Báo cáo khoa học: A novel mechanism of TGFb-induced actin reorganization mediated by Smad proteins and Rho GTPases docx

Here we report that TGFb regulates long-term actin remodeling by increasing the steady-state mRNA levels of the RhoB gene in mouse Swiss 3T3 fibroblasts and human hepatoma HepG2 cells.. I

mediated by Smad proteins and Rho GTPases

Lina Vardouli1, Eleftheria Vasilaki1,2, Elsa Papadimitriou1, Dimitris Kardassis1,2

and Christos Stournaras1

1 Department of Biochemistry, School of Medicine, University of Crete, Heraklion, Greece

2 Institute of Molecular Biology and Biotechnology, Foundation for Research & Technology-Hellas, Heraklion, Greece

It is well established that reorganization of the actin

cytoskeleton is one of the earliest cellular responses to

various extracellular stimuli [1–5] 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 [6–9]

Transforming growth factor b (TGFb) is a pleiotropic

cytokine that regulates homeostasis in various cell types

such as epithelial and endothelial cells, and regulates

other cell functions such as growth, differentiation and

apoptosis [10–12] In addition, TGFb influences epithe-lial–mesenchymal transitions, events critical for normal embryogenesis and also for tumorigenesis, tumor-cell invasiveness and metastasis [13–15] The classical TGFb signaling apparatus consists of a plasma membrane complex of type I (TbRI) and type II (TbRII) receptors and downstream Smad signaling effectors [16] Activa-tion of TbRI by TGFb leads to phosphorylaActiva-tion of the receptor-regulated Smad proteins (R-Smad proteins) Smad2 and Smad3, which in turn oligomerize with the common partner Smad4 and rapidly translocate to the

Keywords

actin; Rho GTPases; Smad; TGFb; a-SMA

Correspondence

C Stournaras, Department of Biochemistry,

School of Medicine, University of Crete,

GR-71110 Heraklion, Greece

Fax: +30 2810 394530

Tel: +30 2810 394563

E-mail: cstourn@med.uoc.gr

(Received 22 April 2008, revised 1 June

2008, accepted 12 June 2008)

doi:10.1111/j.1742-4658.2008.06549.x

In previous studies, we have demonstrated that RhoA⁄ B-dependent signal-ing regulates TGFb-induced rapid actin reorganization in Swiss 3T3 fibro-blasts Here we report that TGFb regulates long-term actin remodeling by increasing the steady-state mRNA levels of the RhoB gene in mouse Swiss 3T3 fibroblasts and human hepatoma HepG2 cells We show that this regu-lation is specific for the RhoB gene and is facilitated by enhanced activity

of the RhoB promoter Adenovirus-mediated gene transfer of Smad2 and Smad3 in Swiss 3T3 fibroblasts induced transcription of the endogenous RhoBgene but not the RhoA gene Interestingly, in JEG-3 choriocarcinoma cells that lack endogenous Smad3, TGFb-induced transcriptional up-regu-lation of the RhoB gene was not observed, but it was restored by adeno-viral Smad3 overexpression In addition, Smad2 and Smad3 triggered activation of RhoA and RhoB GTPases and long-term actin reorganization

in Swiss 3T3 fibroblasts Finally, Smad3, and to a lesser extent Smad2, induced transcription of the a-smooth muscle actin (a-SMA) gene, and enhanced the incorporation of a-SMA into microfilaments in Swiss 3T3 fibroblasts These data reveal a novel mechanism of cross-talk between the classical TGFb⁄ Smad pathway and Rho GTPases, regulating the rapid and the long-term actin reorganization that may control the fibroblast– myofibroblast differentiation program

Abbreviations

ALK5, activin like kinase 5; ca, constitutively active; FITC, fluorescein isothiocyanate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GEF, guanine nucleotide exchange factor; GST, glutathione S-transferase; luc, luciferase; RBD, Rho binding domain; RT-PCR, reverse transcription-polymerase chain reaction; SBE, Smad binding element; a-SMA, a-smooth muscle actin; TbRI, TGFb receptor type I; TGFb, transforming growth factor b; TI, Triton X-100 insoluble; TS, Triton X-100 soluble.

nucleus where they bind to promoters and regulate

the expression of various target genes in a positive or a

negative manner [16] This pathway is negatively

regu-lated by multiple signaling inputs The best understood

is a feedback loop that involves Smad7, an inhibitory

Smad, which blocks R-Smad phosphorylation by TbRI

and directs lysosomal degradation of the receptor, thus

ensuring termination of the pathway [17,18]

Further-more, TGFb was shown to modulate cell morphology

and growth in a concerted manner via mechanisms that

control the actin cytoskeleton in a variety of cell types

[19–22] In Swiss 3T3 fibroblasts, TGFb has been

shown to induce rapid actin polymerization and

formation of stress fibers via a non-genomic RhoA⁄ B ⁄

ROCK⁄ Limk2 ⁄ cofilin signaling cascade downstream of

TbRI [23] Similarly, in other studies, it has been

reported that RhoA signaling is activated early

follow-ing TGFb treatment in smooth muscle cells [24], while

RhoA and Rac1 are regulated by TGFb during aortic

endothelial morphogenesis [25] However, the

mecha-nisms controlling the cross-talk between the classical

TGFb⁄ Smad pathways, regulation of Rho GTPases and

long-term actin cytoskeleton reorganization have not

been addressed so far

In the present work, we focus on the regulatory role

of the TGFb⁄ Smad pathway in the activation and ⁄ or

transcriptional regulation of the Rho GTPases and

long-term actin cytoskeleton restructuring Using

vari-ous cell models, we analyzed the TGFb-induced

tran-scriptional regulation of the RhoA⁄ B GTPases Using

adenoviral overexpression of Smad2⁄ 3, we studied the

role of Smad proteins in regulation of the RhoA⁄ B

genes, activation of these small GTPases and the

induction of actin reorganization Finally, to address

the biological significance of the TGFb-induced actin

reorganization, we assessed the Smad-induced

tran-scriptional regulation of a-SMA, and its expression

and incorporation into the microfilamentous network

of fibroblasts Our results provide evidence for a novel

signaling mechanism in TGFb-induced actin

cytoskele-ton reorganization mediated by Smad proteins and

Rho GTPases that may regulate

fibroblast–myofibro-blast differentiation

Results

TGFb1 induces transcription of the gene coding

for the small GTPase RhoB in Swiss 3T3

fibroblasts and hepatoma HepG2 cells

We have shown previously that TGFb induces rapid

and sustained activation of the small GTPases RhoA

and RhoB, and that this activation is essential for

TGFb-induced actin cytoskeleton reorganization in fibroblasts [23] In the present study, we sought to examine whether TGFb, in addition to inducing Rho protein activation, regulates the expression of these two Rho GTPases

First, we investigated the requirement for active gene transcription in TGFb-induced actin cytoskeleton poly-merization For this purpose, Swiss 3T3 fibroblasts were treated with TGFb for 24 h in the absence or presence

of the general inhibitor of transcription actinomycin D, and changes in the actin cytoskeleton were monitored

by fluorescence using rhodamine phalloidin staining As shown in Fig 1A, TGFb caused potent actin cytoskele-ton reorganization as evidenced by the formation of stress fibers Importantly, long-term TGFb-induced cytoskeleton reorganization was abolished in the pres-ence of actinomycin D, suggesting that, in addition to short-term activation events, TGFb elicits long-term actin cytoskeleton regulation requiring transcriptional induction of TGFb target genes (Fig 1A)

We then examined the effect of the TGFb signaling pathway in transcriptional regulation of the genes coding for the human Rho GTPases A and B by two different approaches: measuring the mRNA levels and measuring the activity of the promoters of the two genes in response to TGFb stimulation First, we determined the effect of TGFb on the mRNA levels of the RhoA and RhoB genes in Swiss 3T3 fibroblasts by RT-PCR experiments For this purpose, Swiss 3T3 cells were serum-starved for 24 h and then stimulated with 5 ngÆmL)1 TGFb1 for various time periods (from

30 min up to a maximum of 24 h) As shown in Fig 1B, treatment of Swiss 3T3 cells with TGFb1 resulted in a rapid increase (1.6-2.2-fold) in the steady-state mRNA levels of the RhoB gene, which was initiated at 30 min and persisted for a period of 24 h post-induction (top panel) In contrast, transcriptional activation of the RhoA gene by TGFb1 was not observed (Fig 1B, middle panel) As expected, TGFb did not affect the mRNA levels of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene used as a control (Fig 1B, bottom panel)

In a similar manner, we showed that TGFb induces transcription of the RhoB gene but not the RhoA gene in human hepatoma HepG2 cells In this experiment, HepG2 cells were serum-starved for 24 h, treated with TGFb1 (5 ngÆmL)1) for various time periods (from 1 h

up to a maximum of 24 h), and then subjected to RT-PCR analysis for determination of the RhoB and RhoAmRNA levels As shown in Fig 1C (top panel), treatment of HepG2 cells with TGFb1 resulted in rapid transcriptional activation of the RhoB gene The induc-tion of transcripinduc-tion started at 1 h of treatment with

TGFb1, reached a maximum (2.6-fold) at 2 h, and

declined thereafter (1.4-fold activation at 24 h) In

agreement with the findings in Swiss 3T3 fibroblasts, no

transcriptional activation of the RhoA gene by TGFb1 was observed in HepG2 cells (Fig 1C, middle panel)

Overexpression of the Smad3 protein via adenovirus-mediated gene transfer induced transcription of the human RhoB gene

By using recombinant adenoviruses expressing Smad2 and Smad3 proteins, we investigated the role of Smad proteins in TGFb-induced transcriptional activation of the RhoA and RhoB genes For this purpose, Swiss 3T3 cells were infected with recombinant adenoviruses expressing Smad2 (ad-Smad), Smad3 (ad-Smad3) or a control adenovirus expressing the b-galactosidase gene (ad-LacZ), and were treated with TGFb1 (5 ngÆmL)1) for 2 h or left untreated RT-PCR analysis revealed that overexpression of Smad3 caused a significant increase in RhoB mRNA levels (2.1-fold) even in the absence of TGFb stimulation (Fig 2A, top panel, lane 5), and treatment with TGFb1 slightly enhanced this effect (2.3-fold) (Fig 2A, top panel, lane 6) In contrast, adenovirus-mediated overexpression of the Smad2 protein did not significantly upregulate expres-sion of the RhoB gene either in the absence (1.3-fold)

or presence of added TGFb (1.9-fold) (Fig 2A, top panel, lanes 3 and 4) In line with our findings in Fig 1, transcription of the RhoA gene was not affected

by overexpression of Smad proteins in Swiss 3T3 cells (Fig 2A, middle panel)

Smad3 is required for TGFb1-induced transcriptional activation of the RhoB gene The results presented in Fig 2A indicate that Smad3, and to a much lesser extent Smad2, are transcriptional activators of the human RhoB gene To further evalu-ate these findings, we used the JEG-3 human chorio-carcinoma cell line that does not express endogenous Smad3 protein [26] We first determined mRNA levels for the RhoA and RhoB genes following treatment of JEG-3 cells with TGFb1 in the absence or presence of exogenous Smad3 expressed via adenovirus-mediated gene transfer For this purpose, JEG-3 cells were adenovirally infected with ad-Smad3 or ad-LacZ as a negative control, serum-starved for 24 h and stimulated

or not with 5 ngÆmL)1TGFb1 for 24 h Cells were then lysed and processed for total RNA extraction and RT-PCR As shown in Fig 2B (top panel), treatment

of JEG-3 cells with TGFb1 resulted in minor (1.6-fold) transcriptional activation of the endogenous RhoB gene Importantly, TGFb caused a potent (4.5-fold) induction of RhoB gene expression when JEG-3 cells were infected with an adenovirus expressing Smad3,

Fig 1 TGFb1 induces rapid and sustained transcriptional

upregula-tion of the RhoB gene but not the RhoA gene in Swiss 3T3 and

HepG2 cells (A) Active transcription is required for TGFb-induced

actin cytoskeleton reorganization Swiss 3T3 fibroblasts were

trea-ted with 5 ngÆmL)1TGFb for 24 h in the absence or presence of

actinomycin D (5 lgÆmL)1) Changes in actin cytoskeleton were

monitored using rhodamine phalloidin staining (B,C) TGFb induces

the transcription of the RhoB gene but not of the RhoA gene.

Swiss 3T3 (B) or HepG2 (C) cells were serum-starved for 24 h and

stimulated with TGFb1 for the indicated time points or left

untreated RT-PCR analysis was performed with primers specific

for the mRNA of the RhoB or RhoA genes The bottom panels

rep-resent the mRNA levels of the housekeeping gene GAPDH at the

same time points The RhoB and RhoA mRNA levels were

normal-ized to the intensity of the corresponding GAPDH mRNA of each

sample Data are representative of two independent experiments.

The fold increase in mRNA levels is shown below each PCR image.

indicating that Smad3 is required for TGFb1-induced

transcriptional upregulation of the RhoB gene In

agreement with the mRNA data, levels of RhoB

pro-tein were also potently increased by TGFb1 only in the

presence of adenovirally expressed Smad3 (Fig 2C, top

panel) In line with our initial findings in Swiss 3T3 and

HepG2 cells, the transcription levels of the RhoA gene

were not affected by Smad3 overexpression in JEG-3

cells (Fig 2B, middle panel)

TGFb and Smad proteins activate the promoter

of the RhoB gene

To further characterize the mechanism of

transcrip-tional upregulation of the RhoB gene by TGFb1, we

cloned the promoters of the human RhoB and RhoA genes in fusion with the firefly luciferase gene (Fig 3A) and used them in transactivation experiments in order

to determine their responsiveness to the TGFb⁄ Smad signaling cascade For this purpose, HepG2 cells were transiently transfected with the reporter plasmids )726 ⁄ +86 RhoB-Luc and )799 ⁄ +166 RhoA-Luc in the absence or the presence of expression vectors for the constitutively active form of the TGFb receptor I (ALK5ca) and the TGFb signaling mediators Smad2, Smad3 and Smad4 As shown in Fig 3B, the constitu-tively active form of the TGFb receptor type I (ALK5ca) enhanced the activity of the )726 ⁄ +86 RhoB promoter 2.2-fold The activity of this promoter was also enhanced by overexpression of Smad3 and Smad4 proteins (3.4-fold) and to a lesser extent by overexpression of Smad2 and Smad4 proteins (2.3-fold), and this activity was stimulated further by simultaneous expression of the ALK5ca receptor (3.6-and 2.6-fold, respectively) In contrast, ALK5ca, Smad2⁄ Smad4 and Smad3 ⁄ Smad4 proteins had a minor effect (1.2-1.6-fold) on the activity of the human )799 ⁄ +166 RhoA promoter (Fig 3C)

In conclusion, the combined data in Figs 2 and 3 indicate that transcriptional activation of the RhoB gene by TGFb is mediated, at least in part, by

TGFb-Fig 2 Adenovirus-mediated overexpression of Smad proteins in Swiss 3T3 cells and JEG-3 choriocarcinoma cells that lack endoge-nous Smad3 confirmed the important role of Smad3 in transcrip-tional upregulation of the RhoB gene (A) RT-PCR analysis of Swiss 3T3 cells infected with ad-LacZ as control (lanes 1 and 2), ad-Smad2 (lanes 3 and 4) or ad-Smad3 (lanes 5 and 6) Cells were serum-starved for 24 h and stimulated with 5 ngÆmL)1 TGFb1 (lanes 2, 4 and 6) for 2 h The bottom panel represents the mRNA levels of the housekeeping gene GAPDH at the same time points The RhoB and RhoA mRNA levels were normalized to the intensity

of the corresponding GAPDH mRNA of each sample Data are rep-resentative of two independent experiments The fold increase in mRNA levels is shown below each PCR image (B) RT-PCR analysis

of JEG-3 (Smad3 ) ⁄ )) cells infected with ad-LacZ as control (lanes

1 and 2) or ad-Smad3 (lanes 3 and 4) Cells were serum-starved for

24 h and stimulated with 5 ngÆmL)1TGFb1 (lanes 2 and 4) for 24 h RT-PCR analysis was performed with primers specific for the RhoB

or RhoA mRNA The bottom panel represents the mRNA levels of the housekeeping gene GAPDH The RhoB and RhoA mRNA levels were normalized to the intensity of the corresponding GAPDH mRNA of each sample Data are representative of two independent experiments (C) Immunoblotting analysis of RhoB and adenovirally overexpressed Smad2 and Smad3 proteins in JEG-3 cells Follow-ing adenovirus infection, cells were serum-starved for 24 h and stimulated with 5 ngÆmL)1TGFb1 for 24 h (+) or left unstimulated ( )) Total extracts from the infected cells were analyzed by SDS– PAGE and Western blotting using antibodies against RhoB and against total or phosphorylated forms of Smad2 and Smad3.

regulated Smad proteins, which act as transcriptional

regulators of the activity of the RhoB promoter

Smad2 and Smad3 proteins activate RhoA and

RhoB GTPases and induce actin polymerization

and microfilament reorganization in Swiss 3T3

fibroblasts

To examine whether Smad proteins, in addition to

serving as transcriptional regulators of RhoB gene

expression in response to TGFb, also play a role in the

activation of Rho proteins as GTPases by this

cyto-kine, we performed affinity precipitation assays using

the rhotekin Rho-binding domain fused to glutathione

S-transferase (GST–RBD) For this purpose, cells

were infected with Smad2- or Smad3-expressing

adeno-viruses or with a LacZ-expressing adenovirus as a

neg-ative control Following serum deprivation for 24 h,

cells were treated with 5 ngÆmL)1 TGFb1 for 10 min,

and GST–RBD was used to isolate GTP-loaded RhoA

and RhoB from cell lysates Both proteins were

moni-tored by immunoblotting using RhoA and

anti-RhoB specific serum, and the protein band intensities

were normalized relative to the total RhoA or RhoB

content of non-adsorbed cell lysates As shown in

Fig 4A (top panel), RhoA was activated by TGFb (3.2-fold) or by overexpression of Smad2 or Smad3 (3.9-fold) in Swiss 3T3 fibroblasts In contrast, RhoB was activated only slightly by Smad2 (1.2-fold) or Smad3 (1.9-fold) compared to the activation of RhoA (Fig 4B, top panel)

The activation of GTPase activity of RhoA and RhoB proteins and the transcriptional induction of the RhoB gene by Smad proteins suggest that the TGFb– Smad pathway may contribute to the long-term actin reorganization induced by TGFb in Swiss 3T3 fibro-blasts To test this hypothesis, we analyzed actin archi-tecture in Swiss 3T3 cells infected with adenoviruses expressing Smad2 and Smad3 In these double staining experiments, the actin cytoskeleton organization was assessed by direct fluorescence using rhodamine phal-loidin, and indirect immunofluorescence against the Flag epitope of Smad2 and Smad3 revealed R-Smad staining Cells were serum-starved for 24 h, and then stimulated, or not, with 5 ngÆmL)1 TGFb1 for 24 h

As shown in Fig 5, control starved cells expressing the LacZ gene exhibited typical morphology, i.e their actin cytoskeleton was restricted to cortical actin and the main cell body was devoid of stress fibers (Fig 5B) Treatment with TGFb1 resulted in cell

Fig 3 TGFb1 increases the activity of the promoter of the RhoB gene in human hepatoma HepG2 cells (A) Schematic representation of the reporter plasmids )726 ⁄ +86 RhoB-Luc and )799 ⁄ +166 RhoA-Luc that were used in the transactivation experiments shown in (B) and (C) Numbers refer to the transcription start site of each gene (+1) (B,C) HepG2 cells were transiently transfected with the )726 ⁄ +86 RhoB-Luc or the )799 ⁄ +166 RhoA-Luc plasmid (1 lg) together with the expression vector for the constitutively active form of TGFb recep-tor I (ALK5ca) independently or in combination with expression vecrecep-tors for Smad2, Smad3 and Smad4 (1 lg each) as indicated beneath each histogram The CMV-b-gal plasmid expressing b-galactosidase (1 lg) was included in each sample for normalization of transfection variability Luciferase activity was determined in cell lysates at 48 h after transfection, and the mean values and SEM from at least two independent experiments performed in duplicate are shown as percentage relative luciferase activity.

flattening and scattering, supported by changes in the organization of the actin cytoskeleton (Fig 5D) In contrast to control adenovirus (LacZ), ectopic expres-sion of Smad2 in serum-starved cells resulted in increased actin polymerization, even in the absence of TGFb1 stimulation (Fig 5F) Likewise, adenoviral overexpression of Smad3 resulted in even more pro-found changes in the organization of the actin cyto-skeleton, resulting in cell flattening and cell shape change Cells appeared elongated or spindle-shaped, with a parallel arrangement of actin bundles (Fig 5J) Addition of TGFb did not cause any additional changes in actin cytoskeleton organization in the ad-Smad3-infected cells (Fig 5, panels J and L), but enhanced the formation of stress fibers in the ad-Smad2-infected cells (Fig 5, panels F and H) These morphological findings were further corrobo-rated by quantitative immunoblot analysis of the Tri-ton X-100-soluble (TS) and -insoluble (TI) actin cytoskeleton fractions of cells overexpressing the R-Smad proteins (Table 1) As calculated from the rel-ative band intensities, the G⁄ total actin ratio of Swiss 3T3 fibroblasts treated with TGFb1 for 24 h was clearly and reproducibly decreased in comparison with control cells serum-starved for 24 h Furthermore, cells ectopically expressing Smad2 or Smad3 revealed a greater decrease in the G⁄ total actin ratio, correspond-ing to a clear shift of the dynamic equilibrium towards polymerized actin Thus, quantitative analysis of actin dynamics revealed that Smad2 and Smad3 overexpres-sion in Swiss 3T3 fibroblasts induces actin polymeriza-tion and microfilament reorganizapolymeriza-tion

To address the contribution of Smad3 protein to the actin reorganization induced by TGFb, we infected JEG-3 cells with adenoviruses expressing Smad3 (or LacZ as a negative control), and assessed the polymeri-zation dynamics of the actin cytoskeleton by

quantita-Fig 4 Overexpression of Smad2 and Smad3 proteins via

adeno-virus-mediated gene transfer induced activation of Rho GTPases in

Swiss 3T3 fibroblasts Rho–GTP loading assay with Swiss 3T3 cells

infected with ad-LacZ as control (lanes 1 and 2), ad-Smad2 (lanes 3

and 4) or ad-Smad3 (lanes 5 and 6) Cells were serum-starved for

24 h and stimulated with 5 ngÆmL)1 TGFb1 (lanes 2, 4 and 6) for

15 min Immunoblots of the GST–RBD pulldown (RhoA–GTP or

RhoB–GTP), or total cell extracts with RhoA antibody (A) or RhoB

antibody (B) and Flag-M5 antibody (Smad2 and Smad3) are shown.

Densitometric analysis of the RhoA–GTP and RhoB–GTP

immuno-blots was performed, and the fold increase of the active ⁄ total ratio

values of each condition is shown These data are representative of

two independent experiments.

Fig 5 Smad2 and Smad3 induce actin

reor-ganization in Swiss 3T3 fibroblasts Swiss

3T3 cells infected with the adenoviruses

ad-LacZ, ad-Smad2 or ad-Smad3 were

sub-sequently serum-starved for 24 h and

stimu-lated (+) or not ( )) with 5 ngÆmL )1TGFb1

for 24 h (+) Indirect immunofluorescence

against the Flag epitope of Smad2 and

Smad3 is shown in the left panels (FITC,

fluorescein isothiocyanate) and direct

fluorescence of actin is shown in the right

panels The bar represents 10 lm.

tive immunoblot analysis of the Triton X-100-soluble

(TS) and -insoluble (TI) actin cytoskeleton fractions of

cells (Table 2) The G⁄ total actin ratio of JEG-3 cells

treated with TGFb1 for 24 h was decreased compared

to control serum-starved cells, indicating slight actin

polymerization Furthermore, the G⁄ total actin ratio

decreased in cells overexpressing Smad3, and this ratio

decreased further following treatment with TGFb1,

corresponding to a clear shift of the dynamic

equilib-rium toward actin polymerization These results

indi-cate that Smad3 is crucial for the TGFb1-induced

actin reorganization mediated by Rho GTPases

Smad3 (and to a lesser extent Smad2) induces

the expression of a-smooth muscle actin

TGFb stimulation of various cell types, including

Swiss 3T3 fibroblasts, resulted in increased a-smooth

muscle actin (a-SMA) expression [24,27–29 and unpublished data] Having established that overexpres-sion of R-Smad proteins results in long-term actin polymerization, we investigated the involvement of Smad2 and Smad3 in TGFb1-induced a-SMA gene regulation and protein incorporation into the actin network of Swiss 3T3 fibroblasts For this purpose, we transiently overexpressed Smad2 and Smad3 using adenoviral cell infections Swiss 3T3 fibroblasts expressing Smad2, Smad3 or LacZ (as a negative con-trol) were serum-starved for 24 h, then stimulated with

5 ngÆmL)1 TGFb1 for 24 h, and the levels of expres-sion of a-SMA were analyzed As shown in Fig 6A, control cells infected with the LacZ adenovirus and stimulated with TGFb1 for 24 h showed a 5.4-fold upregulation of a-SMA gene expression In agreement with previous findings, Smad2 overexpression resulted

in a moderate (1.5-fold) increase in a-SMA mRNA levels, whereas stimulation with TGFb1 led to a robust 4.6-fold upregulation of a-SMA mRNA (Fig 6A) Interestingly, cells infected with ad-Smad3 showed a stronger upregulation of a-SMA mRNA (2.4-fold), which was even more prominent with TGFb1 treat-ment (5.4-fold) Analysis of a-SMA protein levels under the same experimental conditions as described in Fig 6A provided very similar results (Fig 6B) These data indicated that Smad3 rather that Smad2 is involved in a-SMA transcriptional upregulation in Swiss 3T3 fibroblasts

To further evaluate incorporation of the newly synthesized a-SMA to the actin cytoskeleton, we performed immunoblot analysis of the Triton X-100-soluble (TS) and -inX-100-soluble (TI) preparations after sub-cellular fractionation of Swiss 3T3 fibroblasts treated

or not with TGFb and overexpressing Smad2, Smad3

or LacZ (as a negative control) Smad3 overexpression led to incorporation of a-SMA into the insoluble (fila-mentous) part (Fig 6D, compare lanes 1⁄ 2 and 5 ⁄ 6)

In line with the weaker induction of a-SMA protein expression in Smad2-transfected cells, a-SMA incorpo-ration into the insoluble cytoskeleton fraction was very low (Fig 6C, compare lanes 1⁄ 2 and 5 ⁄ 6) These con-clusions were fully supported by the morphological analysis shown in Fig 6E Indirect immunostainning

of Swiss 3T3 fibroblasts with an antibody against a-SMA, and subsequent analysis by fluorescence microscopy revealed increased a-SMA structures after Smad3 (but not Smad2) overexpression, similar to the control cells stimulated with TGFb1 for 24 h (Fig 6E) These results indicate that ectopic expression of Smad3 (and to a lesser extent Smad2) leads to increased a-SMA expression in Swiss 3T3 fibroblasts Interestingly, however, the newly synthesized a-SMA

Table 1 Effect of the ectopic expression of Smad2 and Smad3 on

the polymerization state of actin in Swiss 3T3 fibroblasts Swiss

3T3 fibroblasts were infected with the adenoviruses indicated,

serum-starved for 24 h, and then stimulated with 5 ngÆmL)1TGFb1

for 24 h Triton-soluble (TS) and Triton-insoluble (TI) actin

cytoskele-ton fractions were prepared as described in Experimental

proce-dures, and their actin content was analyzed by immunoblotting.

Data presented correspond to the G ⁄ total actin ratio in each

condi-tion These data are representative of three independent

experi-ments.

* Statistically different from ad-LacZ (control) at P < 0.05.

Table 2 Effect of the ectopic expression of Smad3 on the

poly-merization state of actin in JEG-3 cells JEG-3 (Smad3 ) ⁄ )) cells

were infected with the adenoviruses indicated, serum-starved for

24 h, and then stimulated with 5 ngÆmL)1TGFb1 for 24 h

Triton-soluble (TS) and Triton-inTriton-soluble (TI) actin cytoskeleton fractions

were prepared as described in Experimental procedures, and their

actin content was analyzed by immunoblotting Data presented

correspond to the G ⁄ total actin ratio in each condition These data

are representative of five independent experiments.

* Statistically different from ad-LacZ (control) at P < 0.05.

can be incorporated into the newly formed stress fibers

only when Smad3 is overexpressed, even in the absence

of TGFb

Discussion

In the present study, we provide evidence for the

essential role of Smad proteins and Rho GTPases in

long-term TGFb-induced actin cytoskeleton

reorgani-zation in various cell models We also show that

acti-vation of Smad and Rho proteins by TGFb in

fibroblasts is correlated with potent induction of

a-SMA gene expression and subsequent incorporation

of a-SMA into microfilamentous structures As a-SMA

expression is indicative of a myofibroblast phenotype [30], our findings suggest that these proteins control a TGFb-induced fibroblast to myofibroblast differentia-tion program Fibroblast to myofibroblast conversion

is a pathophysiological feature of various fibrotic dis-eases such as idiopathic pulmonary fibrosis, asthma and chronic obstructive pulmonary diseases (COPD) [31–33] Given that enhanced TGFb concentrations have been detected in patients with various fibrotic dis-eases including idiopathic pulmonary fibrosis [34,35], sarcoidosis [36] and cystic fibrosis [37], in-depth under-standing of the mechanism that underlies this TGFb-induced conversion program, identification of the molecules involved and elucidation of their role or their

Fig 6 Smad3 (and to a lesser extent Smad2) induce the expression of a-smooth muscle actin (a-SMA) (A) RT-PCR of Swiss 3T3 cells infected with control (LacZ) adenovirus or adenoviruses expressing Smad2 and Smad3 Cells were serum-starved for 24 h and stimulated with 5 ngÆmL)1TGFb1 for 3 h RT-PCR analysis was performed with primers specific for the mRNA of the a-SMA gene The bottom panel represents the mRNA levels of the housekeeping gene GAPDH The a-SMA mRNA level was normalized to the intensity of the correspond-ing GAPDH mRNA for each sample Data are representative of two independent experiments The fold increase in a-SMA mRNA levels is shown below each image (B) Immunoblot analysis of total cell extracts of Swiss 3T3 cells infected with control (LacZ) adenovirus or adeno-viruses expressing Smad2 and Smad3 Cells were starved for 24 h and stimulated (+) or not ( )) with 5 ngÆmL )1TGFb1 for 24 h

Immuno-blotting of the corresponding total extracts with the a-SMA antibody and control b-tubulin antibody is shown The fold increase in a-SMA protein levels is shown below each image (C,D) Immunoblot analysis of the Triton-soluble (TS) and Triton-insoluble (TI) actin cytoskeleton fractions of Swiss 3T3 cells infected with adenoviruses expressing LacZ (lanes 1-4, both panels), Smad2 (lanes 5-8, C) or Smad3 (lanes 5-8, D) Following adenovirus infection, cells were starved for 24 h and stimulated (+) or not ( )) with 5 ngÆmL )1TGFb1 for 24 h (+)

Immunoblot-ting of the corresponding total extracts with the a-SMA antibody is shown (E) Indirect fluorescence microscopy of Swiss 3T3 cells infected with the indicated adenoviruses using an antibody against a-SMA Following adenovirus infection, cells were serum-starved for 24 h and stimulated (+) or not ( )) with 5 ngÆmL )1TGFb1 for 24 h Bars correspond to 10 lm.

modes of regulation may lead to the development of

novel therapeutic approaches for these diseases

We show here that TGFb stimulation of fibroblasts

or hepatic cells causes differential regulation of

expres-sion of the genes encoding the two small GTPases

RhoA and RhoB (Fig 1) Thus, treatment of these

cells with TGFb1 resulted in a rapid increase in

steady-state mRNA levels of the RhoB gene that was

initiated at 30 min (Swiss 3T3) or 1 h (HepG2) and

persisted for a period of 24 h post-induction, whereas

no transcriptional activation of the RhoA gene by

TGFb1 was observed (Fig 1) These findings confirm

previous experimental evidence which suggested that

RhoB is the only member of the Rho-related subfamily

of small GTPases that is regulated at the

transcrip-tional level, and this regulation may be important for

its function due to the short half-life of this protein in

the cell [38,39] Of interest was the periodic pattern of

induction of RhoB gene transcription by TGFb As

shown in Fig 1B, TGFb induced RhoB gene

expres-sion 2.6-fold at 2 h of treatment in HepG2 cells, and

this induction declined to 1.8-fold at 4 h, increased

again to 2.2-fold at 8 h and dropped back to 1.4-fold

at 24 h after TGFb addition A similar periodic

pat-tern of RhoB gene transcriptional induction was

observed in Swiss 3T3 cells (Fig 1A) These findings

could account for our previously reported observations

that regulation of the RhoB GTPase activity by

TGFb in Swiss 3T3 cells follows a periodic pattern

[23] The periodic decrease in expression of the RhoB

gene during TGFb stimulation strongly suggests that

RhoB is subject to an auto-inhibitory loop In

agree-ment with this hypothesis, we have shown that

over-expression of RhoB causes a potent reduction in the

activity of its own promoter (E Vasilaki, unpublished

observations)

We speculated that the differential effect of TGFb

on expression of these two genes reflects a difference in

the mechanism by which the TGFb-regulated Smad

proteins (Smad2 and Smad3) regulate the activity of

the two promoters To address this hypothesis, we

cloned the promoters of the human RhoA and RhoB

genes in front of the firefly luciferase gene, and, using

transient transfection experiments and luciferase

assays, did indeed show that the TGFb–Smad pathway

specifically targets the RhoB gene (Fig 3) A

mechanis-tic explanation for this RhoB-specific transcriptional

response to the TGFb–Smad pathway could be that

the promoter of the human RhoA gene lacks Smad

binding elements that could serve as sites of Smad

recruitment in response to TGFb stimulation A search

for transcription factor binding sites in the two

promoters revealed that the human RhoB promoter

contains three putative Smad binding elements (sequence 5¢-CAGAC-3¢) [40] in the proximal )726 ⁄ +86 region that was used in the transactivation experiments (at positions )294 ⁄ )290, )278 ⁄ )274 and +20⁄ +24), whereas the human RhoA promoter, which

is not homologous to the human RhoB promoter, does not contain any of these putative Smad binding ele-ments We are in the process of characterizing these sites further and studying their role and participation

in the transcriptional upregulation of the RhoB promoter in response to TGFb stimulation or Smad overexpression Our preliminary experiments have shown that regulation of the RhoB promoter by the TGFb–Smad pathway is more complex than initially suspected, and that elements additional to the Smad binding elements are required for this regulation (E Vasilaki, E Papadimitriou, C Stournaras &

D Kardassis, unpublished data)

An interesting finding during this work was that Smad proteins, in addition to serving as specific tran-scriptional activators of the RhoB gene, can also act as activators of Rho GTPase function This is in line with previous studies that had provided indications of the involvement of the Smad pathway in the activation of Rho protein activity, and specifically that of RhoA, by TGFb [23,24], We have shown recently that overex-pression of Smad7, a known and potent inhibitor of the TGFb signaling pathway and of Smad function that operates in the context of a feedback inhibitory loop [41], was able to block both the activation of RhoA GTPase activity and reorganization of the actin cytoskeleton by TGFb1 in Swiss 3T3 fibroblasts, sug-gesting cross-talk between Smad signaling and RhoA activation [23] In line with these observations, it was recently reported that dominant-negative RhoA inhib-its the nuclear translocation of Smad2 and Smad3 dur-ing the smooth muscle cell differentiation induced by TGFb, indicating that RhoA is a modulator of Smad activation [24] Moreover, by studying the signaling properties of a type I TGFb receptor with a mutation

in the L45 loop that contains the Smad docking site [16], it was demonstrated that interaction of the recep-tor with Smad proteins is required for signaling to Rho GTPases and the actin cytoskeleton [23] These data suggest that Smad proteins, in addition to their role in long-term RhoB transcriptional regulation, might be directly involved in activation of Rho GTP-ases and the regulation of actin dynamics The results presented in this study clearly support this assumption Indeed, adenovirus-mediated Smad2 and Smad3 over-expression triggered activation of RhoA, and to a lesser extent RhoB, and caused potent long-term actin reorganization (Figs 4 and 5) Activation of Rho

proteins by Smad proteins could be facilitated by

dif-ferent, not necessarily mutually exclusive mechanisms,

as discussed below

Another interesting finding arising from this work

was the differential involvement of the two

reg-ulated Smad proteins (Smad2 and Smad3) in

TGFb-induced Rho gene regulation and actin restructuring

While adenovirus-mediated overexpression of Smad3

largely mimicked the TGFb effects on transcriptional

upregulation of the RhoB and a-SMA genes, as well as

on RhoA and RhoB activation and actin

reorgani-zation, Smad2 was consistently less effective in all of

these processes (Figs 2–6) This may reflect a specific

requirement for Smad3 in transcriptional regulation of

both RhoB and a-SMA genes by TGFb This

hypothe-sis is supported by experiments in a cellular model that

lacks endogenous Smad3 expression (JEG-3

choriocar-cinoma cells) We found that TGFb treatment of this

cell line had no effect on transcriptional upregulation

of the RhoB gene, but RhoB gene expression was

rescued after adenovirus-mediated ectopic expression

of Smad3 (Fig 2B) Taken together, these findings

support a key role for Smad3 in mediating Rho⁄ actin

regulation by TGFb

The combined data from this as well as previous

studies suggest a model of short- and long-term

TGFb-induced actin cytoskeleton reorganization in

fibroblasts and other cell types This model is shown

schematically in Fig 7, and can be summarized as

follows In the short-term activation process, TGFb receptor activation by its ligand induces rapid activa-tion of RhoA and RhoB GTPases (1), which is fol-lowed by activation of the ROCK⁄ LIMK ⁄ cofilin pathway (2–4) and actin cytoskeleton restructuring (5),

as shown previously [23] The mechanism by which Rho activation is linked to activation of the type I TGFb receptor (TbRI) is currently unknown It may

be that the phosphorylated TbRI activates very rapidly, by phosphorylation, a specific guanine nucleo-tide exchange factor (GEF), which in turn activates Rho proteins In a previous study, we showed that dominant-negative forms of either RhoA or RhoB are equally effective in blocking TGFb-induced actin cyto-skeleton reorganization in Swiss 3T3 cells, suggesting that these two GTPases are in the same pathway and activation of the one may precede activation of the other [23] Gene silencing experiments may shed some light into the details of this activation process and the specific role of each Rho protein, but it seems reason-able to suggest, based on the availreason-able data, that RhoA protein plays a more critical role in this short-term activation event

The long-term actin cytoskeleton response to TGFb stimulation involves the Smad pathway and transcrip-tional activation events Thus, TGFb rapidly induces the phosphorylation of R-Smad proteins (a), the for-mation of R-Smad(P)–Smad4 complexes (b), transloca-tion of these complexes to the nucleus (c), and their

Fig 7 Mechanisms of short-term and

long-term actin cytoskeleton reorganization

induced by TGFb in fibroblasts Schematic

representation of the proposed mechanisms

regulating actin cytoskeleton reorganization

in fibroblasts following TGFb stimulation.

Numbers in parentheses (1–5) indicate the

successive steps that lead to short-term

actin reorganization immediately after TGFb

stimulation Letters in parentheses (a–g)

indicate events that contribute to long-term

actin cytoskeleton reorganization Solid

arrows indicate events that have been

experimentally proven in this or previous

studies Dashed arrows indicate

hypotheti-cal events.