The transforming growth factor β (TGF-β) superfamily
encompasses a large variety of signalling proteins, including
TGF-β isoforms, bone morphogenetic proteins (BMPs),
growth and differentiation factors (GDFs), activins, inhibins
and Müllerian inhibiting substance (Roberts and Sporn, 1993;
Piek et al., 1999). TGF-β signalling pathways are important
during embryonic development; in adult organisms, they
regulate tissue homeostasis and are frequently implicated in
diverse pathological conditions (Barolo and Posakony, 2002;
Waite and Eng, 2003). TGF-β superfamily members signal
through receptor serine/threonine kinases and intracellular
Smad proteins (Moustakas et al., 2001; Shi and Massagué,
2003). In addition, several intracellular proteins that mediate
signalling by receptor tyrosine kinases, G-protein-coupled
receptors or cytokine receptors also participate in the TGF-β
signalling network (Derynck and Zhang, 2003; Nohe et al.,
The Smad pathway represents an evolutionarily conserved
signalling module that transmits signals to the nucleus and is
of paramount importance for the precise execution of tissue-
and organ-patterning programmes during animal development.
By contrast, the relative weight and evolutionary or
developmental significance of non-Smad effectors has been
less clear. However, the cellular and genetic evidence for
important roles of these effectors downstream of TGF-β
ligands is increasing. Here, we discuss non-Smad signalling
proteins downstream of the receptors for various TGF-β
superfamily members. We classify their modes of action into
three distinct but interrelated signalling mechanisms (Fig. 1):
(1) non-Smad signalling pathways that directly modify Smad
function; (2) non-Smad proteins whose function is directly
modulated by Smads and which transmit signals to other
pathways; and (3) non-Smad proteins that directly interact with
or become phosphorylated by TGF-β receptors and do not
necessarily affect the function of Smads. The existence of such
pathways raises several important questions. Why are they
needed? Do they promote signalling specificity? Are they truly
independent of Smads? Are non-Smad proteins points of
convergence between signalling by TGF-β and other factors?
Below we examine these questions and discuss the evidence
for non-Smad signalling mechanisms downstream of
serine/threonine kinase receptors.
Overview of the Smad pathway
The dimeric TGF-β superfamily ligands interact with hetero-
tetrameric complexes of type II and type I receptors, which
leads to phosphorylation-dependent activation of the dormant
type I receptor kinase by the constitutively active type II
receptor kinase (Shi and Massagué, 2003). Then, the activated
type I receptor kinase phosphorylates and activates receptor-
activated (R)-Smads (Moustakas et al., 2001). TGF-β
superfamily signalling can be classified into two branches: (1)
the TGF-β branch, represented by ligands such as TGF-β,
activin, nodal or myostatin, which activate Smad2 and Smad3;
and (2) the BMP branch, represented by ligands such as BMPs
and GDFs, which activate Smad1, Smad5 and Smad8
(Moustakas et al., 2001). The phosphorylated R-Smads form
complexes with the common mediator, Smad4, and enter the
nucleus, where they bind to DNA and interact with
transcription factors to regulate gene expression (Fig. 2A). This
basic Smad pathway is conserved throughout evolution and is
regulated by diverse
nucleocytoplasmic shuttling, ubiquitin-mediated proteasomal
degradation and, finally, by inhibitory (I)-Smads (Shi and
During the past 10 years, it has been firmly established that
Smad pathways are central mediators of signals from the
receptors for transforming growth factor β (TGF-β)
superfamily members to the nucleus. However, growing
biochemical and developmental evidence supports the
notion that alternative, non-Smad pathways also
participate in TGF-β signalling. Non-Smad signalling
proteins have three general mechanisms by which they
contribute to physiological responses to TGF-β: (1) non-
Smad signalling pathways
phosphorylate) the Smads and thus modulate the activity
of the central effectors; (2) Smads directly interact and
modulate the activity of other signalling proteins (e.g.
directly modify (e.g.
kinases), thus transmitting signals to other pathways; and
(3) the TGF-β receptors directly interact with or
phosphorylate non-Smad proteins, thus initiating parallel
signalling that cooperates with the Smad pathway in
eliciting physiological responses. Thus, non-Smad signal
transducers under the control of TGF-β
quantitative regulation of the signalling pathway, and serve
as nodes for crosstalk with other major signalling
pathways, such as tyrosine kinase, G-protein-coupled or
Key words: BMP, MAPK, PI3K, Phosphatase, Ras, Rho, Smad,
Non-Smad TGF-β signals
Aristidis Moustakas* and Carl-Henrik Heldin
Ludwig Institute for Cancer Research, Biomedical Center, Uppsala University, Box 595, SE 751 24 Uppsala, Sweden
*Author for correspondence (e-mail: email@example.com)
Journal of Cell Science 118, 3573-3584 Published by The Company of Biologists 2005
Journal of Cell Science
Massagué, 2003). The latter are induced by Smad signalling,
accumulate in the nucleus and are exported from the nucleus
after stimulation of cells with TGF-β or BMP. They then bind
to type I receptors and exert negative feedback by blocking R-
Smad phosphorylation and
formation, stimulating receptor
recruiting phosphatases, and promoting receptor ubiquitylation
and lysosomal degradation (Fig. 2B).
Non-Smad TGF-β signalling
The identification of non-Smad signalling proteins that
participate in TGF-β signal transduction pre-dates the
discovery of Smads (reviewed by Yue and Mulder, 2000). Most
of the experiments were performed in cell models in vitro and
implicated the small GTPase Ras and the mitogen-activated
protein kinases (MAPKs) ERKs, p38 and c-Jun N-terminal
kinases (JNKs) in TGF-β signalling (reviewed by Yue and
Mulder, 2000). However, many in vitro studies of non-Smad
signalling pathways have relied primarily on pharmacological
inhibitors of various intracellular protein or lipid kinases. The
specificity and the effective doses of such inhibitors must be
evaluated with great caution because, for example, ‘specific’
inhibitors of p38 can also lead to inhibition of the TGF-β
receptor kinases (Yakymovych et al., 2001; Fu et al., 2003).
In many cases where a role for a ‘Smad-independent’
pathway downstream of TGF-β, activin or BMP receptors has
been proposed, the link between the activated receptor complex
and the cytoplasmic effector molecule remains to be
elucidated. Below, we review the most prominent examples of
non-Smad signalling whose physical links to the receptor
complex are at least partially understood. We also list some
additional cases where the physical links to the receptors are
more ambiguous, but the potential physiological role of such
non-Smad proteins makes them interesting targets for future
A set of genes regulated by Smads is proposed to mediate the
pro-apoptotic effects of TGF-β. These include those encoding
the phospholipid phosphatase SHIP, death-associated protein
kinase (DAPK) and TGF-β-inducible early response gene 1
(TIEG1) (Fig. 3A) (reviewed by ten Dijke et al., 2002; Siegel
and Massagué, 2003). In addition, through Smad3, TGF-β
induces expression and activation of the Fas receptor, leading
to caspase-8 activation and apoptosis of gastric carcinoma cells
(Kim et al., 2004). However, the most consistent observation
in apoptotic mechanisms downstream of TGF-β ligands is the
involvement of MAPKs, such as p38 and JNK, as outlined
The type II receptor for TGF-β interacts with the pro-
apoptotic adaptor protein Daxx, which leads to activation of
JNK and induction of apoptosis in epithelial cells and
hepatocytes (Perlman et al., 2001). The Daxx-JNK pathway
also involves homeodomain-interacting protein kinase 2
(HIPK2), which interacts with and phosphorylates Daxx; this
activates the MAPK kinases MKK4 and MKK7, which
ultimately activate JNK and induce apoptosis (Fig. 3B)
(Hofmann et al., 2003). Genetic evidence in normal mammary
epithelial cells stably transfected with a mutant TGF-β type I
receptor that cannot bind and thus activate R-Smads, also
implicates the p38 and JNK pathways in mammary cell
apoptosis (Yu et al., 2002; Itoh et al., 2003). This work shows
that the type I receptor kinase activates p38 and JNK
independently of factors (including Smads) that bind to the L45
loop of the receptor. Whether the two distinct TGF-β receptors
signal towards different non-Smad proteins in order to mobilize
the p38 and JNK pathways remains an interesting open
A second direct link between receptor complexes and
intracellular kinases involves the TGF-β-activated kinase 1
(TAK1), which can form a complex with the BMP receptors
through its binding partner TAB1 and the inhibitor of apoptotic
caspases XIAP, an E3 ubiquitin ligase (Fig. 3B) (Yamaguchi
et al., 1999). The complex was reported to promote BMP
signalling during Xenopus embryogenesis. Whether BMP
receptors activate Smad versus non-Smad (e.g. TAK1-p38)
pathways seems to be at least partially regulated by the mode
of oligomerization of the cell-surface receptor (Nohe et al.,
Journal of Cell Science 118 (16)
TGF-β β β β
Fig. 1. Non-Smad signalling. The canonical Smad pathway starting
from the ligand-receptor complex and ending in the nucleus is
illustrated by thick black arrows. Non-Smad signalling mechanisms
are shown by thin blue arrows. The receptor complex activates (by
interaction and/or phosphorylation) protein X, which then modulates
the activity of the Smad (pathway 1). The phosphorylated Smad
activates (by interaction) protein Y, which then transmits further
signals into the cell (pathway 2). The receptor complex activates (by
interaction and/or phosphorylation) protein Z, which then transmits
signals without direct crosstalk with the Smad (pathway 3). Proteins
X, Y and Z can be enzymes (e.g. protein or lipid kinases) or adaptor
Journal of Cell Science
Non-Smad TGF-β signals
2002). In this model, pre-assembled type-I–type-II receptor
complexes activate Smads in response to BMP, whereas ligand-
induced receptor complexes activate the TAK1-p38 module
(reviewed by Nohe et al., 2004).
TAK1 can also act downstream of TGF-β by initiating a
kinase cascade that leads to Stat3 activation during mesoderm
induction in Xenopus (Ohkawara et al., 2004). TAK1, despite
its original name, has also been firmly placed as a crucial
signalling intermediate in many pro-inflammatory cytokine
and Toll-like receptor signalling pathways (reviewed by
Moustakas and Heldin, 2003). In these pathways, TAK1
cooperates with TRAF-mediated signalling to regulate
inhibitor of nuclear factor κB (IκB) function. XIAP was also
found to interact with multiple type I receptors of the TGF-β
superfamily, enhancing their signalling output (Birkey Reffey
et al., 2001). However, downstream of TGF-β receptors, XIAP
seems to activate JNK and nuclear factor κB (NF-κB) through
cooperation with Smad4, leading to regulation of gene
expression but not apoptosis (Birkey Reffey et al., 2001). These
observations underscore two general principles: there is a high
degree of cell-type specificity, and many non-Smad signalling
proteins modulate the activity of the Smad pathway. Thus, the
delineation of true Smad-independent signals is rather
complicated, as we discuss throughout this review.
Another mechanism in which XIAP is implicated in TGF-
β-mediated apoptosis involves
mitochondrial components by TGF-β signalling. TGF-β
induces export of the mitochondrial septin family member
ARTS to the cytoplasm and promotes its association with, and
inactivation of, XIAP (Fig. 3A) (Larisch et al., 2000); XIAP
the mobilization of
inactivation leads to caspase-3 activation and apoptosis
(Gottfried et al., 2004).
An alternative mode of crosstalk is between kinases such as
TAK1 and inhibitory Smads. Thus, BMPs can cause apoptosis
of various cell types through TAK1 and p38, and Smad6 and
Smad7 efficiently block such responses (Yanagisawa et al.,
2001). However, TGF-β induces apoptosis of prostate
carcinoma cells by promoting cooperation between Smad7 and
the TAK1-p38 signalling module (Fig. 3B) (Edlund et al.,
2003). These complex functions of the I-Smads may well
depend on their emerging role as adaptor proteins, whereby
they not only inhibit R-Smad phosphorylation by TGF-β
receptors, but also mediate recruitment of phosphatases and
ubiquitin ligases to the receptor complex (Shi and Massagué,
2003). The link between TAK1 signalling and Smad7 also
includes effects on transcription: the TAK1-p38 module
activates Smad7 expression by regulation of the transcription
factor ER81, a member of the ETS family (Dowdy et al., 2003).
It is therefore possible that even more complex mechanisms
linking TGF-β superfamily receptors to non-Smad signal
transducers through inhibitory Smads will surface in the near
Finally, TGF-β might also antagonize pro-survival signals.
The relative levels of Smad3 and the pro-survival kinase Akt
are proposed to define whether a cell undergoes apoptosis in
response to TGF-β (Fig. 3A) (Conery et al., 2004; Remy et al.,
2004). This model is based on the physical interaction between
Smad3 and Akt and, because of its strictly quantitative nature,
requires further validation in a large set of cell types. The
adaptor molecule CD2-associated protein (CD2AP) activates
TGF-β β β β
TGF-β β β β
Fig. 2. (A) The canonical Smad
pathway. TGF-β bound to the two
receptor serine/threonine kinases
(type II, light green; type I, blue)
initiates signalling by trans-
phosphorylation of the type I receptor
juxtamembrane domain (red arrow).
Signalling ensues with R-Smad
phosphorylation (Smad with black
dot), oligomerization of R-Smad with
Smad4, nuclear translocation and
formation of complexes between
transcription factors (TF) and co-
activators/co-repressors (co) on
chromatin. This leads to positive or
negative regulation of mRNA
synthesis (grey arrow). (B) The
inhibitory I-Smad, together with the
E3 ligase Smurf, exit the nucleus in
response to the incoming TGF-β
signal and bind the receptor complex,
leading to shut-down of R-Smad
phosphorylation and receptor
Journal of Cell Science
the phosphoinositide 3-kinase (PI3K)/Akt pathway, enhancing
cell survival and protecting from TGF-β-induced apoptosis in
kidney podocytes (Schiffer et al., 2004). Interestingly, the
regulatory subunit of PI3K, p85, indirectly associates with type
I and type II TGF-β receptors and the former activates PI3K
in a ligand-dependent manner (Yi et al., 2005). Whether the
bridge between the receptor complex and p85 of PI3K is
CD2AP or another protein remains unknown.
Overall, we can conclude that the apoptotic response of
normal or tumour cells to TGF-β family members involves the
interplay between Smad and non-Smad pathways and, in many
cases, both pro-survival and pro-apoptotic pathways are
activated. Thus, the end result may well depend on other
signalling inputs the cell receives.
Epithelial-mesenchymal transition and migratory
Morphogenetic responses to TGF-β members include cell
migration and epithelial/endothelial-mesenchymal transitions
(EMTs), which are crucial during embryogenesis, fibrotic
diseases and advanced carcinoma spreading (Tosh and Slack,
2002; Condeelis and Segall, 2003; Gotzmann et al., 2004). The
EMT is a characteristic change in polarized epithelia in which
the cell-cell and cell-matrix adhesion is disrupted, the
surrounding matrix is degraded, and the cell phenotype is
changed by rearranging its actin cytoskeleton to become more
motile and invasive. Since the Smad pathway primarily leads
to regulation of gene expression, it was originally thought that
non-Smad effectors signal the rapid or direct effects of TGF-β
on the actin cytoskeleton. Today, however, we appreciate that
Smads are crucial mediators of this process downstream of
TGF-β, because they induce dramatic changes in gene
expression in epithelial cells (Fig. 4A) (Zavadil et al., 2001;
Kowanetz et al., 2004; Valcourt et al., 2005). However, the
EMT also depends on ERK activity, and pathways activated by
oncogenic Ras or physiological Notch receptor signalling
synergize with TGF-β (Oft et al., 1996; Oft et al., 1998; Zavadil
et al., 2001; Jechlinger et al., 2003; Zavadil et al., 2004). The
Smads, especially Smad3 and Smad4, are critical for the EMT
response not only in vitro (Valcourt et al., 2005), but also in
vivo, for aggressive carcinoma metastasis and lens EMT (Oft
et al., 2002; Li et al., 2003; Tian et al., 2003; Saika et al., 2004;
Tian et al., 2004). Mutant TGF-β type I receptors that lack the
Smad-docking site fail to induce EMT but can activate
endogenous p38 or JNK signalling (Yu et al., 2002; Itoh et al.,
2003). These mutant type I receptors also inhibit endogenous
autocrine TGF-β signalling and thus induce a stronger
epithelial phenotype in vitro, and suppress breast carcinoma
metastasis to the lung in vivo (Tian et al., 2004; Valcourt et al.,
A direct link between the TGF-β receptors and the Rho
Journal of Cell Science 118 (16)
TGF-β β β β
BMPTGF-β β β β
Fig. 3. (A) Smads in
apoptosis. TGF-β receptors
induce through Smads the
expression of DAPK, SHIP
and TIEG pro-apoptotic genes.
Smads also bind and inactivate
the survival kinase Akt, thus
promoting apoptosis. TGF-β
can also mobilize the
mitochondrial serpin ARTS to
the nucleus, which blocks
XIAPs, the inhibitors of
caspases, thus leading to
apoptotic events. (B) The
TAK1 pathway leading to
apoptosis. The TGF-β type I
receptor binds Smad7, the type
II receptor binds the pro-
apoptotic protein Daxx,
whereas BMP receptors bind
XIAP and its interacting
partners, TAB and TAK1.
Both TGF-β and BMP
receptors activate TAK1,
leading to MKK3, MKK4 or
MKK7 activation. This
activates JNK or p38, both of
which lead to apoptosis of
various cell types. In the Daxx
phosphorylates Daxx (curved
arrow), which then activates
MKK4 and MKK7.
Journal of Cell Science
Non-Smad TGF-β signals
GTPase was recently uncovered and provides a novel
mechanism by which TGF-β promotes EMT, at least in vitro
(Barrios-Rodiles et al., 2005; Ozdamar et al., 2005). In
polarized epithelial cells, the TGF-β receptor complex is
recruited to tight junctions by the structural protein occludin,
where it also interacts with the polarity protein Par6 (Fig. 4A).
Upon TGF-β signalling, the type II receptor phosphorylates not
only the type I receptor but also the type-I-receptor-tethered
Par6, leading to recruitment of the ubiquitin ligase Smurf1 and
subsequent ubiquitylation and degradation of RhoA (Ozdamar
et al., 2005). This leads to local disassembly of the actin
cytoskeleton and dissolution of tight junctions, which is one of
the hallmarks of EMT (Thiery, 2003). Simultaneously,
activation of Smads by the type I receptor leads to
transcriptional induction of genes involved in EMT such as that
encoding Snail (Peinado et al., 2003), which is a transcriptional
repressor of the gene encoding E-cadherin (Fig. 4A) (Nieto,
2002). When E-cadherin levels drop inside the cell, the
adherens junctions of polarized epithelial cells also dissolve,
which is a second hallmark of EMT (Thiery, 2003). Direct
phosphorylation of Par6 by the type II receptor kinase is the
first example of a non-TGF-β receptor protein substrate for this
receptor kinase. This opens the exciting possibility that a
diverse set of proteins, in addition to the type I receptor of
TGF-β, are phosphorylated and regulated by the type II
receptor serine/threonine kinase.
Two recent reports similarly directly link the cytoplasmic
protein kinase Limk1 to the long cytoplasmic tail of the BMP
type II receptor (BMPR-II) (Foletta et al., 2003; Lee-Hoeflich
et al., 2004). Since Limk1 is a well-studied kinase that signals
downstream of Rho GTPases and regulates reorganization of
the actin cytoskeleton (Raftopoulou and Hall, 2004), this
observation may be relevant to the mechanism by which BMPs
regulate the actin cytoskeleton during neuronal dendrite
morphogenesis (Fig. 4B) (Foletta et al., 2003; Lee-Hoeflich et
al., 2004). Possible misregulation of this pathway during
primary pulmonary hypertension has also been proposed
(Foletta et al., 2003; Lee-Hoeflich et al., 2004). The small
GTPase Cdc42 has also been shown to be required for
activation of Limk1 after BMP stimulation (Foletta et al., 2003;
Lee-Hoeflich et al., 2004). In contrast to BMPR-II, the TGF-β
or activin type II receptors lack the critical motifs of the long
tail of BMPR-II, which suggests a pathway-specific link
between the BMP receptor and Limk1. However, the TGF-β
type I receptor can activate the related Limk2 indirectly
through Rho and its downstream effector ROCK1 (Vardouli et
al., 2005). Whether Limk2 can directly associate with TGF-β
receptors has not yet been examined. Thus, both direct physical
links and multi-effector signalling cascades might regulate the
activation of Limk isoforms by TGF-β family members (Fig.
In addition to the activation of Limk isoforms, Smad3-
Smad4 cooperates with Rho and p38 signalling to drive
expression of NET1 and tropomyosins, respectively, which are
important for long-term establishment of actin stress fibres
(Shen et al., 2001; Bakin et al., 2004). In prostate cancer cells,
TGF-β mobilizes the small GTPases RhoA and Cdc42, as well
as the downstream effector p38, to induce membrane ruffling
(Edlund et al., 2002). Interestingly, the inhibitory Smad7
mediates activation of Cdc42 by TGF-β receptors during the
TGF-β β β β
TGF-β β β β
Fig. 4. Regulation of the
actin cytoskeleton and the
EMT response. (A) TGF-β
induces Smads, which
regulate genes such as that
encoding Snail, the
transcriptional repressor of
E-cadherin gene expression
that leads to the dissolution
of adherens junctions.
Alternatively, the receptors
constitutively associate with
occludin and the polarity
protein Par6. Upon ligand
stimulation, the type II
receptor phosphorylates Par6
directly. This then recruits
the ubiquitin ligase Smurf1,
which ubiquitylates and
degrades RhoA, thus leading
to dissolution of tight junctions. The combined outcome of the two
pathways cooperatively promotes EMT. (B) TGF-β activates Rho
GTPases, which activate ROCK, followed by phosphorylation and
activation of Limk2 and subsequent phosphorylation and inhibition of
cofilin. Cofilin is an actin-binding protein that leads to actin
depolymerization. BMP receptors bind directly to Limk1 and activate it,
leading to inhibition of cofilin. The net effect of both pathways is a shift
towards actin polymerization (thick arrow).
Journal of Cell Science
actin response (Edlund et al., 2004), which mirrors its role in
induction of apoptosis in the same system (Edlund et al., 2003).
In systems where cell migration is a clear-cut response, the
role of Smads remains unclear. Migratory metastatic breast
cancer cells, which produce large amounts of autocrine TGF-
β, activate the PI3K/Akt and ERK pathways to drive their
motility (Dumont et al., 2003). A similar mechanism, which
also involves Rac1, is activated by TGF-β in mammary
epithelial cells transformed by overexpression of the epidermal
growth factor receptor 2 (HER2) (Ueda et al., 2004). Genetic
evidence from MEKK1-knockout mice strongly implicates
MEKK1 and the downstream MAPK JNK in the migratory
properties of the eyelid epithelium and the underlying effects
on the actin cytoskeleton induced by TGF-β or activin (Zhang
et al., 2003). In the same fashion, keratinocytes migrate in
response to activin by using the RhoA-ROCK-MEKK1-
JNK/p38 pathway (Zhang et al., 2005).
The above evidence indicates that a combination of Smad
and non-Smad signals is important for morphogenic responses
of cells to TGF-β. Thus, whereas specific inhibitors of Smad3
have been emphasized for the treatment of tumour metastasis
(Flanders, 2004), a combination of Smad inhibitors and kinase
or GTPase inhibitors may be more efficacious.
TGF-β was discovered as a factor that induces anchorage-
independent growth of fibroblasts (Roberts et al., 1980; Moses
et al., 1981). Thus, initially, it was thought to stimulate cell
proliferation. Subsequently, TGF-β was shown to inhibit
proliferation of epithelial cells and lymphocytes (Tucker et al.,
1984; Pietenpol et al., 1990). Today, it is widely accepted that
TGF-β inhibits the growth of non-transformed epithelial,
endothelial and haematopoetic cells, and also primary
fibroblasts of embryonic origin (reviewed by ten Dijke et al.,
2002). Nevertheless, TGF-β has mitogenic activity in certain
transformed cells and in immortalized fibroblasts (Alexandrow
and Moses, 1995).
The growth inhibitory pathway induced by TGF-β includes
critical regulators of the G1 phase of the cell cycle, whose gene
expression is modulated by Smads (Massagué, 2004). These
include the cell-cycle inhibitors p15, p21 and p57, which are
induced by Smad signals, the proto-oncogene product Myc,
and the inhibitors of differentiation (Id1, Id2 and Id3), which
are repressed by Smads (Fig. 5). p21 is induced rapidly by all
TGF-β superfamily receptor complexes (Pardali et al., 2005).
Smads, together with FoxO, p53 and Sp1, form large
transcriptional complexes on the p21 promoter enhancer (Fig.
5) (Datto et al., 1995; Moustakas and Kardassis, 1998; Pardali
et al., 2000; Cordenonsi et al., 2003; Seoane et al., 2004).
However, p21 is also induced by TGF-β through mechanisms
that involve Ras, MEKK1 and ERK (Hu et al., 1999; Kivinen
and Laiho, 1999). Furthermore, p21 can be induced in tumour
cells that lack Smad4 (Ijichi et al., 2004), which indicates that
the role of Smad4 in the regulation of this gene is not exclusive.
In human keratinocytes and hepatoma cells, TGF-β activates
protein kinase Cα (PKCα) (Miyazaki et al., 2004; Sakaguchi
et al., 2004). PKCα phosphorylates the regulatory protein
S100C/A11, which translocates to the nucleus, binds to
transcription factor Sp1 and recruits it to the promoters of the
p15 and p21 genes. This pathway is very similar to the parallel
Smad pathway, which also induces these genes through
interactions between Smad proteins and Sp1 (Fig. 5) (Feng et
al., 2000; Pardali et al., 2000). Simultaneously, PKCα can
phosphorylate Smad3 close to its DNA-binding domain and
thereby block its transcriptional activity (Yakymovych et al.,
2001). Thus, the two mechanisms might constitute a single
integrated network for quantitative regulation of p21 and
deserve further analysis.
Recent reports shed light on a novel mechanism by which
the Smads themselves can contribute to the activation of non-
Smad signalling proteins (Lee et al., 2004; Zhang et al., 2004).
Smad2 can activate ERK in carcinoma cells growing in
suspension, and Smad3 binds to the regulatory subunit of
protein kinase A (PKA) and activates the enzyme
independently of cAMP levels (Fig. 5). This effect is linked to
the transcriptional activation of p21 and subsequent inhibition
of cell growth.
Alternative pathways in the mechanism of cell growth
control by TGF-β include inhibition of p70 S6 kinase through
dephosphorylation by protein phosphatase PP2A (Petritsch et
al., 2000). The TGF-β receptor complex binds directly to its
regulatory subunit PP2A-Bα (Griswold-Prenner et al., 1998;
Petritsch et al., 2000). This leads to assembly of the tri-subunit
(PP2A-Bα, Aβ, Cα) phosphatase and its association with p70
Journal of Cell Science 118 (16)
TGF-β β β β
Bα α α α
Fig. 5. Epithelial growth suppression induced by TGF-β. The TGF-β
receptors activate Smads, which induce p21 expression in
cooperation with the transcription factors FoxO, p53 and Sp1. The
Smad pathway also induces transcription of p15 or p57 and represses
expression of Myc and Id genes. The net result of all these
transcriptional events is the arrest of the cell cycle in early G1 phase.
Smads can also activate PKA, which leads to Sp1 phosphorylation
and induction of p21 expression. The TGF-β receptor also binds the
regulatory subunit Ba of PP2A phosphatase, leading to inactivation
of p70 S6K kinase, thus indirectly inhibiting cell-cycle progression.
Journal of Cell Science
Non-Smad TGF-β signals
S6 kinase (Fig. 5). Alternatively, TGF-β activates the small
GTPase RhoA, which activates ROCK1, ultimately leading to
phosphorylation and inactivation of the phosphatase Cdc25A
in epithelial cells (Bhowmick et al., 2003). Cdc25A
dephosphorylates cyclin-dependent kinases and thus promotes
cell-cycle progression, whereas inactivation of Cdc25A
contributes to cell-cycle arrest in early G1 phase. Similarly,
Rho and p38 activation in cooperation with the Smad pathway
elicits growth arrest of mammary epithelial cells (Kamaraju
and Roberts, 2005).
The mitogenic response of fibroblasts to TGF-β involves
activation of the cytoplasmic kinase PAK2 (Wilkes et al.,
2003). Small GTPases, such as Rac1 or Cdc42, link TGF-β
receptors to PAK2; Smad2 and Smad3 are possibly dispensable
for this pathway, which seems to operate in fibroblasts but not
in epithelial cells. TGF-β also induces mitogenesis of
carcinoma cells, especially those from advanced, invasive
tumours that harbour mutations in Ras. In such cells, signalling
by the Ras pathway appears to be crucial and, in them, TGF-
β can either induce degradation of the cell cycle inhibitor p21
or fail to induce p15 and p21, thus ultimately leading to cell-
cycle stimulation (Yan et al., 2002). However, in other tumour
cells, TGF-β can stabilize p21 levels independently of its
Smad-mediated transcriptional effects (Gong et al., 2003).
proliferation and self-renewal of stem cells. For example,
BMP-4 signals through ERK and p38 to support such self-
renewal of embryonic stem cells (Qi et al., 2004).
The growth inhibitory response of epithelial cells to TGF-β
thus appears to be governed by gene expression programs
regulated by combinations of Smad and non-Smad signalling
molecules. More rigorous analysis of the mitogenic effects of
TGF-β are warranted, and even more critical is the need to
decipher whether the above models apply to the relevant tissues
members stimulate the
TGF-β induces several genes that encode major constituents of
the extracellular matrix and matrix regulatory enzymes
(reviewed by Siegel and Massagué, 2003; Schiller et al., 2004),
including plasminogen activator inhibitor 1 (PAI-1),
collagenase I and the collagens (Dennler et al., 1998; Qing et
al., 2000; Javelaud et al., 2003). Smads regulate PAI-1
expression in cooperation with transcription factors, such as
Sp1 and TFE3 (Dennler et al., 1998; Hua et al., 1998; Song et
al., 1998; Stroschein et al., 1999; Datta et al., 2000), but signals
from ERK and Rac1 are also required (Mucsi et al., 1996; Kutz
et al., 2001). Fibronectin gene expression was originally shown
to depend on JNK signals activated by TGF-β in a Smad-
independent manner (Hocevar et al., 1999), but subsequently a
role for Smads was recognized (Itoh et al., 2003; Dai and Liu,
2004; Kowanetz et al., 2004). By contrast, regulation of the
urokinase-type plasminogen activator receptor gene by TGF-β
seems to require exclusively the Ras/MKK4/JNK1 pathway
(Yue et al., 2004).
Other non-Smad effectors that regulate expression of genes
encoding matrix components include the calcium-dependent
phosphatase calcineurin and its downstream partner NFATc,
and the tyrosine kinase Abl (Daniels et al., 2004; Gooch et al.,
2004). The latter has clinical importance because an Abl
inhibitor, Imatinib/Glivec, successfully blocks TGF-β-induced
lung and kidney fibrosis. This suggests that also targeting non-
Smad TGF-β pathways can be beneficial for treatment of TGF-
β-induced disease (Daniels et al., 2004; Wang et al., 2005).
Regulation of gene expression by TGF-β superfamily members
is often critically linked to cell differentiation. Osteoblasts, for
example, require BMP inputs to differentiate from pluripotent
progenitor cells, and Smads together with transcription factors
of the Runx family and Id proteins contribute greatly to this
process (reviewed by ten Dijke et al., 2003; Miyazono et al.,
2004). In addition, accumulating evidence implicates the p38,
ERK and JNK pathways in osteoblast differentiation in
response to BMP-2, BMP-4 or BMP-7 (Gallea et al., 2001; Lai
and Cheng, 2002; Vinals et al., 2002; Xiao et al., 2002;
Guicheux et al., 2003). Moreover, regulation of Runx2
expression by TGF-β and BMPs involves both Smad and p38
inputs (Lee et al., 2002). In a parallel scenario, chondrocyte
differentiation involves Smads and non-Smad effectors such as
PKA, p38 and ERK (Lee and Chuong, 1997; Valcourt et al.,
2002; Noth et al., 2003; Seto et al., 2004).
An alternative mode of BMP receptor signalling during
differentiation has been revealed by studies of the inherited
disorder brachydactyly (Sammar et al., 2004). Here, GDF-5, a
member of the BMP family, signals through a receptor
complex that involves the known BMP type I receptor BMPR-
IB and the receptor tyrosine kinase Ror2. BMPR-IB
phosphorylates and activates Ror2, which blocks the BMPR-
IB-induced Smad pathway; these effects are critical for
chondrogenesis, and BMPR-IB and Ror2 are mutated in
alternative forms of brachydactyly. Thus, like cell proliferation,
apoptosis and cell migration, cell differentiation induced by
TGF-β family members frequently utilizes the abundant
MAPK modules and occasionally alternative non-Smad
Evidence from developmental studies
Most of the studies described so far have used in vitro cell
culture models, which are often indispensable for the
elucidation of signalling and gene regulatory mechanisms, but
have obvious limitations. Below, we briefly summarize a few
developmental studies that provide in vivo evidence for non-
Smad signalling mechanisms.
During migration of embryonic epithelia in Drosophila, the
JNK pathway and the small GTPases Dcdc42 and Drac1 are
important regulators of decapentaplegic (Dpp) expression and
secretion, and eventually synergize with Dpp signalling (the
Drosophila BMP pathway) (Glise and Noselli, 1997). During
this migratory process, cytoskeletal regulation is very
important, and the JNK and Drac1 pathways cooperate with
Dpp-activated Dcdc42 and DPAK, which become the key
players (Ricos et al., 1999). This scenario is similar to that
discussed earlier in the context of the MEKK1 knockout
(Zhang et al., 2003). Furthermore, the critical role of
Drosophila JNK in secretion of Dpp resembles that of JNK in
autocrine TGF-β secretion that has been revealed in studies of
JNK1-JNK2 double-knockout mice (Ventura et al., 2004).
During Xenopus development, the XrhoA protein regulates
Journal of Cell Science
formation of the body axis and head in response to BMP
signalling (Wunnenberg-Stapleton et al., 1999). In chicken
neural crest development, BMP signalling similarly induces
RhoB expression, which is necessary for neural crest
delamination (Liu and Jessell, 1998). During chicken limb
development, BMP-5 induces expression of many genes that
drive the process, and both Smads and cooperating p38
pathways mediate this effect (Zuzarte-Luis et al., 2004).
Finally, in a rat model of blood-testis barrier development,
TGF-β3 signals in Sertoli cells through p38 to downregulate
occludin expression (Lui et al., 2003).
The above studies provide some in vivo evidence that non-
Smad signalling contributes to crucial developmental processes
downstream of TGF-β factors. However, the plethora of non-
Smad proteins discussed above remains largely untested by
rigorous in vivo developmental or genetic studies.
Modulation of Smad activity by non-Smad effectors
An interesting feature of non-Smad signalling proteins is their
ability to modulate Smad activity, most frequently negatively.
One of the first clearly demonstrated examples of such
regulation was the discovery that the ERK pathway can lead to
direct phosphorylation of specific serine residues in the linker
domain of R-Smads, blocking their nuclear translocation and
transcriptional output (Kretzschmar et al., 1997). This finding
has been corroborated by in vivo studies in Xenopus (Pera et
al., 2003). In addition, the p38 substrate MSK1 kinase
regulates the transcriptional activity of Smad3 by promoting its
association with the co-activator p300 (Abecassis et al., 2004).
TGF-β-activated JNK can phosphorylate Smad3 and induce its
nuclear translocation and transcriptional activity (Engel et al.,
1999). JNK also phosphorylates Jun, which enhances
formation of complexes by Smad2 and its co-repressor protein
TGIF and thus inhibits Smad2-dependent transcription (Pessah
et al., 2001). A similar effect is evident in TGF-β activation of
the PI3K pathway and the downstream phosphoinositide-
dependent kinase 1 (PDK1), which phosphorylates Smad3 and
enhances its transcriptional activation of the collagen I gene in
mesangial cells (Runyan et al., 2004). The same mechanism
seems to operate downstream of BMPs during osteoblast
differentiation (Ghosh-Choudhury et al., 2002): BMP-2
activates PI3K/Akt, which enhances the transcriptional activity
of the BMP-specific Smad5. Thus, the crosstalk between
Smads and kinase effectors whose activity is stimulated by
TGF-β ligands can be either positive or negative and seems to
permeate many physiological processes.
A commonly used term in the TGF-β field is the discrimination
between Smad-dependent and Smad-independent signalling
pathways. An important tool for the delineation of so-called
‘Smad-independent’ pathways is a collection of mammalian
cells that lack the gene encoding Smad4 because of a deletion
that occurred either in knockout mice or in various tumour cells
(Hocevar et al., 1999; Sirard et al., 2000). For example,
embryonic fibroblasts from the Smad4-knockout mouse were
used to demonstrate that these cells maintain a number of
cellular and gene responses to TGF-β (Sirard et al., 2000).
Comparative gene profiling screens in the Smad4-null human
mammary carcinoma cell line MDA-MB-468 comparing TGF-
β and BMP signalling demonstrated that essentially all
measurable gene expression responses to TGF-β1 and BMP-7
require reconstitution of wild-type Smad4 (Kowanetz et al.,
2004). Smad4-null cellular tools offer the great advantage that
signalling downstream of TGF-β, activin, BMP or other
ligands is in principle disrupted. However, Smad4-null cells
often express higher levels of R-Smads than normal cells
(Maurice et al., 2001). Moreover, conditionally knocking out
Smad4 in the mouse epiblast showed that only a subset of TGF-
β/nodal and BMP responses require Smad4 (Chu et al., 2004).
Thus, data supporting ‘Smad-independent’ signalling solely on
the basis of studies performed in Smad4-deficient cells must
be interpreted with caution. More recent approaches in which
mice lacking Smad2 or Smad3 were used are more indicative
because R-Smads are the primary effectors activated by TGF-
β receptors (Piek et al., 2001; Yang et al., 2003). In addition,
the use of RNA interference (RNAi) technology promises a
potentially more reliable tool that can examine the role of all
Smads downstream of TGF-β members (Kretschmer et al.,
2003; Imamura et al., 2004). Tissue-specific multi-Smad-
knockout experiments in mice or multi-Smad RNAi
approaches are warranted to address this fundamental problem.
Nuclear signals transmitted by non-Smad proteins could
regulate transcription independently or synergize with Smads.
The latter seems to apply for most genes that receive signalling
inputs from the TGF-β superfamily (Massagué, 2000).
Nevertheless, many recent efforts have searched for specific
Smad-dependent and non-Smad transcriptional modules by
gene expression profiling. The pioneering work of Zavadil and
colleagues (Zavadil et al., 2001) has provided an example: they
measured the contribution of ERK signalling to the profile of
gene expression downstream of TGF-β. Similar systematic
approaches should soon shed further light on this area.
Whereas a classical view of TGF-β signalling is based on a
relatively simple and linear Smad pathway that links receptor
complexes on the plasma membrane to gene regulation in the
nucleus, increasing evidence indicates that these signalling
pathways are more complex networks. Multiple effector
proteins can participate and transmit signals to specific cellular
compartments, such as the actin cytoskeleton or the apoptotic
machinery. In addition, non-Smad proteins can affect the
activity of Smads and thus integrate with Smad signalling. A
central feature of all these networks is the cell-type- and
context-dependent mode of activation, which is a principal
characteristic of TGF-β biology. Future studies, aimed at
identification of additional components in such networks, need
to focus more closely on the receptor complexes and identify
novel cytoplasmic proteins whose function is directly regulated
by the receptors. Analysis of such nodal points in the signalling
networks in vitro and in vivo in model organisms should place
non-Smad effectors closer to the heart of TGF-β signalling.
Owing to space limitations, selected literature is cited in this
commentary. Funding of the authors’ work is provided by the
Ludwig Institute for Cancer Research, the Swedish Cancer Society,
the Swedish Research Council and the European Commission
FP6 programme. We thank all current and previous members of
our laboratories for their contributions to the scientific work.
Special thanks to K. Miyazono (Tokyo University) and P. ten Dijke
(Leiden University) for continuous
Journal of Cell Science 118 (16)
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