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Endocrine Reviews 19(3): 349 –363
Copyright © 1998 by The Endocrine Society
Printed in U.S.A.
Molecular Mechanisms of Transforming
Growth Factor-b Signaling
PATRICK PEI-CHIH HU*, MICHAEL B. DATTO,
AND
XIAO-FAN WANG†
Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham,
North Carolina 27710
cellular growth for many cell types, including cells derived
from epithelial, endothelial, neuronal, hematopoietic, and
lymphoid origins. This ability of TGF-b to cause growth
inhibition is thought to play a critical role in its ability to
influence many aspects of cellular functions. In addition,
TGF-b-mediated growth inhibition may also play a more
global regulatory role in complex physiological processes
such as in the immune response and development (5). Other
effects of TGF-b include its ability to modulate wound healing, extracellular matrix deposition, cellular adhesion and
migration, and most recently, synaptic facilitation (1– 4, 6).
Deregulation of TGF-b signaling is implicated in the
pathogenesis of many diseases including arthritis, atherosclerosis, glomerulonephritis, human hereditary telangiectasia, and carcinogenesis. Loss of cellular sensitivity to TGFb-mediated growth inhibition may contribute directly to
these pathological states, specifically carcinogenesis.
With the demonstrated importance of TGF-b signaling in
a variety of biological processes, and loss of TGF-b responsiveness as an important correlate of certain diseases, a tremendous effort has been undertaken in the last decade to
elucidate the mechanisms by which TGF-b propagates its
signal. An important step in understanding TGF-b signaling
came with the identification of three cell surface proteins that
bind to TGF-b ligand with high affinity. These were called
type I, II, and III receptors based on their molecular weight.
The type I and type II receptors belong to a large family of
receptor serine/threonine kinases. Upon TGF-b ligand binding to type II receptor, type I receptor is recruited into a
complex containing both receptors and ligand. This causes
the phosphorylation and subsequent kinase activation of
type I receptor by the constitutively active type II receptor
kinase. Currently, activated type I receptor kinase is thought
to be sufficient to modulate most TGF-b downstream signals,
although it is possible that type II receptor may also contribute to downstream signaling. The type III receptor is not
essential for signal transduction, but may serve to present
TGF-b ligand to the type I and II receptors. In cells with lower
signaling receptor affinities for a particular TGF-b ligand, the
presence of a large amount of type III receptor on the cell
surface may serve to promote a productive TGF-b signal.
The purpose of this review is to focus on the current body
of knowledge of downstream signaling events resulting from
TGF-b treatment. Recently, relevant substrates and effectors
of the TGF-b receptor kinases have been identified at a rapid
pace and will be the subject of this review. The TGF-b ligands
and receptors responsible for propagating this signal will
I. Introduction
II. Ligands and Receptors
A. Ligands
B. Receptors
C. Receptor kinase signaling
III. Smads
A. Cloning of the Smads
B. Smad regulation
C. Smad nuclear function
D. Smad structure
E. Smads as negative regulators
F. Smad-deficient mice
G. Smads: an emerging model
IV. TGF-b and the Cell Cycle
A. TGF-b induction of the CKIs
B. TGF-b-mediated decrease in Cdc25A levels in the
breast epithelial cell line MCF10A
C. TGF-b-mediated cyclin-CDK inhibition may be common strategy to arrest cells in G1
V. TGF-b and Cancer
A. Receptor mutants
B. Smad mutants
C. Cell cycle mutants
D. Multilevel resistance to TGF-b is important in
multistep model of carcinogenesis
VI. Conclusions
I. Introduction
T
HE transforming growth factor-bs (TGF-bs)1 are a family of potent multifunctional cytokines that modulate a
wide variety of cellular activities (1– 4). Originally identified
as a factor that induced the growth of rat kidney fibroblasts
in soft agar, TGF-b was later shown to be an inhibitor of
Address reprint requests to: X.-F. Wang, Ph.D., Assistant Professor,
Department of Pharmacology and Cancer Biology, Duke University
Medical Center, Box 3813, Durham, North Carolina 27710. E-mail:
[email protected]
* Supported by a National Science Foundation Predoctoral Fellowship.
† Leukemia Society Scholar. Supported by NIH Grant DK-45746.
1
Abbreviations: TGF-b, Transforming growth factor-b; CDK, cyclindependent kinase; CKI, cyclin-dependent kinase inhibitor; BMP, bone
morphogenic protein, ARE, activin response element; ARF, activin response factor; PAI-1, plasminogen activator inhibitor; EGF, epidermal
growth factor; DPC-4, deleted in pancreatic cancer, locus 4; DAD, daughters against decapentaplegic; dpp, decapentaplegic; MAD, mothers
against decapentaplegic.
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HU ET AL.
only be reviewed briefly, as an in-depth review of the ligands
and receptors can be found elsewhere in the literature (1– 4).
We will focus our discussion on one family of TGF-b receptor
substrates, the Smads, and discuss the recent advances in our
understanding of Smad signaling (also reviewed in Refs.
7–10). Due to the importance of TGF-b-mediated growth
inhibition on a wide variety of cell types, we will discuss in
detail the molecular mechanisms by which TGF-b can potentiate growth inhibition. Finally, we will discuss recent
members of the TGF-b signaling pathway that have been
discovered to be mutated in human cancers, in particular
Smad-4. Throughout the discussion, we will attempt to address current questions and present challenges that await
researchers in the field.
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served residues found in serine-threonine kinases. A fusion
protein comprised of the cytoplasmic region linked to
glutathione-S-transferase was able to autophosphorylate its
serine residues, with some threonine phosphorylation.
The type I receptor is a 50- to 60-kDa protein composed of
501 amino acids. It contains a 101-amino acid hydrophilic
extracellular domain, a single 23-amino acid transmembrane
domain, and a 355-amino acid intracellular domain. Like the
type II receptor, the type I receptor contains conserved regions resembling serine-threonine kinases. The cytoplasmic
region fused to glutathione-S-transferase was able to autophosphorylate its threonine residues, with some serine phosphorylation. Unlike the type II receptor, the type I receptor
does not bind independently to ligand. The cloning and
identification of both mammalian type II and type I TGF-b
receptors are discussed in detail in other reviews (4, 10).
II. Ligands and Receptors
A. Ligands
C. Receptor kinase signaling
The TGF-b ligands are part of a large superfamily of peptide hormones that are important in many different biological processes. Members of the superfamily include bone
morphogenic proteins (BMPs), activin, decapentaplegic Vgrelated proteins (DVR), dorsalin, nodal, Müllerian-inhibiting
substance (MIS), inhibin, growth and differentiation factors
(GDF), and glial-cell derived neurotrophic factor (GDNF).
Five TGF-b ligands have been cloned (TGF-b1–5). These
ligands are secreted as 100-kDa inactive complexes. The inactive form consists of a dimer of the N-terminal peptide,
noncovalently associated with the 25-kDa dimer of the biologically active form. Structurally, the ligands exhibit a high
degree of amino acid identity (64 – 82%) with nine invariant
cysteine residues. The structures of TGF-b1 and b2 have been
solved (11–13). Eight of the nine cysteines make four intramolecular disulfide bonds, while the ninth cysteine (amino acid 77) forms an intermolecular disulfide bond with the
corresponding ninth cysteine of the other monomer. The
distinguishing feature of the TGF-b structure is the ’cysteine
knot, ’ formed from three of the four intramolecular disulfide
bonds that maintains structural integrity for the monomer.
Aside from the nine invariant cysteines, superfamily members share less than 40% amino acid identity. This may account for the diversity of responses elicited from different
superfamily members.
The type II receptor is a constitutive receptor kinase that
associates with type I receptor on binding to TGF-b. Upon
association, type I receptor kinase is phosphorylated by the
constitutively active type II receptor in a glycine-serine-rich
region known as the GS domain (24). Phosphorylation of the
GS domain subsequently activates the type I receptor kinase.
A constitutively active type I receptor has been created by
mutating residues adjacent to the site of phosphorylation.
This receptor kinase can mediate the TGF-b growth-inhibitory effect and induce certain TGF-b-responsive genes, suggesting that type I receptor is a bona fide mediator of TGF-b
signaling. At present it is not known whether type II has
physiological substrates other than type I receptor with
which it interacts.
Other TGF-b superfamily members also signal through a
type II and type I receptor kinase cascade. Previously, we and
others had proposed a nomenclature whereby type II and
type I receptors are grouped according to both the functional
and structural characteristics they exhibit rather than by size
alone, as the TGF-b receptors were first categorized (4, 7).
Structurally, type II receptors have longer extracellular domains and longer serine-threonine cytoplasmic tails than
type I receptors. In addition, each receptor type contains
certain conserved regions that the other does not possess.
One such region is the GS domain. We further proposed that
receptors containing GS domains be categorized as type I
receptors whereas receptors that either bind ligand independently or lack a GS domain be categorized as type II receptors.
B. Receptors
Like other members of its superfamily, TGF-b ligands
signal by binding to specific receptors on the cell surface. A
breakthrough in the field came with the identification (reviewed in Refs. 10, 14, and 15) and cloning of these receptors
(16 –22). Although four receptors have been cloned (type I, II,
III, endoglin), only two of them, the type II and type I receptors, have been conclusively proven to mediate TGF-b
signaling (23).
The type II receptor is a 75- to 85-kDa glycoprotein composed of 565 amino acids. It contains a signal sequence, a
136-residue hydrophilic extracellular domain, a single transmembrane domain, and a large intracellular domain of 376
amino acids. Its cytoplasmic region contains 18 of 21 con-
III. Smads
Recent advances in the identification of direct substrates
for TGF-b type I receptor include the discovery that Smads
may act as direct effectors for the TGF-b type I receptor. The
Smad family of proteins has been identified as mediators of
the TGF-b signal from the cytoplasm to the nucleus. Seven
Smad genes have been cloned. Of these, Smad-2 and Smad-3
mediate the TGF-b and activin signals, whereas Smad-1 and
Smad-5 mediate the BMP signal. Smad-4 (DPC-4) appears to
be a general partner for these Smads by bringing the cyto-
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plasmic Smads into the nucleus where they can potentially
regulate the transcription of target genes. A schematic is
shown in Fig. 1. In contrast, the most recently identified
members of the Smad family, Smad-6 and Smad-7, negatively regulate receptor action. In this section, we will give a
comprehensive review of the role of Smads in TGF-b signaling. First, we will describe the cloning of the Smads from
their homologs in Drosophila and Caenorhabditis elegans. Second, we will review findings that suggest a model for Smad
activation upon TGF-b treatment. Possible nuclear functions
for Smads as both a transcription factor and coactivator will
be presented. Third, we will focus on the structure and possible function of the different Smad domains. Questions that
need to be addressed to further elucidate the role of Smads
in TGF-b signal transduction will be discussed throughout
the section.
Other substrates of TGF-b receptor kinases that have been
reported in the literature include FKBP12, WD40, and farnesyl transferase. At present, it is still unclear what physiological role each of these interacting proteins plays in TGF-b
signaling. As such, these substrates will not be discussed in
this review.
A. Cloning of the Smads
Smad homologs were first identified using a genetic approach in Drosophila and C. elegans, two organisms in which
a TGF-b-like signaling pathway is present. In Drosophila,
signaling proteins exist that are homologous to their mammalian TGF-b superfamily counterparts. ’Decapentaplegic’
(dpp) is the TGF-b-like ligand (5). The receptor homologs are
’punt’ (type II), ’thick veins,’ and ’saxophone’ (both type I).
In a genetic screen to determine dominant enhancers of a
weak dpp allele, ’mothers against dpp’ (MAD) was isolated
(25). Loss-of-function mutations in MAD result in organisms
that phenotypically resemble those with null alleles of dpp,
and a constitutively active thick veins phenotype is repressed
by null alleles of MAD, suggesting that MAD is in the TGFb-like signaling pathway.
In C. elegans, the story is remarkably similar. The receptor
homologs are daf-4 (type II) and daf-1 (type I). A mutant
daf-4 gives rise to small worms and fused male tail rays.
FIG. 1. Diagram of TGF-b-like signaling in Drosophila, C. elegans, Xenopus,
mouse, and human. The signaling cascade is shown from ligand to Smad homolog. A dendrogram of the Smad homologs is also shown for comparison.
Smad-6 and Smad-7 act as inhibitors of
the TGF-b signal. Smad-6 acts to block
Smad-1 and Smad-2 activation. Smad-7
acts to block Smad-2 and Smad-3 activation (7, 9, 10).
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SMA-2, SMA-3, and SMA-4 were identified as genes that are
downstream of daf-4. Individual null sma alleles result in an
organism that phenotypically resembles that of the mutant
daf-4. The fact that other SMA genes cannot compensate for
inactivation of a single SMA gene suggests that each SMA
allele may play a functionally distinct role in the pathway.
These genes are homologs of MAD, further suggesting the
importance of these genes in TGF-b signaling.
With conservation of these proteins in both Drosophila and
C. elegans, research turned toward identifying homologs in
higher organisms. Using Drosophila MAD or C. elegans SMA
genes, libraries from organisms such as Xenopus, mice, and
humans were screened for homologous genes (26 –33). A
region on chromosome 18q that is homozygously deleted in
human pancreatic carcinomas was found to contain a gene
called Deleted in Pancreatic Cancer-4 (DPC-4), identified as
a MAD homolog (34). These homologs of the SMA and MAD
genes have subsequently been called Smads.
B. Smad regulation
Since the cloning of the Smads, a working model has
emerged as to how Smads are regulated. Upon direct phosphorylation by type I TGF-b receptor, Smad-2 or Smad-3
binds to its Smad-4 partner to form a heteromeric complex
and translocates into the nucleus. Once in the nucleus, the
Smads regulate the transcription of genes important in the
TGF-b response. One such gene may be the plasminogen
activator inhibitor-1 (PAI-1). This working model is the result
of several different lines of investigation that will be reviewed in this section.
Initial work by several groups showed that certain endogenous Smads are phosphorylated upon treatment with
TGF-b or BMP (28, 29, 35). Specifically, Smad-1 and Smad-5
are phosphorylated upon BMP treatment. Smad-2 and
Smad-3 are phosphorylated upon TGF-b or activin treatment
(Fig. 1). This transient phosphorylation is induced within 15
min and peaks at about 1 h before returning to basal levels
by 2 h. Based on this initial observation, work focused on
determining the kinase responsible for phosphorylating the
Smads.
The first clue on the possible candidate kinase that could
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phosphorylate Smads after TGF-b addition came from a
study in which Smad-3, but not Smad-4, was found to be
phosphorylated and associated with the ligand-bound receptor kinase, suggesting that Smad-3 may serve as a direct
substrate for the receptor kinase (31). Subsequently, overexpression of the type I receptor in COS cells led to phosphorylation of overexpressed Smad-2 (MADR2) in the presence of
type II receptor. Furthermore, upon mutation of three serine
residues in the C terminus of Smad-2, the phosphorylation
of Smad-2 by type I receptor kinase was inhibited. Using
two-dimensional tryptic peptide mapping, it was further
shown that these three serines are indeed phosphorylated by
type I receptor (32). The mutant Smad-2 could be coimmunoprecipitated with type I receptor, suggesting that Smad-2
is an in vivo substrate for the type I receptor kinase.
Phosphorylation of the C terminus of Smad-2 is necessary
for its nuclear accumulation (32), but the exact mechanism by
which nuclear localization occurs upon Smad-2 phosphorylation is still unclear. Phosphorylation may change the
conformation of Smad-2 or Smad-3 so that it can form a
heteromeric complex with Smad-4 and/or translocate to the
nucleus and thereby exert its nuclear function.
C. Smad nuclear function
1. Smads as coactivators. A breakthrough in our understanding
of Smad nuclear function came with the discovery that
Smad-2 was in a complex with the transcription factor forkhead activin-induced signal transducer (FAST-1) (36). Upon
activin, TGF-b, or Vg-1 treatment, a rapidly induced complex
forms on the activin response element (ARE) of the Mix.2
promoter. This novel transcription factor, FAST-1, is the major DNA-binding component of the activin response factor
(ARF). Smad-2 is found only in activin-induced ARF complexes, suggesting that Smad-2 plays a role as a mediator of
the activin signal. Overexpression of the MH-2 domain or
Smad-2 was sufficient to activate transcription of the Mix.2
promoter, whereas FAST-1 overexpression could not cause
an increase in transcription, suggesting an essential role for
Smad-2 in transcriptional activation of the Mix.2 promoter.
It is unclear as to how FAST-1 DNA binding ability is induced, although Smad-2 phosphorylation and subsequent
association with FAST-1 may be important in allowing DNA
binding to occur.
The same group further showed that Smad-4 is also contained in ARF complexes (37). They mapped the region of
interaction of FAST-1 with Smad-2/Smad-4 and used a yeast
two-hybrid system to show that FAST-1 directly interacts
with Smad-2 rather than Smad-4. Furthermore, they created
a putative dominant-negative FAST-1 containing 153 amino
acids of the C terminus (a.a. 366 –518) that interacts with the
Smads. Injection of the dominant negative fragment into the
animal pole of two-cell embryos inhibited the formation of
ARF and the mRNA expression of early mesodermal marker
brachyury. This suggests that the Smad complex may be
directly involved in ARF function.
2. Smads bind DNA directly. A new development to the story
came with the discovery that Smads have DNA binding
ability (38). The Drosophila MAD, homolog of Smad-1, can
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bind to a consensus promoter sequence GCCGnCGC in the
fly vg promoter. Our work in mammalian cells indicates that
Smad-4 can also bind DNA in a sequence-specific manner as
discussed below (39). Although the Smads have an intrinsic
DNA binding ability, it is unclear as to the exact contribution
of Smad DNA binding activity on transcriptional activation.
One function for DNA binding might be to cooperate with
other adjacent transcription factors to allow stabilization of
these factors with the transcriptional machinery complex,
thereby resulting in the activation of transcription. This degree of cooperativity may vary in different promoter contexts. For example, in a different Drosophila promoter, Ubx,
which also contains the same MAD/Smad-1 DNA binding
site, the element responsive to dpp activation was mapped
to an adjacent cAMP response/regulatory element (CRE).
Further investigation should clarify the role that Smad DNA
binding may play in transcriptional activation.
3. Smads can activate transcription. Smad phosphorylation and
translocation to the nucleus are thought to effect transcriptional regulation of TGF-b-responsive genes, one of which
may be the gene for PAI-1, a protein important in extracellular matrix deposition. Using a luciferase reporter gene under the transcriptional control of the PAI-1 promoter, it was
determined that: 1) reintroduction of Smad-4 could restore
TGF-b activation of PAI-1 in a Smad-4 mutant cell line
SW480.7 and 2) overexpression of Smad-3 with Smad-4 was
sufficient to activate the PAI-1 promoter in a ligand-independent manner (31). Those initial observations were subsequently substantiated with similar results from studies of
Smad-2 and Smad-4 coexpression in the activation of the
p3TP-lux reporter in another Smad-4 deficient MDA-MB-468
human breast cancer cell line (40, 41). The p3TP-lux reporter
is an artificial promoter consisting of a combination of AP-1
binding site-containing 12-O-tetradecanoylphorbol-13-acetate-responsive elements and a portion of the PAI-1 promoter
(39).
Thus, Smads may potentiate transcription by multiple
mechanisms: as a transcription factor capable of binding to
specific sequences and/or as a coactivator. This conclusion
is supported by the results of a recent study in which Smad3/Smad-4 overexpression was able to activate transcription
from a minimal reporter controlled by four sets of a 31-bp
sequence derived from the 3TP-lux promoter (39). Smad-4
was shown to bind directly to DNA on a site adjacent to the
AP-1 element. To dissect the individual contribution of Smad
DNA binding activity and AP-1 on transcriptional activation
of p3TP-lux by TGF-b, two sets of luciferase constructs were
constructed. The first set of constructs was a series of concatemerized minimal AP-1 elements from the p3TP-lux promoter. Smad-3/Smad-4 overexpression induced transcription driven by the concatemerized minimal AP-1 elements
20-fold but the construct was barely activated by TGF-b (39).
This suggests that although overexpression of Smads may
allow an increase in transactivation, the ability of TGF-b to
mobilize endogenous levels of Smads may not be sufficient
to potentiate transcription through a minimal AP-1 element
alone. Transcriptional activation may require the presence of
a Smad-4 DNA binding site to stabilize the interaction of
AP-1 complex with the core transcriptional apparatus. To
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MOLECULAR MECHANISMS OF TGF-b SIGNALING
address this question, a second set of constructs was generated to contain the wild-type AP-1-responsive element with
the adjacent Smad-4 DNA-binding sites mutated. Despite
mutating the Smad-4 DNA-binding sites, TGF-b treatment
and Smad-3/Smad-4 overexpression could still drive transcription with the same fold induction as in the wild-type
construct.
The unanticipated result from this study, therefore, was
the apparent dispensability of the Smad binding site
within the p3TP-Lux reporter (39). While the lack of correlation between the DNA binding capability of Smads
and their ability to activate transcription makes the interpretation of those findings somewhat difficult, the result
could be explained in several ways. Smad complex binding may cause effects that cannot be assayed in these
transient transfection experiments with the use of a large
quantity of plasmid DNA as the template of transcription.
For example, Smad binding may play a role in the recruitment of other transcription factors to adjacent sites (e.g.,
AP1) or in rearrangement of chromosome structure to
provide accessibility of other transcription factors to their
binding sites so that an effect in the transient transfection
assay may be difficult to observe. The transient nature of
Smad nuclear accumulation and DNA binding would be
consistent with this type of role in transcriptional activation. Alternatively, Smad binding sites may represent enhancer-like regulatory sequences that can function properly only in the context of specific promoters. The proper
promoter context may be essential to allow the appropriate
interactions between the Smads and the core transcription
machinery. Thus, in the context of the artificial p3TP-Lux
promoter constructs, Smad binding may not be required,
but in the context of wild-type promoters, Smad binding
may become indispensable for transcriptional activation.
In this regard, the demonstration in the same study that
the PAI-1 promoter contains a Smad-3/Smad-4 binding
site allows an opportunity to dissect in vivo functions of the
Smad-3/Smad-4 binding site and should provide insight
into these important questions.
4. Smad transcriptional ability effected by other extracellular signals. In a normal cellular context, Smad transcriptional activity may also be influenced by other extracellular signals
that possibly dictate its proclivity for driving TGF-b-mediated transcription. One such target of modulation might be
the ERK (extracellular stimulus responsive kinase/mitogenactivated protein kinase) PXSP phosphorylation motif found
in Smad-1 (42). When four of these consensus sites are mutated to an inactive alanine, the mutant protein remains in the
nucleus. When treated with epidermal growth factor (EGF),
which instigates a receptor-mediated kinase cascade resulting in phosphorylation of PXSP target sites, the wild-type
Smad-1 protein remains in the cytoplasm. BMP treatment
leads to Smad-1 nuclear accumulation, but when treated with
both BMP and EGF, Smad-1 remains in the cytoplasm. These
data suggest that the phosphorylation status of Smads in vivo
may dictate their responsiveness to TGF-b superfamily signaling.
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D. Smad structure
The transcriptional activation of the AP-1 element by
Smads is supported by a growing body of biochemical findings that relate the structure of Smads to their function. The
five cloned Smads have two regions of high homology, MH-1
and MH-2, shared with each other and with both Drosophila
MAD and C. elegans SMA genes. While evolutionarily conserved, these regions contain no known structural or signaling motifs. Figure 1 compares the percent identity of seven
of the known mammalian Smad protein sequences with their
C. elegans and Drosophila homologs. In addition, the specific
Smads involved in a particular TGF-b superfamily signaling
pathway are indicated.
1. Autoinhibitory regulation: MH-1 domain represses MH-2 domain. Studies with BMP have provided clues as to possible
functions for the different domains of the Smads. In these
experiments, the MH-2 region of Smad-1 and Smad-4 possess
a constitutive transcription activity when fused to a GAL4
DNA-binding domain (30, 43). This supports the model in
which Smads are recruited to specific promoters either by
direct DNA binding or through transcription factors and
activate transcription. Full-length Smad-4 fused to the GAL4
DNA-binding domain is normally transcriptionally silent,
but can be inducibly activated upon BMP treatment. This
suggests a model for Smad intramolecular regulation such
that the MH-1 normally functions to silence the transcription
activity of the MH-2 region of the Smad molecule. Upon
ligand treatment, the MH-1 region dissociates from its MH-2
region, allowing the MH-2 region to modulate transcription.
2. Homomeric and heteromeric interactions of Smad-3 and Smad-4.
Using yeast two-hybrid and coimmunoprecipitations, the
domains important for homomerization and heteromerization of Smad-3 and Smad-4 were mapped (44). In Smad-3, the
MH-2 domain mediates homomerization, whereas in
Smad-4 both the MH-1 and MH-2 domain are involved. The
heteromeric interactions between Smad-3 and Smad-4 are
mediated through their MH-2 domains. This correlates with
the functional data showing that Smad-3 and Smad-4 MH-2
domain overexpression is sufficient to induce PAI-1 promoter reporter activity (44, 45).
3. Crystallization of MH-2 domain of Smad-4. The recent crystallization of the C-terminal domain (CTD/MH-2) of Smad-4
helps shed light on the importance of this conserved region
(46). As mentioned above, the MH-2 domain can act as an
effector for the ligand-independent transactivating function.
An in-depth characterization of its structure has provided
insight into the mechanism by which Smad4 interacts with
other Smads and how the interaction is regulated by TGF-b.
The structural information has allowed a determination of
the importance of specific amino acids for Smad function
through a detailed comparative analysis of sequences from
mutant Smads identified in both developmental systems and
human cancers.
Based on the structural analysis, the Smad-4 MH-2 domain
was shown to consist of a core of five and six antiparallel
b-strands forming a b-sandwich. These are flanked on one
side by a three-helix bundle (H3–H5) and a three-loop/one-
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helix (L1–L3/H1) region on the other. The three-helix bundle
and loop/helix region may play an important role in Smad
function because mutations in these regions produce organisms with severe developmental abnormalities. In addition,
five Smad-4 mutants isolated from human tumors contain
mutations in these regions.
Those mutations were postulated to result in the disruption of protein-protein interactions. Therefore, the ability of
wild-type and mutant Smads to homo-oligomerize or heterooligomerize was investigated. Crystal structures showed a
strong interaction between three Smad4 proteins through an
interface composed of one loop/helix region contacting the
helix/bundle region of another Smad. Interestingly, only one
loop (L3) is free from this homomeric interaction. When
oriented, it was discovered that in the homotrimer, the L3
from each protein is positioned on the same side of the
protein complex. The L3 region is mutated in both developmental organisms and some tumors. Mutations in L3 prevent
hetero-oligomerization whereas mutations in other regions
of the loop/helix and three-helix bundle region prevent both
homo-oligomerization and hetero-oligomerization.
4. Proline linker region of Smad-4 important for restoration of
TGF-b response. Further domain studies on Smad-4 showed
an essential region in Smad-4 that is important in restoring
TGF-b transcriptional activation of the promoter reporter
p3TP-lux (41). In the Smad-4 defective cell line, MDA-MB468,
transfected full-length Smad-4 restored TGF-b responsiveness as assayed with the p3TP-lux reporter. Using a combination of chimeric Smad-1/Smad-4 and deletion Smad-4
constructs, an essential region in Smad-4 for restoration of
TGF-b mediated p3TP-lux activity was isolated. This 47amino acid region of Smad-4 is from amino acids 274 –321
and resides in the proline-rich linker region between the
MH-1 and MH-2 domains.
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become phosphorylated by the receptor complex were investigated. In COS cells, Flag-tagged Smad-6 or Smad-7 was
overexpressed and the type I and type II receptor complex
was covalently affinity labeled with [125I]TGF-b1. Flag-antibodies coimmunoprecipitated the receptor-ligand complex
independent of the co-transfected type I receptor kinase status (WT or K3 R), although the integrity of type II receptor
kinase status must be maintained (48, 50). This suggests that
Smad-6 and Smad-7, when overexpressed, interact with the
receptor-Smad complex. Overexpressed Smad-6 appears to
block the ability of the type I receptor to phosphorylate
Smad-1 and Smad-2 whereas Smad-7 blocks the ability of the
type I receptor to phosphorylate Smad-2 and Smad-3 (47, 48,
50). Smad-6 or Smad-7 may directly bind to the receptor
complex to prevent this phosphorylation, or interacts with
the complex through endogenous Smads. A truncated form
of Smad-7, D408, is unable to block TGF-b-mediated induction of p3TP-lux activity, does not associate with the receptor,
and does not prevent Smad-2 interaction with the receptor
(47).
From both the biochemical and functional data it is clear
that these new Smad family members, Smad-6 and Smad-7,
are biochemically regulated differently by TGF-b and its
superfamily members and thus have different functions than
the previously cloned Smads. Given the fact that Smad-7 can
be transcriptionally activated by TGF-b in both mink lung
epithelial cells and human keratinocytes, it is possible that
Smad-7 may play a role as an effector of other uncharacterized effects of TGF-b signaling, in addition to its possible role
in feedback regulation as a negative regulator of Smad-2 or
Smad-3 signaling. As such, these findings represent an additional exciting new area of research for Smad signaling,
and reinforce the potential importance of Smad family members as effectors of the TGF-b signaling cascade.
F. Smad-deficient mice
E. Smads as negative regulators
Recently, two other Smad family members have been
cloned, Smad-6/DAD and Smad-7 (47–50). Both these proteins were identified using expressed sequence tag technology (48, 50). DAD was identified using enhancer trap screening of Dpp-controlled genes (49), and Smad-7 was identified
from proteins induced upon laminar fluid sheer stress (47).
These proteins share MH-2 domain homology with the other
Smad family members but lack an MH-1 domain, suggesting
a potential functional difference. The proteins lack the three
regulatory serines at the C-terminal end conserved in the
other Smads (except Smad-4) suggesting a different mode of
regulation by TGF-b and its family members. Smad-7 RNA
was shown to be induced within 30 min of TGF-b treatment
in both HaCaT and mink lung epithelial cells before peaking
at 90 min (50). Both Smad-6 and Smad-7 may interfere with
Smad-associated gene responses such as p3TP-lux induction
(47, 48, 50). In addition, Smad-7 can block GAL4-Smad2
transactivation by TGF-b (47). From genetic studies in Drosophila, a distinct wing phenotype caused by overexpression
of MAD can be rescued by overexpression of DAD, the
Drosophila homolog for Smad-6 (49).
The abilities of Smad-6 and Smad-7 to associate with and
Recently, homozygous Smad-4 mutant mice were created
(51). The mice were embryonic lethal before embryonic day
7.5. The mutant embryos have developmental defects that
include failure to gastrulate or express mesodermal markers,
an abnormal visceral endoderm, and a reduced size. Mutant
embryos were rescued with wild-type visceral endoderm,
suggesting that Smad-4 is required for visceral endoderm
differentiation. Smad-4-deficient embryos exhibit a similar
phenotype as those of the BMP4- and BMPR-I deficient embryos which exhibit a reduction in size and inability to express mesodermal markers.
Because Smad-4 homozygous mutant mice were embryonic lethal, the effect of Smad-4 loss on TGF-b signaling in
adult tissues could not be studied. Other Smad-deficient
mice that are being generated include Smad-1, 2, 3, and 5.
Work is proceeding apace to analyze these mutants.
G. Smads: an emerging model
A model has emerged as to how Smads are activated by
TGF-b family members. Researchers in the field have made
a comparison between Smad signaling and signal transducers and activators of transcription (STAT) signaling. Upon
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MOLECULAR MECHANISMS OF TGF-b SIGNALING
355
the role of Smads in TGF-b superfamily signaling. In addition, Smad-6/DAD and Smad-7 may represent a completely
new class of Smads with different functions in TGF-b signaling. Analysis of Smad-deficient mice should soon bring a
great deal of insight into the physiological role various
Smads play in TGF-b signaling.
IV. TGF-b and the Cell Cycle
FIG. 2. Proposed model for the Smad-dependent TGF-b signal transduction pathway. Upon binding of ligand to the constitutive Type II
receptor serine-threonine kinase, Type I receptor is recruited into the
complex and phosphorylated at its GS domain by Type II receptor.
This activates the Type I receptor kinase, which phosphorylates
Smad-2 or Smad-3. Upon phosphorylation, the Smad-2 or Smad-3
homotrimer associates with the Smad-4 homotrimer to form a heterohexamer that translocates into the nucleus. The Smad complex
then activates transcription of target genes through an intermediary
transcription factor or by binding to DNA directly. Smad-6 can prevent the phosphorylation of Smad-1 and Smad-2 whereas Smad-7 can
prevent the phosphorylation of Smad-2 and Smad-3.
phosphorylation by the receptor tyrosine kinases, Janus kinases (JAKS) phosphorylate the STATs, which in turn dimerize and translocate to the nucleus, where they bind to DNA
and activate transcription. Figure 2 depicts a proposed model
for TGF-b activation of Smads from the cytoplasm to the
nucleus. TGF-b treatment causes the formation of heteromeric type II receptor/type I receptor/ligand complexes.
Type II receptor phosphorylates the type I receptor at the GS
domain, thereby activating it. Type I receptor, in turn, phosphorylates its Smad-2 or Smad-3 substrate. When phosphorylated, this homotrimeric Smad forms a hexaheteromer with
Smad-4. This event most likely allows entry of the Smad
complex into the nucleus where the Smads can make contacts
with specific DNA elements and/or other sequence-specific
transcription activators, perhaps as well as components of
the core transcriptional machinery, to modulate transcription. This model is highly simplified for a number of reasons.
At the cytoplasmic level, it is unclear whether the Smads are
phosphorylated by type I receptor in a homotrimer complex.
Whether this phosphorylation triggers a Smad-4 interaction
concomitant with translocation into the nucleus is also unknown. In the nucleus, even more questions remain unanswered. What are other Smad-inducible target genes, in addition to the PAI-1 gene? What dictates the different modes
of transcriptional activation, as a coactivator or through
DNA binding, the Smads may use? How does Smad localization into the nucleus precipitate transcriptional activation? Do Smads bind to members of the basal transcription
machinery? With what other transcription factors might the
Smads interact? These are just some of the more important
Smad-related questions whose answers will further define
Perhaps one of the best characterized and most important
functions of TGF-b is its ability to arrest certain cells in the
G1 phase of the cell cycle. This growth-inhibitory effect may
be central to much of the effect of TGF-b on a wide variety
of cells. The deregulation of the ability of TGF-b to effect a
G1 arrest may contribute to diseases such as human hereditary telangiectasia and oncogenesis. The inability of many
transformed cells to respond to TGF-b suggests that overcoming this negative signaling pathway may bring the cell
one step further in the development and progression of cancer. In this section, we will discuss the mechanism by which
TGF-b causes cell cycle arrest. The three cell lines discussed
in detail, mink lung epithelial cells, human keratinocytes,
and human breast epithelial cells, halt the cell cycle in a
similar fashion, suggesting a common strategy by which
TGF-b may impinge on the cell cycle.
Cytoplasmic events that propagate the TGF-b growtharrest signal into the nucleus are not well understood, and
the involvement of Smads in this process has not been determined (our unpublished results). The nuclear signal by
which TGF-b halts the cell cycle is well characterized and can
be best explained by beginning with TGF-b’s link to the
retinoblastoma protein, Rb (52, 53).
Rb is a cell cycle protein that is differentially phosphorylated during the cell cycle. About two-thirds of the way
through G1, at the restriction point, it changes from a hypophosphorylated state to a hyperphosphorylated state that is
maintained through the cell cycle until the cell emerges from
mitosis. Cells are only responsive to TGF-b when they are in
the window of G1 before the restriction point. Once past the
restriction point, the cell is committed to completing the cell
cycle (54). Thus, TGF-b mediated growth arrest was found to
be correlated with an accumulation of hypophosphorylated
Rb, although the precise mechanism of action was not known
at that time.
Further work in the cell cycle field showed that the kinase
activity of specific enzyme complexes was required for G1 to S
phase transition (55–57). Importantly, the kinase activity of
these complexes was regulated in a cell-cycle dependent manner. The regulatory subunit of these complexes are known as
’cyclins,’ and their catalytic subunits are the ’cyclin-dependent
kinases’ (CDKs). In addition to cyclin association, the kinase
activity of the CDKs can be regulated in three other ways. First,
the cyclins can be regulated through transcription. Second, activating and deactivating phosphorylations can regulate CDK
activity (e.g., CDK4 has an activating phosphorylation site on
threonine-160 and inactivating phosphorylation sites on threonine-14 and tyrosine-15). The kinases and phosphatases that
regulate CDK phosphorylation can also be regulated. In the case
of CDK4, the activating kinase is CAK, and the activating phos-
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HU ET AL.
phatase is cdc25A. Third, a group of low molecular wt molecules known as the cyclin-dependent kinase inhibitors (CKIs)
have been shown to associate with and concomitantly block
CDK activity.
It was hypothesized that one of the targets for cyclin-CDK
phosphorylation was Rb. Indeed, certain cyclin-CDK complexes could phosphorylate Rb. The phosphorylation of Rb
prevents its association with the transcription factor, E2F. Without Rb binding to it, E2F can transcriptionally activate genes
necessary for S phase progression (reviewed in Ref. 58). Recent
studies suggest the existence of two rate-limiting steps during
the G1 to S phase transition (59). The first step is dictated by the
ability of CDK4 to phosphorylate in vivo substrates, including
Rb, thereby allowing E2F to transactivate S phase-required
genes. The second step is dictated by the ability of CDK2 to
phosphorylate a much broader range of in vivo targets, which
may include other proteins in addition to Rb and Rb family
members. At present, other targets of the cyclin-CDK complexes are not known. To arrest cells in G1, TGF-b would potentially have to block the activities of both CDKs.
A. TGF-b induction of the CKIs
Of the four strategies by which CDK activity can be regulated as mentioned above, TGF-b was first shown to be able
to increase the amount of functional cyclin-dependent kinase
inhibitors in the cell. These CKIs can be grouped into two
subsets based on homology and function. The first group of
proteins include p21 (WAF1/Cip1), p27 (Kip1), and p57
(Kip2). The second group of inhibitors includes p15 (INK4B/
MTS2), p16 (INK4A/MTS1), p18, and p19. The first group of
CKIs bind to cyclin-CDK complexes and inhibit complex
activity while the second group of CKIs bind to CDKs alone,
which sequesters the catalytic CDKs from their regulatory
cyclin partners, thereby preventing activity.
1. In the mink lung epithelial cell line CCL64. The first CKI to
be implicated in TGF-b G1 arrest was p27. It was found to be
associated with Cyclin E-CDK2 complexes in mink lung epithelial cells and subsequently cloned by both biochemical
purification and the two-hybrid system in yeast (60 – 62). p27
Binds to and inhibits the activity of cyclin E-CDK2, indicating
that it plays a negative regulatory role in the cell cycle.
Recently, the crystal structure of the protein complex between Cyclin A-CDK2 and p27 has been resolved. p27 Makes
contacts with both Cyclin A and CDK2 so that ATP can no
longer bind to the active site of CDK2 enzyme (63). In functional assays, overexpression of p27 can arrest Saos-2 cells.
Although p27 is placed functionally in the TGF-b pathway
due to its ability to inhibit cyclin E-CDK2 activity, TGF-b
does not increase its transcription or translation.
In mink lung epithelial cells, TGF-b can potently increase
the transcription of another CKI, p15 (64). This inhibitor
functions by sequestering CDK4 and CDK6 (64). The current
model for TGF-b growth arrest in mink lung epithelial cells
is that upon TGF-b treatment, p15 transcription is induced.
This causes an increase in p15 protein levels, leading to their
association and sequestration of CDK4. p27 Protein, which
normally associates with cyclin D1-CDK4, is now displaced
and binds to its lower affinity partner Cyclin E-CDK2. The
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combined inactivation of both CDK2 and CDK4 corresponds
with a decrease in Rb phosphorylation and arrest in G1.
2. In the human keratinocyte cell line HaCaT. The mechanism by
which TGF-b increases the expression of the CKIs p15 and
p21 is more easily understood in human keratinocytes.
TGF-b treatment causes a rapid increase in p15 and p21 RNA
levels within 1 h (64 – 66). For p21, this corresponds to an
increase in protein levels, an increased association with cyclin E-CDK2 and cyclin D1-CDK4, and a decrease in the
activity of those cyclin-CDK complexes. The association of
p21 with CDK complexes is thought to prevent activating
phosphorylations on CDKs (T-160). Recent crystallography
studies suggest that the p21 family of inhibitors (including
p21 and p27) function by masking the ATP-binding domain
of CDK2. For p15, the increase in protein level allows its
sequestration of CDK4 and CDK6, thereby inhibiting kinase
activity.
To determine the mechanism by which TGF-b regulates
the transcription of p15 and p21, a comprehensive analysis
of the TGF-b response elements in the promoters of both p15
and p21 was conducted. Both TGF-b response elements
mapped to a 6-bp GC-rich element (67, 68). Electromobility
shift analysis confirmed that Sp1 family members could bind
to this element, although no changes in binding affinities
were detected for any of the complexes upon TGF-b treatment. Further functional assays with different GAL4-Sp1
fusions showed that GAL4-Sp1 proteins can confer TGF-b
responsiveness. These experiments demonstrate that, in addition to physically associating with the TGF-b response
element, Sp1 is also functionally important in modulating the
TGF-b-mediated response, although TGF-b does not regulate its level of expression. The protein synthesis inhibitor
cycloheximide does not inhibit TGF-b’s ability to induce p21
or p15 RNA accumulation, suggesting that the level of regulation by TGF-b is posttranslational. Overall phosphorylation of Sp1 does not change upon TGF-b treatment, suggesting another possible scenario involving the differential
modification of an adaptor molecule, which may activate Sp1
to allow transactivation of the p15 and p21 promoters.
a. E1A blocks TGF-b-mediated growth inhibition at multiple levels.
To determine the identity of this hypothetical adaptor protein,
the adenoviral immediate early gene product, 12S E1A, proved
to be a valuable tool. Both E1A of adenovirus and the large T
antigen of SV40 prevent TGF-b-mediated growth arrest, presumably due to the ability of both of these proteins to bind Rb
and mimic the hyperphosphorylation state of Rb (52, 69–74).
The dissociation of Rb allows E2F to transactivate genes important for S phase progression (58). Although the ability of
E1A and large T to bind Rb appears to be sufficient to abrogate
TGF-b signaling, cellular infections with a mutant E1A adenovirus, Ad 928, which is severely attenuated in its ability to bind
Rb, demonstrate that it too is able to overcome TGF-b-mediated
growth arrest in mink lung epithelial cells (75). If the phosphorylation status of Rb dictates the ability of a cell to enter S
phase, it was hypothesized that E1A might be able to intercept
Rb function at a point upstream of physical sequestration of Rb,
such as at the level of the CKIs. This model was particularly
attractive since the suppression of TGF-b-mediated transcriptional induction of the CKIs could prevent the hyperphospho-
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MOLECULAR MECHANISMS OF TGF-b SIGNALING
rylation of Rb, and hence provide E1A with an additional mechanism by which to abrogate TGF-b signal and create an S phase
environment necessary for viral replication. This model proved
to also apply in mink lung epithelial cells, where E1A has
recently been shown to bind directly to p27, but not p21 (76).
b. E1A blocks CKI induction by binding to p300. In HaCaT,
ribonuclease (RNase) protection analysis showed that p15
mRNA levels are not induced upon TGF-b treatment when
HaCaT cells are infected with adenovirus expressing 12S
E1A. To further isolate the proteins involved in abrogation
of TGF-b signaling, cotransfections were performed with
different 12S E1A mutants. Of these, an N-terminal deletion
mutant of E1A displayed an attenuated ability to block TGFb-mediated induction. This implicated proteins bound to the
N-terminal region of E1A, such as p300, as part of the TGF-bsignaling pathway. Additional luciferase experiments with
pCMV-p300 cotransfected with pCMV-12S-E1A demonstrated that expression of p300 could partially rescue the
inhibition of response to TGF-b caused by E1A. In addition
to demonstrating a novel function for E1A by blocking CKI
induction, these experiments directly point to p300 as a potential mediator of TGF-b activation of the p15 and p21
promoters (77).
p300 Was first discovered in anti-E1A immunoprecipitations of radioactively labeled cells. It associates with the
N-terminal region of E1A, which by itself is sufficient to
stimulate DNA synthesis. A different region of E1A containing the pocket-domain that binds to Rb and Rb family members is also capable of inducing DNA synthesis (78 – 80).
However, both regions of E1A were necessary to form colonies in soft agar assays (81). Ubiquitously expressed, the
p300 protein contains three cysteine-histidine rich regions
(two of which are putative zinc fingers), a bromo domain,
and a nuclear localization sequence (82). p300 Has a functional homolog, CREB-binding protein (CBP), which also
binds to E1A (83– 85). p300 And CBP can both overcome
E1A-mediated expression of the SV40 enhancer activity. p300
And CBP contact both transcription factors and the basal
machinery, suggesting a role for them as transcriptional
adaptors or coactivators. Recently, p300 and CBP have been
implicated in nuclear receptor signaling (86, 87), cAMP signaling (88, 89), STAT signaling (90), calcium-dependent differentiation (74a), and myogenesis (91).
c. A working hypothesis for the mechanism of TGF-b mediated
transcriptional activation of the p15 and p21 promoters. As discussed above, the mechanism by which TGF-b acts to induce
transcription of the p15 and p21 promoters remains largely
unknown. Studying this mechanism could lead to further
insight into what factors are modified by TGF-b that allow
it to differentially regulate gene transcription and thereby
engineer its cellular activities. The implication that p300 and
CBP are involved in TGF-b signaling creates an opportunity
to further dissect its signaling mechanism. Furthermore, the
Smads may be found to act as one of the effectors transducing
the TGF signal from the activated receptor complex directly
to the transcriptional apparatus assembled onto the p15 and
p21 promoters, thus linking the tumor suppressor activitity
of Smads to the cell cycle control machinery. Based on available evidence, a working hypothesis can be proposed, which
consists of the following two aspects: 1) p300/CBP may
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respond to the TGF-b signal to form a functional complex
with Sp1, thus leading to transcriptional activation of the p15
and p21 promoters. Our studies with NGF mediated transcriptional induction of the p21 promoter through a NGFinduced interaction between p300 and Sp1 (our unpublished
results) support this model in which a similar interaction
between the two proteins can be induced by TGF-b. 2) The
Smads may form a complex with p300/CBP and/or Sp1 to
activate transcription once they translocate into the nucleus.
This hypothesis is supported by our preliminary findings
that coexpression of “dominant negative” forms of either
Smad3 or Smad4 lacking their conserved functional MH1
domain with the p15 and p21 promoter-controlled reporter
constructs result in a significant reduction in TGF-b-induced
transcriptional activity. Although we cannot rule out the
possibility that the truncated Smads can sequester some common transcription factors other than the coactivator p300/
CBP and/or Sp1, this attractive model needs to be further
explored. One important feature of this potentially Smadmediated transactivating event, however, lies in the finding
that overexpression of the Smads does not lead to the activation of the p15 and p21 promoters (our unpublished results), unlike the scenario with the AP1 elements-controlled
transcriptional activation of PAI-1 promoter mediated by the
overexpression of Smads as discussed in the last section. It
is possible that Smads with proper structural conformation,
achieved through receptor-mediated phosphorylation, are
required to physically interact with p300/CBP and/or Sp1.
Alternatively but not exclusively, a separate signaling cascade, also initiated at the receptor complex, may be required
in conjuction with Smads to activate transcription at the p15
and p21 promoters. Further analysis in this direction will
undoubtly yield valuable information in elucidating the
mechanism through which the p15 and p21 promoters are
regulated by TGF-b.
B. TGF-b-mediated decrease in Cdc25A levels in the breast
epithelial cell line MCF10A
Recently, a different mechanism for cell cycle arrest was
observed in a spontaneously immortalized epithelial line,
MCF10A (92). In these cells, a rapid decrease in mRNA levels
of the cell cycle phosphatase, cdc25A, is observed upon
TGF-b treatment. Consequently, protein levels for cdc25A
decrease by 8 h. As mentioned above, cdc25A is thought to
regulate CDK activity. Based on an analogous model with
cdc2 and the wee1 kinase/cdc25 phosphatase in the G2/M
transition, dephosphorylation of an inhibitory tyrosine on
CDK4, presumably by cdc25A, is thought to be required for
cell cycle progression. In the MCF10A cells, the authors correlate a decrease in cdc25A activity with an increase in tyrosine phosphorylation of CDK4 and CDK6. As such, this
may be another mechanism by which TGF-b can cause an
arrest in G1. E1A can also increase cdc25A activity in quiescent fibroblasts, suggesting another possible level of regulation by which E1A can overcome TGF-b growth inhibition
(93).
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HU ET AL.
C. TGF-b-mediated cyclin-CDK inhibition may be common
strategy to arrest cells in G1
TGF-b appears to halt cell cycle progression in a cell typespecific manner. This may reflect the intrinsic differential
protein profiles that separate one cell type from another.
Thus, the entire cell cycle-arrest programming may vary
from cell type to cell type. One example is the difference
between TGF-b-mediated CKI induction of mink lung epithelial cells and human keratinocytes. In other cells, TGF-b
may employ one of the remaining three strategies for influencing CDK activity to effect a G1 cell cycle arrest. The breast
epithelial cell line, MCF10A, is one such example. Figure 3 is
a schematic summarizing the known strategies by which
TGF-b inhibits CDK activity. In this section, we have reviewed some of the documented strategies by which TGF-b
impinges on the cell cycle to effect an arrest in G1 depending
on cell type. Other cell types that are also responsive to the
TGF-b growth-inhibitory signal, such as fibroblasts, may not
use these mechanisms. However, we suggest that to arrest
the cells in G1, TGF-b may employ other strategies limiting
cyclin-CDK activities to reach the same growth inhibition
endpoint. Restricting cyclin-CDK kinase activity may be a
common denominator by which TGF-b can arrest cells in G1
phase of the cell cycle.
V. TGF-b and Cancer
A delicate balance exists between positive and negative
growth regulators in a normal cycling cell. In carcinogenesis,
this homeostasis can be destroyed in two ways—increasing
the signal from the positive growth regulators (mitogens) or
decreasing the signal from the negative growth regulators
Vol. 19, No. 3
(apoptosis, tumor suppressors). Oncogenes are often proteins downstream of mitogenic signals such as mutated ras.
Tumor suppressors, on the other hand, often function as
checkpoints to ensure that the cell cycle is arrested when the
cell is treated with inhibitory growth factors such as TGF-b
or subjected to a variety of insults such as DNA damage.
In this review, we have focused on characterizing the
known components of the TGF-b pathway. Components that
are necessary for the negative-regulatory TGF-b signal to
propagate, such as the receptors, are commonly mutated in
cancer. Blockage of TGF-b signaling may disrupt this cellular
steady state, biasing the cell toward inappropriate growth
that ultimately results in tumor formation. In this section, we
will briefly describe more mutations in type II receptor that
have recently been discovered in cancer patients. The primary focus of this section will be on the discovery of mutated
Smad family members in cancers. In addition, other cell cycle
components in the TGF-b growth arrest pathway that are
mutated in cancers will be briefly mentioned.
A. Receptor mutants
In recent years, an overwhelming body of evidence shows
that mutations or loss of expression of the type II receptor are
found in a number of gastrointestinal cancer lines (94 –102).
In most of the cell lines assayed, type II receptor was either
mutated or not expressed correlating with an unresponsiveness to the TGF-b growth-inhibitory signal. Southern blot
analysis revealed deletions in the type II receptor gene. Sequencing of other mutants revealed 1-bp frame shifts. To
verify the importance of the type II receptor mutant on carcinogenesis, wild-type receptor II was transfected into cells
and the cells were assayed for fibronectin expression, foci
formation, and clonogenicity in soft agar (98). In the receptor
II-transfected cells, a TGF-b-dependent increase in fibronectin expression and a reduction in clonogenicity were observed, suggesting not only that the block in TGF-b signaling
correlated directly with an increase in clonogenicity, but that
the downstream signaling components were still functional.
Similar experiments have also been published in small cell
lung cancer and breast cancer (103–105), suggesting that mutations in receptor type II expression are not specific for
gastrointestinal cancers. These data strongly support the notion that type II receptor dysfunction is an important event
in cancer progression. Although some cancer lines have type
I receptor mutations, they are less frequent (105).
B. Smad mutants
FIG. 3. TGF-b causes G1 cell cycle arrest by inhibiting cyclin-CDK
activity. In the human keratinocyte line, HaCaT, TGF-b transcriptionally increases the CKIs p15 and p21, which leads to the sequestration of CDK4/6 by p15 and the inactivation of cyclin D1-CDK4 and
cyclin E-CDK2 by p21 binding. In mink lung epithelial cells, p27 is
normally bound to cyclin D1-CDK4, but, upon TGF-b treatment, induced p15 protein sequesters CDK4, displacing p27 and allowing it to
bind and inhibit its lower affinity target cyclin E-CDK2. In MCF10A
cells, TGF-b treatment decreases the amount of cdc25A transcript,
preventing the phosphatase from activating CDK4. The ability of
TGF-b to block cyclin-CDK activity appears to be a common strategy
by which it causes growth arrest.
Many of the events downstream of TGF-b signaling have
been shown to be mutated in cancers. Two of these are
Smad-2 and Smad-4. Using PCR, researchers showed that a
region on chromosome 18q that maps to Smad-4 is homozygously deleted in pancreatic carcinomas. This region is specific for Smad-4 and not the gene DCC (Deleted in Colorectal
Cancer), which is located adjacent to Smad-4. The deleted
fragment was isolated from yeast artificial chromosomes
(YAC) and hybridized to a human fetal brain cDNA library
leading to the cloning of Smad-4 (34). In 84 pancreatic carcinomas screened, Smad-4 was homozygously deleted in 25
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MOLECULAR MECHANISMS OF TGF-b SIGNALING
cases. Of 27 pancreatic carcinomas without homozygous deletions, six had additional mutations. Ninety percent of all
pancreatic tumors screened have deletions in chromosome
18, but only 50% are deletions for Smad-4. Researchers hypothesized that another dysfunctional gene on chromosome
18 may also contribute to pancreatic carcinomas.
The discovery that Smad-2 mapped to 18q21 just two
genes away from Smad-4 suggests that Smad-2 may be the
other mutational target contributing to pancreatic carcinomas (32). Based on our current knowledge of Smad
signaling, it is an attractive model to suggest that only one
Smad-2 or one Smad-4 homozygous deletion is necessary
to abrogate their physiological role in TGF-b signaling
since their cooperation is needed for appropriate signal
transduction. At this time, the loss of Smad-2 in pancreatic
carcinoma has yet to be characterized. Of 66 colon carcinomas screened, four were found to contain point mutations in Smad-2. Of these, three could not be phosphorylated by TGF-b. Since phosphorylation of Smad-2
initiates their localization to the nucleus, these mutations
may prevent the proper Smad-2 homotrimerization and
subsequent heterohexamerization with Smad-4 to allow
nuclear entry. Recently, it was discovered that the MH1
region of Smad 2 and Smad 4 bind to and inhibit the MH2
region from signaling. Phosphorylation of the C-terminal
region may prevent the Smad-2 MH2 domain from interacting with its MH1 domain, thereby activating it. Overexpression of the MH1 domain of either Smad-2 or Smad-4
can prevent interaction between full-length Smad-2 and
Smad-4 (43). However, homotrimerization of the MH2
domain was not prevented by the MH1 domain of either
Smad-2 or Smad-4. Functionally, the Smad-4 R100T mutant or the Smad-2 R133C mutant may be preventing wildtype Smad-2 from interacting with wild-type Smad-4,
since cotransfection of wild-type Smads in conjunction
with Smad mutants blocks the ability of the overexpressed
Smads to transactivate the 3TP-lux reporter. In addition to
pancreatic cancers, Smad-4 mutations have also been discovered in breast, ovary, head and neck, and esophagus
cancers (34, 106 –109), whereas Smad-2 mutations have
only been found in colon, head, and neck cancers (32, 33,
110).
C. Cell cycle mutants
Mutations in basic cell cycle components are a frequent
target of cancers, since a change in a single cell-cycle protein
may provide a significant growth advantage for the cell.
Recently, CBP and p300 mutations have also been discovered
in gastric and colon carcinomas and leukemias (111, 112).
Gastric and colon carcinomas frequently contain loss of heterozygosity on chromosome 22q. Although p300 is mutated
in both of these cancers, the frequency is low (one of six) in
colon cancers, and thus the overall contribution of p300 to
these tumors is unclear. Other downstream targets of TGF-b
action include Rb and p15. Mutations and deletions in these
genes have been found in wide variety of cancers (113). In
addition to mutations of the negative regulators mentioned
above, oncogenic mutations in positive cell cycle regulators
can also give the cell a tremendous growth advantage. In
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certain cancers, mutations have been discovered in cyclin D1,
cdk4, and the cdc25 family of phosphatases. The contribution
of both types of cell cycle mutants has been reviewed extensively in the literature.
D. Multilevel resistance to TGF-b is important in multistep
model of carcinogenesis
Resistance to TGF-b growth inhibition may occur at multiple levels in a cancerous cell. Receptor mutations create an
unresponsive phenotype, and mutations in different downstream components of the TGF-b-signaling pathway may
also generate the same phenotype. Mutations in basic cell
cycle regulators such as Rb, p15, p21, and p300/CBP may
prevent negative growth regulators in addition to TGF-b
from regulating cell growth. These mutations may also cooperate with oncogenic mutations such as cdk4 or cyclin D
to allow rapid cellular proliferation. At present, it is unclear
whether the Smads are part of the cell cycle machinery since
their overexpression does not lead to an increase in CKIs p15
and p21 (our unpublished results). However, the Smads are
clearly involved in extracellular matrix deposition as their
overexpression results in the transcriptional induction of
PAI-1. Mutations in this pathway could lead to differences in
cell-cell contact that may provide a growth advantage to the
cell. These multiple levels of deregulation ultimately result
in an aggressive, rapidly growing, highly metastatic tumor
that is unresponsive to negative growth signals.
VI. Conclusions
Upon TGF-b treatment, type I receptor is recruited to the
type II receptor/ligand complex on the cell surface and is
subsequently activated by the type II receptor kinase upon
phosphorylation of its GS domain. Activated type I receptor
kinase can phosphorylate its Smad-2 or Smad-3 target. This
phosphorylation is associated with the nuclear accumulation
of Smad-2 and Smad-3 and is thought to occur by dimerization with Smad-4. In the nucleus, Smad-2/4 and Smad3/4 can activate transcription of certain genes to modulate a
certain subset of TGF-b gene responses such as PAI-1. This
activation may occur through direct Smad binding to elements in the DNA, or indirectly by binding to other transcription factors such as FAST-1. Smad-7 protein levels are
up-regulated by TGF-b and block the ability of the Smad
signal to enter the nucleus by preventing the phosphorylation of Smad-2 and Smad-3 by type I receptor. This may
represent a negative feedback signal by which the cell can
control the degree of Smad signaling. The elucidation of this
signal cascade within the last 2 yr is the result of tremendous
efforts from researchers throughout the field.
The propagation of the Smad signal from the cytoplasm to
the nucleus is the only translocation event known for TGF-b
signaling. Thus, it is interesting to speculate on a possible role
for the Smads as effectors for TGF-b-mediated growth inhibition. Although Smad overexpression can lead to a higher
percentage of cells arresting (28), whether this arrest pathway is molecularly identical to the well characterized TGFb-mediated growth inhibition, mediated by either an increase in p15 or p21 protein levels or a decrease in cdc25A
360
HU ET AL.
levels to decrease cyclin-CDK activity, remains to be determined. Smad overexpression does not appear sufficient to
cause an increase in p15 and p21 promoter activity, suggesting the requirement of another TGF-b-mediated signal to
mediate growth inhibition (see discussion in Section IV.A).
Whether Smads are required for TGF-b-mediated growth
inhibition also remains to be determined. Furthermore, the
molecular mechanism by which the p15 and p21 promoters
are activated through the functional interactions between
p300/CBP, Sp1, and possibly Smads, will be the focus of
immediate intensive research.
In cancer, not only are the genes involved in the cell cycle
often mutated, genes involved in cell-cell contact or extracellular matrix deposition such as Smads, when mutated,
may induce a morphological change on the cell surface such
that these cells have a growth advantage or are more easily
able to metastasize. This may be the case with Smad-4/DPC,
which has been shown to be mutated in many pancreatic
cancers. Although the Smad-4 heterozygous null mice do not
appear more predisposed to cancer compared with their
wild-type counterparts, it is possible that other mutations are
also required for tumorigenesis (51). The loss of Smad-4 may
represent a relatively late event in carcinogenesis that triggers the beginning of a more malignant and aggressive form
of cancer.
In addition to its potential deregulation in diseases such as
cancer, TGF-b may cause more global effects in multicellular
systems such as in the immune response, wound healing,
and synaptic facilitation. Whether Smad signaling and/or
TGF-b mediated growth inhibition is important in these cells
remains to be determined. The Smad-deficient mice may also
prove to be very useful in addressing these questions.
With the collaborative efforts of so many researchers focused on the dissection of the TGF-b-signaling circuitry,
more answers should be forthcoming. With the recent discovery of the Smad family of proteins and new insights into
the mechanism of TGF-b cell cycle arrest, a new era in the
field of serine-threonine receptor kinase signaling is beginning.
Acknowledgments
We would like to thank C. Bassing, J. Frederick, B. Gilmour, P.-C. Hu,
S.-C. Hu, C.-H. Huang, Y.-S. Huang, J.-M. Li, N. Liberati, I. Liu, E.-H.
Park, J. Rich, E. M. Scurry, H. Symonds, X. Shen, C. Wong, J. Yingling,
Y. Yu, M. Zhang, members of the A. M. Pendergast laboratory, and the
family of P.H. (C. Hu, H. Hu, P. Hu, F.-M.C., and Y.-Y. Hu) for helpful
discussions, constant support, and encouragement.
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