REVIEW ARTICLE
Control of transforming growth factor b signal
transduction by small GTPases
Dimitris Kardassis
1,2
, Carol Murphy
3
, Theodore Fotsis
3,4
, Aristidis Moustakas
5
and
Christos Stournaras
1
1 Department of Biochemistry, University of Crete Medical School, Heraklion, Greece
2 Institute of Molecular Biology and Biotechnology, Foundation for Research & Technology-Hellas, Heraklion, Greece
3 Biomedical Research Institute, Foundation for Research & Technology-Hellas, Ioannina, Greece
4 Laboratory of Biological Chemistry, University of Ioannina Medical School, Greece
5 Ludwig Institute for Cancer Research, Uppsala University, Sweden
Transforming growth factor b (TGFb) is the prototype
member of a large, evolutionarily conserved, superfam-
ily of pleiotropic cytokines that also includes activins,
bone morphogenetic proteins (BMPs) and growth and
differentiation factors, among others [1]. TGFb con-
trols various physiological processes during embryo-
genesis and is an important homeostatic regulator in
various cell types, for example, epithelial and endo-
thelial cells in adult organisms [1–3]. TGFb is a growth
suppressor because of its cytostatic program [4].
However, during the late stages of cancer and metasta-
sis, TGFb acts as a tumor promoter because of its

and their regulation by the small GTPases Rab, RalA ⁄ Ral-binding protein
1 and Rap2. Second, we focus on the mechanisms and regulation of Smad
trafficking in the cytoplasm, through the nuclear pores and into the
nucleus, and the contribution of Ran GTPase to these events. Third, we
summarize the role of Rho small GTPases in early and late cytoskeleton
remodeling in various cell models and diseases, and the positive and nega-
tive cross-talk between Rho GTPases and the TGFb ⁄ Smad pathway. The
biological significance of this exciting research field, the perspectives and
critical open questions are discussed.
Abbreviations
AP1, activating protein 1; ARIP2, activin receptor interacting protein 2; BMP, bone morphogenetic proteins; CCVMR, clathrin-coated vesicle-
mediated route; CRM1, chromosome region maintenance 1; EH, Eps15 homology; EMT, epithelial to mesenchymal transition; Endofin,
endosome-associated FYVE-domain protein; GAP, GTPase activating protein; GEF, guanine exchange factor; NES, nuclear export signal;
NLS, nuclear localization signal; RalBP1, Ral-binding protein 1; ROCK, Rho coiled-coiled kinase; SARA, Smad anchor for receptor activation;
TGFb, transforming growth factor b;TbRI, TGFb type I receptor; TbRII, TGFb type II receptor.
FEBS Journal 276 (2009) 2947–2965 ª 2009 The Authors Journal compilation ª 2009 FEBS 2947
cytoplasmic effector proteins termed Smads [8,9].
TGFb promotes receptor oligomerization which leads
to the phosphorylation of its type I receptor (TbRI) by
the constitutively active type II receptor (TbRII). Acti-
vated TbRI (also called ALK5), phosphorylates Smad2
and Smad3 at their C-terminal SSXS motifs [8–10].
The R-Smads, in turn, oligomerize with the common
partner Smad4 and rapidly translocate to the nucleus
where they bind to the promoters of a large variety of
target genes and regulate their expression in a positive
or negative manner [8–10]. TGFb target genes code for
proteins involved in cell-cycle regulation, apoptotic
regulation, extracellular matrix production, cytokine
signaling, transcriptional regulation, differentiation

To date, five emerging transport routes for proteins
that become internalized have been identified: the
clathrin-coated vesicle-mediated route (CCVMR),
macropinocytosis ⁄ phagocytosis, the APPL route, the
caveolar route and the nonclathrin and noncaveolar
pathways [22]. Therefore, it is clear that understanding
the endocytic route followed by TGFb receptor–ligand
complexes will allow a systems-level molecular dissec-
tion of the signaling regulators of TGFb.
Since the discovery and molecular cloning of Smad
proteins, it has been known that Smads rapidly accu-
mulate in the cell nucleus upon activation of the TGFb
receptors [23–26]. The original studies gave a static
view of the pathway, whereby Smads were thought to
reside firmly in the cytoplasm and translocate rapidly
into the nucleus upon activation via receptor-mediated
phosphorylation. Twelve years later, we appreciate that
Smad proteins show a very dynamic behavior within
the cell because they constantly shuttle in to and out
of the nucleus [27].
Furthermore, both TGFb receptor endocytosis and
Smad trafficking seem to rely on interactions and
cross-talk with the cytoskeleton, including micro-
tubules and actin-based microfilaments [10]. Such
cross-talk facilitates the timely movement and accurate
transport of signaling components to their various des-
tinations. In addition, TGFb signaling has a profound
impact on the regulation of the actin cytoskeleton,
which supports various physiological and developmen-
tal processes such as cell motility, differentiation

TGFb signaling and small GTPases D. Kardassis et al.
2948 FEBS Journal 276 (2009) 2947–2965 ª 2009 The Authors Journal compilation ª 2009 FEBS
interlinked [34,35]. Furthermore, transport from early
to late endocytic compartments is controlled by the
cargo, and activated receptors may alter the kinetics to
modulate their signaling duration [36].
Is internalization required for TGFb family
signaling?
The presence of SARA, Hrs and Endofin in early end-
ocytic compartments questions whether signaling can
occur from the plasma membrane or whether internali-
zation is required to bring activated receptors to the
endosome which is enriched in SARA and Endofin.
This issue remains controversial, reflecting differences
in experimental approaches and their limitations.
TbRII undergoes constitutive internalization in the
absence of ligand via clathrin-coated pits. This process
is dependent on a short sequence (I
218
-I
219
-L
220
) that
conforms to the di-leucine family of internalization sig-
nals [37,38] and the direct binding of type I and II
receptors to b2-adaptin [39]. No di-leucine motifs have
been found in type I receptors. Interestingly, the
NANDOR box is well conserved throughout type I
receptors [40] and appears to play a role in type I

extracellular matrix polysaccharide, attenuated TGFb
signaling by increasing the segregation of TGFb recep-
tors into a lipid raft–caveolar compartment [48],
whereas ADAM12 (a disintegrin and metalloprotein-
ase) facilitated signaling by inducing the accumulation
of TbRII in early endosomal vesicles and counteract-
ing the internalization of TbRII into a caveolin1-posi-
tive compartment [49]. Likewise, interleukin-6
augmented TGFb signaling by increasing partitioning
of TGFb receptors to the nonlipid raft fraction (early
endosomal) [50]. No significant caveolar internalization
was observed in the study by Mitchell et al. [42], in
which nystatin (used at lower, more specific doses) had
no effect on receptor internalization and degradation.
Moreover, TGFb receptors did not exhibit consi-
derable co-localization in compartments positive for
caveolin-1 [42].
What about the role of endocytosis in the signaling
of other members of the TGFb receptor family? With
regard to activin A signaling, an ALK4 mutant,
Alk4W477A, that was unable to undergo activin-
dependent internalization, retained the ability to signal,
demonstrating that ALK4 can signal without receptor
internalization [51]. However, in another detailed study
addressing the memory of Xenopus embryonic cells to
activin A exposure, the critical step in determining the
duration of activin A signaling was the time spent by
the ligand ⁄ receptor complexes in the endo-lysosomal
pathway. Activin A internalization was required for
correct signaling, suggesting that the localization of

a means of rapidly and dynamically regulating surface
receptor number and thus sensitivity to TGFb [42].
Recent biochemical data has shed more light on the
link between BMP signaling and endocytic trafficking.
BMPRI and BMPRII appear to be continuously inter-
nalized via CCVMR endocytosis, and BMPRII is also
endocytosed via a caveolae- and cholesterol-dependent
route [58]. Smad1 ⁄ 5 phosphorylation seems to occur at
the plasma membrane; however, continuation of
Smad1 ⁄ 5-dependent signaling requires internalization
via the CCVMR. The BMP receptor population that
resides in cholesterol-enriched, detergent-resistant
membrane fractions is required for Smad-independent
BMP signaling [58]. However, downregulation of cave-
olin-1 via siRNA resulted in a loss of BMP-dependent
Smad phosphorylation and gene regulation, and was
not linked only to Smad-independent signaling [59].
Rab GTPases
Rab GTPases are master regulators of vesicular trans-
port and are distributed in distinct intracellular com-
partments. Rab5 is a key regulator of endocytosis that,
by interacting with multiple effectors [60], regulates
organelle-tethering, fusion and motility. Rab7 localizes
to the late endocytic compartment and controls the
trafficking of late endosomes [61]. Therefore, conver-
sion of Rab5 to Rab7 controls the progression of
cargo from the early to the late endocytic compart-
ment, but the cargo itself can also modulate the kinet-
ics of this transport step [36]. Thus, inputs from the
Rab5 ⁄ 7 machinery or cargo (activated growth-factor

once trafficked there. This is important because the
signaling outcome is proportional to the residence time
in this compartment. Trafficking of TGFb family
receptors via early endosomes with extremely fast
kinetics will most likely have a minimal enhancing
effect on signaling compared with early endosomal
trafficking that is accompanied by blocking of further
trafficking. Indeed, several studies on activin A and
BMPs have revealed that the enhancing effect on sig-
naling of various proteins was dependent on how long
the relevant receptors resided on early endosomes [52–
54]. Whether conversion of Rab5 to Rab7 or other
mechanisms are responsible remains open. Our previ-
ous results suggest that Rab5 cycling between the GTP
and GDP forms may influence the length and intensity
of TGFb ⁄ activin signaling cascades by regulating
TGFb–activin type I ⁄ II receptor trafficking via the
early endocytic compartment [17]. Indeed, in endothe-
lial cells, Rab5S34N, a Rab5 mutant locked in the
GDP form, caused augmented Smad3-dependent tran-
scription in the absence of ligand. Because RN-tre, a
specific Rab5 GTPase-activating protein (GAP) that
blocks plasma membrane endocytosis, did not influ-
ence Smad3-dependent transcription, we concluded
that the effect of Rab5S34N should have been the con-
sequence of decreased degradative or recycling traffick-
ing, leading to an accumulation of constitutively
formed TGFb–activin type I ⁄ II receptor complexes on
early endosomal membranes.
Certainly, the station after early endosomes in the

[68–70]. Activated Ral associates with the Ral effector
Ral-binding protein 1 (RalBP1), a cytosolic protein
that is recruited to membranes following Ral activa-
tion [71] and activates hydrolysis of GTP bound to
Rac1 and Cdc42. RalA has been implicated in many
intracellular trafficking events [72] from the regulation
of the endocytosis of EGF and insulin receptors [73]
to secretion [74]. Indeed, RalA, via its effector protein
RalBP1, interacts with the l2 subunit of the AP-2
complex [75] as well as with REPS1 [76] and POB1
[77] which are EGF receptor substrates containing
Eps15 homology (EH) domains. POB1 interacts
directly with the EH-containing proteins epsin and
eps15, which have been reported to be involved in the
regulation of EGF and transferin receptor endocytosis
[67,78,79]. Thus, activation of RalA by EGF and insu-
lin suggests that RalA ⁄ RalBP1 and its interactions
with the l2 chain of AP-2, REPS1, POB1, epsin and
eps15 act as a scaffold that conveys signals from recep-
tors to the endocytic machinery, thereby regulating
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RalA and RalBP1 appear to be involved in activin
A receptor trafficking and signaling (Fig. 1B). It has
been shown that activin receptor interacting protein 2
(ARIP2) interacts with ActRIIs and regulates their
endocytosis via a PDZ domain-mediated interaction,
concentrating them in a perinuclear compartment.
Thus, ARIP2 reduces the response to ligands by
decreasing the levels of ActRII at the plasma
membrane [81]. ARIP2 triggers the endocytosis of
ActRIIs via Ral ⁄ RalBP1. Indeed, ARIP2 associates
with ActRIIA and RALBP1 via its PDZ domain and
C-terminal region, respectively. Because ARIP2C, the
C-terminal deletion mutant of ARIP2 that does not
bind RalBP1, failed to induce ActRII endocytosis, it
appears that endocytosis of ActRIIs by ARIP2 is
RalA ⁄ RalBP1 dependent. Moreover, activin A acti-
vates GDP–GTP exchange in RalA [81]. Activation of
RalA ⁄ RalBP1 by activin A is calcium dependent, in
contrast to activation by EGF and insulin, which
occurs via a Ras-dependent cascade [73]. Interestingly,
because only ActRIIs among all the serine ⁄ threonine
kinase receptors for BMP ⁄ TGFb ⁄ activin have the
PDZ-binding sequence (ESSL for ActRIIA and ESSI
for ActRIIB) [82], PDZ protein-regulated endocytosis
and sorting is expected to influence only ActRIIs.
Because ActRIIs bind both activins and also nodal
and BMP7, ARIP2 is likely to play a role in shaping
the activin ⁄ nodal ⁄ BMP gradient by regulating the
endocytosis of ActRIIs.
Rap2

Growing evidence links the progression of TGFb
receptor signaling to key regulatory steps in endocytic
trafficking. These steps involve the active regulation of
GDP-to-GTP exchange by various small GTPases of
the Rab ⁄ Ral and Rap families. These mechanisms
ensure optimal signal transduction from active receptor
complexes to activated Smads.
Intracellular Smad trafficking – the role
of the Ran GTPase
Most current evidence on the mechanisms that govern
the dynamic shuttling of Smad proteins in the cell is
based on the behavior of engineered GFP–Smad2 and
GFP–Smad4 fusion proteins which are stably
expressed in human cells cultured in vitro. The evi-
dence supports a model whereby Smads shuttle con-
stantly, although each specific Smad seems to obey
distinct kinetic properties during its movements [86].
Mathematical modeling of Smad protein shuttling has
recently suggested that the strength of Smad signaling
depends directly upon the length of time a certain
Smad molecule spends in the nucleus [87]. Such kinetic
analysis also emphasized that the nuclear export of
Smads is highly regulated, whereas the nuclear import
of Smads may act as a default pathway.
The evidence from the in vitro cell system is comple-
mented by pioneering in vivo studies first developed in
Xenopus embryos [88,89]. Continuous shuttling of
Smad2 could be observed in developing Xenopus and
zebrafish embryos [88]. Furthermore, Smad2 and
Smad4 proteins fused to fluorescent protein fragments

on microtubules [90]. Another motor-like protein that
associates with Smad2 is the dynein light chain km23-1,
which assists in the nuclear accumulation of Smad2,
and also regulates trafficking of the TbRI [91]. Accord-
ing to this new evidence, cytoplasmic Smads traffic
towards the signaling receptors with the help of kinesin
motors that slide on microtubules. The signaling recep-
tors most likely reside on endosomes, as discussed
above. However, cytoplasmic Smad trafficking towards
the nucleus involves the dynein motor–microtubule
machinery. Although it makes sense to consider micro-
tubules as trafficking highways that facilitate the
movement of Smad proteins, microtubules have also
been shown to act as cytoplasmic traps for Smads [92].
According to this model, connexin 43 is a regulatory
protein that competes with Smads for binding to
microtubules. However, the latter mechanism needs to
be further clarified as it is important to understand
which factor regulates the residence of Smads on
microtubules versus their mobility along microtubules
and towards neighboring cellular locations.
The association of Smads with microtubules pro-
vides additional insight into the functional regulation
of these proteins. In dividing cells, such as those of the
Xenopus embryo, Smads can associate with the spindle
and decorate the metaphase chromosomes [89]. This
evidence is compatible with a role for microtubules in
trapping Smads and protecting their integrity, thus
delivering them safely to the daughter cells after mito-
sis. It remains unclear as to whether Smad signaling

Smad2 isoform that incorporates exon 3 fails to bind
to importin-b, whereas the shorter Smad2 isoform that
lacks exon 3 binds to importin-b similar to Smad3
[95]. In addition, the importin moleskin mediates the
nuclear entry of the Drosophila R-Smad Mad, and its
human orthologues, importin-7 and importin-8,
mediate the nuclear translocation of Smad1, Smad2,
Smad3 and Smad4 in human cancer cells in response
to BMP or TGFb signaling [99]. Future work may
explain why Smads utilize multiple importins for their
entry to the nucleus (Fig. 2).
Importins are known to move through the pore by
consecutive contacts with the phenylalanine ⁄ glycine
(F ⁄ G)-rich repeats of specific nucleoporins. Such step-
wise translocation is energetically demanding and
requires GTP expenditure. Similar to the role of Rab
GTPases that control the trafficking of endocytic vesi-
cles during TGFb signaling in the cytoplasm (Fig. 1),
the small GTPase Ran controls Smad3 trafficking via
the nuclear pore (Fig. 2) [95]. Ran is a small GTPase
D. Kardassis et al. TGFb signaling and small GTPases
FEBS Journal 276 (2009) 2947–2965 ª 2009 The Authors Journal compilation ª 2009 FEBS 2953
dedicated to the control of nucleocytoplasmic traffick-
ing and chromosomal segregation during mitosis [100].
A Ran activity gradient is established through the
nuclear pore with high Ran–GDP concentrations in
the cytoplasmic phase of the pore which gradually
decrease along the pore [101]. In the nuclear phase of
the pore, the Ran-specific GEF RCC1 loads Ran with
GTP, thus establishing a high Ran–GTP concentration

replenishing the cytoplasmic pool of Smads with mole-
cules that are ready to become activated again, as long
as the receptors remain active. The importance of
nuclear export is underscored by the presence of nuclear
export signals (NES) in all Smads examined to date.
Smad4 carries a leucine-rich NES in its linker domain,
which mediates export via exportin-1 ⁄ chromosome
region maintenance 1 (CRM1) (Fig. 2) [105,106]. Muta-
tion of hydrophobic amino acids within the Smad4 NES
or exposure of cells to the pharmacological inhibitor of
CRM1 leptomycin-B, lead to an exclusive nuclear distri-
bution of Smad4, independent of the presence or
absence of ligand. Smad3 is exported from the nucleus
in a CRM1-independent manner and an extended
peptide surface of the MH2 domain has been identified
as critical for this export by exportin-4 [107]. In the case
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to the cytoplasm (not shown). Nuclear Smads associate with exprotins (Exp) and Ran–GTP and translocate to the cytoplasm by making con-
tacts with nucleoporins. The cytoplasmic Smad–exportin–Ran–GTP complex is disrupted by the action of RanGAP, which releases Smad,
exportin and Ran–GDP, and free orthophosphate after the hydrolysis of GTP. Completion of the Ran cycle is shown in the middle for Smad3
because the role of Ran has only been analyzed in detail in the case of Smad3. Cytoplasmic Ran–GDP (grey symbol) diffuses through the
nuclear pore where it meets the nuclear GEF RCC1, which exchanges GDP for GTP and restores nuclear Ran–GTP (black symbol) levels.
TGFb signaling and small GTPases D. Kardassis et al.
2954 FEBS Journal 276 (2009) 2947–2965 ª 2009 The Authors Journal compilation ª 2009 FEBS
of Smad3, the role of Ran has been studied and it was
clearly demonstrated that, similar to many other
exported proteins, Ran supports the movement of
Smad3 via the nuclear pore towards the cytoplasm
(Fig. 2). The Smad3 NES has no obvious resemblance
to a bipartite leucine-rich motif identified in the MH2
domain of Smad1, the R-Smad of the BMP pathways,
which is thought to be recognized by CRM1 based on
leptomycin-B inhibitor experiments [108]. The role of
Ran in mediating the export of proteins from the
nucleus follows the inverse biochemical steps used for
import of proteins to the nucleus [100,102]. Ran–GTP
promotes the association of Smad3 with exportin-4 in
the nuclear phase of the pore [107]. Upon trafficking
via the nuclear pore, Ran–GTP in complex with cargo
is attacked by Ran GAP, which is associated on the
cytoplasmic phase of the nuclear pore, and activates
the GTPase activity of Ran so that GTP is hydrolyzed
to GDP and orthophosphate (Fig. 2) [100,102]. This
leads to conformational changes in Ran that facilitate
disruption of the complex between exportin and
its cargo, and the ultimate release of cargo to the
cytoplasm.

homolog, the WW domain protein YAP, which might
also be involved in a similar mechanism. Thus, we
await significant developments in Smad nuclear traf-
ficking that might provide a more comprehensive view
of how the entry and exit of Smads from the nucleus
coordinates with transcription. It will also be interest-
ing to examine the role of additional nuclear small
GTPases as regulators of nuclear Smad function,
because this class of proteins offers a versatile regula-
tory system that empowers biological processes with
the ability to switch on and off.
The role of small GTPases of the Rho
subfamily in TGFb-induced actin
cytoskeleton remodeling
Actin cytoskeleton remodeling is one of the earliest
cellular responses to extracellular stimuli [110–115].
Binding of ligands to the appropriate receptors triggers
specific signaling cascades, which may generate rapid
and long-term modifications of actin polymerization
dynamics and microfilament organization [116–120].
Among the specific signaling effectors regulating actin
architecture, the family of small Rho GTPases has a
prominent role. Classically, plasma membrane recep-
tors activate specific guanine-exchange factors often
via phosphorylation, which leads to the subsequent
activation of Rho GTPases [121]. Rho GTPases have
been implicated in many cellular processes, including
actin and microtubule cytoskeleton organization, cell
division, motility, cell adhesion, cell-cycle progression,
vesicular trafficking, phagocytosis and transcriptional

The ability of TGFb to regulate actin cytoskeleton
remodeling has been demonstrated in a variety of cell
systems, and specific members of the Rho subfamily of
small GTPases including RhoA, RhoB, Rac and cdc42
have been found to play essential roles (Fig. 3). The
contribution of individual Rho GTPases and their
downstream effectors in TGFb-induced actin remodel-
ing has been studied using a variety of experimental
tools. These tools include constitutively active and
dominant-negative mutants of Rho proteins or their
target proteins, siRNA-mediated gene silencing or gen-
eral inhibition of Rho function using molecules such
as the C3 exoenzyme, which selectively ADP-ribosy-
lates and inactivates low molecular mass G proteins of
the Rho subfamily at an asparagine residue within the
effector domain. Rho GTPase activation is generally
measured by affinity precipitation using appropriate
GST–fusion peptides that bind only to GTP-bound
Rho proteins such as GTP–Rhotekin binding domain
for RhoA and RhoB or GST–p21-activated kinase and
GST–Wiskott–Aldrich syndrome protein for Rac1 and
cdc42 [135]. Changes in the actin cytoskeleton are
monitored by immunofluorescence microscopy of
rhodamin ⁄ phalloidin-labelled actin or by calculating
the ratio of total versus polymerized actin by immuno-
blotting Triton-soluble (globular actin) and Triton-
insoluble (filamentous actin) cell extracts [136].
TGFb-induced cytoskeleton rearrangements
involving Rho activation in EMT
The most extensively investigated TGFb-induced cyto-

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cell type) and is followed by the activation of down-
stream target kinases such as ROCK. Rapid RhoA
activation was found to operate in a variety of cell
models of TGFb-induced EMT under physiological or
pathological conditions. (a) In the atrioventricular
canal of the embryonic chicken heart, TGFb was
found to promote the conversion of endothelial cells to
mesenchymal cells via a pathway that requires the acti-
vation of RhoA [139]. (b) During tubulointerstitial
fibrosis, TGFb promotes the differentiation of tubular
epithelium to mesenchymal cells via a biphasic activa-
tion of RhoA and its downstream target ROCK; a
rapid and transient elevation of RhoA-GTP levels
which was detectable as early as 1 min after TGFb
stimulation and lasted for 5 min, and a chronic eleva-
tion at 24 h of stimulation. Chronic activation was
correlated with the upregulation of a-SMA gene
expression via activating protein 1 (AP1) factors [140].
(c) In proliferative vitroretinopathy, TGFb leads to the
transformation of retinal pigment epithelial cells to
contractile fibroblasts via rapid activation of RhoA
and Rac1 GTPases and their downstram effectors
ROCK kinase, LIMK and cofilin, and the concomitant
upregulation of a-SMA gene expression [141].
TGFb-induced Rho GTPase activation and actin
remodeling in various cell systems
In addition to their role in EMT, Rho GTPases and
their downstream effectors are activated by TGFb and
contribute to cytoskeletal rearrengements in other cell
systems. (a) TGFb promotes the differentiation of neu-

mouse embryo fibroblasts and Swiss3T3 fibroblasts
[145–148]. In fibroblasts transformed by inducible
expression of the H-Ras oncogene, TGFb induced the
formation of new stress fibers from focal adhesions as
early as 15 min post TGFb addition and this
reorganization was associated with an increase in the
polymerization state of actin and in protein levels of
RhoA and RhoB [146].
In Swiss3T3 fibroblasts, TGFb induced rapid activa-
tion of both RhoA and RhoB small GTPases as early
as 5 min post TGFb1 addition which remained high
for 3 h before decreasing [147]. Activation of RhoA
and RhoB was accompanied by phosphorylation of
the downstream effectors LIMK2 and cofilin, whereas
inhibition of ROCK1 completely blocked TGFb1-
induced LIMK2 ⁄ cofilin phosphorylation and down-
stream stress fiber formation (Fig. 3). In these cells,
TGFb induced fibroblast to myofibroblast differentia-
tion, which was evidenced by enhanced expression of
a-SMA and the subsequent incorporation of a-SMA
into microfilamentous structures [148]. Fibroblast to
myofibroblast conversion is a pathophysiological
feature of various fibrotic diseases such as idiopathic
pulmonary fibrosis, asthma and chronic obstructive
pulmonary diseases [149–151]. Given that enhanced
TGFb concentrations have been detected in various
fibrotic diseases, including idiopathic pulmonary fibro-
sis [152,153], sarcoidosis [154] and cystic fibrosis [155],
understanding the mechanism that underlies this
TGFb-induced conversion may lead to the develop-

Although the extremely rapid activation of Rho
GTPases in response to TGFb stimulation implies the
involvement of non-Smad pathways, in certain cases it
was found that the Smad pathway may also play a
role in the early activation of Rho proteins by TGFb.
By studying the signaling properties of a TbRI bearing
a mutation in its L45 loop, which contains the Smad
docking site [8], Vardouli et al. [147] demonstrated that
interaction of TbRI with R-Smads is required for
signaling towards Rho GTPases and the actin cyto-
skeleton. The role of Smads in TGFb-induced actin
remodeling is further supported by experiments in a
cellular model lacking endogenous Smad3 expression
(JEG3 choriocarcinoma cells) [148]. In addition,
TGFb-induced Rho activation and cytoskeleton orga-
nization was abolished by overexpression of the inhibi-
tory Smad7 protein which blocks the TGFb ⁄ Smad
signaling pathway [147,157]. This observation is in
contrast to a study showing that Smad7 is required for
TGFb-induced activation of Cdc42 and the concomi-
tant reorganization of the actin filament system, as
discussed below.
The ability of TGFb to affect both rapid and sus-
tained actin cytoskeleton remodeling in various cell
types [144,147,148] implies that genomic actions of
TGFb may be involved in long-term cytoskeleton reor-
ganization (Fig. 3). In support of this, it was shown that
treatment of Swiss3T3 fibroblasts with actinomycin D, a
well-established inhibitor of active gene transcription,
abolished TGFb-induced actin reorganization in

upregulated. In epithelial cells, RhoB overexpression
antagonized TGFb for the transcriptional activation of
a Smad-responsive promoter, whereas dominant-
negative RhoB mutant enhanced TGFb signaling
towards this promoter [159]. In a different study and
system, it was shown that ectopic expression of RhoB,
but not RhoA, caused a decrease in the expression
TbRII and in the activity of the TbRII promoter in
HaCaT keratinocytes and pancreatic carcinoma cells,
and antagonized the TGFb-mediated anti-proliferative
responses [160]. Downregulation of the TbRII gene by
RhoB was mediated by inhibition of AP1 transcription
factors that bind to an AP1 site in the proximal TbRII
promoter [160]. Rho proteins might also play a posi-
tive regulatory role in TGFb ⁄ Smad signaling, as dem-
onstrated by Chen et al. [142] who showed that ectopic
expression of a dominant-negative RhoA mutant in
Monc-1 neural crest stem cells blocked the phospho-
rylation of Smad2 and Smad3 by TbRI, their
TGFb signaling and small GTPases D. Kardassis et al.
2958 FEBS Journal 276 (2009) 2947–2965 ª 2009 The Authors Journal compilation ª 2009 FEBS
translocation to the nucleus and the activation of a
Smad-specific reporter gene. Chen et al. [142] also
showed that general inhibition of Rho activity by C3
exotoxin attenuated Smad-mediated transactivation.
The positive role of R-Smads and the negative role
of the inhibitory Smad7 in Rho GTPase activation by
TGFb is discussed above. A novel, positive role for
Smad7 in actin remodeling was reported by Edlund
et al. [156]. This study showed that increased expres-

The balance of evidence suggests that the endocytosis
of TGFb family receptors plays an enhancing role in
TGFb family signaling. However, the magnitude and
duration of the effect on the signaling output depend
on the cell type. Embryonic stem cells and differenti-
ated cells of various types are not expected to conform
to the same mechanisms. Indeed, it has been reported
that there are fundamental differences in the endocytic
sorting of TGFb receptors between fibroblasts and epi-
thelial cells [164]. Moreover, in some cell types, endo-
cytosis of TGFb receptors might not be interconnected
with signaling, as observed in some studies. However,
many questions remain. Which endocytic routes are
taken by TGFb receptor complexes and what is the
contribution of each pathway to the final signal? Of
the five emerging transport routes, internalization of
TGFb receptors has been reported to occur via the
CCVMR and caveolar routes. There are no studies
regarding the contribution and significance of the other
routes. What dictates which route the receptor will fol-
low, and more interestingly which are the effec-
tors ⁄ regulators with which TGFb family receptors will
interact along the various endocytic routes? These
questions are more or less unanswered. It is anti-
cipated that understanding the endocytic route fol-
lowed by a receptor–ligand complex will allow for a
more detailed dissection of the molecular mechanisms
of TGFb family signaling and the functional conse-
quences thereof on cell responses.
As far as Smad trafficking is concerned, although

of Rho GTPases is beginning to be elucidated.
However, the complexity of TGFb signaling towards
Rho-governed actin cytoskeleton reorganization and
cellular responses leave several exciting open ques-
tions to be addressed.
D. Kardassis et al. TGFb signaling and small GTPases
FEBS Journal 276 (2009) 2947–2965 ª 2009 The Authors Journal compilation ª 2009 FEBS 2959
Acknowledgements
DK and CS acknowledge funding by the Greek Secre-
tariat for Research and Technology (PENED03ED688)
and the Research Council of the Greek Ministry of
Health (KESY03KA2396). TF and CM acknowledge
funding from EndoTrack FP6 Integrated Project and
PENED03ED688 and thank Savvas Christoforidis for
comments on the manuscript. AM acknowledges fund-
ing by the Ludwig Institute for Cancer Research, the
Atlantic Philanthropies ⁄ Ludwig Institute for Cancer
Research Clinical Discovery Program, the Swedish
Cancer Society, the Swedish Research Council and
the Marie Curie Research Training Network (RTN)
‘EpiPlastCarcinoma’ under the European Union FP6
program.
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