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 Pdf file on Cortactin is involved in transforming growth factor-b1-induced epithelial-mesenchymal transition in AML-12 cells
Kehua Zhang, Dongmei Wang, and Jianguo
Song*
Laboratory of Molecular Cell Biology, *Correspondence address. Tel: + 86-21-54921167; Fax: + 86-21-54921011; E-mail: [email protected]
Cortactin is an F-actin binding protein,
regulating cell movement and adhesive junction assembly. However, the function
of cortactin in epithelial-mesenchymal
transition (EMT) remains elusive. Here we found that during transforming growth
factor-b1 (TGF-b1)-induced EMT in AML-12 murine hepatocytes, cortactin underwent tyrosine dephosphorylation.
Inhibition of the dephosphorylation of cortactin by sodium vanadate
blocked TGF-b1-induced EMT. Knockdown of cortactin
by RNAi led to decrease of intercellular junction
proteins E-cadherin and Zonula
occludens-1 and induced expression of mesenchymal
protein fibronectin. Additionally, knockdown of cortactin further promoted TGF-b1-induced EMT in
AML-12 cells, as determined by EMT markers and cell morphological changes.
Moreover, migration assay showed that cortactin
knockdown promoted the migration of AML-12 cells, and also enhanced TGF-b1-induced
migration. Our study showed the involvement of cortactin
in the TGFb1- induced EMT.
Keywords epithelial-mesenchymal transition; cortactin;
transforming growth factor-b; migration
Received: April 3, 2009 Accepted: May 31, 2009
Introduction
Epithelial-mesenchymal transition (EMT)
describes a switch in which epithelial cells acquire mesenchymallike
characteristics, including increased motility and invasiveness. EMT was
originally defined as the formation of mesenchymal
cells from epithelia during different embryonic development stages. It is now
known to occur in a variety of diseases including the fibrotic diseases in
lung, kidney, and liver [1–4], and malignant
tumor progression [5]. Epithelial cells are characterized by the formation of
intercellular junctions, which connect and immobilize adjacent cells. During
EMT, intercellular junctions of epithelial cells are interrupted or decreased
by downregulation of adhesion molecules E-cadherin and tight junction component Zonula
occludens-1 (ZO-1). The subsequent disassembly of adhesion junction increases
cell motility. Epithelial tight junction participates in the maintenance of
epithelial integrity that protects multicellular
organisms from the external environment. ZO-1 belongs to a family of
membrane-associated guanylate kinase
homologues that function as an essential tight junctional
plaque protein. During EMT, the loss of tight junctions and, accordingly, the
delocalization of their structural components (e.g. ZO-1) from cell–cell contacts were implicated in the depolarization of epithelial cells
[6]. EMT is also characterized by morphological changes from epithelial to
fibroblast-like, with upregulation of mesenchymal markers, including fibronectin
and vimentin.
Cortactin is an F-actin binding protein that stabilizes
F-actin networks and stimulates Arp2/3 (Actin related proteins 2/3)-mediated actin
polymerization and branched actin assembly [7,8]. Cortactin was identified as
one of the major substrates for tyrosine kinases such
as Src and Fer. Tyrosine phosphorylation modification of cortactin
regulates F-actin cross-link [9] and mediates the actin filament-driven centrosome
separation at G2-M transition [10]. Because cortactin
distributes mainly in cell motility structures such as lamellipodia
and invadopodia, the role of cortactin
in cell movement was studied. Overexpression of cortactin has been shown to enhance cell motility in a
variety of assays [11,12], mainly due to its roles in actin assembly and promotion of the persistence of lamellipodial protrusion [13]. Cortactin
is also reportedly involved in the formation of adhesion junction after
recruitment to cell–cell adhesive contacts
[14], suggesting that cortactin has a potential
function in maintaining epithelial properties. Transforming growth factor
(TGF)-b1 is extensively involved in EMT induction in various epithelial cells
[15]. However, whether cortactin participates in the
disruption of intercellular junction during TGF-b1-induced EMT still
remains unclear.
In this study, we examined the status of phosphorylation
and the expression level of cortactin during TGF-b1-induced EMT. We also
investigated the role of cortactin in TGF-b1-induced EMT using
RNA interference technique in AML-12 murine hepatocyte cell line. The results revealed that TGF-b1 induced a decrease
in the levels of tyrosine phosphorylated cortactin during EMT. Knockdown of cortactin
led to a disruption of tight junction and adhesion junction, causing an
enhancement of TGF-b1-induced EMT and increased the migratory capacity of AML-12 cells.
Materials and Methods
Materials
Cell culture and transfection reagents were
purchased from Invitrogen (
Cell culture
AML-12 cells were grown in a 1:1 mixture of Dulbecco’s modified Eagle’s medium and Ham’s F12 medium
containing 10% fetal calf serum and supplemented with insulin-transferrin-selenium-X, dexamethasone
(40 ng/ml), penicillin (100 U/ml), and streptomycin
(100 mg/ml). The cells were incubated at 37ºC in a humidified
atmosphere of 5% CO2 until 30– 50% confluence was reached.
RNA interference
The target sequence of cortactin is 5‘-GGATCGGATGGACAAGAAT-3‘, which
was inserted into the pPGK super vector at the BglII and HindIII sites. An empty
vector was used as negative control.
Transfection
Cells (30–50% confluence) in
35-mm plates were transfected with the plasmids as
indicated using LipofectamineTM reagent according to the manufacturer’s instructions. For transient transfection, the expression of the indicated plasmids was
examined 48 h after transfection. Stable transfection was proceeded with G418 (800 mg/ml) selection after
the transient transfection.
Examination of morphological change
The morphological changes of the cells were observed under an inverted
phase-contrast microscope (
Migration assays
Cell migration assays were performed using Transwell
migration chambers (8 mm pore size; Costar,
Statistical analysis
Quantitative data are presented as mean±SD. Statistical significance was determined
by the two-tailed student’s t-test or one-way ANOVA followed by the LSD t-test for multiple comparisons. A P-value of < 0.05 was considered
statistically significant.
Results
TGF-b1 downregulated
the levels of tyrosine phosphorylated cortactin during EMT
AML-12 murine hepatocytes
have a typical epithelial phenotype with polygonal morphology and tight
arrangement. In response to TGF-b1, AML-12 cells acquired a spindlelike mesenchymal
morphology, which could be detected after 12 h, and became highly prominent
after 48 h [Fig. 1(A)] [4,16]. Furthermore, downregulation
of E-cadherin and ZO-1, the two well-known EMT
markers, was observed during TGF-b1 treatment in time-dependent manner [Fig. 1(B)], suggesting a
dissolution of adhesion and tight junctions. TGF-b1 treatment also
increased fibronectin and vimentin
levels in AML-12 cells [Fig. 1(B)], indicating that these cells acquired a mesenchymal
phenotype. The results demonstrate that AML-12 cells could undergo EMT in
response to TGF-b1.
In order to know whether TGF-b1 regulates cortactin,
we detected the protein levels of cortactin by immunoblotting. The results showed that the protein levels
of cortactin remained unchanged before and after TGF-b1 treatment from 6 to
48 h [Fig. 1(C)]. Because the tyrosine phosphorylation of cortactin was reported to modify its activity and function,
we also examined the effect of TGF-b1 on the phosphorylated-cortactin
levels. As shown in Fig. 1(D), a decrease in the levels of tyrosinephosphorylated
cortactin was detected by immunoblotting
with PY99 or
Knockdown of cortactin induced
EMT-like changes
The alterations of the phosphorylated cortactin levels during EMT raises the possibility that cortactin may play a role in TGF-b1-induced EMT. To test
this possibility, we knocked down cortactin by RNAi in AML-12 cells, and selected two cortactin
RNAi clones [Fig. 3(A)]. E-cadherin and
ZO-1 were dramatically downregulated in cortactin RNAi cells, as compared
with control cells. As an important mesenchymal
marker, the level of fibronectin was also found
elevated in cortactin RNAi
cells. Moreover, a change from well-arranged rectangle shape to irregular
morphology was observed [Fig. 3(B)]. These data suggest that knockdown of cortactin
induced molecular and phenotypic changes essential for EMT in AML-12 cells.
Knockdown of cortactin promoted
TGF-b1-induced EMT
We next examined the effect of knockdown of cortactin
on TGF-b1-induced EMT in AML-12 cells. As shown in [Fig. 4(A)], TGF-b1 induced decrease in
the levels of E-cadherin and ZO-1 was significantly
enhanced in cortactin RNAi
cells. Similarly, the increase of vimentin, another
EMT marker, was also enhanced in cortactin RNAi cells. In addition, TGF-b1-induced apparent
morphological changes were accelerated in cortactin RNAi cells [Fig. 4(B)], indicating that knockdown of cortactin
promoted TGF-b1-induced EMT.
Knockdown of cortactin enhances
cell migratory capacity
Since EMT can increase the cell motility, we examined whether the downregulation of cortactin by RNAi has any effect on the cell migration. We performed transwell assay to compare the migration of cortactin RNAi cells with control
cells. The results showed that cortactin RNAi promoted the migration of AML-12 cells, and enhanced
TGF-b1-induced migration (Fig. 5).
Discussion
As an important cytoskeleton regulator protein, cortactin
was reported implicated in various biological events such as migration and
matrix remodeling [17], mitosis [10], and cell endocytosis
[18]. It has been reported that cortactin is required
for adhesive contacts formation through interaction with E-cadherin
and promoting F-actin accumulation in adhesive
complex; inhibition of cortactin activity
significantly reduced cadherin adhesive contact zone
extension [14]. Using yeast two-hybrid screening, Katsube
et al. [19] found that cortactin also can bind to tight junction protein ZO-1. Our
study showed that after knockdown of cortactin in
AML-12 cells, the protein levels of E-cadherin and
ZO-1 dramatically decreased, suggesting that cortactin
is required for maintaining the levels of these important molecules. Furthermore,
as we have shown, inhibition of cortactin enhanced
the expression of fibronectin, which is a specific mesenchymal marker, indicating that cortactin
is also involved in maintaining the epithelial properties of cells. However,
knockdown of cortactin did not induce the full EMT
morphology as that induced by TGF-b treatment. These
results indicate that the regulation of cortactin by
TGF-b1 was mainly involved in the process of dissociation of intercellular
junctions during EMT. Our recent studies have shown that PKA, STAT3, labile
iron pool/ROS, and other signaling events are involved in the control of EMT
[20,21]. Therefore, the data presented also suggest
that cortactin only partially involved in the
mediation of the maintenance of the epithelial phenotype.
Overexpression of cortactin and amplification of the cortactin gene, EMS1, have been found in cancer cells [22–24], and have been shown to enhance the cell motility and invasion [12,25,26]. However, there was a discrepancy in the published
reports. It has been reported that knockdown of cortactin
impaired the cell motility [27] or showed no significant effect on the cell
motility [28,29]. Jia et al. [30] reported that
knockdown of cortactin in cells with high level of cortactin tyrosine phosphorylation
enhanced cell migration in gastric cancer cells and breast cancer cells MCF7.
In the present study, we found that cortactin RNAi in AML-12 cells enhanced the cell migration in the
absence or presence of TGF-b1. Our results also suggest that the effect
of cortactin knockdown on cell migration is dependent
on a disruption of intercellular junctions and is related to EMT process. We
concluded that cortactin plays an important role in
intracellular junction formation. In tumor cells, cortactin
knockdown reportedly impaired the persistence of lamellipodial,
leading to a selective defect in motility [13]. The effect of disruption of
intercellular junctions was not evident because of the dispersive property of
tumor cells. However, in normal epithelial cells such as AML-12 cells, the
disruption of intercellular junctions may account for the dominant effect of cortactin knockdown. These observations provide an
explanation for the paradoxical effects of cortactin
knockdown.
Tyrosine phosphorylation is an important
posttranscriptional modification of cortactin. Phosphorylation of cortactin by Src kinase occurs at tyrosine
residues 421, 466, and 482 through a progressive manner with initial phosphorylation at tyrosine 421 followed by 466 [31]. The
tyrosine phosphorylation has been shown to attenuate
its ability to cross-link F-actin in vitro [32] and to inhibit
its activation on N-WASP [33], an important protein involving in actin polymerization. However, it is not clear whether
tyrosine phosphorylation of cortactin
is involved in assembly of intercellular junctions. In cortactin
over-expressing cells, cortactin has been shown to
redistribute from the cytoplasm to contact sites at the margins of cells,
concurrent with an increase in its levels of phosphorylation
[34]. Tyrosine phosphorylation of cortactin
and p130Cas (Crk-associated substrate) have been
shown to coincide with the tyrosine phosphorylation
of focal adhesion kinase during integrin-mediated
cell adhesion to extracellular matrix [35]. Our results demonstrated that TGF-b1 reduced the phosphorylated cortactin levels,
which was accompanied by the disruption of adhesion junction and tight junction
in AML-12 cells. These results suggested that the tyrosine phosphorylation
of cortactin might play a role in cell adhesion and
assembly of intercellular junctions.
One of the critical processes of EMT is disruption of adhesive junction
and tight junction, which relieves cells from mutual restriction and makes free
migration possible. As the main component of adhesive junction, the downregulation of E-cadherin is
generally accepted as a hall marker of EMT. The mechanism of downregulation of E-cadherin was
intensively studied during TGF-b1-induced EMT. Many key transcription
factors such as snail family proteins and zinc finger E-box binding family
proteins activated directly or indirectly by Smad2/3 were identified to transcriptionally inhibit the expression of E-cadherin [36]. In fact, the assembly and disruption of
adhesive junction is far more complicated than regulation of the level of E-cadherin. The ectodomain of E-cadherin interacts with other E-cadherin
in neighbor cells in homotypic manner. The cytoplasmic domain of E-cadherin
binds to b–catenin, which interacts with a–catenin
and cortactin and anchors to the actin
cytoskeleton [14,37]. Considering the important role
of cortactin in assembly of adhesion junction
complex, regulation of cortactin may be a way
involving in disruption of adhesion junction during EMT. Our study showed that
during EMT, TGF-b1 indeed decreased the phosphorylated cortactin levels, which can affect the activity of cortactin. Knockdown of cortactin
accelerated TGF-b1-induced EMT. This study suggested a new mechanism of regulating of
disassembly of adhesive junction.
Acknowledgements
We thank our lab members Weiwei Lei and Guangwen Shu for their helpful
suggestions.
Funding
This work was supported by grants from the Natural Science Foundation of
References
1 Kalluri R and Neilson EG. Epithelial-mesenchymal transition and its implications for fibrosis.
J Clin Invest 2003, 112: 1776–1784.
2 Nitta T, Kim JS, Mohuczy D and Behrns KE. Murine cirrhosis induces hepatocyte
epithelial mesenchymal transition and alterations in
survival signaling pathways. Hepatology 2008, 48: 909–919.
3 Zeisberg M, Yang C, Martino M, Duncan MB, Rieder F, Tanjore H and Kalluri R. Fibroblasts derive from hepatocytes
in liver fibrosis via epithelial to mesenchymal
transition. J Biol Chem
2007, 282: 23337–23347.
4 Dooley S, Hamzavi J, Ciuclan
L, Godoy P, Ilkavets I, Ehnert
S and Ueberham E, et al. Hepatocyte-specific
Smad7 expression attenuates TGF-b-mediated fibrogenesis
and protects against liver damage. Gastroenterology 2008, 135: 642–659.
5 Thiery JP. Epithelial-mesenchymal
transitions in tumour progression. Nat Rev Cancer
2002, 2: 442–454.
6 Grunert S, Jechlinger
M and Beug H. Diverse cellular and molecular
mechanisms contribute to epithelial plasticity and metastasis. Nat Rev Mol Cell
Biol 2003, 4: 657–665.
7 Uruno T, Liu J, Zhang P, Fan Y, Egile C, Li R and Mueller SC, et al. Activation of Arp2/3 complex-mediated actin polymerization by cortactin.
Nat Cell Biol 2001, 3: 259–266.
8 Weaver AM, Karginov AV, Kinley
AW, Weed SA, Li Y, Parsons JT and Cooper JA. Cortactin
promotes and stabilizes Arp2/3-induced actin filament
network formation. Curr Biol
2001, 11: 370–374.
9 Lua BL and Low BC. Cortactin phosphorylation
as a switch for actin cytoskeletal
network and cell dynamics control. FEBS Lett
2005, 579: 577–585. 10 Wang W, Chen
L, Ding Y, Jin J and Liao K. Centrosome separation
driven by actin-microfilaments during mitosis is
mediated by centrosome-associated tyrosine-phosphorylated cortactin. J Cell Sci 2008, 121: 1334–1343.
11 Hill A, McFarlane S, Mulligan K, Gillespie H, Draffin JE, Trimble A and Ouhtit
A, et al. Cortactin
underpins CD44-promoted invasion and adhesion of breast cancer cells to bone
marrow endothelial cells. Oncogene 2006, 25: 6079–6091.
12 Rothschild BL, Shim AH, Ammer AG, Kelley LC,
Irby KB, Head JA and Chen L, et al. Cortactin overexpression
regulates actin-related protein 2/3 complex activity,
motility, and invasion in carcinomas with chromosome 11q13 amplification.
Cancer Res 2006, 66: 8017–8025.
13 Bryce NS, Clark ES, Leysath JL, Currie JD,
Webb DJ and Weaver AM. Cortactin promotes cell motility by enhancing lamellipodial persistence. Curr Biol 2005, 15: 1276–1285.
14 Helwani FM, Kovacs EM, Paterson AD, Verma S, Ali RG, Fanning AS and Weed SA, et al. Cortactin
is necessary for E-cadherin-mediated contact
formation and actin reorganization. J Cell Biol 2004, 164: 899–910.
15 Zavadil J and Bottinger
EP. TGF-beta and epithelial-to-mesenchymal
transitions. Oncogene 2005, 24: 5764–5774.
16 Yang Y, Pan X, Lei W, Wang J and Song J. Transforming growth factor-b1 induces
epithelial-to-mesenchymal transition and apoptosis
via a cell cycle-dependent mechanism. Oncogene 2006,
25: 7235–7244.
17 Weaver AM. Cortactin in tumor invasiveness. Cancer Lett
2008, 265: 157–166.
18 Chen L, Wang ZW, Zhu JW and Zhan X. Roles of cortactin,
an actin polymerization mediator, in cell endocytosis. Acta Biochim Biophys Sin 2006, 38: 95–103.
19 Katsube T, Takahisa
M, Ueda R, Hashimoto N, Kobayashi M and Togashi S. Cortactin associates with the cell-cell junction protein ZO20 Yang Y, Pan X, Lei W, Wang J, Shi J, Li F and Song J. Regulation of
transforming growth factor-b1-induced apoptosis and epithelial-to-mesenchymal transition by protein kinase
A and signal transducers and activators of transcription 3. Cancer Res 2006,
66: 8617–8624.
21 Zhang KH, Tian HY, Gao
X, Lei WW, Hu Y, Wang DM and Pan XC, et al. Ferritin
heavy chain-mediated iron homeostasis and subsequent increased reactive oxygen
species production are essential for epithelialmesenchymal
transition. Cancer Res 2009, 16: 5340–5348.
22 Campbell DH, deFazio A, Sutherland RL and
Daly RJ. Expression and tyrosine phosphorylation of EMS23 Freier K, Sticht C,
Hofele C, Flechtenmacher C,
Stange D, Puccio L and Toedt G, et al. Recurrent co-amplification of cytoskeletonassociated
genes EMS1 and SHANK2 with CCND24 Zhang LH, Tian B, Diao
LR, Xiong YY, Tian SF,
Zhang BH and Li WM, et al. Dominant expression of 85-kDa form of cortactin
in colorectal cancer. J Cancer Res Clin Oncol 2006, 132: 113–120.
25 Patel AS, Schechter GL, Wasilenko WJ and
Somers KD. Overexpression of EMS1/cortactin in NIH3T3
fibroblasts causes increased cell motility and invasion in vitro. Oncogene
1998, 16: 3227–3232.
26 Kowalski JR, Egile C, Gil S, Snapper SB, Li R
and Thomas SM. Cortactin regulates cell migration
through activation of N-WASP. J Cell Sci 2005, 118:
79–87.
27 van Rossum AG, Moolenaar WH and Schuuring E. Cortactin affects cell migration by regulating
intercellular adhesion and cell spreading. Exp Cell Res 2006, 312: 1658–1670.
28 Lua BL and Low BC. BPGAP1 interacts with cortactin and facilitates its translocation to cell
periphery for enhanced cell migration. Mol Biol Cell
2004, 15: 2873–2883.
29 Barroso C, Rodenbusch
SE, Welch MD and Drubin DG. A role
for cortactin in Listeria
monocytogenes invasion of NIH 3T3 cells, but
not in its intracellular motility. Cell Motil
Cytoskeleton 2006, 63: 231–243.
30 Jia L, Uekita T and
Sakai R. Hyperphosphorylated cortactin
in cancer cells plays an inhibitory role in cell motility. Mol Cancer Res 2008,
6: 654–662.
31 Head JA, Jiang D, Li M, Zorn LJ, Schaefer EM, Parsons JT and Weed SA. Cortactin tyrosine phosphorylation
requires Rac1 activity and association with the cortical actin
cytoskeleton. Mol Biol Cell 2003, 14: 3216–3229.
32 Huang C, Ni Y, Wang T, Gao Y, Haudenschild CC and Zhan X. Down-regulation of the
filamentous actin cross-linking activity of cortactin by Src-mediated
tyrosine phosphorylation. J Biol
Chem 1997, 272: 13911–13915.
33 Martinez-Quiles N, Ho HY, Kirschner
MW, Ramesh N and Geha RS. Erk/Src
phosphorylation of cortactin
acts as a switch on-switch off mechanism that controls its ability to activate
N-WASP. Mol Cell Biol 2004, 24: 5269–5280.
34 van Damme H, Brok
H, Schuuring-Scholtes E and Schuuring
E. The redistribution of cortactin into cell-matrix
contact sites in human carcinoma cells with 11q13 amplification is associated
with both overexpression and post-translational
modification. J Biol Chem
1997, 272: 7374–7380.
35 Vuori K and Ruoslahti
E. Tyrosine phosphorylation of p130Cas and cortactin accompanies integrin-mediated
cell adhesion to extracellular matrix. J Biol Chem 1995, 270: 22259–22262.
36 Xu J, Lamouille S
and Derynck R. TGF-b-induced epithelial to
mesenchymal transition. Cell Res 2009, 19:
156–172.
37 Niessen CM and Gottardi
CJ. Molecular components of the adherens
junction. Biochim Biophys
Acta 2008, 1778: 562–571.
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