or another
element of the TGF- signaling cascade has been performed. Homozygous
TGF-1 knockout mice have a
50% intrauterine death rate because of severe developmental
retardation. Defective hematopoiesis, resulting in a reduced number of
erythroid cells128 as well as a lack of Langerhans
dendritic cells,129 has been correlated with the absence
of TGF-1. However, TGF-1
knockout mice also present defects in liver development130
and bone formation,131 as well as many other
dysregulations including autoimmune manifestations.132,133 The phenotype of TR-II
knockout mice has been reported to be indistinguishable from that of
TGF-1 knockout
mice.134 Mice lacking endoglin show defective
angiogenesis,135 providing a good animal model of
HHT.136 TGF-3
knockout mice show abnormal lung and craniofacial development due to
altered epidermal-mesenchymal interactions,137 but these
mice do not provide clear information concerning the role of this gene
in the development of hematopoietic tissue. Knockout mice for the
TGF-2 gene exhibit a wide
range of developmental defects that do not overlap with those of the other TGF- knockout
phenotypes.138 Concerning the Smad genes, Smad3 knockout mice demonstrate defects in immune
function,139 whereas Smad4 and Smad5
knockout mice have multiple embryonic and extraembryonic
defects.140,141
| |
Control of human and murine stem/progenitor cell proliferation by
TGF-: in vitro studies |
Human and murine hematopoietic stem/progenitor cells are
usually in a quiescent or slow cycling state in
adults.142,143 As suggested by in vivo studies, TGF- is
a good candidate for controlling this quiescence. This possible
function of TGF- has been studied extensively in vitro using clonal
semisolid colony-forming assays, stroma-supported culture systems, and
single-cell liquid cultures in both the murine and human
hematopoietic systems.
TGF- exerts a preferential growth-inhibitory effect on the most
primitive stem/progenitor cells
A first approach has been to study the effects of exogenous
TGF- added to clonal cultures of hematopoietic progenitors. The first TGF- isoform, TGF-1, has been shown to inhibit colony formation by early murine144-146 and human hematopoietic
progenitors in semisolid media,147-149 but not that of
late progenitors.145,147-149
In these studies, the effects of TGF- on colony formation have often
been tested in combination with only 1 or 2 other exogenous growth
factors, added to serum-containing or conditioned culture media that
contain undefined combinations of other growth factors. The cytokines
used were mainly interleukin (IL)-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), and/or erythropoietin (Epo). In such culture
conditions, TGF-1 was found to efficiently inhibit colony formation
by early human multipotential progenitors (CFU-Mix) at concentrations
between 10 and 100 pg/mL, whereas later progenitors were less affected or were, in contrast, stimulated by these low TGF-1
concentrations.149
An alternative approach has been to use blocking antibodies or
antisense oligonucleotides to neutralize TGF- secreted by cells and
present in the culture media. A study performed at a clonal level in
semisolid or in single-cell liquid assays has revealed that the
quiescence of human stem/progenitor cells is controlled in part through
an autocrine loop involving TGF-1 with the retinoblastoma
susceptibility gene Rb as a downstream effector.150 In this study, blocking of autocrine TGF-1
was sufficient to release from quiescence primitive erythro-myeloid (CFU-Mix), myeloid (CFU-GM), and erythroid progenitors (BFU-E), which
then gave rise to macroscopic colonies, in the presence of IL-3, IL-6,
granulocyte colony-stimulating factor (G-CSF), and Epo. Using the same
antisense oligonucleotide, it was observed that autocrine TGF-1
inhibits colony formation by early murine and human hematopoietic
stem/progenitor cells stimulated with KIT ligand
(SF).151,152
Since the first studies on TGF-, many others have subsequently
addressed its effects on progenitor cell growth when present in
association with various other cytokines. For example, in a single-cell
liquid assay, it has been demonstrated that this factor directly
inhibits early human hematopoietic progenitor cell proliferation in the
presence of Epo, SF, GM-CSF, and IL-3.153 Moreover, in a
semisolid culture system containing a combination of 7 stimulatory growth factors (IL-3, IL-6, IL-11, Epo, SF, GM-CSF, and G-CSF), it has
been shown that whereas 10 to 30 pg/mL of TGF-1 is sufficient to
inhibit 90% of primitive high proliferative potential (HPP)-Mix, 100 to 300 pg/mL of TGF-1 is required to inhibit 70% of the bipotent HPP-GM and the HPP-BFU-E. Concentrations of up to 1000 to 3000 pg/mL
of TGF-1 had little or no effect on the development of late CFU-G
and CFU-M.154
Preferential growth inhibition of the most primitive human
hematopoietic cells has also been reported in studies in which the
effects of anti-TGF-1 antisense oligonucleotides, which neutralize autocrine TGF-1 production155 or exogenous
TGF-1,156 were investigated on
CD34+CD38 cells and on
CD34+CD38+ cells. It was indeed observed that
primitive CD34+CD38 cells show a high
sensitivity to cell-cycle inhibition by TGF-1, whereas more mature
CD34+CD38+ cells are poorly affected or are
even stimulated by TGF-1. Interestingly, the concentrations of
TGF-1 for which these inhibitory effects were observed on primitive
cells correspond to those detected in human plasma in an active form
(usually less than 300 pg/mL).157
The second isoform, TGF-2, has also been shown to inhibit early
progenitor cell proliferation in both the human148,149,158 and murine hematopoietic systems,144 but with a lower
efficiency.149,158 The third isoform, TGF-3, also
inhibits colony formation by early human hematopoietic
progenitors149 or slows their rate of
proliferation159 at least as efficiently as does TGF-1.
However, only TGF-1 and -2 were shown to exert bidirectional
effects on proliferation of early and late hematopoietic progenitors,
whereas the effects of TGF-3 were only inhibitory.149
TGF- in stroma-supported cultures of hematopoietic
cells
Stroma-supported cultures containing both hematopoietic
progenitors and nonhematopoietic accessory cells allow the
proliferation and differentiation of primitive stem/progenitor cells
for several weeks without the addition of exogenous factors (for
reviews see Dexter,160 1979; Eaves et al,161
1991). In these systems, progenitor cell development is regulated
through a complex interaction between positive and negative factors
that are secreted by both stromal and hematopoietic cells.
Studies performed on adherent bone marrow primary stromal cells or cell
lines have shown that these cells can produce a variety of growth
factors, including cytokines such as G-CSF; GM-CSF162,163; IL-1, IL-6, and IL-7162,164; SF and
M-CSF165; and thrombopoietin (TPO).166
They also produce chemokines such as the monocyte chemoattractant protein-1 and the interferon-inducible protein-10,165 as
well as amounts of active TGF- sufficient to control the
proliferation of hematopoietic progenitors.162,167 Two
aspects render the presence of TGF- in stroma-supported cultures
critical for the development of hematopoietic cells. First, TGF-
acts directly on these cells. Second, TGF- is able to modulate the
growth factor production by stromal cells,163-166 a
process that indirectly controls their development.
The term "long-term culture initiating cell" (LTC-IC) has been
assigned to a subpopulation of primitive human hematopoietic stem/progenitor cells that possess the potential to sustain continuous production of progenitors for at least 8 weeks in the presence of
stroma.161 Anti-TGF- added to stroma-supported
cultures of human hematopoietic stem/progenitor cells was able to
prolong or reactivate the proliferation of LTC-ICs,162
implicating TGF- as an endogenous inhibitor of primitive
hematopoietic cells. The proliferation of these cells was also
selectively inhibited when exogenous TGF- was added.168
Similar observations have been reported for murine hematopoietic
stem/progenitor cells cultured according to the Dexter method. Dexter-type culture systems consist of total marrow cultures in which
an expansion of primitive hematopoietic stem/progenitor cells is
maintained for several months because of the presence of a bone
marrow-derived adherent layer consisting of different types of
nonhematopoietic accessory cells including adipocytes.169 The addition of antibodies neutralizing the biologic activity of
TGF-1, -2, and -3 in such cultures resulted in a significant increase in early hematopoietic cells, demonstrating the ability of
these factors to inhibit their cell cycling.170
These stroma-supported in vitro culture systems have provided important
information concerning the role of TGF- in the control of the
cell-cycle status of primitive murine and human hematopoietic stem/progenitor cells. However, they do not permit discrimination between the effect of TGF- |
Top
Abstract
Introduction
s